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WO2004035819A2 - Oligonucleotides utiles dans la detection d'acides nucleiques d'interet - Google Patents

Oligonucleotides utiles dans la detection d'acides nucleiques d'interet Download PDF

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Publication number
WO2004035819A2
WO2004035819A2 PCT/DK2003/000715 DK0300715W WO2004035819A2 WO 2004035819 A2 WO2004035819 A2 WO 2004035819A2 DK 0300715 W DK0300715 W DK 0300715W WO 2004035819 A2 WO2004035819 A2 WO 2004035819A2
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nucleic acid
nucleic acids
population
lna
exon
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PCT/DK2003/000715
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WO2004035819A8 (fr
WO2004035819A3 (fr
Inventor
Sakari Kauppinen
Carsten Alsbo
Peter Stein Nielsen
Daniel Charlton Jeffares
Tobias Mourier
Søren MØRK
Peter Arctander
Niels Tommerup
Niels Tolstrup
Henrik Vissing
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Exiqon A/S
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Priority to AU2003273778A priority Critical patent/AU2003273778A1/en
Priority to EP03757735A priority patent/EP1556510A2/fr
Publication of WO2004035819A2 publication Critical patent/WO2004035819A2/fr
Publication of WO2004035819A3 publication Critical patent/WO2004035819A3/fr
Publication of WO2004035819A8 publication Critical patent/WO2004035819A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the invention relates to nucleic acids and methods for expression profiling of mRNAs, identi-fying and profiling of particular mRNA splice variants, and detecting mutations, deletions, or duplications of particular exons, e.g., alterations associated with a disease such as cancer, in a nucleic acid sample, e.g., a patient sample.
  • the invention furthermore relates to methods for detecting nucleic acids by fluorescence in situ hybridization.
  • oligonucleotides e.g., oligonucleotide arrays
  • DNA chip technology utilizes minituarized arrays of DNA molecules immobilized on solid surfaces for biochemical analyses.
  • the power of DNA microarrays as experimental tools relies on the specific molecular recognition via complementary base-pairing, which makes them highly useful for massive parallel analyses. In the post-genomic era, microarray technology has thus become the method of choice for many hybridization-based assays, such as expression profiling, SNP detection, DNA re-sequencing, and genotyping on a genomic scale.
  • Expression microarrays are capable of profiling gene expression patterns of tens of thousands of genes in a single experiment. Hence, this technology provides a powerful tool for deciphering complex biological systems, and thereby greatly facilitates research in basic biology and living processes, as well as disease diagnostics, theranostics, and drug development.
  • the mRNAs are distributed in three frequency classes: (i) superprevalent (10-20% of the total mRNA mass); (ii) intermediate (40-45%); and (iii) low- abundant mRNAs (40-45%). It is therefore of utmost importance that the dynamic range and sensitivity of the expression arrays are optimal, especially when analyzing expression levels of messages or mRNA splice variants belonging to the low-abundant class.
  • GeneChip manufacturing begins with a 5 -inch square quartz wafer. Initially the quartz is washed to ensure uniform hydroxylation across its surface. The wafer is placed in a bath of silane, which reacts with the hydroxyl groups of the quartz and forms a matrix of covalently linked molecules. The distance between these silane molecules determines the probes' packing density, allowing arrays to hold over 500,000 features within 1.28 square centimeters.
  • the principal disadvantage of this method is that a significant amount of chip design work and cost is associated with the mask design. Once a set of masks has been made, a large number of chips can be produced at a reasonable cost.
  • the current pricing of oligonucleotide arrays available from Affymetrix are in the range of 5-10 fold more expensive than cDNA microarrays.
  • DNA-DNA hybridization using oligonucleotide chips is clearly different from that of cDNA microarrays.
  • Hybridizations involving oligos are much more sensitive to the GC content of individual heteroduplexes.
  • single base mismatches have a pronounced effect on the hybridization reassociation of short oligos, and point mutations can thus be readily detected using oligo chips.
  • cDNA microarrays cDNA microarrays containing large DNA segments such as cDNAs are generated by physically depositing small amounts of each DNA of interest onto known locations on glass surfaces. Two technologies for printing microarrays are (1) mechanical microspotting, and (2) ink-jetting.
  • Microspotting has been extensively used at, e.g., Stanford University, and it utilizes pins or capillaries to deposit small quantities of DNA onto known addresses using motion control systems. Recent advances in microspotting technology using modern arraying robots allow for the preparation of 100 microarrays containing over 10,000 features in less than 12 hours. A DNA arrayer is relatively easy to set up, and the cost is usually low compared to on-chip oligoarrayers. cDNA microarrays are capable of profiling gene expression patterns of tens of thousands of genes in a single experiment.
  • the two samples are first labeled using two different fluorescent dyes such as Cy-3 and Cy-5.
  • the labeled samples are mixed and hybridized to the clones on the array slide.
  • laser excitation of the incorporated, fluorescent target molecules yields an emission with a characteristic spectra, which is measured with a confocal laser scanner.
  • the monochrome images from the scanner are imported to the software in which the images are pseudo-colored and merged. Data from a single hybridization is viewed as a normalized ratio in which significant deviations from the ratio are indicative of either increased or decreased expression levels relative to the reference sample. Data from multiple experiments can be examined using any number of data mining tools.
  • Current status of array technology e.g., the total mRNA isolated from two different cell populations.
  • cDNA microarrays originally developed by Pat Brown and co-workers at the Stanford University, are sensitive, but may not be sufficiently specific with respect to, e.g., discrimination of homologous transcripts in gene families and alternatively spliced isoforms.
  • the Affymetrix GeneChip system is specific, but may not be sensitive enough. This lack of sensitivity may explain why
  • Affymetrix uses 16x 26-mer perfect match capture probes together with 16x25-mer mismatch probes per transcript in its expression profiling chips resulting in enormous data sets in genome- wide arrays. Therefore, the functional genomics field is in the process of switching, as they run out of samples, from existing PCR-amplified cDNA fragment libraries for microarraying to custom longmer oligonucleotide arrays comprising transcript-specific oligonucleotide capture probes typically in the range of 30-mers to 80-mers, thus addressing both specificity and sensitivity.
  • RNA and protein sequences in order to elucidate gene expression in all its complexity.
  • a common feature for eukaryotic genes is that they are composed of protein-encoding exons and introns. Introns (intra-genic-regions) are non-coding DNA which interrupt the exons. Introns are characterized by being excised from the pre-mRNA molecule in RNA splicing, as the sequences on each side of the intron are spliced together. RNA splicing not only provides functional mRNA, but is also responsible for generating additional diversity.
  • RNA-mediated gene regulation is widespread in higher eukaryotes and complex genetic phenomena like RNA interference, co-suppression, transgene silencing, imprinting, methylation, and possibly position-effect variegation and transvection, all involve intersecting pathways based on or connected to RNA signalling (Mattick 2001; EMBO reports 2, 11 : 986-991).
  • RNA interference co-suppression
  • transgene silencing co-suppression
  • imprinting imprinting
  • methylation imprinting
  • methylation possibly position-effect variegation and transvection
  • the present invention provides novel tools, in which non-naturally occurring nucleic acids, such as LNA oligonucleotides, can be designed to silence or modulate the regulation of a given mRNA by non-coding antisense RNA, by designing a complementary sense LNA oligonucleotide for the regulatory antisense RNA.
  • This has a high potential in target identification, target validation and therapeutic use of LNA oligonucleotides as modulating and silencing sense nucleic acid agents.
  • Desirable methods can distinguish between mRNA splice variants and quantitate the amount of each variant in a sample.
  • Other desirable methods can detect differences in expressions patterns between patient nucleic acid samples and nucleic acid standards.
  • the present invention demonstrates the usefulness of LNA-modified oligonucleotides in the construction of highly specific and sensitive microarrays for expression profiling (e.g., mRNA splice variant detection) and comparative genomic hybridization.
  • the invention provides novel technology platforms based on nucleic acids with LNA or other high affinity nucleotides for sensitive and specific assessment of alternative splicing using microarray technology.
  • LNA microarrays are able to discriminate between highly homologous as well as differentially spliced transcripts.
  • the invention furthermore provides methods for highly sensitive and specific nucleic acid detection by fluorescence in situ hybridization using LNA-modified oligonucleotides.
  • the present methods greatly facilitate the analysis of gene expression patterns from a particular species, tissue, cell type.
  • the analysis of the human spliceome provides important information for pharmacogenetics.
  • the present methods are highly valuable in medical research and diagnostics as well as in dmg development and toxicological studies.
  • the invention features populations of high affinity nucleic acids that have duplex stabilizing properties and thus are useful for a variety of nucleic acid detection, amplification, and hybridization methods (e.g., expression or mRNA splice variant profiling).
  • oligonucleotides contain novel nucleotides created by combining specialized synthetic nucleobases with an LNA backbone, thus creating high affinity oligonucleotides with specialized properties such as reduced sequence discrimination for the complementary strand or reduced ability to form intramolecular double stranded structures.
  • the invention also provides improved methods for identifying nucleic acids in a sample and for classifying a nucleic acid sample by comparing its pattern of hybridization to an array to the corresponding pattern of hybridization of one or more standards to the array (e.g., comparative genomic hybridization).
  • modified bases have decreased ability to self-anneal or to form duplexes with oligonucleotides containing one or more modified bases.
  • the invention also provides arrays of nucleic acids containing these modified bases that have a decreased variance in melting temperature and/or an increased capture efficiency compared to naturally-occurring nucleic acids. These arrays as well as the oligonucleotides in solution can be used in a variety of applications for the detection, characterization, identification, and/or amplification of one or more target nucleic acids. These oligonucleotides and oligonucleotides of the invention in general can also be used for solution assays, such as homogeneous assays. Merged Probes
  • the invention features a non-naturally-occurring nucleic acid with a melting temperature that is at least 3, 5, 8, 10, 12, 15, 20, 25, 30, 35, or 40°C higher than that of the corresponding control nucleic acid with 2'-deoxynucleotides.
  • the nucleic acid is capable of hybridizing to a first region within a first exon of a target nucleic acid and to a second region within a second exon of the target nucleic acid that is adjacent to the first exon.
  • the invention provides a non-naturally-occurring nucleic acid with a melting temperature that is at least 3, 5, 8, 10, 12, 15, 20, 25, 30, 35, or 40°C higher than that of the corresponding control nucleic acid with 2'-deoxynucleotides.
  • the nucleic acid hybridizes to a first region within an exon of a target nucleic acid and to a second region within an intron of the target nucleic acid that is adjacent to the exon.
  • the invention features a non-naturally-occurring nucleic acid with a melting temperature that is at least 3, 5, 8, 10, 12, 15, 20, 25, 30, 35, or 40°C higher than that of the corresponding control nucleic acid with 2'-deoxynucleotides.
  • the nucleic acid hybridizes to a first region within a first intron of a target nucleic acid and to a second region within a second intron of the target nucleic acid that is adjacent to the first intron.
  • the invention provides a nucleic acid that is a non-naturally- occurring nucleic acid with a capture efficiency that is at least 10, 25, 50, 100, 150, 200, 500, 800, 1000, or 1200% greater than that of a corresponding control nucleic acid with 2'- deoxynucleotides at the temperature equal to the melting temperature of the nucleic acid.
  • the nucleic acid hybridizes to a first region within a first exon of a target nucleic acid and to a second region within a second exon of the target nucleic acid that is adjacent to the first exon.
  • the invention features a nucleic acid that is a non-naturally- occurring nucleic acid with a capture efficiency that is at least 10, 25, 50, 100, 150, 200, 500, 800, 1000, or 1200% greater than that of a corresponding control nucleic acid with 2'- deoxynucleotides at the temperature equal to the melting temperature of the nucleic acid.
  • the nucleic acid hybridizes to a first region within an exon of a target nucleic acid and to a second region within an intron of the target nucleic acid that is adjacent to the exon.
  • the invention provides a nucleic acid that is a non-naturally- occurring nucleic acid with a capture efficiency that is at least 10, 25, 50, 100, 150, 200, 500, 800, 1000, or 1200% greater than that of a corresponding control nucleic acid with 2'- deoxynucleotides at the temperature equal to the melting temperature of the nucleic acid.
  • the nucleic acid hybridizes to a first region within a first intron of a target nucleic acid and to a second region within a second intron of the target nucleic acid that is adjacent to the first intron.
  • the nucleic acids of the invention featuring a non-naturally occurring nucleic acid exhibit increased duplex stability due to slower rates of dissociation of the nucleic acid complexes (the off-rate) (Christensen et al. 2001, Biochem. J. 354: 481-484).
  • the structure of desirable adenosine, thymine, guanine and cytosine analogs are those disclosed in PCT Publication No. WO 97/12896, Formula 5, 6, 7, 8, 9, 10, 11, 12 and 13. These modified bases may be incorporated as part of an LNA, DNA, or RNA unit and used any of the oligomers of the invention.
  • the invention features a nucleic acid that is an LNA (i.e., a nucleic acids with one or more LNA units) and that hybridizes to a first region within a first exon of a target nucleic acid and to a second region within a second exon of the target nucleic acid that is adjacent to the first exon.
  • LNA i.e., a nucleic acids with one or more LNA units
  • the invention features a nucleic acid that is an LNA and that hybridizes to a first region within an exon of a target nucleic acid and to a second region within an intron of the target nucleic acid that is adjacent to the exon.
  • the invention provides nucleic acid that is an LNA and that hybridizes to a first region within a first intron of a target nucleic acid and to a second region within a second intron of the target nucleic acid that is adjacent to the first intron.
  • the length of the segment of the nucleic acid hybridizing to the first region and the length of the segment of the nucleic acid hybridizing to the second region are between 3 and 50 nucleotides, 10 and 40 nucleotides, or 20 and 30 nucleotides, inclusive.
  • the length of the segment of the nucleic acid hybridizing to the first region and the length of the segment of the nucleic acid hybridizing to the second region may be the same length or different lengths.
  • the nucleic acid containing LNA units are symmetrically spaced on both sides of a junction between either two exons, an exon and an intron, or two introns, or alternatively, the nucleic acid containing LNA units are spaced on both sides of a junction based on equalized duplex melting temperatures of the segments.
  • the nucleic acid has one or more LNA units within 5, 4, 3, 2, or 1 nucleotides of a junction between either two exons, an exon and an intron, or two introns.
  • the invention features a population of nucleic acids that includes one or more nucleic acids of any one of the above aspects.
  • the invention features a non-naturally-occurring nucleic acid with a melting temperature that is at least 3, 5, 8, 10, 12, 15, 20, 25, 30, 35, or 40°C higher than that of the corresponding control nucleic acid with 2'-deoxynucleotides.
  • the nucleic acid hybridizes to only one exon or to only one intron of a target nucleic acid.
  • the invention features a non-naturally-occurring nucleic acid with a capture efficiency that is at least 10, 25, 50, 100, 150, 200, 500, 800, 1000, or 1200% greater than that of a corresponding control nucleic acid with 2'-deoxynucleotides at the temperature equal to the melting temperature of the nucleic acid.
  • the nucleic acid hybridizes to only one exon or to only one intron of a target nucleic acid.
  • the invention features a nucleic acid that is an LNA and that hybridizes to only one exon or to only one intron of a target nucleic acid.
  • nucleic acid does not hybridize to both an exon and an intron.
  • the invention features a population of nucleic acids that includes one or more nucleic acids of any one of the above aspects.
  • the invention features a pharmaceutical composition that includes one or more of the nucleic acids of the invention and a pharmaceutically acceptable carrier, such as one of the carriers described herein.
  • the invention features a population of two or more nucleic acids of the invention.
  • the populations of nucleic acids of the invention may contain any number of unique molecules.
  • the population may contain as few as 10, 10 2 , 10 4 , or 10 5 unique molecules or as many as 10 , 10 , 10 or more unique molecules.
  • at least 1, 5, 10, 50, 100 or more of the polynucleotide sequences are a non- naturally-occurring sequence.
  • at least 20, 40, or 60% of the unique polynucleotide sequences are non-naturally-occurring sequences.
  • the nucleic acids are all the same length; however, some of the molecules may differ in length. Desirable Embodiments of Any of the Above Aspects
  • the length of one or more nucleic acids is between 15 and 150 nucleotides, 5 and 100 nucleotides, 20 and 80 nucleotides, or 30 and 60 nucleotides in length, inclusive.
  • the nucleic acid is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 nucleotides or at least 60, 70, 80, 90, 100, 120, or 130 nucleotides in length.
  • the nucleic acid is between 8 and 40 nucleotides, such as between 9 and 30, or 12 and 25, or 15 and 20 nucleotides.
  • At least 5, 10, 15, 20, 30, 40, 50, 60, or 70% of the nucleotides in the nucleic acid are LNA units.
  • every second nucleotide, every third, every fourth nucleotide, every fifth nucleotide, or every sixth nucleotide in the nucleic acid is an LNA unit.
  • every second and every third nucleotide, (ii) every second and every fourth nucleotide, (iii) every second and every fifth nucleotide, (iv) every second and every sixth nucleotide, (v) every third and every fourth nucleotide, (vi) every third and every fifth nucleotide, (vii) every third and every sixth nucleotide, (viii) every fourth and every fifth nucleotide, (ix) every fourth and every sixth nucleotide, and/or (x) every fifth and every sixth nucleotide in the nucleic acid is an LNA unit.
  • every second, every third, and every fourth nucleotide in the nucleic acid is an LNA unit.
  • the nucleic acids of the invention have one or more of the following substitution patterns which is repeated throughout the nucleic acids: XxXx, xXxX, XxxXxx, xXxxXx, xxXxxX, XxxxXxxx, xXxxxXxx, xxXxxxXx, or xxxXxxxX in which "X” denotes an LNA unit and "x" denotes a DNA or RNA unit.
  • the nucleotides that are not LNA units are naturally-occuring DNA or RNA nucleotides.
  • the nucleic acid comprises two or more contiguous LNA units. Desirably, the nucleic acid comprises at least 2, 3, 4, 5, 6, 7, or 8 contiguous LNA units. In desirable embodiments, the number of contiguous LNA units is between 5 and 20% or 10 and 15% of the total length of the nucleic acid. In a particular embodiment, 5 contiguous nucleotides of a 50-mer merged probe are LNA units. In one embodiment, the nucleic acid does not have greatly extended stretches of modified DNA or RNA residues, e.g. greater than about 4, 5, 6, 7, or 8 consecutive modified DNA or RNA residues. According to this embodiment, one or more non-modified DNA or RNA units are present after a consecutive stretch of about 3, 4, 5, 6, 7, or 8 modified nucleic acids.
  • nucleic acids have an LNA substitution pattern that results in the formation of negligible secondary structure by the nucleic acids with itself.
  • the nucleic acids do not form hairpins or do not form other secondary structures that would otherwise inhibit or prevent their binding to a target nucleic acid.
  • opposing nucleotides in a palindrome pair or opposing nucleotides in inverted repeats or in reverse complements are not both LNA units.
  • the nucleic acids in the first population form less than 3, 2, or 1 intramolecular base-pairs or base-pairs between two identical molecules.
  • the nucleic acid does not have LNA-5- nitroindole: LNA-5-nitroindole intramolecular base-pairs.
  • At least one LNA unit (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA units) in the nucleic acid hybridizes to a first region within a first exon of a target nucleic acid and at least one LNA unit (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA units) in the nucleic acid hybridizes to a second region within a second exon of the target nucleic acid that is adjacent to the first exon.
  • the number of LNA units that bind to each region can be the same or different.
  • the 5' terminal nucleotide of the nucleic acid is or is not an LNA unit.
  • the 3' terminal nucleotide of the nucleic acid is not an LNA unit (e.g., the nucleic acid may contain a 3' terminal naturally-occurring nucleotide).
  • the nucleic acid can distinguish between different nucleic acids (e.g., mRNA splice variants) that cannot be distinguished using a naturally-occurring control nucleic acid (e.g., a control nucleic acid that consists of only 2'-deoxynucleotides such as a control nucleic acid of the same length as the nucleic acid of the invention).
  • a naturally-occurring control nucleic acid e.g., a control nucleic acid that consists of only 2'-deoxynucleotides such as a control nucleic acid of the same length as the nucleic acid of the invention.
  • the hybridization intensity of the nucleic acid for an exon of interest is at least 2, 3, 4, 5, 6, or 10 fold greater than the hybridization intensity of the nucleic acid for another exon in the same target nucleic acid (e.g., mRNA) or in another nucleic acid.
  • the hybridization intensity of the nucleic acid for target nucleic acid is at least 2, 3, 4, 5, 6, or 10 fold greater than the hybridization intensity for a non-target nucleic acid with less than 99, 95, 90, 80, 70, or 60% sequence identity to the target nucleic acid.
  • nucleic acids of the population or all of the nucleic acids of a subpopulation of the population are the same length.
  • the population includes one or more nucleic acids of a different length.
  • longer nucleic acids contain one or more nucleotides with universal bases.
  • nucleotides with universal bases can be used to increase the thermal stability of nucleic acids that would otherwise have a thermal stability lower than some or all of the nucleic acids in the population.
  • one or more nucleic acids have a universal base located at the 5' or 3' terminus of the nucleic acid.
  • one or more (e.g., 2, 3, 4, 5, 6, or more) universal bases are located at the 5' and 3' termini of the nucleic acid. Desirably, all of the nucleic acids in the population have the same number of universal bases. Desirable universal bases include inosine, pyrene, 3-nitropyrrole, and 5-nitroindole.
  • the nucleic acid has at least one LNA A or LNA T. In some embodiments, each nucleic acid has at least one LNA A or LNA T. Desirably, all of the adenine and thymine-containing nucleotides in the LNA are LNA A and LNA T, respectively.
  • a nucleic acid with a increased capture efficiency or melting temperature compared to a control nucleic acid has at least one LNA T or LNA C. In some embodiments, all of the thymine and cytosine-containing nucleotides in the LNA are LNA T and LNA C, respectively. In some embodiments, a nucleic acid with an increased specificity or decreased self-complementarity compared to a control nucleic acid has at least one LNA A or LNA C. In some embodiments, all of the adenine and cytosine-containing nucleotides in the LNA are LNA A and LNA C, respectively. Desirably, at least 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100% of the nucleic acids in the population have one ore more LNA units.
  • the LNA has at least one 2,6,-diaminopurine, 2- aminopurine, 2-thio-thymine, 2-thio-uracil, inosine, or hypoxanthine base.
  • the LNA has a nucleotide with a 2'0, 4'C-methylene linkage between the 2' and 4' position of a sugar moiety.
  • one or more nucleic acids in the first population are LNA/DNA, LNA/RNA, or LNA/DNA/RNA chimeras.
  • the variance in the melting temperature of the population is at least 10, 20, 30, 40, 50, 60, or 70% less than the variance in the melting temperature of the corresponding control population of nucleic acids of the same length with 2'-deoxynucleotides (e.g., DNA nucleotides) instead of LNA units or other modified or non-naturally-occurring units.
  • the standard deviation in melting temperature is less than 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, or 6.
  • the range in melting temperatures for nucleic acids in the population is less than 70, 60, 50, 40, 30, or 20°C.
  • the variance in the melting temperature of the population is less than 59, 50, 40, 30, 25, 20, 15, 10, or 5.
  • the nucleic acids are covalently bonded to a solid support.
  • the nucleic acids are in a predefined arrangement.
  • the first population has at least 10; 100; 1,000; 5,000; 10,000; 100,000; or 1,000,000 different nucleic acids.
  • the nucleic acids in the population together hybridize to at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% of the exons of a target nucleic acid.
  • the population includes nucleic acids that together hybridize to at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% of the nucleic acids expressed by a particular cell or tissue.
  • the population includes nucleic acids that together hybridize to at least one exon from at least 1, 5, 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100% of the nucleic acid sequences expressed by a particular cell or tissue at a given point in time (e.g., an expression array with sequences corresponding to the sequences of mRNA molecules expressed by a particular cell type or a cell under a particular set of conditions).
  • the plurality of nucleic acids are used as PCR primers or FISH probes.
  • Desirable modified bases of the present invention when incorporated into the central position of a 9-mer oligonucleotide (all other eight residues or units being natural DNA or RNA units with natural bases) exhibit a T m difference equal to or less than about 15, 12, 10, 9, 8, 7, 6, 5, 4, 3 or 2°C upon hybridizing to the four complementary oligonucleotide variants that are identical except for the unit corresponding to the LNA unit, where each variant has one of the natural bases uracil, cytosine, thymine, adenine or guanine. That is, the highest and the lowest T m (referred to herein as the T m differential) obtained with such four complementary sequences is 15, 12, 10, 9, 8, 7, 6, 5, 4, 3 or 2°C or less.
  • Modified nucleic acid oligomers of the invention desirably contain at least one LNA unit, such as an LNA unit with a modified nucleobase.
  • Modified nucleobases or nucleosidic bases desirably base-pair with adenine, guanine, cytosine, uracil, or thymine.
  • Exemplary oligomers contain 2 to 100, 5 to 100, 4 to 50, 5 to 50, 5 to 30, or 8 to 15 nucleic acid units.
  • one or more LNA units with natural nucleobases are incorporated into the oligonucleotide at a distance from the LNA unit having a modified base of 1 to 6 (e.g., 1 to 4) bases.
  • At least two LNA units with natural nucleobases are flanking an LNA unit having a modified base.
  • at least two LNA units independently are positioned at a distance from the LNA unit having the modified base of 1 to 6 (e.g., 1 to 4 bases).
  • Desirable modified nucleobases or nucleosidic bases for use in nucleic acid compositions of the invention include optionally substituted carbon alicyclic or carbocyclic aryl groups (i.e., only carbon ring members), particularly multi-ring carbocyclic aryl groups such as groups having 2, 3, 4, 5, 6, 7, or 8 linked, particularly fused carbocyclic aryl moieties.
  • Optionally substituted pyrene is also desirable.
  • Such nucleobases or nucleosidic bases can provide significant performance results, as demonstrated in the examples which follow.
  • Heteroalicyclic and heteroaromatic nucleobases or nucleosidic bases also are suitable.
  • the carbocyclic moiety is linked to the 1 '-position of the LNA unit through a linker (e.g., a branched or straight alkylene or alkenylene).
  • a linker e.g., a branched or straight alkylene or alkenylene.
  • Desirable LNA units have a carbon or hetero alicyclic ring with four to six ring members, e.g. a furanose ring, or other alicyclic ring structures such as a cyclopentyl, cycloheptyl, tetrahydropyranyl, oxepanyl, tetrahydrothiophenyl, pyrrolidinyl, thianyl, tliiepanyl, piperidinyl, and the like.
  • At least one ring atom of the carbon or hetero alicyclic group is taken to form a further cyclic linkage to thereby provide a multi- cyclic group.
  • the cyclic linkage may include one or more, typically two atoms, of the carbon or hetero alicyclic group.
  • the cyclic linkage also may include one or more atoms that are substituents, but not ring members, of the carbon or hetero alicyclic group.
  • Other desirable LNA units are compounds having a substituent on the 2'-position of the central sugar moiety (e.g., ribose or xylose), or derivatives thereof, which favors the C3'-endo conformation, commonly referred to as the North (or simply N for short) conformation.
  • LNA units include ENA (2'-O,4'-C-ethylene-bridged nucleic acids such as those disclosed in WO 00/47599) units as well as non-bridged riboses such as 2'-F or 2'-O-methyl.
  • an exemplary control nucleic acid has ⁇ -D-2- deoxyribose instead of one or more bicyclic or sugar groups of a LNA unit or other modified or non-naturally-occurring units in a nucleic acid of the first population.
  • the nucleic acid or population of the invention and the control nucleic acid or population only have naturally-occurring nucleobases.
  • the capture efficiency of the corresponding control nucleic acid is calculated as the average capture efficiency for all of the nucleic acids that have either A, T, C, G or mC (methyl Cytosin) in each position corresponding to a non-naturally-occurring nucleobase in the nucleic acid in the first population.
  • A, T, C, G or mC methyl Cytosin
  • the invention features a complex of one or more target nucleic acids and nucleic acid of the invention (e.g., nucleic acid probes) in which one or more target nucleic acids are hybridized to a plurality of nucleic acids of the invention. Desirably, at least 2, 3, 4, 5, 6, 7, 10, 15, 20, 30, or 40 different target nucleic acids are hybridized.
  • the target nucleic acids are cDNA molecules reverse transcribed from a patient sample or cRNA molecules amplified from a patient sample using a T7 RNA polymerase-based linear amplification system or the like. The target nucleic acids are labeled prior to hybridization to the nucleic acids of invention.
  • the invention features a method for detecting the presence of one or more target nucleic acids in a sample.
  • This method involves incubating a nucleic acid sample with one or more nucleic acids of the invention under conditions that allow at least one target nucleic acid to hybridize to at least one of the nucleic acids of the invention. Desirably, hybridization is detected for at least 2, 3, 4, 5, 6, 8, 10, or 12 target nucleic acids.
  • the method further includes contacting the target nucleic acid with a second nucleic acid or a population of second nucleic acids that binds to a different region of the target molecule than the first nucleic acid.
  • the method further involves identifying one or more hybridized target nucleic acids and/or determining the amount of one or more hybridized target nucleic acids.
  • the method further includes determining the presence or absence of an mRNA splice variant of interest in the sample and/or determining the presence or absence of a mutation, deletion, and/or duplication of an exon of interest.
  • the mutation, deletion, and/or duplication is indicative of a disease, disorder, or condition, such as cancer.
  • the method is repeated under one or more different incubation conditions.
  • the method is repeated at 1, 3, 5, 8, 10, 15, 20, 30, 40 or more different temperatures, cation concentrations (e.g., concentrations of monovalent cations such as Na + and K + or divalent cations such as Mg 2+ and Ca 2+ ), denaturants (e.g., hydrogen bond donors or acceptors that interfere with the hydrogen bonds keeping the base- pairs together such as formamide or urea).
  • cation concentrations e.g., concentrations of monovalent cations such as Na + and K + or divalent cations such as Mg 2+ and Ca 2+
  • denaturants e.g., hydrogen bond donors or acceptors that interfere with the hydrogen bonds keeping the base- pairs together such as formamide or urea.
  • the method also includes identifying the target nucleic acid hybridized to the nucleic acids of the invention and/or determining the amount of the target nucleic acid hybridized to the nucleic acids of the invention.
  • the target nucleic acids are labeled with a fluorescent group.
  • the labeling is repeated using different fluorescent groups (e.g., labelling for so-called dye-swap labeling experiments).
  • the determination of the amount of bound target nucleic acid involves one or more of the following: (i) adjusting for the varying intensity of the excitation light source used for detection of the hybridization, (ii) adjusting for photobleaching of the fluorescent group, and/or (iii) comparing the fluorescent intensity of the target nucleic acid(s) hybridized to the nucleic acids of the invention of nucleic acids to the fluorescent intensity of a different sample of nucleic acids hybridized to the nucleic acids of the invention (e.g., a different sample hybridized to the same population of nucleic acids of the invention on the same or a different solid support such as the same chip or a different chip).
  • this comparison in fluorescent intensity involves adjusting for a difference in the amount of the nucleic acids of the invention used for hybridization to each sample and/or adjusting for a difference in the
  • 9+ buffer e.g., a difference in Mg concentration
  • the target nucleic acids are cDNA molecules reverse transcribed from a patient sample or cRNA molecules amplified using a T7 RNA polymerase-based linear amplification system or the like from a patient sample.
  • the sample has nucleic acids that are amplified using one or more primers specific for an exon of a target nucleic acid, and the method involves determining the presence or absence of an mRNA splice variant with the exon in the sample.
  • one or more of the primers are specific for an exon or exon-exon junction of a pathogen of interest, and the method involves determining the presence or absence of a nucleic acid with the exon in the sample.
  • the nucleic acids of the invention are covalently bonded to a solid support by reaction of a nucleoside phosphoramidite with an activated solid support, and subsequent reaction of a nucleoside phosphoramide with an activated nucleotide or nucleic acid bound to the solid support.
  • the solid support or the growing nucleic acid bound to the solid support is activated by illumination, a photogenerated acid, or electric current.
  • the invention features a method for amplifying a target nucleic acid molecule.
  • the method involves (a) incubating a first nucleic acid of the invention with a target nucleic acid under conditions that allow the first nucleic acid to bind the target nucleic acid; and (b) extending the first nucleic acid with the target nucleic acid as a template.
  • the method further involves contacting the target nucleic acid with a second nucleic acid (e.g., a second nucleic acid of the invention) that binds to a different region of the target nucleic acid than the first nucleic acid.
  • the sequence of the target nucleic acid is known or unknown.
  • the invention features a method of detecting a nucleic acid of a pathogen (e.g., a nucleic acid in a sample such as a blood or urine sample from a mammal).
  • a nucleic acid probe of the invention e.g., a probe specific for an exon or a mRNA from a particular pathogen or family of pathogens
  • the probe is desirably at least 60, 70, 80, 90, 95, or 100% complementary to a nucleic acid of a pathogen (e.g., a bacteria, virus, or yeast such as any of the pathogens described herein).
  • Hybridization between the probe and a nucleic acid in the sample is detected, indicating that the sample contains the corresponding nucleic acid from a pathogen.
  • the method is used to determine what strain of a pathogen has infected a mammal (e.g., a human) by determining whether a particular nucleic acid is present in the sample.
  • the probe has a universal base in a position corresponding to a nucleotide that varies among different strains of a pathogen, and thus the probe detects the presence of a nucleic acid from any of a multiple of pathogenic strains.
  • the invention features a method for classifying a test nucleic acid sample including target nucleic acids.
  • This method involves (a) incubating a test nucleic acid sample with a one or more nucleic acids of the invention under conditions that allow at least one of the nucleic acids in the test sample to hybridize to at least one nucleic acid of the invention, (b) detecting a hybridization pattern of the test nucleic acid sample, and (c) comparing the hybridization pattern to a hybridization pattern of a first nucleic acid standard, whereby the comparison indicates whether or not the test sample has the same classification as the first standard.
  • the method also includes comparing a hybridization pattern of the test nucleic acid sample to a hybridization pattern of a second standard.
  • a hybridization pattern of the test nucleic acid sample is compared to at least 3, 4, 5, 8, 10, 15, 20, 30, 40, or more standards.
  • the method also includes identifying the hybridized target nucleic acid and/or determining the amount of hybridized target nucleic acid.
  • the target nucleic acids are labeled with a fluorescent group.
  • the first nucleic acid standard is labeled with a different fluorescent group. The fluorescence of the target nucleic acids and the first nucleic acid standard can be detected simultaneously or sequentially.
  • the method further includes determining the presence or absence of an mRNA splice variant of interest in the sample and/or determining the presence or absence of a mutation, deletion, and/or duplication of an exon of interest.
  • the mutation, deletion, and/or duplication is indicative of a disease, disorder, or condition, such as cancer.
  • the determination of the amount of bound target nucleic acid involves one or more of the following: (i) adjusting for the varying intensity of the excitation light source used for detection of the hybridization, (ii) adjusting for photobleaching of the fluorescent group, andor (iii) comparing the fluorescent intensity of the target nucleic acid(s) hybridized to the nucleic acids of the invention to the fluorescent intensity of a different sample of nucleic acids hybridized to the nucleic acids of the invention (e.g., a different sample hybridized to same set of nucleic acids of the invention on the same or a different solid support such as the same chip or a different chip).
  • this comparison in fluorescent intensity involves adjusting for a difference in the amount of the plurality used for hybridization to each sample and/or adjusting for a difference in the buffer (e.g., a difference in Mg concentration) used for hybridization to each sample.
  • the nucleic acids in the population together hybridize to at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% of the exons of a target nucleic acid.
  • the population includes nucleic acids that together hybridize to at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% of the nucleic acids expressed by a particular cell or tissue.
  • the population includes nucleic acids that together hybridize to at least one exon from at least 1, 5, 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100% of the nucleic acid sequences expressed by a particular cell or tissue at a given point in time (e.g., an expression array with sequences corresponding to the sequences of mRNA molecules expressed by a particular cell type or a cell under a particular set of conditions).
  • the method further includes using a nucleic acid or a region of a nucleic acid that is present in a first test sample but not present in a first standard or not present in a second test sample as a probe or primer for the detection, amplification, or characterization of the nucleic acid.
  • the method is repeated under one or more different incubation or hybridization conditions.
  • the method is repeated at 1, 3, 5, 8, 10, 15, 20, 30, 40 or more different temperatures, cation concentrations (e.g., concentration of monovalent cations such as Na + and K + or divalent cations such as Mg 2+ and Ca 2 ); denaturants (e.g., hydrogen bond donors or acceptors that interfere with the hydrogen bonds keeping the base-pairs together such as formamide or urea).
  • the sample has nucleic acids that are amplified using one or more primers specific for an exon of a target nucleic acid, and the method involves determining the presence or absence of an mRNA splice variant with the exon in the sample.
  • one or more of the primers are specific for an exon or exon-exon junction of a pathogen of interest, and the method involves determining the presence or absence of a nucleic acid with the exon in the sample.
  • the comparison of the hybridization pattern of a patient nucleic acid sample to that of one or more standards is used to determine whether or not a patient has a particular disease, disorder, condition, or infection or an increased risk for a particular disease, disorder, condition, or infection.
  • the comparison is used to determine what pathogen has infected a patient and to select a therapeutic for the treatment of the patient.
  • the comparison is used to select a therapeutic for the treatment or prevention of a disease or disorder in the patient.
  • the comparison is used to include or exclude the patient from a group in a clinical trial.
  • the comparison is used to compare the expression of nucleic acids (e.g., mRNA splice forms associated with toxicity) in the presence and absence of a candidate compound (e.g., a lead compound for drug development).
  • a candidate compound e.g., a lead compound for drug development
  • the comparison is used to determine differences in expression of nucleic acids (e.g., mRNA splice variants) under particular conditions (e.g., under different environmental stress conditions) or at different developmental time points.
  • the expression of one or more members from a particular enzyme class e.g., protein kinase splice variants is measured.
  • the nucleic acids of the invention are covalently bonded to a solid support by reaction of a nucleoside phosphoramidite with an activated solid support, and subsequent reaction of a nucleoside phosphoramide with an activated nucleotide or nucleic acid bound to the solid support.
  • the solid support or the growing nucleic acid bound to the solid support is activated by illumination, a photogenerated acid, or electric current.
  • the invention features the use of one ore more nucleic acids of the invention for the detection, amplification, or classification of a nucleic acid of interest or a population of nucleic acids of interest.
  • the invention features the use of one or more nucleic acids of the invention for alternative mRNA splice variant detection, expression profiling, comparative genomic hybridization, or real-time PCR.
  • the nucleic acids are used to determine the amount of one or more target nucleic acids (e.g., mRNA splice variants) in a sample.
  • fluorescently labeled RT- PCR products from the amplification of a test nucleic acid sample are hybridized to a population of nucleic acids of the invention.
  • the amount of one or more RT-PCR products is measured to determine the amount of the corresponding nucleic acid in the initial sample.
  • the invention features the use of a nucleic of the invention as a PCR primer or FISH probe.
  • the invention features a method of selecting a nucleic acid for a population of nucleic acids. This method involves (a) determining the melting temperature of a nucleic acid of the invention, determining the ability of the nucleic acid to self-anneal, determining the ability of the nucleic acid to hybridize to one or more exons or introns of a target nucleic acid, and/or determining the ability of the nucleic acid to hybridize to a non- target nucleic acid, and (b) selecting the nucleic acid for inclusion or exclusion from the population based on the determination in step (a).
  • step (a) is performed for at least 2, 3, 4, 5, 6, 10, 20, 50, 100, 200, 500, 1,000, 5,000 or more nucleic acids, and a subset of the nucleic acids are selected for inclusion in the population based on the determination in step (a).
  • the nucleic acids with the highest melting temperatures and/or ability to hybridize to one or more exons or introns of a target nucleic acid are selected.
  • the nucleic acids with the lowest ability to self-anneal and/or hybridize to a non-target nucleic acid are selected.
  • the invention also features a variety of databases. These databases are useful for storing the information obtained in any of the methods of the invention. These databases may also be used in the diagnosis of disease or an increased risk for a disease or in the selection of a desirable therapeutic for a particular patient or class of patients.
  • the invention provides an electronic database including at least 1, 10, 10 2 , 10 3 , 5 x 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 records of a nucleic acid of interest or a population of nucleic acids of interest (e.g., one or more nucleic acids in a standard or in a test nucleic acid sample) correlated to records of its hybridization pattern to a plurality of nucleic acids of the invention under one or more incubation conditions (e.g., one or more temperatures, denaturant concentrations, or salt concentrations).
  • a nucleic acid of interest e.g., one or more nucleic acids in a standard or in a test nucleic acid sample
  • incubation conditions e.g., one or more temperatures, denaturant concentrations, or salt concentrations.
  • the invention features computer including the database of the above aspect and a user interface (i) capable of displaying a hybridization pattern for a nucleic acid of interest or a population of nucleic acids of interest whose record is stored in the computer or (ii) capable of displaying a nucleic acid of interest (e.g., displaying the polynucleotide sequence or another identifying characteristic of the nucleic acid of interest) or a population of nucleic acids of interest that produces a hybridization pattern whose record is stored in the computer.
  • a user interface capable of displaying a hybridization pattern for a nucleic acid of interest or a population of nucleic acids of interest whose record is stored in the computer or a user interface (i) capable of displaying a hybridization pattern for a nucleic acid of interest or a population of nucleic acids of interest whose record is stored in the computer or (ii) capable of displaying a nucleic acid of interest (e.g., displaying the polynucleotide sequence or
  • oligonucleotides which are complementary to a specific target messenger RNA (mRNA) sequence, such as a specific mRNA splice variant.
  • mRNA target messenger RNA
  • oligonucleotides with a modified backbone such as LNA or phosphorothioate
  • the invention features the use of a nucleic acid of the invention for the manufacture of a pharmaceutical composition for treatment of a disease curable by an antisense or RNAi technology.
  • the invention provides a method for inhibiting the expression of a target nucleic acid in a cell.
  • the method involves introducing into the cell a nucleic acid of the invention in an amount sufficient to specifically attenuate expression of the target nucleic acid.
  • the introduced nucleic acid has a nucleotide sequence that is essentially complementary to a region of desirably at least 20 nucleotides of the target nucleic acid.
  • the cell is in a mammal.
  • the invention provides a method for preventing, stabilizing, or treating a disease, disorder, or condition associated with a target nucleic acid in a mammal.
  • This method involves introducing into the mammal a nucleic acid of the invention in an amount sufficient to specifically attenuate expression of the target nucleic acid, wherein the introduced nucleic acid has a nucleotide sequence that is essentially complementary to a region of desirably at least 20 nucleotides of the target nucleic acid.
  • the invention provides a method for preventing, stabilizing, or treating a pathogenic infection in a mammal by introducing into the mammal a nucleic acid of the invention in an amount sufficient to specifically attenuate expression of a target nucleic acid of a pathogen.
  • the introduced nucleic acid has a nucleotide sequence that is essentially complementary to a region of desirably at least 20 nucleotides of the target nucleic acid.
  • the mammal is a human.
  • the introduced nucleic acid is single stranded or double stranded.
  • nucleic acids may be administered to the mammal in a single dose or multiple doses.
  • the doses may be separated from one another by, for example, one week, one month, one year, or ten years. It is to be understood that, for any particular subject, specific dosage regimes should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.
  • Exemplary mammals that can be treated using the methods of the invention include humans, primates such as monkeys, animals of veterinary interest (e.g., cows, sheep, goats, buffalos, and horses), and domestic pets (e.g., dogs and cats).
  • Exemplary cells in which one or more target genes can be silenced using the methods of the invention include invertebrate, plant, bacteria, yeast, and vertebrate (e.g., mammalian or human) cells.
  • Optimum dosages for gene silencing applications may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC 5 o values found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.001 ug to 100 g per kg of body weight (e.g., 0.001 ug/kg to 1 g/kg), and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years (U.S.P.N. 6,440,739). Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drag in bodily fluids or tissues.
  • oligonucleotide is administered in maintenance doses, ranging from 0.001 ug to 100 g per kg of body weight (e.g., 0.001 ug/kg to 1 g/kg), once or more daily, to once every 20 years.
  • conventional treatments may be used in combination with the nucleic acids of the present invention.
  • Suitable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the composition can be adapted for the mode of administration and can be in the form of, for example, a pill, tablet, capsule, spray, powder, or liquid.
  • the pharmaceutical composition contains one or more pharmaceutically acceptable additives suitable for the selected route and mode of administration.
  • compositions may be administered by, without limitation, any parenteral route including intravenous, intra-arterial, intramuscular, subcutaneous, intradermal, intraperitoneal, infrathecal, as well as topically, orally, and by mucosal routes of delivery such as intranasal, inhalation, rectal, vaginal, buccal, and sublingual.
  • the pharmaceutical compositions of the invention are prepared for administration to vertebrate (e.g., mammalian) subjects in the form of liquids, including sterile, non-pyrogenic liquids for injection, emulsions, powders, aerosols, tablets, capsules, enteric coated tablets, or suppositories.
  • the invention features a method of synthesizing a nucleic acid.
  • This method involves synthesizing a 2-thio-uridine nucleoside or nucleotide of formula IV using a compound of formula VIII, IX, X, XI, or XII as shown in Figure 6.
  • the nucleoside, nucleoside phosphoramidite, or nucleotide is incorporated into a nucleic acid of the invention.
  • nucleobase thiolation is performed on the 02 position of compound XI to form compound IV.
  • sulphurization on both O2 and O4 in compound VIII generates a 2,4-dithio-uridine nucleoside or nucleotide of formula X which is converted into compound IV.
  • a cyclic ether of formula XI is transferred into compound IV or a 2-O-alkyl-uridine nucleoside or nucleotide of formula XII through reaction with the 5' position.
  • a 2-O-alkyl- uridine nucleoside or nucleotide of formula XII is generated by direct alkylation of a uridine nucleoside or nucleotide of formula VIII.
  • R 4 and R 2 are each independently alkyl (e.g., methyl or ethyl), acyl (e.g., acetyl or benzoyl), or any appropriate protecting group such as silyl, 4,4'- dimethoxytrityl, monomethoxytrityl, or trityl(triphenylmethyl).
  • R 5 is any appropriate protecting group such as silyl, 4,4'-dimethoxytrityl, monomethoxytrityl, trityl(triphenylmethyl), acetyl, benzoyl, or benzyl.
  • R 5 is hydrogen, alkyl (e.g., methyl or ethyl), 1-propynyl, thiazol-2-yl, pyridine-2-yl, thien-2-yl, imidazol-2-yl, (4/5-methyl)-thiazol-2-yl, 3-(iodoacetamido)propyl, 4- /V,N-bis(3- aminopropyl)amino]buty ⁇ ), or halo (e.g., chloro, bromo, iodo, fluoro).
  • alkyl e.g., methyl or ethyl
  • 1-propynyl thiazol-2-yl
  • pyridine-2-yl thien-2-yl
  • imidazol-2-yl (4/5-methyl)-thiazol-2-yl
  • 3-(iodoacetamido)propyl 4- /V,N-bis(3- aminopropyl)amin
  • the group -OR 3' in the formulas IV, VIII, IX, X, XI, and XII is any of the groups listed for R 3 or R 3' in formula la or formula lb or listed for R 3 or R 3* in formula Ila, Scheme A, or Scheme B, or the group -OR 3 ' or R 3' in the formulas IV, VIII, IX, X, XI, and XII is selected from the group consisting of H, -OH, P(O(CH 2 ) 2 C ⁇ ) ⁇ (iPr) 2 ,P(O(CH 2 ) 2 C ⁇ ) ⁇ (iPr) 2 , phosphate, phosphorothioate, phosphorodithioate, phosphoramidate, phosphoroselenoate, phosphorodiselenoate, alkylphosphotriester, methyl phosphonate, halo (e.g., chloro, fluoro, iodo, or bromo), optionally substituted ary
  • acetyl or benzoyl aroyl, aralkyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acylamino, aroylamine, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, arylsulfmyl, heteroarylsulfinyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio,amidino, amino, carbamoyl, sulfamoyl, alkene, alkyne, protecting groups (e.g., silyl, 4,4 , -dimethoxytrityl, monomethoxytrityl, or tr
  • the group -OR 5 in the formulas IV, and VIII, IX, X, and XII is any of the groups listed for R 5 or R 5 in formula la or formula lb or listed for R 5 orR 5 in formula Ila, Scheme A, or Scheme B, or the group -OR 5 ' or R 5 ' in the formulas IV, and VIII, IX, X, and XII is selected from the group consisting of H, -OH, P(O(CH 2 ) 2 CN)N(iPr) 2j P(O(CH 2 ) 2 CN)N(iPr) 2, phosphate, phosphorothioate, phosphorodithioate, phosphoramidate, phosphoroselenoate, phosphorodiselenoate, alkylphosphotriester, methyl phosphonate, halo (e.g., chloro, fluoro, iodo, or bromo), optionally substituted aryl, (e.g., pheny
  • acetyl or benzoyl aroyl, aralkyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acylamino, aroylamine, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, arylsulfmyl, heteroarylsulfinyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio,amidino, amino, carbamoyl, sulfamoyl, alkene, alkyne, protecting groups (e.g., silyl, 4,4'-dimethoxytrityl, monomethoxytrityl, or trityl
  • the invention features a method of synthesizing a nucleic acid.
  • This method involves synthesizing a 2-thiopyrimidine nucleoside or nucleotide of formula IV using a compound of formula III or compounds of the formula I, II, and III as shown in Figure 7.
  • the nucleoside, nucleoside phosphoramidite, or nucleotide is incorporated into a nucleic acid of the invention.
  • lewis acid-catalyzed condensation of a substituted sugar of formula I and a substituted 2-thio-uracil of formula II results in a substituted 2-thio-uridine nucleoside or nucleotide of the formula III.
  • a compound of formula III is converted into a LNA 2-thiouridine nucleoside or nucleotide of formula IV.
  • R 4 and R 5 are, e.g., methanesulfonyloxy, p- toluenesulfonyloxy, or any appropriate protecting group such as silyl, 4,4'-dimethoxytrityl, monomethoxytrityl, trityl(triphenylmethyl), acetyl, benzoyl, or benzyl
  • R 1 is, e.g., acetyl, benzoyl, alkoxy (e.g., methoxy).
  • R 2 is, e.g.,acetyl or benzoyl
  • R 3 is any appropriate protecting group such as silyl, 4,4'-dimethoxvtrityl, monomethoxytrityl, trityl(triphenylmethy ⁇ ), acetyl, or benzoyl.
  • R 5 is hydrogen, alkyl (e.g.
  • the group -OR 3 ' in the formulas I, III, and IV is any of the groups listed for R 3 or R 3 in formula la or formula lb or listed for R 3 orR 3 in formula Ila, Scheme A, or Scheme B, or the group -OR 3 orR 3 in the formulas I, III, and IV is selected from the group consisting of H, -OH, P(O(CH 2 ) 2 C ⁇ ) ⁇ (iPr) 2; phosphate, phosphorothioate, phosphorodithioate, phosphoramidate, phosphoroselenoate, phosphorodiselenoate, alkylphosphotriester, methyl phosphonate, halo (e.g., chloro, fluoro, iodo, or bromo), optionally substituted aryl, (e.g., phenyl or benzyl), alkyl (e.g, methyl or ethyl), alkoxy (e.g., methoxy), acyl (
  • acetyl or benzoyl aroyl, aralkyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acylamino, aroylamine, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio,amidino, amino, carbamoyl, sulfamoyl, alkene, alkyne, protecting groups (e.g., silyl, 4,4'-dimethoxytrityl, monomethoxytrityl, or trity
  • R 5 in the formulas I, III, and IV is any of the groups listed for R 5 orR 5 ' in formula la or formula lb or listed for R 5 orR 5* in formula Ila, Scheme A, or Scheme B, or R 5' in the formulas I, III, and IV is selected from the group consisting of H, -OH, P(O(CH 2 ) 2 CN)N(iPr) 2 , phosphate, phosphorothioate, phosphorodithioate, phosphoramidate, phosphoroselenoate, phosphorodiselenoate, alkylphosphotriester, methyl phosphonate, halo (e.g., chloro, fluoro, iodo, or bromo), optionally substituted aryl, (e.g., phenyl or benzyl), alkyl (e.g, methyl or ethyl), alkoxy (e.g., methoxy), acyl (e.g.
  • acetyl or benzoyl aroyl, aralkyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acylamino, aroylamine, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, arylsulfmyl, heteroarylsulfinyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio,amidino, amino, carbamoyl, sulfamoyl, alkene, alkyne, protecting groups (e.g., silyl, 4,4'-dimethoxytrityl, monomethoxytrityl, or trityl
  • This method involves synthesizing a 2-thiopyrimidine nucleoside or nucleotide of formula IV using a compound of formula VII, compounds of the formula V, VI, and VII, or compounds of the formula I, V, VI, and VII as shown in Figure 8.
  • the nucleoside, nucleoside phosphoramidite, or nucleotide is incorporated into a nucleic acid of the invention.
  • a 2-thio-uridine nucleoside or nucleotide of the formula IV is synthesized through ring-synthesis of the nucleobase by reaction of an amino sugar of the formula V and a substituted isothiocyanate of the formula VI.
  • R 4 and R 5 are each idenpendently, e.g., methanesulfonyloxy, p-toluenesulfonyloxy, or any appropriate protecting group such as silyl, 4,4'-dimethoxytrityl, monomethoxytrityl, trityl(triphenylmethyl), acetyl, benzoyl, or benzyl.
  • R 1 is, e.g., acetyl or benzoyl or alkoxy (e.g., methoxy), and R 2 is, e.g., acetyl or benzoyl, R 3 is any appropriate protecting group such as silyl, 4,4'-dimethoxytrityl, monomethoxytrityl, trityl(triphenylmethyl), acetyl, or benzoyl.
  • R 5 are R 6 each idenpendently, e.g., hydrogen or alkyl (e.g. methyl or ethyl).
  • R 6 can also be, e.g., an appropriate protecting group such as silyl, 4,4'-dimethoxytrityl, monomethoxytrityl, or trityl(triphenylmethyl).
  • R 5 is hydrogen or methyl
  • R 6 is methyl or ethyl.
  • the group -OR 3 in the formulas I, V, VII, and IV is any of the groups listed for R 3 or R 3 in formula la or formula lb or listed for R 3 orR 3* in formula Ila, Scheme A, or Scheme B, or the grou -OR orR m the formulas I, V, VII, and IV is selected from the group consisting of H, -OH, P(O(CH 2 ) 2 CN)N(iPr) 2j phosphate, phosphorothioate, phosphorodithioate, phosphoramidate, phosphoroselenoate, phosphorodiselenoate, alkylphosphotriester, methyl phosphonate, halo (e.g., chloro, fluoro, iodo, or bromo), optionally substituted aryl, (e.g., phenyl or benzyl), alkyl (e.g, methyl or ethyl), alkoxy (e.g., methoxy),
  • acetyl or benzoyl aroyl, aralkyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acylamino, aroylamine, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, arylsulfmyl, heteroarylsulfinyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio,amidino, amino, carbamoyl, sulfamoyl, alkene, alkyne, protecting groups (e.g., silyl, 4,4'-dimethoxytrityl, monomethoxytrityl, or trityl
  • R in the formulas I, V, VII, and IV is any of the groups listed for R or R in formula la or formula lb or listed for R 5 orR 5* in formula Ila, Scheme A, or Scheme B, or R 5' in the formulas I, V, VII, and IV is selected from the group consisting of H, -OH, P(O(CH 2 ) 2 CN)N(iPr) 2j phosphate, phosphorothioate, phosphorodithioate, phosphoramidate, phosphoroselenoate, phosphorodiselenoate, alkylphosphotriester, methyl phosphonate, halo (e.g., chloro, fluoro, iodo, or bromo), optionally substituted aryl, (e.g., phenyl or benzyl), alkyl (e.g, methyl or ethyl), alkoxy (e.g., methoxy), acyl (e.g.
  • acetyl or benzoyl aroyl, aralkyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acylamino, aroylamine, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio,amidino, amino, carbamoyl, sulfamoyl, alkene, alkyne, protecting groups (e.g., silyl, 4,4'-dimethoxytrityl, monomethoxytrityl, or trity
  • the invention features a method of synthesizing a nucleic acid.
  • This method involves synthesizing a 2-thiopyrimidine nucleoside as shown in Figure 5.
  • the method further comprises reacting one or both compounds of the formula 4 with a phosphodiamidite (e.g., 2-cyanoethyl tetraisopropylphosphorodiamidite) to produce the corresponding nucleoside phosphoramidite.
  • a phosphodiamidite e.g., 2-cyanoethyl tetraisopropylphosphorodiamidite
  • the nucleoside, nucleoside phosphoramidite, or nucleotide is incorporated into a nucleic acid of the invention.
  • a glycosyl-donor is coupled to a nucleobase as shown in pathway A.
  • ring synthesis of the nucleobase is performed as show in pathway B.
  • LNA-T diol is modified as shown in pathway C.
  • Ris hydrogen, methyl, 1-propynyl, thiazol-2-yl, pyridine- 2-yl, thien-2-yl, imidazol-2-yl, (4/5-methyl)-thiazol-2-yl, 3-(iodoacetamido)propyl, 4-[N,N- bis(3-aminopropyl)amino]butyl, or halo (e.g., chloro, bromo, iodo, fluoro).
  • halo e.g., chloro, bromo, iodo, fluoro
  • R ⁇ ,R 2 , and R 3 are each any appropriate protecting group such as acetyl, benzyl, silyl, 4,4'- dimethoxytrityl, monomethoxytrityl, or trityl(triphenylmethyl).
  • the invention features a method of synthesizing a nucleic acid.
  • This method involves synthesizing a 2-thiopyrimidine nucleoside or nucleotide of formula 4 using a compound of formula 3, compounds of the formula 2 and 3, or compounds of the formula 1, 2, 3, and 4 as shown in Figure 28.
  • the nucleoside, nucleoside phosphoramidite, or nucleotide is incorporated into a nucleic acid of the invention.
  • This method can also be performed using any other appropriate protecting groups instead of Bn (benzyl), Ac (acetyl), or Ms (methansulfonyl).
  • the method further comprises reacting one or both compounds of the formula 4 with a phosphodiamidite (e.g., 2-cyanoethyl tetraisopropylphosphorodiamidite) to produce the corresponding nucleoside phosphoramidite.
  • a phosphodiamidite e.g., 2-cyanoethyl tetraisopropylphosphorodiamidite
  • the invention features a method of synthesizing a nucleic acid. This method involves synthesizing a nucleoside or nucleotide of formula 10 or 11 using a compound of any one of the formula 6-9, compounds of the formula 5 and any one of the formulas 6-9, or compounds of the formula 4, 5, and any one of the formulas 6-9 as shown in Figure 48.
  • the nucleoside, nucleoside phosphoramidite, or nucleotide is incorporated into a nucleic acid of the invention. This method can also be performed using any other appropriate protecting groups instead of DMT, Bn
  • a compound of formula 4 is used as a glycosyl donor in a coupling reaction with silylated hypoxantine to form a compound of the formula 5.
  • a compound of the formula 5 is used in a ring closing reaction to forma compound of the formula 6.
  • deprotection of the 5'-hydroxy group of compound 6 is performed by displacing the 5'-O-mesyl group with sodium benzoate to produce a compound of the formula 7 that is converted into a compound of the formula 8 after saponification of the 5 '-benzoate.
  • compound 8 is converted to a DMT- protected compound 9 prior to debenzylation of the 3'-O-hydroxy group.
  • a phosphoramidite of the formula 11 is generated by phosphitylationof a nucleoside of the formula 10.
  • the R is H or P(O(CH 2 ) 2 CN)N(iPr) 2 .
  • the group Ri or -OR t is any of the groups listed for R 3 orR 3 in formula la or formula lb or listed for R 3 or R 3 in formula Ila, Scheme A, or Scheme B, or the group -ORi or Ri is selected from the group consisting of-OH, P(O(CH 2 ) 2 CN)N(iPr) 2; phosphate, phosphorothioate, phosphorodithioate, phosphoramidate, phosphoroselenoate, phosphorodiselenoate, alkylphosphotriester, methyl phosphonate, halo (e.g., chloro, fluoro, iodo, or bromo), optionally substituted aryl, (e.g., phenyl or benzyl), alkyl (e.g, methyl or ethyl), alkoxy (e.g., methoxy), acyl (e.g.
  • acetyl or benzoyl aroyl, aralkyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acylamino, aroylamine, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, arylsulfmyl, heteroarylsulfinyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio,amidino, amino, carbamoyl, sulfamoyl, alkene, alkyne, protecting groups (e.g., silyl, 4,4'-dimethoxytrityl, monomethoxytrityl, or trityl
  • the invention features a method of synthesizing a nucleic acid.
  • This method involves synthesizing a nucleoside or nucleotide of formula 20 or 21 as shown in Figure 3, in wliich compound 4 is the same sugar shown in the above aspect.
  • the nucleoside, nucleoside phosphoramidite, or nucleotide is incorporated into a nucleic acid of the invention.
  • This method can also be performed using any other appropriate protecting groups instead of DMT, Bn, Bz (benzoyl), Ac, or Ms. Additionally, the method can be performed with any other halogen (e.g., fluoro or bromo) instead of chloro.
  • a solution of compound 14 in aqueous 1,4-dioxane is treated with sodium hydroxide to give a bicyclic compound 15.
  • sodium benzoate is used for displacement of 5'- mesylate of compound 15 to give compound 16.
  • compound 17 is formed by reaction of compound 16 with sodium azide.
  • compound 18 is produced by saponification of the 5'-benzoate of compound 17.
  • hydrogenation of compound 18 produces compound 19.
  • the peracelation method is used to benzolylate the 2- and 6-amino groups of compound 19, yielding 20, which is desirably converted into the phosphoramidite compound 21.
  • the invention features a derivative of a compound of the formula 20 or 21 as described in the above aspect in which 3' -OH or -OP(O(CH 2 ) 2 CN)N(iPr) 2 group is replaced by any other group is selected from the group consisting of phosphorothioate, phosphorodithioate, phosphoramidate, phosphoroselenoate, phosphorodiselenoate, alkylphosphotriester, methyl phosphonate, halo (e.g., chloro, fluoro, iodo, or bromo), optionally substituted aryl, (e.g., phenyl or benzyl), alkyl (e.g, methyl or ethyl), alkoxy (e.g., methoxy), acyl (e.g.
  • acetyl or benzoyl aroyl, aralkyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acylamino, aroylamine, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, arylsulfmyl, heteroarylsulfinyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio,amidino, amino, carbamoyl, sulfamoyl, alkene, alkyne, protecting groups (e.g., silyl, 4,4'-dimethoxytrityl, monomethoxytrityl, or trityl
  • the invention features a method of synthesizing a nucleic acid.
  • This method involves synthesizing a nucleoside or nucleotide of formula 20 or 21 as shown in Figure 10.
  • the nucleoside, nucleoside phosphoramidite, or nucleotide is incorporated into a nucleic acid of the invention.
  • This method can also be performed using any other appropriate protecting groups instead of DMT.
  • compound 17 is formed by reaction of compound 7 with! ,3 - dichloro-l,l,3,3-tetraisopropyldisiloxane.
  • compound 18 is formed by reaction of compound 17 with phenoxyacetic anhydride.
  • compound 19 is generated by reaction of compound 18 with acid.
  • compound 20 is produced by reacting compound 19 with DMT-C1.
  • compound 20 is reacted with 2-cyanoethyl tetraisopropylphosphorodiamidite to give the phosphoramidite 21.
  • the R is H or P(O(CH 2 ) 2 CN)N(iPr) 2 .
  • the Ror -OR is any of the groups listed for R 3 orR 3 in formula la or formula lb or listed for R 3 orR 3* in formula Ila, Scheme A, or Scheme B, or the group -OR orR is selected from the group consisting of-OH, P(O(CH 2 ) 2 CN)N(iPr) 2j phosphate, phosphorothioate, phosphorodithioate, phosphoramidate, phosphoroselenoate, phosphorodiselenoate, alkylphosphotriester, methyl phosphonate, halo (e.g., chloro, fluoro, iodo, or bromo), optionally substituted aryl, (e.g., phenyl or benzyl), alkyl (e.g, methyl or ethyl), alkoxy (e.g., methoxy), acyl (e.g.
  • acetyl or benzoyl aroyl, aralkyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acylamino, aroylamine, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, arylsulfmyl, heteroarylsulfinyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio,amidino, amino, carbamoyl, sulfamoyl, alkene, alkyne, protecting groups (e.g., silyl, 4,4'-dimethoxytrityl, monomethoxytrityl, or trityl
  • the invention features a method of synthesizing a nucleic acid.
  • This method involves synthesizing a nucleoside or nucleotide of formula 24 or 25 as shown in Figure 54.
  • the nucleoside, nucleoside phosphoramidite, or nucleotide is incorporated into a nucleic acid of the invention.
  • This method can also be performed using any other appropriate protecting groups instead of Bz, Bn, and DMT. Additionally, the method can be performed with any other halogen (e.g., fluoro or bromo) instead of chloro.
  • the compound 16 is formed from compounds 4, 14, and 15 as illustrated in an aspect above.
  • the 5'-O-benzoyl group of compound 16 is hydrolyzed by aqueous sodium hydroxyde to give compound 22.
  • Compound 23 is desirably produced by incubation of compound 22 in the presence of paladium hydroxide and ammonium formate.
  • the 2-amine of compound 23 is selectively protected with an amidine group after treatment with N,N-dimethylformamide dimethyl acetal to yield compound 24.
  • the diol 24 is 5'-O-DMT protected and 3'-O- phosphitylated produce the phosphoramidite L ⁇ A-2AP compound 25.
  • compound 25 has one of the following groups instead of the
  • P(O(CH 2 ) 2 CN)N(iPr) 2 group any of the groups listed for R 3 orR 3 in formula la or formula lb or listed for R 3 orR 3 in formula Ila, Scheme A, or Scheme B, or a group selected from the group consisting of-OH, phosphate, phosphorothioate, phosphorodithioate, phosphoramidate, phosphoroselenoate, phosphorodiselenoate, alkylphosphotriester, methyl phosphonate, halo (e.g., chloro, fluoro, iodo, or bromo), optionally substituted aryl, (e.g., phenyl or benzyl), alkyl (e.g, methyl or ethyl), alkoxy (e.g., methoxy), acyl (e.g.
  • acetyl or benzoyl aroyl, aralkyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acylamino, aroylamine, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, arylsulfmyl, heteroarylsulfinyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio,amidino, amino, carbamoyl, sulfamoyl, alkene, alkyne, protecting groups (e.g., silyl, 4,4'-dimethoxytrityl, monomethoxytrityl, or trityl
  • the invention features a nucleic acid of the invention that includes a compound of the formula 6pCor the product of a compound of the formula 6pC treated with ammonia as described herein.
  • the invention features a method of synthesizing a nucleic acid that involves performing one or more of the steps described herein for the synthesis of a compound of the formula 6pCor the product of a compound of the fo ⁇ nula 6pC treated with ammonia.
  • the invention features a method of synthesizing a nucleic acid.
  • This method involves one or more of any of the nucleosides or nucleotides of the invention with (i) any other nucleoside or nucleotide of the invention, (ii) any other nucleoside or nucleotide of formula la, formula lb, formula Ila, Scheme A, or Scheme B, and/or (iii) any naturally-occurring nucleoside or nucleotide.
  • the method involves reacting one or more nucleoside phosphoramidites of any of the above aspects with a nucleotide or nucleic acid.
  • the invention provides a method for the synthesis of a population of nucleic acids (e.g., a population of nucleic acids of the invention) on a solid support.
  • This method involves the reaction of a plurality of nucleoside phosphoramidites with an activated solid support (e.g., a solid support with an activated linker) and the subsequent reaction of a plurality of nucleoside phosphoramidites with activated nucleotides or nucleic acids bound to the solid support.
  • an activated solid support e.g., a solid support with an activated linker
  • the solid support or the growing nucleic acid bound to the solid support is activated by illumination, a photogenerated acid, or electric current.
  • one or more spots or regions e.g., a region with an area of less than 1 cm , 0.1 cm , 0.01 cm , 1 mm , or 0.1 mm that desirably contains one particular nucleic acid monomer or oligomer
  • the solid support are irradiated to produce a photogenerated acid that removes the 5'-OH protecting group of one or more nucleic acid monomers or oligomers to which a nucleotide is subsequently added.
  • an electric current is applied to one or more spots or regions (e.g., a region with an area of less than 1 cm 2 , 0.1 cm 2 , 0.01 cm 2 , 1 mm 2 , or 0.1 mm 2 that desirably contains one particular nucleic acid monomer or oligomer) on the solid support to remove an electrochemically sensitive protecting group of one or more nucleic acid monomers or oligomers to which a nucleotide is subsequently added.
  • one or more spots or regions e.g., a region with an area of less than 1 cm 2 , 0.1 cm 2 , 0.01 cm 2 , 1 mm 2 , or 0.1 mm 2 that desirably contains one particular nucleic acid monomer or oligomer
  • the solid support e.g., chip, coverslip, microscope glass slide, quartz, or silicon
  • the solid support is less than 1, 0.5, 0.1. or 0.05 mm thick.
  • the invention features a method of reacting a population of nucleic acids
  • This method involves incubating an immobilized population of nucleic acids of the invention with a solution that includes one or more probes (e.g., at least 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, or 150 different nucleic acids) and one or more target nucleic acids (e.g., at least 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, or 150 different target nucleic acids).
  • probes e.g., at least 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, or 150 different nucleic acids
  • target nucleic acids e.g., at least 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, or 150 different target nucleic acids
  • ligase 15 presence of a ligase under conditions that allow the ligase to covalently react one or more immobilized nucleic acids with one or more nucleic acid probes in solution that hybridize to the same target nucleic acid.
  • at least 2, 5, 10, 15, 20, 30, 40, 50, 80, or 100 pairs of immobilized nucleic acids and nucleic acid probes are ligated.
  • the incubation occurs between 15 and 45°C, such as between 20 and 40°C or between 25 and
  • a nucleic acid probe or primer specifically hybridizes to a target nucleic acid but does not substantially hybridize to non-target molecules, which include other nucleic acids in a cell or biological sample
  • the amount of the these non-target molecules hybridized to, or associated with, the nucleic acid probe or primer, as measured using standard assays is 2-fold, desirably 5-fold, more desirably 10-fold, and most desirably 50-fold lower than the amount of the target nucleic acid hybridized to, or associated with, the nucleic acid probe or
  • the amount of a target nucleic acid hybridized to, or associated with, the nucleic acid probe or primer, as measured using standard assays is 2- fold, desirably 5-fold, more desirably 10-fold, and most desirably 50-fold greater than the amount of a control nucleic acid hybridized to, or associated with, the nucleic acid probe or primer.
  • the nucleic acid probe or primer is substantially complementary (e.g., at least 80, 90, 95, 98, or 100% complementary) to a target nucleic acid or a group of target nucleic acids from a cell.
  • the probe or primer is homologous to multiple RNA or DNA molecules, such as RNA or DNA molecules from the same gene family. In other embodiments, the probe or primer is homologous to a large number of RNA or DNA molecules. In desirable embodiments, the probe or primer binds to nucleic acids which have polynucleotide sequences that differ in sequence at a position that corresponds to the position of a universal base in the probe or primer. Examples of control nucleic acids include nucleic acids with a random sequence or nucleic acids known to have little, if any, affinity for the nucleic acid probe or primer. In some embodiments, the target nucleic acid is an RNA, DNA, or cDNA molecule.
  • the association constant (K a ) of the nucleic acid toward a complementary target molecule is higher than the association constant of the complementary strands of the double stranded target molecule.
  • the melting temperature of a duplex between the nucleic acid and a complementary target molecule is higher than the melting temperature of the complementary strands of the double stranded target molecule.
  • the LNA-pyrene is in a position corresponding to the position of a non-base (e.g., a unit without a base) in another nucleic acid, such as a target nucleic acid.
  • a non-base e.g., a unit without a base
  • incorporation of pyrene in a DNA strand in a position opposite a non-base only decreases the T m by -2.3°C to -4.6°C, most likely due to the better accommodation of the pyrene in the B-type duplex (Matray and Kool, J. Am. Chem. Soc.
  • incorporation on LNA-pyrene into a nucleic acid in a position opposite a non-base (e.g., a unit without a base or a unit with a small group such as a noncyclic group instead of a base) in a target nucleic acid may also minimize any potential decrease in T m due to the pyrene substitution
  • the number of molecules in the plurality of nucleic acids of the invention is at least 2, 4, 5, 6, 7, 8, or 10-fold greater than the number of molecules in the test nucleic acid sample.
  • a LNA is a triplex-forming oligonucleotide.
  • the target nucleic acids e.g., cDNA molecules reverse transcribed from a patient sample or cRNA molecules amplified from a patient sample using a T7 RNA polymerase-based amplification system or the like
  • an enzyme such as a uracil-DNA glycosylase (e.g., E.
  • the average size of the fragmented nucleic acids is between 300 and 50 nucleic acids, such as approximately 300, 200, 100, or 50 nucleotides.
  • Advantages The present invention has a variety of advantages related to nucleic acid analysis methods.
  • the ability to equalize melting temperatures of a series of nucleic acids is generally applicable and desirable in all situations where more than one sequence is used simultaneously (e.g. DNA arrays with more than one capture probe, PCR and especially multiplex PCR, homogeneous assays such as Taqman and Molecular beacon).
  • Sample preparation of specific sequences e.g., DNA or RNA extraction using capture probes on filters or magnetic beads
  • melting temperature equalization of specific probe sequences is useful.
  • the invention provides high affinity nucleotides (e.g., LNA and other high affinity nucleotides with a modified base and/or backbone) that can be used, e.g., arrays of the invention.
  • the nucleic acids of the invention containing LNA units exhibited a suprising ability to discriminate between different mRNA splice variants compared to naturally-occurring nucleic acids.
  • universal bases can be added as part of flanking regions in capture probes (e.g., probes of an array) to stabilize hybridization with high affinity nucleotides in the capture probes.
  • Replacement of one or more DNA-t nucleotides with LNA-T and/or replacement of one or more DNA-a nucleotides with LNA-A reduces the variability of melting temperatures for capture probes of similar length but different GC and AT content by desirably at least 10, 20, 30, 40 or 50%. Additionally, replacement of one or more DNA-t nucleotides with LNA-T and or replacement of one or more DNA-c with LNA-C increases the stability of a large number of capture probes, while desirably avoiding self-complementary sequences with LNA:LNA base-pairs within a capture probe that would otherwise reduce or eliminate the binding of target molecules to the probe.
  • the invention also provides a general substitution algorithm for enhancement of the hybridization signal of a test nucleic acid sample by inclusion of high affinity monomers (e.g., LNA and other high affinity nucleotides with a modified base and/or backbone) in the array.
  • high affinity monomers e.g., LNA and other high affinity nucleotides with a modified base and/or backbone
  • This method increases the stability and binding affinity of capture probes while avoiding substitutions in positions that may form self-complementary base-pairs which may otherwise inhibit binding to a target molecule.
  • the substitution algorithm is broadly useful for specialized arrays, as well as for PCR primers and FISH probes.
  • LNA Locked Nucleoside Analogues
  • nucleoside analogues e.g., bicyclic nucleoside analogues, e.g., as disclosed in WO 9914226
  • LNA nucleoside and LNA nucleotide e.g., LNA nucleoside and LNA nucleotide
  • LNA includes the compounds as described in the present specificatiion including the compounds described in Example 17.
  • the term “monomeric LNA” may, e.g., refer to the monomers LNA A, LNA T, LNA C, or any other LNA monomers.
  • LNA unit is meant an individual LNA monomer (e.g., an LNA nucleoside or LNA nucleotide) or an oligomer (e.g., an oligonucleotide or nucleic acid) that includes at least one LNA monomer.
  • LNA units as disclosed in WO 99/14226 are in general particularly desirable modified nucleic acids for incorporation into an oligonucleotide of the invention.
  • the nucleic acids may be modified at either the 3' and/or 5' end by any type of modification known in the art. For example, either or both ends may be capped with a protecting group, attached to a flexible linking group, attached to a reactive group to aid in attachment to the substrate surface, etc.
  • Desirable LNA units and their method of synthesis also are disclosed in WO 0056746, WO 0056748, WO 0066604, Morita et al, Bioorg. Med. Chem. Lett. 12(l):73-76, 2002; Hakansson et al. , Bioorg. Med. Chem. Lett. l l(7):935-938, 2001; Koshkin et al, J. Org. Chem. 66(25):8504-8512, 2001; Kvaemo et al, J. Org. Chem. 66(16):5498-5503, 2001; Hakansson etal, J. Org. Chem.
  • LNA modified oligonucleotide a oligonucleotide comprising at least one LNA monomeric unit of the general scheme A, described infra, having the below described illustrative examples of modifications:
  • X is selected from -O-, -S-, -N(R N , -C(R°R 6 )-, -O-C(R 7 R 7 )-, -C(R 6 R 6 )-O-, -S- C(R 7 R 7* )-, -C(R 6 R 6* )-S-, -N(R N* )-C(R 7 R 7* )-, -C(R 6 R 6* )-N(R N *)-, and -C(R 6 R 6* )-C(R 7 R 7* ).
  • B is selected from a modified base as discussed above e.g.
  • an optionally substituted carbocyclic aryl such as optionally substituted pyrene or optionally substituted pyrenylmethylglycerol, or an optionally substituted heteroalicylic or optionally substituted heteroaromatic such as optionally substituted pyridyloxazole, optionally substituted pyrrole, optionally substituted diazole or optionally substituted triazole moieties; hydrogen, hydroxy, optionally substituted C ⁇ -alkoxy, optionally substituted C ⁇ -alkyl, optionally substituted Ci. 4 -acyloxy, nucleobases, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands.
  • P designates the radical position for an intemucleoside linkage to a succeeding monomer, or a 5'-terminal group, such intemucleoside linkage or 5'-terminal group optionally including the substituent R .
  • One of the substituents R , R , R , and R is a group P* which designates an intemucleoside linkage to a preceding monomer, or a 273 '-terminal group.
  • 6 -alkyl-aminocarbonyl mono- and 6 -alkyl-aminocarbonyl, Ci- ⁇ -alkyl-carbonylamino, carbamido, Ci. 6 -alkanoyloxy, sulphono, Ci- 6 -alkylsulphonyloxy, nitro, azido, sulphanyl, C ⁇ .
  • Each of the substituents R 1* , R 2 , R 2* , R 3 , R 4* , R 5 , R 5* , R 6 and R 6* , R 7 , and R 7* which are present and not involved in P, P or the biradical(s), is independently selected from hydrogen, optionally substituted C ⁇ _ ⁇ 2 -alkyl, optionally substituted C 2 _ ⁇ 2 -alkenyl, optionally substituted C 2 _ 1 -alkynyl, hydroxy, C 2 .
  • Exemplary 5', 3', and/or 2' terminal groups include -H, -OH, halo (e.g., chloro, fluoro, iodo, or bromo), optionally substituted aryl, (e.g., phenyl or benzyl), alkyl (e.g, methyl or ethyl), alkoxy (e.g., methoxy), acyl (e.g.
  • acetyl or benzoyl aroyl, aralkyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acylamino, aroylamine, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, arylsulfmyl, heteroarylsulfinyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio,amidino, amino, carbamoyl, sulfamoyl, alkene, alkyne, protecting groups (e.g., silyl, 4,4'-dimethoxytrityl, monomethoxytrityl, or trityl
  • references herein to a nucleic acid unit, nucleic acid residue, LNA unit, or similar term are inclusive of both individual nucleoside units and nucleotide units and nucleoside units and nucleotide units within an oligonucleotide.
  • a “modified base” or other similar term refers to a composition (e.g., a non-naturally occuring nucleobase or nucleosidic base) which can pair with a natural base (e.g., adenine, guanine, cytosine, uracil, and/or thymine) and/or can pair with a non-naturally occurring nucleobase or nucleosidic base.
  • the modified base provides a T m differential of 15, 12, 10, 8, 6, 4, or 2°C or less as described herein.
  • Exemplary modified bases are described in EP 1 072 679 and WO 97/12896.
  • nucleobase is meant the naturally occurring nucleobases adenine (A), guanine
  • G cytosine
  • C cytosine
  • T thymine
  • U uracil
  • nucleobases such as xanthine, diaminopurine, 8-oxo-N 6 -methyladenine, 7-deazaxanthine, 7-deazaguanine, N 4 ,N 4 -ethanocytosin, N 6 ,N 6 -ethano-2,6-diaminopurine, 5-methylcytosine (mC), 5-(C 3 -C 6 )- alkynyl-cytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4- triazolopyridin, isocytosine, isoguanine, inosine and the "non-naturally occurring" nucleobases described in Benner et al., U.S.
  • nucleobase thus includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof. Further naturally and non-naturally occurring nucleobases include those disclosed in U.S. Pat. No. 3,687,808 (Merigan, et al.), in Chapter 15 by Sanghvi, in Antisense Research and Application, Ed. S. T. Crooke and B.
  • nucleosidic base or “base unit” is further intended to include compounds such as heterocyclic compounds that can serve like nucleobases including certain "universal bases” that are not nucleosidic bases in the most classical sense but serve as nucleosidic bases.
  • universal bases are 3-nitropyrrole, optionally substituted indoles (e.g., 5-nitroindole), and optionally substituted hypoxanthine.
  • Other desirable universal bases include, pyrrole, diazole or triazole derivatives, including those universal bases known in the art.
  • various groups of an LNA unit may be optionally substituted.
  • substituted group such as a nucleobase or nucleosidic base and the like may be substituted by other than hydrogen at one or more available positions, typically 1 to 3 or 4 positions, by one or more suitable groups such as those disclosed herein.
  • suitable groups that may be present on a "substituted” group include e.g.
  • alkanoyl such as a C ⁇ - 6 alkanoyl group such as acyl and the like; carboxamido; alkyl groups including those groups having 1 to about 12 carbon atoms, or 1, 2, 3, 4, 5, or 6 carbon atoms; alkenyl and alkynyl groups including groups having one or more unsaturated linkages and from 2 to 12 carbon, or 2, 3, 4, 5 or 6 carbon atoms; alkoxy groups including those having one or more oxygen linkages and from 1 to about 12 carbon atoms, or 1, 2, 3, 4, 5 or 6 carbon atoms; aryloxy such as phenoxy; alkylthio groups including those moieties having one or more thioether linkages and from 1 to about 12 carbon atoms, or 1, 2, 3, 4, 5 or 6 carbon atoms; alkylsulfinyl groups including those moieties having one or more s
  • coumarinyl quinolinyl, pyridyl, pyrazinyl, pyrimidyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl, benzofuranyl, benzothiazolyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino and pyrrolidinyl.
  • oxy-LNA monomer or unit is meant any nucleoside or nucleotide which contains an oxygen atom in a 2'-4' linkage.
  • non-oxy-LNA monomer or unit is broadly defined as any nucleoside or nucleotide which does not contain an oxygen atom in a 2'-4'- linkage.
  • non-oxy- LNA monomers include 2'-deoxynucleotides (DNA) or nucleotides (RNA) or any analogues of these monomers which are not oxy-LNA, such as for example the thio-LNA and amino- LNA described herein with respect to formula la and in Singh et al. J. Org. Chem. 1998, 6, 6078-9, and the derivatives described in Susan M. Freier and Karl-Heinz Altmann, Nucleic Acids Research, 1997, vol 25, pp 4429-4443.
  • universal base is meant a naturally-occurring or desirably a non-naturally occurring compound or moiety that can pair with a natural base (e.g., adenine, guanine, cytosine, uracil, and/or thymine), and that has a T m differential of 15, 12, 10, 8, 6, 4, or 2°C or less as described herein.
  • oligonucleotide oligomer
  • oligo is meant a successive chain of monomers (e.g., glycosides of heterocyclic bases) connected via intemucleoside linkages.
  • ucceeding monomer is meant the neighboring monomer in the 5'-terminal direction
  • preceding monomer is meant the neighboring monomer in the 3 '-terminal direction
  • LNA spiked oligo an oligonucleotide, such as a DNA oligonucleotide, wherein at least one unit (and preferably not all units) has been substituted by the corresponding LNA nucleoside monomer.
  • T m is used in reference to the "melting temperature.”
  • the melting temperature is the temperature at which 50% of a population of double-stranded nucleic acid molecules becomes dissociated into single strands.
  • the equation for calculating the T m of nucleic acids is well-known in the art.
  • T m differential of a specified amount (e.g., less than 15, 12, 10, 8, 6, 4, 2, or 1°C) means the nucleic acid exhibits that specified T m differential when incorporated into a specified 9-mer oligonucleotide with respect to the four complementary variants, as defined immediately below.
  • a T m differential provided by a particular modified base is calculated by the following protocol (steps a) through d)): a) incorporating the modified base of interest into the following oligonucleotide 5'-d(GTGAMATGC), wherein is the modified base; b) mixing 1.5 x 10 "6 M of the oligonucleotide having incorporated therein the modified base with each of 1.5x10 "6 M of the four oligonucleotides having the sequence 3'- d(CACTYTACG), wherein Y is A, C, G, T, respectively, in a buffer of lOmM sodium phosphate, 100 mM sodium chloride, 0.1 mM EDTA, pH 7.0; c) allowing the oligonucleotides to hybridize; and d) detecting the T m for each of the four hybridized nucleotides by heating the hybridized nucleotides and observing the temperature at which the maximum of the first derivative of the melting
  • a T m differential for a particular modified base is determined by subtracting the highest T m value determined in steps a) tlirough d) immediately above from the lowest T m value determined by steps a) through d) immediately above.
  • T m is meant the variance in the values of the melting temperatures for a population of nucleic acids.
  • the T m for each nucleic acid is determined by experimentally measuring or computationally predicting the temperature at which 50% of a population double-stranded molecules with the sequence of the nucleic acid becomes dissociated into single strands.
  • the T m is the temperature at which 50% of a population of 100% complementary double-stranded molecules with the sequence of the nucleic acid becomes dissociated into single strands.
  • the T m of this "modified" nucleic acid is approximated by determining the T m for each possible double stranded molecule in which one strand is the modified nucleic acid and the other strand has either A, T, C, or G in each position corresponding to a nucleobase other than A, T, C, G, or U in the modified nucleic acid.
  • the modified nucleic acid has the sequence XMX in which X is 0, 1, or more A, T, C, G, or U bases and M is any other nucleobase or nucleosidic base
  • the T m is calculated for each possible double stranded molecule in which one strand is XMX and the other strand is X' YX' in which X' is the base complementary to the corresponding X base and Y is either A, T, C, or G.
  • the average is then calculated for the T m values for each possible double stranded molecule (i.e., four possible duplexes per modified nucleobase or nucleoside base in the modified nucleic acid) and used as the approximate T m value for the modified nucleic acid.
  • capture efficiency is meant the amount of target nucleic acid(s) bound to a particular nucleic acid or a population of nucleic acids. Standard methods can be used to calculate the capture efficiency by measuring the amount of bound target nucleic acid(s) and/or measuring the amount of unbound target nucleic acid(s).
  • the capture efficiency of a nucleic acid or nucleic acid population of the invention is typically compared to the capture efficiency of a control nucleic acid or nucleic acid population under the same incubation conditions (e.g., using same buffer and temperature).
  • a control nucleic acid may have ⁇ -D-2-deoxyribose instead of one or more bicyclic or sugar groups of a LNA unit or other modified or non-naturally-occurring units in a nucleic acid of the invention.
  • the nucleic acid of the invention and the control nucleic acid only have naturally-occurring nucleobases.
  • the capture efficiency of the corresponding control nucleic acid is calculated as the average capture efficiency for all of the nucleic acids that have either A, T, C, or G in each position corresponding to a non-naturally-occurring nucleobase in the nucleic acid of the invention.
  • Monomers are referred to as being "complementary” if they contain nucleobases that can form hydrogen bonds according to Watson-Crick base-pairing rules (e.g., G with C, A with T, or A with U) or other hydrogen bonding motifs such as for example diaminopurine with T, inosine with C, and pseudoisocytosine with G.
  • substantially complementarity is meant having a sequence that is at least 60, 70, 80, 90, 95, or 100% complementary to that of another sequence. Sequence complementarity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). This software program matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.
  • the term “homology” refers to a degree of complementarity. There can be partial homology or complete homology (i.e., identity). A partially complementary sequence that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid is referred to using the functional term "substantially homologous.”
  • substantially homologous refers to a probe that can hybridize to a strand of the double-stranded nucleic acid sequence under conditions of low stringency, e.g. using a hybridization buffer comprising 20% formamide in 0.8M saline/0.08M sodium citrate (SSC) buffer at a temperature of 37°C and remaining bound when subject to washing once with that SSC buffer at 37°C.
  • SSC sodium citrate
  • substantially homologous refers to a probe that can hybridize to (i.e., is the complement of) the single-stranded nucleic acid template sequence under conditions of low stringency, e.g. using a hybridization buffer comprising 20% fomiamide in 0.8M saline/0.08M sodium citrate (SSC) buffer at a temperature of 37°C and remaining bound when subject to washing once with that SSC buffer at 37°C.
  • SSC sodium citrate
  • nucleic acid e.g., a probe or primer
  • the internal probe may hybridize to the 5' end of the exon or intron, the 3' end of the exon or intron, or between the 5' end and the 3' end of the exon or intron.
  • the internal probe is at least 90, 95, 96, 97, 98, 99, or 100% identical to the corresponding region of a target nucleic acid.
  • merged probe is meant a nucleic acid (e.g., a probe or primer) that hybridizes to more than one exon and/or intron of a nucleic acid (e.g., mRNA). Desirably, the merged probe hybridizes to two consecutive exons (e.g., exons in a mature mRNA transcript that may or may not be consecutive in the corresponding DNA molecule). In another desirable embodiment, the merged probe hybridizes to an exon and the consecutive intron. In desirable embodiments, the merged probe hybridizes to the same number of nucleotides in each exon or to the same number of nucleotides in the exon and intron.
  • a nucleic acid e.g., a probe or primer
  • the length of the region of the merged probe that hybridizes to one exon differs by less than 60, 40, 20, 10, or 5% from the length of the region of the merged probe that hybridizes to the other exon or to the intron.
  • the merged probe is at least 90, 95, 96, 91, 98, 99, or 100% identical to the corresponding region of a target nucleic acid.
  • poly-T 2 o tail is meant a DNA polymer consisting of 20 DNA-t units added by polymerase chain reaction as a tail to a nucleic acid sequence, which is subsequently cloned in a plasmid vector allowing in vitro synthesis of poly(A) 2 o polyadenylated RNA.
  • RNA or DNA unit e.g., a naturally-occurring RNA or DNA unit.
  • corresponding unmodified reference nucleobase is meant a nucleobase that is not part of an LNA unit and is in the same orientation as the nucleobase in an LNA unit.
  • mutation is meant an alteration in a naturally-occurring or reference nucleic acid sequence, such as an insertion, deletion, frameshift mutation, silent mutation, nonsense mutation, or missense mutation.
  • the amino acid sequence encoded by the nucleic acid sequence has at least one amino acid alteration from a naturally-occurring sequence.
  • selecting is meant substantially partitioning a molecule from other molecules in a population.
  • the partitioning provides at least a 2-fold, desirably, a 30-fold, more desirably, a 100-fold, and most desirably, a 1, 000-fold enrichment of a desired molecule relative to undesired molecules in a population following the selection step.
  • the selection step may be repeated a number of times, and different types of selection steps may be combined in a given approach.
  • the population desirably contains at least 10 9 molecules, more desirably at least 10 11 , 10 13 , or 10 14 molecules and, most desirably, at least 10 15 molecules.
  • apopulation is meant more than one nucleic acid.
  • a “population” according to the invention desirably means more than 10 1 , 10 2 , 10 3 , or 10 different molecules.
  • photochemically active groups is meant compounds which are able to undergo chemical reactions upon irradiation with light.
  • functional groups are quinones, especially 6-methyl-l,4-naphtoquinone, anthraquinone, naphtoquinone, and 1,4- dimethyl-anthraquinone, diazirines, aromatic azides, benzophenones, psoralens, diazo compounds, and diazirino compounds.
  • thermochemically reactive group is meant a functional group which is able to undergo thermochemically-induced covalent bond formation with other groups.
  • functional parts of thermochemically reactive groups are carboxylic acids, carboxylic acid esters such as activated esters, carboxylic acid halides such as acid fluorides, acid chlorides, acid bromide, acid iodides, carboxylic acid azides, carboxylic acid hydrazides, sulfonic acids, sulfonic acid esters, sulfonic acid halides, semicarbazides, thiosemicarbazides, aldehydes, ketones, primary alcohols, secondary alcohols, tertiary alcohols, phenols, alkyl halides, thiols, disulphides, primary amines, secondary amines, tertiary amines, hydrazines, epoxides, maleimides, and boronic acid derivatives.
  • chelating group is meant a molecule that contains more than one binding site and frequently binds to another molecule, atom, or ion through more than one binding site at the same time.
  • functional parts of chelating groups are iminodiacetic acid, nitrilotriacetic acid, ethylenediamine tetraacetic acid (EDTA), and aminophosphonic acid.
  • reporter group is meant a group which is detectable either by itself or as a part of an detection series. Examples of functional parts of reporter groups are biotin, digoxigenin, fluorescent groups (e.g., groups which are able to absorb electromagnetic radiation, e.g.
  • Ligand is meant a compound which binds.
  • Ligands can comprise functional groups such as aromatic groups (such as benzene, pyridine, naphthalene, anthracene, and phenanthrene), heteroaromatic groups (such as thiophene, furan, tetrahydrofuran, pyridine, dioxane, and pyrimidine), carboxylic acids, carboxylic acid esters, carboxylic acid halides, carboxylic acid azides, carboxylic acid hydrazides, sulfonic acids, sulfonic acid esters, sulfonic acid halides, semicarbazides, thiosemicarbazides, aldehydes, ketones, primary alcohols, secondary alcohols, tertiary alcohols, phenols, alkyl halides, thiols, disulphides, primary amines, secondary amines, tertiary amines, hydrazines, epoxide
  • Affinity ligands include functional groups or biomolecules that have a specific affinity for sites on particular proteins, antibodies, poly- and oligosaccharides, and other biomolecules. It should be understood that the above-mentioned specific examples under DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands correspond to the "active/functional" part of the groups in question. For the person skilled in the art it is furthermore clear that DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands are typically represented in the form M-K- where M is the
  • the group B in the case where B is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, has the form M-K-, where M is the "active/functional" part of the DNA intercalator, photochemically active group, thermochemically active group, chelating group, reporter group, and ligand, respectively, and where K is an optional spacer comprising 1-50 atoms, desirably 1-30 atoms, in particular 1-15 atoms, between the 5- or 6-membered ring and the "active/functional" part.
  • spacer is meant a thermochemically and photochemically non-active distance- making group and is used to join two or more different moieties of the types defined above. Spacers are selected on the basis of a variety of characteristics including their hydrophobicity, hydrophilicity, molecular flexibility and length (e.g., Hermanson et. al, "Immobilized Affinity Ligand Techniques," Academic Press, San Diego, California (1992). Generally, the length of the spacers is less than or about 400 A, in some applications desirably less than 100 A.
  • the spacer thus, comprises a chain of carbon atoms optionally interrupted or terminated with one or more heteroatoms, such as oxygen atoms, nitrogen atoms, and/or sulphur atoms.
  • the spacer K may comprise one or more amide, ester, amino, ether, and/or thioether functionalities, and optionally aromatic or mono/polyunsaturated hydrocarbons, polyoxyethylene such as polyethylene glycol, oligo/polyamides such as poly- ⁇ -alanine, polyglycine, polylysine, peptides, oligosaccharides, or oligo/polyphosphates.
  • the spacer may consist of combined units thereof.
  • the length of the spacer may vary, taking into consideration the desired or necessary positioning and spatial orientation of the "active/functional" part of the group in question in relation to the 5- or 6-membered ring.
  • the spacer includes a chemically cleavable group. Examples of such chemically cleavable groups include disulphide groups cleavable under reductive conditions and peptide fragments cleavable by peptidases.
  • target nucleic acid or “nucleic acid target” is meant a particular nucleic acid sequence of interest. Thus, the "target” can exist in the presence of other nucleic acid molecules or within a larger nucleic acid molecule.
  • solid support any rigid or semi-rigid material to which a nucleic acid binds or is directly or indirectly attached.
  • the support can be any porous or non-porous water insoluble material, including without limitation, membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, strips, plates, rods, polymers, particles, microparticles, capillaries, and the like.
  • the support can have a variety of surface forms, such as wells, trenches, pins, channels and pores.
  • an “array” is meant a fixed pattern of at least two different immobilized nucleic acids on a solid support.
  • the array includes at least 10 , more desirably, at least 10 3 , and, most desirably, at least 10 4 different nucleic acids.
  • antisense nucleic acid is meant a nucleic acid, regardless of length, that is complementary to a coding strand or mRNA of interest. In some embodiments, the antisense molecule inhibits the expression of only one nucleic acid, and in other embodiments, the antisense molecule inhibits the expression of more than one nucleic acid. Desirably, the antisense nucleic acid decreases the expression or biological activity of a nucleic and or encoded protein by at least 20, 40, 50, 60, 70, 80, 90, 95, or 100%. An antisense molecule can be introduced, e.g., to an individual cell or to whole animals, for example, it may be introduced systemically via the bloodstream.
  • a region of the antisense nucleic acid or the entire antisense nucleic acid is at least 70, 80, 90, 95, 98, or 100% complementary to a coding sequence, regulatory region (5' or 3' untranslated region), or an mRNA of interest.
  • the region of complementarity includes at least 5, 10, 20, 30, 50, 75,100, 200, 500, 1000, 2000 or 5000 nucleotides or includes all of the nucleotides in the antisense nucleic acid.
  • the antisense molecule is less than 200, 150, 100, 75, 50, or 25 nucleotides in length.
  • the antisense molecule is less than 50,000; 10,000; 5,000; or 2,000 nucleotides in length. In certain embodiments, the antisense molecule is at least 200, 300, 500, 1000, or 5000 nucleotides in length. In some embodiments, the number of nucleotides in the antisense molecule is contained in one of the following ranges: 5-15 nucleotides, 16-20 nucleotides, 21-25 nucleotides, 26-35 nucleotides, 36-45 nucleotides, 46-60 nucleotides, 61-80 nucleotides, 81-100 nucleotides, 101-150 nucleotides, or 151-200 nucleotides, inclusive. In addition, the antisense molecule may contain a sequence that is less than a full-length sequence or may contain a full-length sequence.
  • double stranded nucleic acid is meant a nucleic acid containing a region of two or more nucleotides that are in a double stranded conformation.
  • the double stranded nucleic acids consist entirely of LNA units or a mixture of LNA units, ribonucleotides, and/or deoxynucleotides.
  • the double stranded nucleic acid may be a single molecule with a region of self-complementarity such that nucleotides in one segment of the molecule base-pair with nucleotides in another segment of the molecule.
  • the double stranded nucleic acid may include two different strands that have a region of complementarity to each other.
  • the regions of complementarity are at least 70, 80, 90, 95, 98, or 100% identical.
  • the region of the double stranded nucleic acid that is present in a double stranded conformation includes at least 5, 10, 20, 30, 50, 75,100, 200, 500, 1000, 2000 or 5000 nucleotides or includes all of the nucleotides in the double stranded nucleic acid.
  • Desirable double stranded nucleic acid molecules have a strand or region that is at least 70, 80, 90, 95, 98, or 100% identical to a coding region or a regulatory sequence (e.g., a transcription factor binding site, a promoter, or a 5' or 3' untranslated region) of a nucleic acid of interest.
  • the double stranded nucleic acid is less than 200, 150, 100, 75, 50, or 25 nucleotides in length. In other embodiments, the double stranded nucleic acid is less than 50,000; 10,000; 5,000; or 2,000 nucleotides in length.
  • the double stranded nucleic acid is at least 200, 300, 500, 1000, or 5000 nucleotides in length.
  • the number of nucleotides in the double stranded nucleic acid is contained in one of the following ranges: 5-15 nucleotides, 16-20 nucleotides, 21-25 nucleotides, 26-35 nucleotides, 36-45 nucleotides, 46-60 nucleotides, 61- 80 nucleotides, 81-100 nucleotides, 101-150 nucleotides, or 151-200 nucleotides, inclusive.
  • the double stranded nucleic acid may contain a sequence that is less than a full- length sequence or may contain a full-length sequence.
  • the double stranded nucleic acid inhibits the expression of only one nucleic acid, and in other embodiments, the double stranded nucleic acid molecule inhibits the expression of more than one nucleic acid.
  • the nucleic acid decreases the expression or biological activity of a nucleic acid of interest or a protein encoded by a nucleic acid of interest by at least 20, 40, 50, 60, 70, 80, 90, 95, or 100%.
  • a double stranded nucleic acid can be introduced, e.g., to an individual cell or to whole animals, for example, it may be introduced systemically via the bloodstream.
  • the double stranded nucleic acid or antisense molecule includes one or more LNA nucleotides, one or more universal bases, and/or one or more modified nucleotides in wliich the 2' position in the sugar (e.g., ribose or xylose) contains a halogen (such as fluorine group) or contains an alkoxy group (such as a methoxy group) which increases the half-life of the double stranded nucleic acid or antisense molecule in vitro or in vivo compared to the corresponding double stranded nucleic acid or antisense molecule in which the corresponding 2' position contains a hydrogen or an hydroxyl group.
  • a halogen such as fluorine group
  • alkoxy group such as a methoxy group
  • the double stranded nucleic acid or antisense molecule includes one or more linkages between adjacent nucleotides other than a naturally-occurring phosphodiester linkage.
  • linkages include phosphoramide, phosphorothioate, and phosphorodithioate linkages.
  • the double stranded or antisense molecule is purified.
  • a factor is substantially pure when it is at least 50%, by weight, free from proteins, antibodies, and naturally-occurring organic molecules with which it is naturally associated. Desirably, the factor is at least 75%, more desirably, at least 90%, and most desirably, at least 99%, by weight, pure.
  • a substantially pure factor may be obtained by chemical synthesis, separation of the factor from natural sources, or production of the factor in a recombinant host cell that does not naturally produce the factor. Nucleic acids and proteins may be purified by one skilled in the art using standard techniques such as those described by Ausubel et al.
  • the factor is desirably at least 2, 5, or 10 times as pure as the starting material, as measured using polyacrylamide gel electrophoresis, column chromatography, optical density, HPLC analysis, or western analysis (Ausubel et al, supra). Desirable methods of purification include immunoprecipitation, column chromatography such as immunoaffinity chromatography, magnetic bead immunoaffinity purification, and panning with a plate-bound antibody.
  • treating, stabilizing, or preventing a disease, disorder, or condition is meant preventing or delaying an initial or subsequent occurrence of a disease, disorder, or condition; increasing the disease-free survival time between the disappearance of a condition and its reoccurrence; stabilizing or reducing an adverse symptom associated with a condition; or inhibiting or stabilizing the progression of a condition.
  • at least 20, 40, 60, 80, 90, or 95% of the treated subjects have a complete remission in which all evidence of the disease disappears.
  • the length of time a patient survives after being diagnosed with a condition and treated with a nucleic acid of the invention is at least 20, 40, 60, 80, 100, 200, or even 500% greater than (i) the average amount of time an untreated patient survives or (ii) the average amount of time a patient treated with another therapy survives.
  • treating, stabilizing, or preventing cancer is meant causing a reduction in the size of a tumor, slowing or preventing an increase in the size of a tumor, increasing the disease-free survival time between the disappearance of a tumor and its reappearance, preventing an initial or subsequent occurrence of a tumor, or reducing an adverse symptom associated with a tumor.
  • the number of cancerous cells surviving the treatment is at least 20, 40, 60, 80, or 100% lower than the initial number of cancerous cells, as measured using any standard assay.
  • the decrease in the number of cancerous cells induced by administration of a nucleic acid of the invention is at least 2, 5, 10, 20, or 50-fold greater than the decrease in the number of non- cancerous cells.
  • the number of cancerous cells present after administration of a nucleic acid of the invention is at least 2, 5, 10, 20, or 50-fold lower than the number of cancerous cells present prior to the administration of the compound or after administration of a buffer control.
  • the methods of the present invention result in a decrease of 20, 40, 60, 80, or 100% in the size of a tumor as determined using standard methods.
  • at least 20, 40, 60, 80, 90, or 95% of the treated subjects have a complete remission in which all evidence of the cancer disappears.
  • the cancer does not reappear or reappears after at least 5, 10, 15, or 20 years.
  • Exemplary cancers that can be treated, stabilized, or prevented using the above methods include prostate cancers, breast cancers, ovarian cancers, pancreatic cancers, gastric cancers, bladder cancers, salivary gland carcinomas, gastrointestinal cancers, lung cancers, colon cancers, melanomas, brain tumors, leukemias, lymphomas, and carcinomas. Benign tumors may also be treated or prevented using the methods and nucleic acids of the present invention.
  • infection is meant the invasion of a host animal by a pathogen (e.g., a bacteria, yeast, or virus).
  • the infection may include the excessive growth of a pathogen that is normally present in or on the body of an animal or growth of a pathogen that is not normally present in or on the animal.
  • an infection can be any situation in which the presence of a pathogen population(s) is damaging to a host.
  • an animal is "suffering" from an infection when an excessive amount of a pathogen population is present in or on the animal's body, or when the presence of a pathogen population(s) is damaging the cells or other tissue of the animal.
  • the number of a particular genus or species of pathogen is at least 2, 4, 6, or 8 times the number normally found in the animal.
  • a bacterial infection may be due to gram positive and/or gram negative bacteria. In desirable embodiments, the bacterial infection is due to one or more of the following bacteria: Chlamydophila pneumoniae, C. psittaci, C.
  • abortus Chlamydia trachomatis, Simkania negevensis, Parachlamydia acanthamoebae, Pseudomonas aeruginosa, P. alcaligenes, P. chlororaphis, P. fluorescens, P. luteola, P. mendocina, P. monteilii, P. oryzihabitans, P. pertocinogena, P. pseudalcaligenes, P. putida, P.
  • a nucleic acid is administered in an amount sufficient to prevent, stabilize, or inhibit the growth of a pathogenic bacteria or to kill the bacteria.
  • the viral infection relevant to the methods of the invention is an infection by one or more of the following viruses: West Nile viras (e.g., Samuel, "Host genetic variability and West Nile virus susceptibility,” Proc. Natl. Acad. Sci.
  • West Nile viras e.g., Samuel, "Host genetic variability and West Nile virus susceptibility," Proc. Natl. Acad. Sci.
  • mammal in need of treatment is meant a mammal in which a disease, disorder, or condition is treated, stabilized, or prevented by the administration of a nucleic acid of the invention.
  • Figure 1 shows the structures of selected nucleotide monomers: DNA (T), LNA (T L ), pyrene DNA (Py), 2'-OMe-RNA [2'-OMe(T)], abasic LNA (Ab L ), phenyl LNA (17a), and pyrenyl LNA (17d).
  • Figure 2 illustrates the chemical structures of Selective Binding Complementary (SBC) nucleotides.
  • SBC Selective Binding Complementary
  • Figure 3 illustrates a synthetic route
  • Figure 4 shows the base-pairing between modified bases and naturally-occurring 5 nucleotides. These modified bases may be incorporated as part of an LNA, DNA, or RNA unit and used any of the oligomers of the invention.
  • Figures 5-8 illustrate various synthetic routes.
  • Figure 9 is a schematic illustration of the use of an exemplary synthesis for LNA- furanoPyr-SBC-C.
  • Figures 10 and 11 illustrate various synthetic routes.
  • Figure 12 shows the sensitivity of 50-mer LNA capture probes compared to 50-mer DNA capture probes at 5, 10 and 20 ⁇ M, respectively.
  • SW15-specific 50-mer DNA oligonucleotides (1 st , 3 rd and 5 th bars) and 50-mer capture probes with an LNA nucleotide incorporated at every third nucleotide position (2 n , 4 , and 6th bars) were printed at the oligo 15 concentration indicated below.
  • the slides were hybridized at 65°C in 3x SSC ( Figure 12 (left)) and at 70°C in 3xSSC ( Figure 12 (right)).
  • Figure 13 shows the specificity of 40-mer LNA capture probes (bar 7-12) compared to DNA capture probes (bar 1-6).
  • the hybridizations were carried out at 65°C in 3XSSC. Bars 1 and 7 represent perfectly matched duplexes, bars 2 and 8, 3, and 9, 4 and 10, 5 and 11, 20 and 6 and 12 represent duplexes with 1, 2, 3, 4, 5 mismatches, respectively.
  • the in vitro RNA used was SW15 in Figure 13 (left) and TH14 in Figure 13 (right).
  • Figure 14 shows the detection principle for alternative exon skipping in the C. elegans let-2 gene using LNA oligonucleotide capture probes and comparative expression profiling.
  • Figure 15 shows the detection of alternative splicing of C. elegans Let-2 exon 9 and 25 10 using LNA-modified capture probes.
  • Figure 16 shows the comparison of DNA and LNA-modified oligonucleotide capture probes in the specific capture of the C. elegans T01D3.3 mRNA, exon 4.
  • Figure 17 illustrates the LNA exon-exon junction (merged) probe concept.
  • Figure 18 shows the capture probe specificity for the C. elegans T01D3.3 mRNA, 30 exon 4 ( Figure 18 (upper)) and exon 5 ( Figure 18 (lower)) as validated by short complementary target oligonucleotides.
  • Figure 19 shows the construction of the recombinant splice variants in the in vitro transcription vector.
  • the small bars show the location of the hybridization for the oligonucleotide capture probes used in this example. The sequences of the capture probes are described herein.
  • Figure 20 shows the detection of splice variant #1 and #2, respectively using merged capture probes in a comparative, two- color hybridization.
  • Figure 21 shows the sensitivity of 50-mer LNA capture probes compared to 50-mer DNA capture probes.
  • the slides were hybridized at 65°C in 3xSSC.
  • Figure 22 is a bar graph of the signal intensities of a patient DNA sample hybridized to an array of the invention.
  • the names of the probes in Figure 22 and in the table in Example 7 match, although the numbers used in Figure 22 are abbreviated, e.g., probe No.
  • Figure 23 is a graph comparing the spot intensity for probes of the invention with different LNA substitution patterns.
  • Figure 24 is a bar graph of the spot intensity for LNA probes for different exons.
  • Figure 25 illustrates a synthetic route
  • Figure 26 is a flow chart of the steps of oligo design software of the invention.
  • the OligoDesign software features LNA modified oligonucleotide secondary structure prediction, LNA spiked oligonucleotide melting temperature prediction, genome wide cross hybridization prediction, secondary stracture prediction of the target, and recognition and filtering of the target in the genome. These features are determined for each possible probe of the query gene and presented to an artificial neural network. The probes are then ranked according to the neural network prediction and the top scoring probes are returned.
  • Figure 27 is a schematic illustration of the OligoDesign software of the invention.
  • Figure 28 illustrates a synthetic route
  • Figure 29 illustrates photo-activated immobilization of nucleic acids of the invention, which enables polarized coupling of anthraquinone (AQ)-linked LNA oligonucleotides onto the polymer surface. No pretreatment of the slide is needed. A covalent bond is formed between the oligonucleotide and the polymer using a UN source, e.g. Stratalinker.
  • Figure 30 illustrates an injection-molded polymer slide. Finger indents ease slide handling. The slide has a well-defined printing and hybridization window, frosted surface for identification and orientation, and space for barcodes.
  • Figure 31 illustrates spot quality on different slides that can be used to immobilize nucleic acids of the invention.
  • the hydrophobic slide surface ensures that extremely homogenous spots are generated when hydrophilic spotting solution is applied to the surface.
  • a high spot quality is obtained on the ImmobilizerTM polymer slide compared to a glass slide when using a spot-to-spot distance of 150 :M.
  • the high-quality arrays simplify downstream image analyses.
  • Figure 32 is a schematic illustration of a method of the invention.
  • Figure 33 is a table of exemplary target nucleic acids (Holstege et al. (1998)( Cell 95, 717-728, and Causton et al. (2001) Mol. Biol. Cell 12, 323-337).
  • Figure 34 is a graph of on-chip melting profiles of 50-mer oligonucleotide probes of the invention.
  • Yeast actin 1 -specific 50-mer capture probes were synthesized as DNA and DNA/LNA mixmer oligonucleotides.
  • LNA-substituted mixmer capture probes contain an LNA at every 4 th , 5 th , and 6 th nucleotide position (LNA_4, LNA_5, LNA_6).
  • On-chip melting profiles demonstrate a 8-10 °C increase in T m obtained with LNA capture probes.
  • Figure 35 illustrates the heat-shock response in yeast.
  • the array was hybridized with Cy3-labeled standard and Cy5-labelled heat-shock yeast cDNA.
  • Figure 35 (lower) also illustrates the heat-shock response in yeast.
  • the microarray data were normalized using yeast actin 1.
  • the ssa4 gene encoding heat shock protein HSP70 is upregulated over 2-fold. Expression of the gual gene is down-regulated.
  • Figure 36 compares expression of wild-type and ssa4 mutant yeast. The array was hybridized with Cy 3 -labeled wild-type and Cy 5 -labelled ssa4 mutant yeast cDNA. Figure 36 (lower) also compares wild-type and ssa4 yeast. The hybridization data were normalized using yeast actin 1. ssa4 is detected in the wild-type yeast strain, but not in the ssa4 knock-out strain.
  • Figure 37 illustrates mRNA splicing.
  • Figure 38 is a picture showing gel electrophoresis of fragmented cDNA from the yeast wild-type strain.
  • the molecular marker (lane 1 and 9) is from Life technologies, USA.
  • Lanes 2-8 represents the UDG-fragmented cDNA 1-7 according to the different dUTP/dTTP ratios in Table 18.
  • Figure 39 is a graph of the log ratios of the normalized fluorescence intensities from the wild-type yeast strain (signal) and those from the Assa4 yeast strain (noise) as a function of capture probe position in the 3' region of the SSA4 mRNA.
  • Figure 40 is a schematic illustration of mRNA splicing.
  • Figure 41 is a schematic illustration of alternative mRNA splicing.
  • Figure 42 is a schematic illustration of probes of the invention.
  • Figure 43 is a schematic illustration of probes of the invention.
  • Figure 44 illustrates an exemplary computer for use in the methods of the invention.
  • Figure 45 shows the sensitivity and specificity of LNA oligonucleotide capture probes (black solid bars) compared to DNA capture probes (white, open bars) on expression microarrays. Fluorescence intensity is shown in arbitrary units (relative measurements).
  • the arrays comprising 50-mer and 40-mer perfect match and 1-5 mismatch capture probes were hybridized at 65°C in 3xSSC with Cy3-labelled cDNA from 10 ⁇ g C. elegans total RNA spiked with yeast a) SWI5 RNA and c) THI4 RNA.
  • Figure 46 shows the expected (black, solid bars) and observed (white, open bars) fold-of-change in the expression levels of the Cy3-ULS-labelled yeast HSP78 spike RNA as measured by on-chip capture using three different 25-mer oligonucleotide capture probes (DNA control, LNA-T substituted, LNA_3 substituted in which every third nucleotide was substituted with an LNA monomer).
  • DNA control DNA control
  • LNA-T substituted LNA_3 substituted in which every third nucleotide was substituted with an LNA monomer.
  • the fold change of the HSP78 RNA in the two hybridizations in the comparison is 5-fold.
  • Figure 47 shows the measured intensity levels by on-chip capture using three different 25-mer oligonucleotide capture probe designs ( DNA control, LNA_T substituted and LNA 5 C and T substituted probes).
  • DNA control DNA control
  • LNA_T substituted and LNA 5 C and T substituted probes DNA control
  • One (1) ng biotin-labeled HSP78 target was used in the hybridization experiments, followed by staining with Streptavidin Phycoerythrin.
  • the LNA_T and LNA_TC substituted 25-mer capture probes show a significantly enhanced on- chip capture of the HSP78 RNA target, compared to the DNA 25-mer control probes under four different hybridization stringency conditions in dicated on the graph.
  • Figure 48 illustrates a synthetic route
  • Figure 49 shows the detection of alternatively spliced mRNAs using LNA-substituted 50-mer oligonucleotide capture probes. Parts per million (ppm) calculations indicate spike transcripts per total transcripts in the hybridisation mix. Calculations are based on an average C. elegans RNA being 1000 nucleotides as in Hill et al. (2000) Science 290:809-812. The 50-
  • Figure 50 shows the detection of alternatively spliced mRNAs using LNA-substituted 25 40-mer oligonucleotide capture probes. Parts per million (ppm) calculations indicate spike transcripts per total transcripts in the hybridisation mix. Calculations are based on an average C. elegans RNA being 1000 nucleotides as in Hill et al. (2000) Science 290:809-812.
  • the 40- mer LNA-DNA mixmer capture probes, substituted with an LNA nucleotide at every third nucleotide position, are able to provide highly accurate measurements for fold-changes in the 30 expression of three homologous, alternatively spliced mRNA variants in the concentration range of 1000 ppm to 10 ppm.
  • the quantification of the splice isoforms was carried out using a set of both internal, exon-specific probes and merged, splice junction specific probes, printed onto microarrays and hybridized with complex cDNA target pools spiked with different cloned artificial splice isoforms in which the middle exon was either alternatively skipped or excluded completely resulting in the three different splice isoforms; 01-INS3-03, 01-INS4-03 and 01-03.
  • Figure 51 shows the comparison of different LNA/DNA mixmer oligonucleotide probes in the detection of human satellite-2 repeats by fluorescence in situ hybridization.
  • Figure 52 illustrates the hybridisation of the Cy3-labelled human telomere repeat specific, LNA-2 substituted oligonucleotide probe on human metaphase chromosomes, which resulted in prominent signals on the telomeres.
  • Figure 53 illustrates examples of LNA units.
  • Figure 54 illustrates a synthetic route.
  • Alternative splicing is the process by which different mature messenger RNAs are produced from the same pre-mRNA. Because the mRNA composition of a given cell determines the proteins present in a cell, this process is an important aspect of a cells gene expression profile.
  • Current investigations of transcriptomes i.e., the total complexity of RNA transcripts produced by an organism indicate that at least 50-60 % of the genes of complex eukaryotes produce more than one splice variant.
  • the present invention provides a novel method for detecting and quantifying the levels of splice variants in complex mRNA pools using LNA discriminating probes and high-throughput LNA oligonucleotide microarray technology.
  • the detection concept which uses internal LNA exon probes and/or splice-variant specific exon-exon junction or exon-intron or intron-exon (so-called merged) probes is depicted in Figure 17.
  • exon-specific (or intron-specific) LNA oligonucleotide probes are designed and used to detect the relative levels of a given exon (or intron) in complex mRNA pools using oligonucleotide microarray technology or similar techniques.
  • Exon-exon LNA junction probes are designed for multiple or all possible exon-exon combinations or exon- intron combinations.
  • the LNA discriminating probes are highly specific and superior compared to DNA oligonucleotides due to the higher ⁇ Tm of LNA probes.
  • each sub-element i.e., exon structure or exon- intron structure
  • a given alternatively spliced mRNA isoform thus giving the exact composition of the mRNA.
  • the ratios of each splice variant can be quantified using the combined readouts from both internal and merged LNA probes and control probes.
  • the invention is applicable both in single fluor (single channel) or comparative two-fluor (two channel) microarray hybridizations.
  • RNA molecules may be constructed in an in vitro transcription vector for the production of clean INT R A.
  • Both internal and junction-specific L ⁇ A oligonucleotide capture probes are designed, synthesized, and spotted onto, e.g., Exiqon's polymer microarray platform.
  • the resulting splice-specific microarray is used to validate the L ⁇ A discriminating probe concept by spiking the in vitro R ⁇ As individually as well as in different ratios into a complex R ⁇ A background for fluorochrome- labelling and array hybridization.
  • the internal and merged probes of the invention can also be used in any standard method for the analysis of mRNA splice variants (see, for example, Yeakley et al, Nature Biotechnology 20:353-358, 2002; Clark et al, Science 296:907-910, 2002; Mutch etal, Genome Biology 2(12):preprint00009.1-0009.31, 2001).
  • the internal and/or merged probes of the invention can also be used for gene expression profiling of alternative splice variants, oligonucleotide expression microarrays, real-time PCR, and profiling of alternatively spliced mRNAs using microtiterplate assays or fiber-optic arrays.
  • RNA splicing is now widely recognized as a means to generate protein diversity.
  • Comparative Genomic Hybridization is a powerful technology for detection of unbalanced chromosome rearrangements and holds much promise for screening and identification of interstitial submicroscopic rearrangements that otherwise cannot be detected using classical cytogenetic or FISH technologies.
  • the adaptation of CGH onto an oligo microarray platform allows detection of small single exon deletion/duplications on a genome wide scale.
  • microarrays that can detect, e.g., single exon aberrations. This detection can be achieved by employing LNA mixmer oligos as capture probes for individual exons in selected genes.
  • Menkes disease is a lethal-X linked recessive disorder associated with copper metabolism disturbance leading to death in early childhood.
  • the Menkes locus has been mapped to Xql3.
  • the gene spans about 150 kb genomic region, contains 23 exons, and encodes a 8.5 kb gene transcript.
  • the gene for Menkes disease (now designated as A TP 7 A) encodes a 1500 amino acid membrane-bound Cu-binding P-type ATPase (ATP7A).
  • ATP7A P-type ATPase
  • the 8.5 kb transcript is expressed in all tissues from normal individuals (though only trace amounts are present in liver), but is diminished or absent in Menkes disease patients.
  • the Cy5 signal is higher than Cy3 if the patient genome has a deletion, and is lower if there is duplication. In regions that are unchanged, the Cy5:Cy3 ratio is 1:1.
  • LNA oligonucleotide-based CGH makes it possible to assess a large number of chromosomal aberrations that are being screen for in the cytogenetic clinic.
  • standard FISH analysis typically only detects large chromosomal rearrangements.
  • an array that contains a series of overlapping probes is used to detect a chromosomal deletion in a nucleic acid sample, such as a patient sample.
  • Clinical diagnosis is a key element in healthcare management and point-of-care.
  • a large number of analyses in the hospitals are based on the use of robust, cost efficient, sensitive and highly specific diagnostic tests.
  • diagnosis of various diseases is performed with a high selectivity and reliability, resulting in confirmation of medical diagnosis, choice of therapy and follow-up treatment as well as prevention.
  • clinical diagnosis also contributes to the control of healthcare costs.
  • the field of clinical diagnostics involves analyzing biological fluid samples (blood, urine, etc.) or biopsies collected from patients in order to establish the diagnosis of diseases, whether of infectious, metabolic, endocrine or cancerous origin.
  • Medical analysis of infectious diseases involves testing and identifying the micro-organisms causing the infection e.g.
  • micro-organism testing for and identifying a micro-organism in blood and determining its susceptibility to antibiotics or detecting an antigen-antibody reaction produced as a response to an attack by a micro-organism in the human body, e.g. testing for antibodies for the diagnosis of hepatitis.
  • the accurate diagnosis of metabolic and endocrine diseases and cancers, resulting in a disease phenotype with a bodily imbalance involves the measurement of diagnostic substances or elements present in the biological fluids or biopsies. These substances are examined and results are interpreted with reference to known normal values.
  • diagnostic kits in microbiological control
  • RNA levels have been used successfully in accurate quantification of RNA levels in clinical diagnosis as well as in microbiological control.
  • the applications are wide-ranging and include methods for quantification of the regulation and expression of drug resistance markers in tumour cells, monitoring of the responses to chemotherapy, measuring the biodistribution and transcription of gene-encoded therapeutics, molecular assessment of the tumor stage in a given cancer, detecting circulating tumor cells in cancer patients and detection of bacterial and viral pathogens.
  • RT-PCR reverse transcription polymerase chain reaction
  • RNA levels are real-time nucleic acid sequence based amplification (NASBA) combined with molecular beacon detection molecules.
  • NASBA real-time nucleic acid sequence based amplification
  • NASBA is a singe-step isothermal RNA-specific amplification method that amplifies mRNA in a double stranded DNA environment, and this method has recently proven useful in the detection of various mRNAs and in the detection of both viral and bacterial RNA in clinical samples.
  • van't Veer et al. (Nature 2002: 415, 31) describe the successful use of microarrays in obtaining digital mRNA signatures from breast tumors and the use of these signatures in the precise prediction of the clinical outcome of breast cancer in patients.
  • Locked nucleic acid (LNA) oligonucleotides constitute a novel class of bicyclic RNA analogs having an exceptionally high affinity and specificity toward their complementary DNA and RNA target molecules. Besides increased thermal stability, LNA- containing oligonucleotides show significantly increased mismatch discrimination, and allow full control of the melting temperature across microarray hybridizations.
  • the LNA chemistry is completely compatible with conventional DNA phosphoramidite chemistry and thus LNA substituted oligonucleotides can be designed to optimize performance.
  • LNA oligonucleotides would be well-suited for large-scale clinical studies providing highly accurate genotyping by direct competitive hybridization of two allele-specific LNA probes to e.g. microarrays of immobilized patient amplicons.
  • the use of LNA substituted oligonucleotides would increase both sensitivity and specificity in detection and quantification of mRNA levels in clinical samples, either by quantitative RT-PCR, quantitative NASBA or oligonucleotide microarrays, compared with DNA probes.
  • Application of LNA oligonucleotides into diagnostic kits would thus significantly enhance their performance.
  • LNA substituted oligonucleotides would increase the sensititity and specificity in the detection of alternatively spliced mRNA isoforms and non-coding RNAs either by homogeneous assays (Taqman assay, Lightcycler assay, NASBA) or by oligonucleotide microarrays in a massive parallel analysis setup.
  • T m melting temperatures
  • Similar melting temperatures also allow the same hybridization conditions to be used for multiple experiments, wliich is particularly useful for assays involving hybridization to nucleic acids of varying "AT" content.
  • current methods often require less stringent conditions for hybridization of nucleic acids with high "AT” content compared to nucleic acids with low “AT” content. Due to this variation in hybridization stringency, current methods may require significant trial and error to optimize the hybridization conditions for each experiment.
  • LNA nucleotide analogs increase their binding affinity for DNA and RNA.
  • the stability of duplexes can generally be ranked as follows: DNA:DNA ⁇ DNA:RNA ⁇ RNA:RNA ⁇ LNA:DNA ⁇ LNA:RNA ⁇ LNA:LNA.
  • the DNA:DNA duplex is thus the least stable and the LNA:LNA duplex the most stable.
  • the affinity of the LNA nucleotides A and T corresponds approximately to the affinity of DNA G and C to their complementary bases.
  • the variance in T m of all 9-mers furthermore decreases from 59.6 °C for DNA oligonucleotides to only 4.7 °C for the LNA substituted oligonucleotides.
  • the estimations are based on the latest LNA T m prediction algorithms such as those disclosed herein, which have a variance of 6-7 °C.
  • the capture efficiency of one or more nucleic acids can be increased by including any of the high affinity nucleotides (e.g., LNA units) described herein within the nucleic acids.
  • the examples herein also provide algorithms for optimizing the substitution patterns of the nucleic acids to minimize self-complementarity that may otherwise inhibit the binding of the nucleic acids to target molecules.
  • LNA A and LNA T substitutions are made to equalize the melting temperatures of the nucleic acids.
  • LNA A and LNA C substitutions are made to minimize self- complementarity and to increase specificity.
  • LNA C and LNA T substitutions also minimize self-complementarity.
  • oligonucleotides containing LNA C and LNA T are desirable because these modified nucleotides are easy to synthesis and are especially useful for applications such as antisense technology in which minimizing cost is especially desirable.
  • Example 1 The Use of LNA-modified Oligonucleotides in Microarrays Provide Significantly Improved Sensitivity and Specificity in Expression Profiling
  • Capture probes for the Saccharomyces cerevisiae genes SWI5 (YDR146C) and THI4 (YGR144W) were designed as 50-mer standard DNA and different LNA/DNA "mixmer" oligonucleotides (i.e., oligonucleotides containing both LNA and DNA nucleotides) respectively, for comparison (Table 2).
  • LNA/DNA "mixmer" oligonucleotides i.e., oligonucleotides containing both LNA and DNA nucleotides
  • 40-mer oligonucleotides were designed as truncated versions of the 50-mer capture probes (Table 2).
  • LNA oligoarrays The specificity of the LNA oligoarrays was addressed by introducing 1-5 consecutive mismatches positioned in the middle of 40-mer LNA/DNA mixmer capture probes with LNA in every fourth position.
  • in vitro synthesized yeast RNA for either SWI5 or THI4 was spiked into Caenorhabditis elegans total RNA for cDNA target synthesis.
  • Amplification of the yeast genes was performed using standard PCR with yeast genomic DNA as the template.
  • a forward primer containing a restriction enzyme site and a reverse primer containing a universal linker sequence were used.
  • the reverse primer was exchanged with a nested primer containing a poly-T20 tail and a restriction enzyme site.
  • the DNA fragments were ligated into the pTRIampl ⁇ vector (Ambion, USA) using the Quick Ligation Kit (New England Biolabs, USA) according to the supplier's instructions and transformed into E. coli DH-5 ⁇ by standard methods.
  • the PCR clones were sequenced using Ml 3 forward and Ml 3 reverse primers on an ABI 377 (Applied Biosystems, USA). Synthesis of in vitro RNA was carried out using the MEGAscriptTM T7 Kit (Ambion, USA) according to the manufacturer's instructions. Design and synthesis of the LNA capture probes
  • microarrays were printed on ImmobilizerTM Micro Array Slides (Exiqon, Denmark) using the Biochip One Arrayer from Packard Biochip technologies (Packard, USA). The arrays were printed with a spot volume of 2 x 300 pi of a 10 ⁇ M capture probe solution. Four replicas of the capture probes were printed on each slide. Synthesis offluorochrome labelled first strand cDNAfrom total RNA
  • RNA Ten ng of S. cerevisiae in vitro synthesized RNA (either SWI5 or THI4) was combined with 10 ⁇ g of C. elegans total RNA and 5 ⁇ g oligo dT primer (T20VN) in an RNase free, pre-siliconized 1.5 mL tube, and the final volume was adjusted with DEPC- ater to 8 ⁇ L.
  • the reaction mixture was heated at +70°C for 10 minutes, quenched on ice for 5 minutes, and spun for 20 seconds, followed by addition of 1 ⁇ L SUPERase-InTM (20U/ ⁇ L, RNAse inhibitor, Ambion, USA), 4 ⁇ L 5xRTase buffer (Invitrogen, USA), 2 ⁇ L 0.1 M DTT (Invitrogen, USA), 1 ⁇ L dNTP (20mM dATP, dGTP, dTTP; 0.4 mM dCTP in DEPC-water, Amersham Pharmacia Biotech, USA), and 3 ⁇ L Cy3TM-dCTP or Cy5TM-dCTP (Amersham Pharmacia Biotech, USA).
  • First strand cDNA synthesis was carried out by adding 1 ⁇ L of SuperscriptTM II (Invitrogen, 200 U/mL), mixing, and incubating the reaction mixture for one hour at 42°C. An additional 1 ⁇ L of SuperscriptTM II was added, and the cDNA synthesis reaction mixture was incubated for an additional one hour at 42°C; the reaction was stopped by heating at 70°C for 5 minutes, and quenching on ice for 2 minutes. The RNA was hydrolyzed by adding 3 ⁇ L of 0.5 MNaOH, and incubating at 70°C for 15 minutes.
  • SuperscriptTM II Invitrogen, 200 U/mL
  • the samples were neutralized by adding 3 ⁇ L of 0.5 M HC1, and purified by adding 450 ⁇ L lxTE buffer, pH 7.5 to the neutralized sample and transferring the samples onto a Microcon-30 concentrator.
  • the samples were centrifuged at 14000xg in a microcentrifuge for ⁇ 8 minutes, the flow-through was discarded and the washing step was repeated twice by refilling the filter with 450 ⁇ l lxTE buffer and by spinning for -12 minutes. Centrifugation was continued until the volume was reduced to 5 ⁇ L, and finally the labelled cDNA probe was eluted by inverting the Microcon-30 tube and spinning at lOOOxg for 3 minutes.
  • the arrays were hybridized overnight using the following protocol.
  • the Cy3TM or Cy5TM-labelled cDNA samples were combined in one tube followed by addition of 3 ⁇ L 20 ⁇ SSC (3xSSC final), 0.5 ⁇ L 1 M HEPES, pH 7.0 (25 mM final), 25 ⁇ g yeast tRNA (1.25 5 ⁇ g/ ⁇ L final), 10 ⁇ g PolyA blocker (0.5 ⁇ g/ ⁇ L final), 0.6 ⁇ L 10% SDS (0.3% final), and DEPC-treated water to 20 ⁇ L final volume.
  • the labelled cDNA target sample was filtered in a Millipore 0.22 micron spin column according to the manufacturer's instructions (Millipore, USA), and the probe was denatured by incubating the reaction at 100°C for 2 minutes. The sample was cooled at 20-25 °C for 5 minutes by spinning at maximum speed in a
  • the Lifterslip coverslip was washed off in 2 x SSC, pH 7.0 containing 0.1% SDS at room temperature for one minute, followed by washing of the microarrays subsequently in 1.0 x SSC, pH 7.0 at room temperature for one minute, and then in 0.2 x SSC, pH 7.0 at room temperature for one
  • the LNA-spiked (LNA modification at every fourth nucleotide position) 40-mer triple mismatch oligos showed a 3-fold signal intensity decrease relative to the perfectly matched duplexes, whereas the corresponding 40-mer standard DNA capture probes did not form duplexes under standard hybridization stringency. Further, the 40-mer perfect match LNA capture probes showed a 5 -fold to 14-fold increase in the intensity levels compared to DNA oligonucleotides under standard hybridization conditions. Capture probes of other lengths and/or with other LNA substitution patterns can be used similarly. Table 2. DNA and LNA-modified SWI5 (YDR146C) and THI4 (YGR144W) oligonucleotide capture probes.
  • LNA modifications are depicted by uppercase letters in the sequence, mt denotes the number of mismatches (bolded) in the center of the oligonucleotide with respect to its target cDNA (mRNA), and "mC” denotes LNA methyl cytosine.
  • YDR146C-50_mt4 tgggaatggaacggggattatggaaaggccaatgaaaactaatcaaaggt
  • YDR146C-50_mt5 tgggaatggaacggggattatggaaagcccaatgaaaactaatcaaaggt
  • Example 2 Detection of Alternative Splice Isoforms Using Exon-Specific, Internal LNA Capture Probes in the Caenorhabditis elegans Gene let-2 Capture probe design Finding the regions of interest
  • Exons of interest e.g., exons being skipped and their flanking exons
  • Exons of interest in the C. elegans gene T01D3.3 desirably exceed 70 base-pairs.
  • Other genes of interest may be selected using one or more of the above criteria or using other criteria, such as the medical relevance of the gene.
  • an oligonucleotide with a palindromic value below 100 (default value in PICK70, value based on Smith- Waterman algorithm) and with a melting temperature closest to 80°C was picked.
  • the location of the oligonucleotide within the region was then compared to the list made using the above BLAST search. If the oligonucleotide did not coincide with a BLAST hit exceeding around 25 (consecutive) base-pairs, this oligonucleotide sequence was chosen as a 50-mer capture probe. Otherwise, a new oligo sequence was picked from the PICK70 output.
  • the selected 50-mer oligonucleotide sequences were "BLASTed” against the C. elegans transcripts again, as described above. Accounting for the Introns.
  • the oligonucleotide sequences were "BLASTed” against the complete C. elegans genome. The matches were run against a list made from the GenBank reports of the complete genome, indexing whether positions in the genome were genie or intergenic.
  • every fourth DNA nucleotide was substituted with an LNA nucleotide, as shown in Table 3.
  • the oligonucleotides were synthesized with an anthraquinone (AQ) moiety at the 5'-end of each oligonucleotide (e.g., as described in allowed U.S.S.N. 09/611,833), followed by a hexaethyleneglycol tetramer linker region and the LNA/DNA mixmer capture oligonucleotide sequence.
  • AQ anthraquinone
  • CE42.08-0HEG4 GgctGgatmCcccAggaAaccmCaggAatcGgaaGcatTggamCcaaAaggAg
  • CE42.09-0HEG4 mCaccGgatmCcggmCtcaAttgTcggAcctmCgcgGaaamCcctGgagAaaaGg
  • C. elegans wild-type strain (Bristol-N2) was maintained on nematode growth medium (NG) plates seeded with Escherichia coli strain OP50 at 20°C, and the eggs and LI larvae were prepared as described in Hope, I. A. (ed.) " C. elegans - A Practical Approach ", Oxford University Press 1999. The samples were immediately flash frozen in liquid N 2 and stored at - 80°C until RNA isolation. Isolation of Total RNA
  • a 100 ⁇ l aliquot of packed C. elegans worms from a LI larvae population was homogenized using the FastPrep Bio 101 from Kem-En-Tec for 1 minute at speed 6 followed by isolation of total RNA from the extracts using the FastPrep Biol 01 kit (Kem-En-Tec) according to the manufacturer's instructions.
  • a 50 ⁇ l aliquot of packed C. elegans eggs was homogenized in lysis buffer (RNeasy total RNA purification kit, QIAGEN) containing quartz sand for 3 minutes using a Pellet Pestle Motor followed by isolation of total RNA according to the manufacturer's manual.
  • RNA from worms (LI larvae) as well as eggs was ethanol precipitated for 24 hours at- 20°C by addition of 2.5 volumes of 96% EtOH and 0.1 volume of 3M Na-acetate, pH 5.2 (Ambion, USA), followed by centrifugation of the total RNA sample for 30 minutes at 13200 rpm.
  • the total RNA pellet was air-dried and redissolved in 6 ⁇ l (worms) or 2.5 ⁇ l (eggs) of diethylpyrocarbonate (DEPC)-treated water (Ambion, USA) and stored at - 80°C.
  • RNA 1.5 ⁇ g from eggs or 1 ⁇ g total RNA from worms (LI larvae) were mixed with 5 ⁇ g oligo(dT)12-18 primer (Amersham Pharmacia Biotech, USA) and 0.5 ⁇ g of random hexamers, pd(N) 6 (Amersham Pharmacia Biotech, USA) and DEPC-treated water to a final volume of 7 ⁇ l.
  • the mixture was heated at 70°C for 10 minutes, quenched on ice for 5 minutes, followed by addition of 20 units of Superasin RNase inhibitor (Ambion, USA), 4 ⁇ l of 5 x Superscript buffer (Life Technologies, USA), 2 ⁇ l of 100 mM DTT, 1 ⁇ l of dNTP solution (20 mM each dATP, dGTP, dTTP and dCTP, Amersham Pharmacia Biotech, USA), and 3 ⁇ l of DEPC-treated water.
  • the primers were pre-annealed at 37°C for 5 minutes, followed by addition of 400 units of Superscipt II reverse transcriptase (Invitrogen, USA).
  • First strand cDNA synthesis was carried out at 37°C for 30 minutes, followed by 2 hours at 42°C, and the reaction was stopped by incubation at 70°C for 5 minutes, followed by incubation on ice for 5 minutes.
  • Unincorporated dNTPs were removed by gel filtration using MicroSpin S-400 HR columns as described below. The column was pre-spun for 1 minute at 735 x g in a 1.5 ml tube, and the column was placed in a new 1.5 ml tube.
  • the cDNA sample was slowly applied to the top center of the resin and spun at 735 x g for 2 minutes. The eluate was collected. The volume of the eluate was adjusted to 50 ⁇ l with TE-buffer pH 7.0 before being used as the template for linear PCR.
  • Four ⁇ l template (RT from eggs or worms) was combined with 1 ⁇ l dNTP solution (lOmM each dATP, dGTP, dTTP and dCTP, Amersham Phamacia Biotech, USA), 1 ⁇ l of each primer ( 20 ⁇ M CE42.07 sense: gatcgaattcctccaggagagaagggagatg, and CE42.12 antisense:
  • PCR reactions were carried out using the following program: 95 °C for 5 minutes followed by 30 cycles of PCR using the following cycling program (denaturation at 95°C for 45 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute) followed by a final extension step at 72°C for 10 minutes and incubation on ice for 5 minutes.
  • Purification of the PCR amplicons from eggs as well as worms was performed using a
  • PCR reactions were carried out using the following program: 95°C for 5 minutes followed by 30 cycles of PCR using the following cycling program (denaturation at 95°C for 45 seconds annealing at 60°C for 30 seconds extension at 72°C for 1 minute) followed by a final extension step at 72°C for 10 minutes and incubation on ice for 5 minutes.
  • the C. elegans gene Let-2/exon 8-11 capture probes were synthesized with a 5' anthraquinone (AQ)-modification, followed by a hexaethyleneglycol-4 (HEG4) linker (Table 3).
  • the capture probes were first diluted to a 10 ⁇ M final concentration in 100 mM Na- phosphate buffer pH 7.0 and spotted on Euray COP microarray slides using the Biochip Arrayer One (Packard Biochip Technologies) with a spot volume of 300 pi and 300 ⁇ m between the spots.
  • the capture probes were immobilized onto the microarray slide by UN irradiation in a
  • the slides were hybridized with 2.5 ⁇ l of the Cy3-labelled and 2.5 ⁇ l of the Cy5- labelled target preparation from eggs and worms, respectively, as described above (see "Reverse transcription (RT)-PCR” section) in 25 ⁇ l of hybridization solution, containing 25 mM HEPES, pH 7.0, 3 x SSC, 0.3% SDS, and 25 ⁇ g of yeast tR ⁇ A.
  • the target probe was filtered in a Millipore 0.22 micron spin column (Ultrafree-MC, Millipore, USA), denatured by incubation at 100°C for 5 minutes, cooled at room temperature for 5 minutes, and then carefully applied onto the prepared microarray.
  • Example 3 Improved Sensitivity in the Specific Detection of the C. elegans Gene T01D3.3
  • Capture Probe Design The design method of exon-specific capture probes for the C. elegans gene T01D3.3 exon 4 has been described in example 2.
  • Poly(A) + RNA was isolated from the worm samples using the Pick-Pen (Bio-Nobile, Finland) Starter kit combined with the KingFisher mRNA purification kit (ThermoLabsystems, Finland) according to the manufacturer's instructions. The yield was 1- 2 ⁇ g poly(A) + RNA from approximately 50 mg of C. elegans worms. Synthesis of fluorochrome labelled first strand cD A from C. elegans mRNA
  • C. elegans poly(A) + RNA was combined with 2 ⁇ g oligo dT primer (T20VN) in an RNase free, pre-siliconized 1.5 mL tube, and the final volume was adjusted with DEPC- ater to 8 ⁇ L.
  • the reaction mixture was heated at +70°C for 10 minutes, quenched on ice 5 minutes, spun for 20 seconds, followed by addition of 1 ⁇ L SUPERase- InTM (20U/ ⁇ L, RNAse inhibitor, Ambion, USA), 4 ⁇ L 5xRTase buffer (Invitrogen, USA), 2 ⁇ L 0.1 M DTT (Invitrogen, USA), 1 ⁇ L dNTP (20mM dATP, dGTP, dTTP; 4 mM dCTP in DEPC-water, Amersham Pharmacia Biotech, USA), and 3 ⁇ L Cy3TM-dCTP (Amersham Pharmacia Biotech, USA).
  • First strand cDNA synthesis was carried out by adding 1 ⁇ L of SuperscriptTM II (Invitrogen, 200 U/mL), mixing, and incubating the reaction mixture for one hour at 42°C. An additional 1 ⁇ L of SuperscriptTM II was added and the cDNA synthesis reaction mixture was incubated for an additional one hour at 42°C; the reaction was stopped by heating at 70°C for 5 minutes, and quenching on ice for 2 minutes. The RNA was hydrolyzed by adding 3 ⁇ L of 0.5 M NaOH and incubating at 70°C for 15 minutes.
  • SuperscriptTM II Invitrogen, 200 U/mL
  • the samples were neutralized by adding 3 ⁇ L of 0.5 M HC1 and purified by adding 450 ⁇ L lxTE buffer, pH 7.5 to the neutralized sample and transferring the samples onto a Microcon-30 concentrator.
  • the samples were centrifuged at 14000xg in a microcentrifuge for ⁇ 8 minutes, the flow-through was discarded, and the washing step was repeated twice by refilling the filter with 450 ⁇ l lxTE buffer and by spinning for -12 minutes. Centrifugation was continued until the volume was reduced to 5 ⁇ L, and finally the labelled cDNA probe was eluted by inverting the Microcon-30 tube and spinning at lOOOxg for 3 minutes. Printing and Coupling of the C. elegans Microarrays
  • the C. elegans gene T01D3.3/exon 4 capture probes were synthesized with a 5' anthraquinone (AQ)-modification, followed by either a hexaethyleneglycol-2 or a hexaethyleneglycol-4 (HEG2/HEG4) linker (Table 4).
  • the capture probes were first diluted to a 10 ⁇ M final concentration in 100 mM Na-phosphate buffer pH 7.0, followed by a twofold dilution series (10 ⁇ M, 5 ⁇ M, 2.5 ⁇ M, 1.25 ⁇ M, 0.625 ⁇ M, 0.31 ⁇ M, and 0.155 ⁇ M) and spotted on Exiqon's polycarbonate microarray slides using the Biochip Arrayer One (Packard Biochip Technologies, USA) with a spot volume of 3x 300 pi and 400 ⁇ m between the spots.
  • the capture probes were immobilized onto the microarray slide by UN irradiation in a Stratalinker for 90 seconds at full power (Stratagene, USA).
  • ⁇ on-immobilized capture probe oligonucleotides were removed from the slides by washing the slides for 24 hours in milli-Q H O. After washing, the slides were dried in an oven at 37°C for 30 minutes and stored in a slide box until microarray hybridization. Hybridization with CyS-labelled cDNA The arrays were hybridized overnight using the following protocol.
  • the Cy3TM- labelled cD ⁇ A sample was combined with 3 ⁇ L 20xSSC (3xSSC final), 0.5 ⁇ L 1 M HEPES, pH 7.0 (25 mM final), 25 ⁇ g yeast tR ⁇ A (1.25 ⁇ g/ ⁇ L final), 10 ⁇ g PolyA blocker (0.5 ⁇ g/ ⁇ L final), 0.6 ⁇ L 10% SDS (0.3% final), and DEPC-treated water to 20 ⁇ L final volume.
  • the labelled cD ⁇ A target sample was filtered in a Millipore 0.22 micron spin column according to the manufacturer's instructions (Millipore, USA), and the probe was denatured by incubating the reaction at 100°C for 2 minutes.
  • the sample was cooled at 20-25 °C for 5 minutes by spinning at maxium speed in a microcentrifuge, and then carefully applied on top of the microarray.
  • a cover slip was laid over the microarray and the hybridization was performed for 16 hours at 63 °C in a hybridization chamber (Corning, USA) submerged in a water bath, with an aliquot of 30 ⁇ L of 3xSSC added to both ends of the hybridization chamber to prevent evaporation. After hybridization, the slide was removed carefully from the hybridization chamber and washed using the following protocol.
  • the coverslip was washed off in 2 x SSC, pH 7.0 containing 0.1% SDS at room temperature for one minute, followed by washing of the microarrays subsequently in 1.0 x SSC, pH 7.0 at room temperature for one minute, and then in 0.2 x SSC, pH 7.0 at room temperature for one minute. Finally, the slides were washed for 5 seconds in 0.05 x SSC, pH 7.0. The slides were then dried by centrifugation in a swinging bucket rotor at approximately 600 rpm for 5 minutes. Microarray data analysis
  • the C. elegans gene T01D3.3 exon 4 array was scanned in an Array WoRx Scanner (Applied Precision, USA) using an exposure time of 5 seconds, resolution of 5.0, and high (high level) sensitivity.
  • the hybridization data were analyzed using the ArrayNision image analysis software package 5.1 (IMAGING Research Inc., USA).
  • analysis of the hybridization data from the C. elegans gene 26/T01D3.3 exon 4 array demonstrates that the use of LNA-modified capture probes for the C. elegans T01D3.3 exon results in 5 -fold increased sensitivity in exon 4 capture compared to the corresponding DNA oligonucleotide capture probe controls printed on the same microarray. Capture probes of other lengths and/or with other LNA substitution patterns can be used similarly.
  • Capture probe design Exon-specific capture probes for the C. elegans gene T01D3.3 exons 4 and 5 were designed as described in Example 2.
  • Table 5 C. elegans gene T01D3.3/exons 4 and 5-specific capture probes and synthetic target oligonucleotides. Table 5.
  • the C. elegans gene T01D3.3/exon 4-5 capture probes were synthesized with a 5' anthraquinone (AQ)-modification, followed by either a hexaethyleneglycol-2 or a hexaethyleneglycol-4 (HEG2/HEG4) linker (Table 5).
  • the capture probes were first diluted to a 10 ⁇ M final concentration in 100 mM Na-phosphate buffer pH 7.0, followed by a twofold dilution series (10 ⁇ M, 5 ⁇ M, 2.5 ⁇ M, 1.25 ⁇ M, 0.625 ⁇ M, 0.31, ⁇ M, and 0.155 ⁇ M) and spotted on Euray polycarbonate microarray slides using the Biochip Arrayer One (Packard Biochip Technologies) with a spot volume of 3x 300 pi and 400 ⁇ m between the spots.
  • the capture probes were immobilized onto the microarray slide by UN irradiation in a Stratalinker for 90 seconds at full power (Stratagene, USA).
  • ⁇ on-immobilized capture probe oligonucleotides were removed from the slides by washing the slides for 24 hours in milli-Q H 2 O. After washing, the slides were dried in an oven at 37°C for 30 minutes, and stored in a slide box until microarray hybridization.
  • the slides were hybridized with a high (saturated) concentration of 1 ⁇ M of each gene T01D3.3, exon 4 or 5 target oligo (Table 5) in 50 ⁇ l of hybridization solution, containing 25 mM HEPES, pH 7.0, 3 x SSC, 0.22 % SDS, and 0.8 ⁇ g/ ⁇ l of poly(A) blocker.
  • the target probes were filtered in a Millipore 0.45 micron spin column (Ultrafree-MC, Millipore, USA), denatured by incubation at 100 °C for 2 minutes, cooled at room temperature for 5 minutes, and then carefully applied onto the prepared microarray. One-half of a cover slip was laid over the microarray, and the hybridization was performed for 16-18 hours at 63 °C in a hybridization chamber (Coming, USA).
  • the slides were washed sequentially by plunging gently in 1 x SSCT (150 mM NaCl, 15 mM Sodium Citrate + Tween 20) at room temperature for one minute, then in 0.2 x SSCT (30 mM NaCl, 3 mM Sodium Citrate + Tween 20) at room temperature for one minute, and finally in Milli Q water, followed by drying of the slides in an oven at 37°C for 30 minutes.
  • the slides were Cy5 labelled using a Cy5-straptavidin target.
  • the C. elegans gene T01D3.3 exon 4-5 microarray was scanned in an ArrayWoRx Scanner (Applied Precision, USA) using an exposure time of 5 seconds, resolution of 5.0, and high (high level) sensitivity.
  • the hybridization data were analyzed using the ArrayNision image analysis software package 5.1 (IMAGING Research Inc., USA).
  • analysis of the hybridization data from the C. elegans gene 26/T01D3.3 array demonstrates that both the DNA as well as the LNA capture probes for the C.
  • Example 5 Detection of Alternatively Spliced Isoforms using Internal Exon-specific, and Exon-Exon Junction-Specific (merged) LNA-modified Capture Probes Oligonucleotide design for microarrays.
  • Example 2 Methods for designing exon-specific internal oligonucleotide capture probes has been described in Example 2.
  • Design of the LNA-modified capture probes For the LNA-modified oligonucleotide capture probes, every third DNA nucleotide was substituted with an LNA nucleotide.
  • the probes designed to capture the junction of the recombinant splice variants were designed with LNA modifications in a block of five consecutive LNAs nucleotides, two on the 5' side of the splice junction and three on the 3' side of the splice junction. All capture probes are shown in Table 6.
  • the splice variant capture probes were synthesized with a 5' anthraquinone (AQ)- modification, followed by a hexaethyleneglycol-2 (HEG2) linker.
  • the capture probes were first diluted to a 20 ⁇ M final concentration in 100 mM ⁇ a-phosphate buffer pH 7.0, and spotted on the Immobilizer polymer microarray slides (Exiqon, Denmark) using the Biochip Arrayer One (Packard Biochip Technologies, USA) with a spot volume of 2x 300 pi and 300 ⁇ m between the spots.
  • the capture probes were immobilized onto the microarray slide by UV irradiation in a Stratalinker with 2300 ⁇ joules (Stratagene, USA).
  • Non-immobilized capture probe oligonucleotides were removed from the slides by washing the slides two times 15 minutes in lxSSC. After washing, the slides were dried by centrifugation at lOOOx g for 2 minutes, and stored in a slide box until microarray hybridization. Construction of Splice Variant Vectors
  • Triampl ⁇ vector The recombinant splice variant constructs were cloned into the Triampl ⁇ vector (Ambion, USA). The constructs were sequenced to confirm their construction. The plasmid clones were transformed into E. coli XLIO-Gold (Stratagene, USA). Triampl8/SWI5 vector construct
  • Genomic DNA was prepared from a wild-type standard laboratory strain of Saccharomyces cerevisiae using the Nucleon MiY DNA extraction kit (Amersham Biosciences, USA) according to the supplier's instmctions. Amplification of the partial yeast gene was performed using standard PCR with yeast genomic DNA as the template. In the first step of amplification, a forward primer containing a restriction enzyme site and a reverse primer containing a universal linker sequence were used. In this step, 20 base-pairs were added to the 3 '-end of the amplicon, next to the stop codon. In the second step of amplification, the reverse primer was exchanged with a nested primer containing a poly-T 2 o tail and a restriction enzyme site.
  • the SWI5 amplicon contains 730 bp of the SWIS ORF plus a 20 bp universal linker sequence and a ⁇ oly-A 2 o tail.
  • the PCR primers used were YDR 146C-For-EcoRI (acgtgaattcaaatacagacaatgaaggagatga), YDR146C-Rev-Uni (gatccccgggaattgccatgttacctttgattagttttcattggc), and Uni-polyT-BamHI (acgtggatcctttttttttttttttttttttttgatccccgggaattgccatg).
  • the PCR amplicon was cleaved with the restriction enzymes EcoRI and BamHI.
  • the DNA fragment was ligated into the pTRIampl8 vector (Ambion, USA) using the Quick Ligation Kit (New England Biolabs, USA) according to the supplier's instructions and transformed into E. coli DH-5 ⁇ by standard methods. Construction of the Recombinant Splice Variant #1 (Triampl8/swi5-rubisco)
  • the Arabidopsis thaliana Rubisco small subunit ssu2b gene fragment (gil7064721) was amplified from genomic DNA using primers named DJ305 (5'- ACTATGATGGACGATACTGGAC-3') and DJ306 (5'- ATTGGATCGATCCGATGATCCTAATGAAGGC-3'), containing CM restriction site linkers.
  • the purified PCR fragment was digested with Clal and then cloned into the swi5 (gl:7839148) vector at the unique Clal site (atcgat) giving each insert a flanking sequence from the original yeast SWI5 insert (named exonOl and exon 03, Figure 19).
  • the product was inserted in the reverse orientation, so that the insert sequence is as follows: atcgatCCGATGATCCTAATGAAGGCGCCCGGGTACTCCTTCTTGCATTCTTCAACTT CCTTCAACACTTGAGCGGAGTCGGTGCATCCGAACAATGGAAGCTTCCACATTGT CCAGTATCGTCCATCATAGTatcgat
  • Nucleotide sequence analysis revealed a difference between the sequence of A. thaliana rabisco expected from the GenBank database and that obtained from all sequenced constructs and PCR products. Position 30 in the Rubisco insert is “C” rather than the expected "A.” This SNP was probably created by PCR. None of the oligonucleotide capture probes used in the example cover this region.
  • the Rubisco sequence in Genbank is TCCTAATGAAGGCGCCA, and the sequence obtained from the plasmid contract is TCCTAATGAAGGCGCCC.
  • the Arabidopsis thaliana Lea gene (gi 1526423) was amplified from genomic DNA with primers named DJ307 (5'-GGAATTATCGATGTGTGATAGGATCAGTGTTCAG-3'), andDJ308 (5'-AATTGGATCGATATTAGCAGTCTCCTTCGCC-3'), including the Clal linker sites as above.
  • the PCR fragment was digested with Clal cloned into the yeast SWI5 INT constract as above at the unique Clal site.
  • C. elegans wild-type strain (Bristol-N2) was maintained on nematode growth medium (NG) plates seeded with Escherichia coli strain OP50 at 20°C, and the mixed stages of the nematode were prepared as described by Hope (ed.) ("C. elegans - A Practical Approach", Oxford University Press 1999). The samples were immediately flash frozen in liquid N 2 and stored at -80°C until RNA isolation.
  • NG nematode growth medium
  • a 100 ⁇ l aliquot of packed C. elegans worms from a mixed stage population was homogenized using the FastPrep Biol 01 from Kem-En-Tec for one minute, speed 6 followed by isolation of total RNA from the extracts using the FastPrep Biol 01 kit (Kem-En-Tec) according to the manufacturer's instructions.
  • RNA was ethanol precipitated for 24 hours at - 20°C by addition of 2.5 volumes of 96% EtOH and 0.1 volume of 3M Na-acetate, pH 5.2 (Ambion, USA), followed by centrifugation of the total RNA sample for 30 minutes at 13200 rpm.
  • the total RNA pellet was air-dried and redissolved in 10 ⁇ l of diethylpyrocarbonate (DEPC)-treated water (Ambion, USA) and stored at - 80°C.
  • DEPC diethylpyrocarbonate
  • the mixture was heated at 70°C for 10 minutes, quenched on ice for 5 minutes, followed by addition of 20 units of Superasin RNase inhibitor (Ambion, USA), 1 ⁇ l dNTP solution (lOmM each dATP, dGTP, dTTP and 0.4 mM dCTP, and 3 ⁇ l Cy5-dCTP, Amersham Biosciensces, USA), 4 ⁇ l 5 x RTase buffer (Invitrogen), 2 ⁇ l 0.1 mM DTT (Invitrogen), 400 units of Superscript II reverse transcriptase (Invitrogen, USA), and DEPC- treated water to 20 ⁇ l final volume.
  • a parallel set-up was made with 10 ⁇ g total RNA from C. elegans and 1 ng of in vitro
  • RNA from Splice variant #2 labelling with Cy3-dCTP. Both cDNA syntheses were carried out at 42°C for 2 hours, and the reactions were stopped by incubation at 70°C for 5 minutes, followed by incubation on ice for 5 minutes.
  • Unincorporated dNTPs were removed by gel filtration using MicroSpin S-400 HR columns as described below.
  • the column was pre-spun for one minute at 1500 x g in a 1.5 ml tube, and the column was placed in a new 1.5 ml tube.
  • the cDNA sample was slowly applied to the top center of the resin and spun 1500-x g for 2 minutes. The eluate was collected.
  • RNA was degraded by adding 3 ⁇ l of 0.5 M NaOH.
  • the solution was mixed well and incubated at 70 °C for 15 minutes.
  • the solution was neutralized by adding 3 ⁇ l of 0.5 M HC1 and mixed well.
  • the cDNA probes were cooled at room temperature for 2-5 minutes by spinning at maxium speed in a microcentrifuge.
  • a LifterSlip (Erie Scientific Company, USA) was carefully placed on top of the microarray spotted on lmmobilizerTM MicroArray Slide, and the hybridization mixture was applied to the array from the side.
  • An aliquot of 30 ⁇ L of 3xSSC was added to both ends of the hybridization chamber, and the lmmobilizerTM MicroArray Slide was placed in the hybridization chamber (DieTech, USA). The chamber was sealed watertight and incubated at 65°C for 16-18 hours submerged in a water bath. After hybridization, the slide was removed carefully from the hybridization chamber and washed using the following protocol.
  • the slides were washed sequentially by plunging gently in 2 x SSC/0.1% SDS at room temperature until the cover slip falls of into the washing solution, then in lx SSC pH 7.0 (150 mM NaCl, 15 mM Sodium Citrate) at room temperature for one minute, then in 0.2 x SSC, pH 7.0 (30 mM NaCl, 3 mM Sodium Citrate) at room temperature for one minute, and finally in 0.05 x SSC (7.5 mM NaCl, 0.75 mM Sodium Citrate) for 5 seconds, followed by drying of the slides by spinning at 1000 x g for 2 minutes.
  • the slides were stored in a slide box in the dark until scanning.
  • Microarray data analysis The splice variant microarray was scanned in a ScanArray 4000XL confocal laser scanner (Packard Instruments, USA). The hybridization data were analyzed using the GenePix Pro 4.01 microarray analysis software (Axon, USA).
  • Example 6 The Use of LNA-modified Oligonucleotides in Microarrays Provides Significantly Improved Sensitivity in Expression Profiling
  • Capture probes for the Saccharomyces cerevisiae gene SWI5 were designed as 50-mer standard DNA and two different LNA- modified oligonucleotides with LNA substitutions at every second or every third nucleotide position, respectively, for comparison (Table 7).
  • the arrays were hybridized overnight using the following protocol.
  • the desired amount of biotin-labelled oligonucleotide was combined in one tube followed by addition of 3 ⁇ L 20xSSC (3xSSC final), 0.5 ⁇ L 1 M HEPES, pH 7.0 (25 mM final), 25 ⁇ g yeast tRNA (1.25 ⁇ g/ ⁇ L final), 0.6 ⁇ L 10% SDS (0.3% final), and DEPC-treated water to 20 ⁇ L final volume.
  • the biotin-labelled target sample was filtered in a Millipore 0.22 micron spin column according to the manufacturer's instmctions (Millipore, USA), and the probe was denatured by incubating the reaction at 100°C for 2 minutes.
  • the sample was cooled at 20- 25°C for 5 minutes by spinning at maxium speed in a microcentrifuge.
  • a LifterSlip (Erie Scientific Company, USA) was carefully placed on top of the microarray spotted on lmmobilizerTM MicroArray Slide, and the hybridization mixture was applied to the array from the side.
  • An aliquot of 30 ⁇ L of 3xSSC was added to both ends of the hybridization chamber, and the lmmobilizerTM MicroArray Slide was placed in the hybridization chamber.
  • the chamber was sealed watertight and incubated at 65 °C for 16-18 hours submerged in a water bath. After hybridization, the slide was removed carefully from the hybridization chamber and washed using the following protocol.
  • the Lifterslip coverslip was washed off in 2xSSC, pH 7.0 containing 0.1% SDS at room temperature for 1 minute, followed by washing of the microarrays subsequently in l.OxSSC, pH 7.0 at room temperature for 1 minute, and then in 0.2xSSC, pH 7.0 at room temperature for 1 minute. Finally, the slides were washed for 5 seconds in 0.05xSSC, pH 7.0. The slides were then dried by centrifugation in a swinging bucket rotor at approximately 200 G for 2 minutes.
  • YDR146C-50_LNA2 TgGgAaTgGaAcGgGgAtTaTgGtTtmCgmCcAaTgAaAamCtAaTcAaAgGt
  • YDR146C-50_LNA3 TggGaaTggAacGggGatTatGgtTtcGccAatGaaAacTaaTcaAagGt
  • Example 7 The Use of LNA-modified Oligonucleotides in Microarrays Provides Significantly Improved Sensitivity in Comparative Genome Hybridization (CGH).
  • Fluorescent Cy3 labelled patient genomic DNA was hybridized to microarrays spotted with the CGH capture probes listed in the table below. Compared to DNA capture probes, capture probes with LNA in every second position (LNA-2) had a significantly better capture rate of non-amplified labelled genomic patient DNA as shown in Figure 22, Figure
  • Capture probes of other lengths and/or with other LNA substitution patterns can be used similarly.
  • Example 8 Expression Profiling of Stress and Toxicity in Caenorhabditis elesans using LNA Oligonucleotide Microarrays
  • This example demonstrates the use of the C. elegans LNA tox oligoarray in gene expression profiling experiments in the nematode Caenorhabditis elegans.
  • the C. elegans tox oligoarray monitors the expression of a selection of 110 genes relevant for general stress response and for the metabolism of toxic compounds. Two different capture probes for each of these target genes were designed and included in the LNA tox array.
  • the C. elegans LNA tox oligoarray contained capture probes providing control for cDNA synthesis efficiency and the developmental stage of the nematode. Capture probes for constitutively expressed genes for data set normalization were also included on the C. elegans LNA tox oligoarray.
  • the sample was divided into two, and one half of the sample was used as the control, the other was used as the treated sample. Worm samples were harvested and sucrose cleaned by standard methods.
  • heat shock treatment the heat shock sample was added to S-media preheated to 33°C in a 1 L flask suspended in a water bath at 33°C, the other sample was added to a 1 L flask with S-media at 25°C. Both samples were shaken at approximately 100 rpm for an hour.
  • For Lansoprazole treatment 0.5 mL of 10 mg/mL Lansoprazole (Sigma) in DMSO was added to each 500 mL volume of S-media culture after 28 hours of growth from LI. At the same time, 0.5 mL of DMSO was added to the control. Incubation was for 24 hours. Samples were then harvested by centrifugation at 3000xg suspended in RNAE terTM (Ambion) and immediately frozen in liquid nitrogen.
  • T m self-complementarity, and secondary stracture were selected.
  • LNA modifications were incorporated to increase affinity and specificity.
  • microarrays were printed on lmmobilizerTM MicroArray Slides (Exiqon, Denmark) using the Biochip One Arrayer from Packard Biochip technologies (Packard,
  • the arrays were printed with a spot volume of 2x300 pi of a 10 ⁇ M capture probe solution. Four replicas of the capture probes were printed on each slide. Synthesis of Fluorochrome Labelled First Strand cDNA from Total RNA
  • RNA 15 ⁇ g of C. elegans total RNA was combined with 5 ⁇ g oligo dT primer (T20VN) in an RNase free, pre-siliconized 1.5 mL tube, and the final volume was adjusted with DEPC- water to 8 ⁇ L.
  • the reaction mixture was heated at +70°C for 10 minutes, quenched on ice 5 5 minutes, spin 20 seconds, followed by addition of 1 ⁇ L SUPERase-InTM (20U/ ⁇ L, Ambion, USA), 4 ⁇ L 5xRTase buffer (Invitrogen, USA), 2 ⁇ L 0.1 M DTT (Invitrogen, USA), 1 ⁇ L dNTP (20mM dATP, dGTP, dTTP; 0.4 mM dCTP in DEPC-water, Amersham Pharmacia Biotech, USA), and 3 ⁇ L Cy3TM-dCTP or Cy5TM-dCTP (Amersham Pharmacia Biotech, USA).
  • First strand cDNA synthesis was carried out by adding 1 ⁇ L of SuperscriptTM II
  • the arrays were hybridized overnight using the following protocol.
  • the Cy3TM and Cy5TM-labelled cDNA samples were combined in one tube followed by addition of 3 ⁇ L
  • the Lifterslip coverslip was washed off in 2xSSC, pH 7.0 containing 0.1% SDS at room temperature for 1 minute, followed by washing of the microarrays subsequently in l.OxSSC, pH 7.0 at room temperature for 1 minute, and then in 0.2xSSC, pH 7.0 at room temperature for 1 minute. Finally, the slides were washed for 5 seconds in 0.05xSSC, pH 7.0. The slides were then dried by centrifugation in a swinging bucket rotor at approximately 200 G for 2 minutes. The slide was then ready for scanning.
  • LNA-modified oligonucleotide capture probes in the C. elegans LNA tox oligoarray clearly allows the identification of distinct expression profiles for C. elegans genes relevant for general stress response and for the metabolism of toxic compounds.
  • HSP90 C47E8.5
  • nd -1.17 Table 13.
  • LNA-modified oligonucleotide capture probes LNA modifications are depicted by uppercase letters in the sequence; "mC” denotes LNA methyl cytosine.
  • CEABC_C34G6.4_U293_LNA3 TgcmCatTgcAcgGgcActTgtTcgAtcTccTtcTgtTttActTttGgaTg
  • CEABC_C34G6.4_u375_LNA3 TcaTtcTagGatTgcmCagAtgGttAtgAtamCtcAtgTcgGagAgaAagGa CEABC_F57C12.4_u15_LNA3 mCcaAtgTtgTttAatTggTtgTaaTgtmCttGatGacmCtgmCatAatmCatAt CEABC_F57C12.4_u480_LNA3 mCacAagAtcmCtgTgtTgtTctmCcgGaamCaaTgaAaaTgaActTagAtcmC
  • CEATPase_C17H12.14_u89_LNA3 mCcgTttAgaGctTatTgcTaamCcaGatTgtmCccAcaAgtmCagAacAgcTc
  • CEATPase_F55F3.3_u215_LNA3 TgamCggAcgmCtamCtamCccAtaTgtAttTgtTccAtcTtamCcaGcaAccAa AgcTacTtcAttmCgamCaaGgaAcaTctmCggAaaAgtmCaaGtamCatmCccG
  • CEATPase_Y49A3A.2_u103_LNA3 AaaTtcAagGatmCcaGttGccGatGgtGaaGccAagAttmCgcAagGatTa mCgaTcgTttmCtgmCccAttmCtamCaaGacTgtmCggTatGctmCaaGaaTatG
  • CECC_Y46G5A.2_u385_LNA3 AatGagmCggTtgTgcmCgtGtgAcgTcamCttmCgtmCacAgtGttGctmCtamCt
  • CECoA_C29F3.1_u316_LNA3 AaaTtgAcamCcaAtcAaaTctGtcTcaTctmCctGagGacmCgtmCaamCttmCg
  • CECoA_C29F3.1_u392_LNA3 AatmCttTgtGtamCggAgaTggGgcAaaAggmCagmCaaGaaAgtAaamCcaAg
  • CECoA_F08A8.4_u1094_LNA3 AggAcaAggGgcActActGgcAcaGgcTttGatTatTgcAgtGagAtaTt
  • CECoA_F59F4.1_u424_LNA3 AaaGctTcgAgaTggmCacGttmCgtmCtgTatmCtcGtgAagAacTtaTtgmCa
  • CECyclin_R02F2.1 a_u24_LNA3 AtgAg CECyclin_R02F2.1a_u312_LNA3 TctmCatTgcTcgTcgAggmCtamCcaAcaAacActGgcAatAccmCaaTtaAt CECyclin_ZC168.4_u203_LNA3 TaaGaaAgtmCatTgaGgaTgcTgtmCgcTttGctmCgcmCgaAgtmCtcGtaTa CECyclin_ZC168.4_u273_LNA3 AagTtcAtcmCtgTtgAcgGaaTcgAggmCggAgaAtgmCtgTatmCggTcaTt CECYP_B0213.15_u133_LNA3 AcaGgaAatAtgAttTtgGatT
  • F42A9.5 u55_LNA3 GgaGtgActAtgAaaGcaAagAgtTacmCgaTtgAaamCtgAaaGacAgamCa CECYP. K07C6.3. u3_LNA3 AatmCttTaaTgaTaaTttAtgGgaTctGtaTttmCtcTttmCtgTcaAtaAa CECYP.
  • CECYP_K07C6.4_u87_LNA3 mCt CECYP_K07C6.5_u7_LNA3 AttTaaAggAatTcamCagmCtcAaaAaaTaaTaamCtamCcgGttmCagAgaTt
  • CECYP_K07C6.5_u99_LNA3 AatTtgAgcmCacAtgGcaAgtTatmCaamCagAggAgamCaaTgcmCgtAcaGt
  • CEDC_W02A2.3_u374_LNA3 mCc CEDC_W05G11.3_u153_LNA3 AagAcgGagAggmCtgGagAgaAcgGtamCcgAtgGagAgcmCagGaamCtgAt
  • CEHSP_F44E5.4/5_u123_LNA3 t CEHSP_F44E5.4/5_u380_LNA3 TcaTgaAgcTaaAcaAttmCgaAaaGgaAgaTggTgaAcaAcgGgaAcgTg
  • CEHSP_F52E1.7_u175_LNA3 AagTatAacmCttmCcaAcaGggGtcmCgtmCcaGaamCaaAtcAagTccGaaTt
  • CEHSP_F52E1.7_u448_LNA3 TttAacmCatGgcmCgcAgaTtcTtcGatGacGtcGacTttGatmCgcmCacAt
  • CEHSP_F54D5.8_u252_LNA3 GcgTcgAaaAgaTctmCcc
  • CEPPGB_F13D12.6_u44_LN A3 Ag CEPPGB_F13D12.6_u440_LN A3 TgaTgaGagmCccAgtAacmCaaTtaTttGaamCcgTcaGgaTgtGcgTaaGg mCgtmCtaAtcGaaGaaGggGatmCgtGggmCaaTcaTaamCtaAttAacmCttmCttm
  • CERAD_Y41 C4A.14_u731_LNA3 9 CERAD_Y43C5A.6_u131_LNA3 mCagAttGtamCctTcgAaaAggAaaAggAgaGaaTcgmCgtmCgcAaaAatGg CERAD_Y43C5A.6_u429_LNA3 TgaTggmCttTgaTtaTtcGagmCagGagmCaaTgaTgtmCcgAgaGtcGttAt CERFC_F31 E3.3_u128_LNA3 mCaaTgamCgaGaaTatTggAgrAatGggGaaActGgtTgcGacTtgmCgaAa CERFC_F31 E3.3_u55_LNA3 TtgGaaAacAatmCtcmCtc
  • CESLC F52F12.1a u76 LNA3 ggAg CESLC _K11 G9.5_u400_LNA3 GttGttmCttTttTccGtgAtcTttTcaTgtTtaTgtmCtgAacGtgGcaGg CESLC_K11 G9.5_u462_LNA3 GacTcgTtgGtgTctTgcTagGatGtcTtgGgtTcaTtcmCtcAatmCgtTg CESLC_Y32F6B.1_u179_LNA3 GtamCtgGgcTcgAggGctGaaActAatmCgaAgaAgaAaAacTccAgaAgaTa CESLC_Y32F6B.1_u280_LNA3 GgaTcaTgcTctGttTacGacActG
  • CESULT_EEED8.2_u82_LNA3 AagAagAttmCctGacmCagAgaGacTcamCgtGctTacmCcaAgaAgcAtcTa
  • CETOPO_K12D12.1_u449_LNA3 AaaAccTcgTacTggAaaAggAgcTgcGaaAgcGgaAgtTatmCgaTttGt
  • CETOPO_M01 E5.5b_u256_LNA3 GagAagGccmCagAagAagTacGacAgamCtgAagGagmCagTtgAaaAagTt
  • Example 9 Performance Analysis of LNA Oligonucleotide Capture Probes Designed to Detect Ratios of Splice Variants in mRNA Pools. Oligonucleotide Design for Microarrays. The methods for designing exon-specific internal oligonucleotide capture probes are described above. Design of the LNA-modified Capture Probes
  • every third DNA nucleotide was substituted with an LNA nucleotide.
  • the probes designed to capture the junction of the recombinant splice variants were designed with LNA modifications in a block of five consecutive LNAs nucleotides, two on the 5' side of the splice junction and three on the 3' side of the splice junction. All capture probes are shown in Table 14.
  • the splice variant capture probes were synthesized with a 5' anthraquinone (AQ)- modification, followed by a hexaethyleneglycol-2 (HEG2) linker.
  • the capture probes were first diluted to a 20 ⁇ M final concentration in 100 mM Na-phosphate buffer pH 7.0, and spotted on the lmmobilizer polymer microarray slides (Exiqon, Denmark) using the Biochip Arrayer One (Packard Biochip Technologies, USA) with a spot volume of 2x 300 pi and 300 ⁇ m between the spots.
  • the capture probes were immobilized onto the microarray slide by UN irradiation in a Stratalinker with 2300 ⁇ joules (Stratagene, USA).
  • ⁇ on-immobilized capture probe oligonucleotides were removed from the slides by washing the slides two times 15 minutes in lxSSC. After washing, the slides were dried by centrifugation at lOOOxg for 2 minutes, and stored in a slide box until microarray hybridization.
  • Genomic D ⁇ A was prepared from a wild type standard laboratory strain of Saccharomyces cerevisiae using the ⁇ ucleon MiY D ⁇ A extraction kit (Amersham
  • Amplification of the partial yeast gene was performed using standard PCR using yeast genomic D ⁇ A as template.
  • a forward primer containing a restriction enzyme site and a reverse primer containing a universal linker sequence were used.
  • 20 bp was added to the 3 '-end of the amplicon, next to the stop codon.
  • the reverse primer was exchanged with a nested primer containing a poly-T 2 o tail and a restriction enzyme site.
  • the SWI5 amplicon contains 730 bp of the SWI5 ORF plus 20 bp universal linker sequence and a poly-A 2 o tail.
  • PCR primers used were; YDR146C-For- ⁇ coRI: acgtgaattcaaatacagacaatgaaggagatga YDRl 46C-Rev-Uni : gatccccgggaattgccatgttacctttgattagttttcattggc
  • the PCR amplicon was cut with the restriction enzymes, EcoRI + BamHI.
  • the DNA fragment was ligated into the pTRIampl ⁇ vector (Ambion, USA) using the Quick Ligation Kit (New England Biolabs, USA) according to the supplier's instmctions and transformed into E. coli DH-5 ⁇ by standard methods.
  • the Arabidopsis thaliana Rubisco small subunit ssu2b gene fragment (gil7064721) was amplified from genomic DNA by primers named DJ305 5'- ACTATGATGGACGATACTGGAC-3' and DJ306 5'-
  • the Arabidopsis thaliana Lea gene (gi 1526423) was amplified from genomic DNA with primers named DJ307 5'-GGAATTATCGATGTGTGATAGGATCAGTGTTCAG-3' and DJ308 5'-AATTGGATCGATATTAGCAGTCTCCTTCGCC-3' including the Clal linker sites as above.
  • the PCR fragment was digested with Clal cloned into the yeast S I5 INT construct as above at the unique Clal site.
  • the fragment was inserted in the forward orientation, resulting in the following insert sequence: atcgatGTGTGATAGGTTCAGTGTTCAGGGCTGTCCAAGGAACGTATGAGCATGCGA GAGACGCTGTAGTTGGAAAAACCCACGAAGCGGCTGAGTCTACCAAAGAAGGA GCTCAGATAGCTTCAGAGAAAGCGGTTGGAGCAAAGGACGCAACCGTCGAGAA AGCTAAGGAAACCGCTGATTATACTGCGGAGAAGGTGGGTGAGTATAAAGACTA TACGGTTGATAAAGCTAAAGAGGCTAAGGACACAACTGCAGAGAAGGCGAAGG AGACTGCTAATatcgat
  • C. elegans wild-type strain (Bristol- ⁇ 2) was maintained on nematode growth medium
  • the following fluorochrome-labelled cDNA targets were synthesized to test the performance of 'merged' probes that span exon borders.
  • Synthetic RNAs corresponding to the splice variant #1 (exon01-INS3-exon03 (1-INS3-3) and splice variant #2 (exon01-INS4- exon03 (1-INS3-3) were spiked into lO ⁇ g of C. elegans reference total RNA sample in two different ratios.
  • the first target pool (KU007) contained 10 ng of splice variant #1 (1-INS3- 3) transcript and 2 ng of variant #2 (1-INS4-3) transcript, a ratio of 5:1.
  • the second target pool contained 2 ng variant #1 (1-INS3-3) transcript and 10 ng of splice variant #2 5 (1-INS4-3) transcript, a ratio of 1 :5. Both mRNA pools were combined in separate labeling reactions with 5 ⁇ g anchored oligo(dT 2 o) primer and DEPC-treated water to a final volume of 8 ⁇ l. The mixture was heated at 70°C for 10 minutes, quenched on ice for 5 minutes, followed by addition of 20 units of Superasin RNase inhibitor (Ambion, USA), 1 ⁇ l dNTP solution (lOmM each dATP, dGTP, dTTP and 0.4 mM dCTP, and 3 ⁇ l Cy5-dCTP,
  • Microarray Hybridization 30 The fluorochrome-labelled cDNA samples, respectively, were combined (the two different ratios separately). The following were added: 3.75 ⁇ l 20x SSC (3x SSC final, which was passed through a 0.22 ⁇ filter prior to use to remove particulates) yeast tRNA (1 ⁇ g/ ⁇ l final) 0.625 ⁇ l 1 M HEPES, pH 7.0 (25 mM final, which was passed through 0.22 ⁇ filter prior to use to remove particulates) 0.75 ⁇ l 10 % SDS (0.3 % final) and DEPC-water to 25 ⁇ l final volume.
  • the labelled cDNA target samples were filtered in Millipore 0.22 ⁇ filter spin column (Ultrafree-MC, Millipore, USA) according to the manufacturer's instructions, followed by incubation of the reaction mixture at 100 °C for 2-5 minutes.
  • the cDNA probes were cooled at room temp for 2-5 minutes by spinning at maximum speed in a microcentrifuge.
  • a LifterSlip (Erie Scientific Company, USA) was carefully placed on top of the microarray spotted on Immobilizer TM MicroArray Slide, and the hybridization mixture was applied to the array from the side.
  • An aliquot of 30 ⁇ L of 3xSSC was added to both ends of the hybridization chamber, and the lmmobilizerTM MicroArray Slide was placed in the hybridization chamber (DieTech, USA).
  • the chamber was sealed watertight and incubated at 65 °C for 16-18 hours submerged in a water bath. After hybridization, the slide was removed carefully from the hybridization chamber and washed using the following protocol. The slides were washed sequentially by plunging gently in 2 x SSC/0.1% SDS at room temperature until the cover slip falls off into the washing solution, then in lx SSC pH 7.0 (150 mM NaCl, 15 mM Sodium Citrate) at room temperature for 1 minute, then in 0.2 x SSC, pH 7.0 (30 mM NaCl, 3 mM Sodium Citrate) at room temperature for 1 minute, and finally in 0.05 x SSC (7.5 mM NaCl, 0.75 mM Sodium Citrate) for 5 seconds, followed by drying of the slides by spinning at 1000 xg for 2 minutes. The slides were stored in a slide box in the dark until scanning. Microarray data analysis
  • the splice variant microarray was scanned in a ScanArray 4000XL confocal laser scanner (Packard Instruments, USA). The hybridization data were analysed using the GenePix Pro 4.01 microarray analysis software (Axon, USA). Only the Cy5 (650 nm) data were examined as both hybridizations produced comparable, and acceptably low, signal from the C. elegans reference RNA alone (Cy3 channel). Normalization
  • the ratio of signal in hybridization KU007/KU008 should be 5 for probes designed to exon junctions of the LNS3 splice variant #1 and 0.2 for probes corresponding to 1-INS4 splice variant #2.
  • Results are summarized in Table 15. 50-mer capture probes containing LNA in a block spanning exon-exon junctions were consistent in producing the expected ratios.
  • the capture probes were designed to a 602-nucleotide sequence in the 3 '-region of the Yeast (S. cerevisiae) 70 kDa heat shock protein (SSA4) gene.
  • the 602-base pair sequence is shown in Table 16.
  • For the LNA-spiked oligonucleotide capture probes every third DNA nucleotide was substituted with a LNA nucleotide. All capture probes are shown in Table 17.
  • Table 16 Six hundred and two (602) base pair sequence stretch of theS. cerevisiae ssa4 gene.
  • the underlined segments indicate the position of the capture probes. First underline is equal to capture probe YER103W-554, second underline is equal to capture probe YER103W-492 and so forth.
  • the SSA4 capture probes were synthesized with a 5' anthraquinone (AQ)- 5 modification, followed by a hexaethyleneglycol-2 (HEG2) linker.
  • the capture probes (Table 17) were first diluted to a 20 ⁇ M final concentration in 100 mM Na-phosphate buffer pH 7.0, and spotted on the lmmobilizer microarray slides (Exiqon, Denmark) using the Biochip Arrayer One (Packard Biochip Technologies) with a spot volume of 2x 300 pi and 400 ⁇ m between the spots.
  • the capture probes were immobilized onto the microarray slide by UV 10 irradiation in a Stratalinker with 2300 ⁇ joules (Stratagene, USA).
  • Non-immobilized capture probe oligonucleotides were removed from the slides by washing the slides two times 15 minutes in lxSSC. After washing, the slides were dried by centrifugation at lOOOxg for 2 minutes, and stored in a slide box until microarray hybridization.
  • Yeast Cultures 15 Saccharomyces cerevisiae wild-type (BY4741 , MATa; his3 ⁇ l ; leu2 ⁇ 0; metl 5 ⁇ 0; ura3 ⁇ 0) and Assa4 (MATa; his3 ⁇ l; leu2 ⁇ 0; metl5 ⁇ 0; ura3 ⁇ 0; YER103w::kanMX4) mutant strains (EUROSCARF) were grown in YPD at 30°C until the A 6 oo density of the cultures reached 0.8. Half of the cultures were collected by centrifugation and resuspended in one volume of 40°C preheated YPD. Incubation was continued for an additional 30 minutes at
  • RNA Extraction 20 30°C or 40°C for the standard and heat-shocked cultures, respectively. Cells were harvested by centrifugation and stored at -80°C. RNA Extraction
  • a total of seven cDNA assay mixtures were produced; each with ten (10) ⁇ g total RNA from wtand combined with 5 ⁇ g anchored oligo(dT 2 o) primer and DEPC-treated water to a final volume of 8 ⁇ l.
  • the mixtures were heated at 70°C for 10 minutes, quenched on ice for 5 minutes, followed by addition of 20 units of Superasin RNase inhibitor (Ambion, USA), 3 ⁇ l Cy3-dCTP (Amersham Biosciences), lOmM final concentration of dATP and dGTP, 4 ⁇ l 5 x RTase buffer (Invitrogen), 2 ⁇ l 0.1 mM DTT (Invitrogen), 400 units of Superscript II reverse transcriptase (Invitrogen, USA), dUTP and dTTP accordingly to Table 18, and DEPC-treated water to 20 ⁇ l final volume.
  • Superasin RNase inhibitor Ambion, USA
  • 3 ⁇ l Cy3-dCTP Amersham Biosciences
  • lOmM final concentration of dATP and dGTP 4 ⁇ l 5 x RTase buffer
  • 2 ⁇ l 0.1 mM DTT Invitrogen
  • 400 units of Superscript II reverse transcriptase Invit
  • the fluorochrome-labelled cDNA samples were combined (the different UDG-fragmented samples separately). The following were added: 3.75 ⁇ l 20x SSC (3x SSC final, pass through 0.22 ⁇ filter prior to use to remove particulates) yeast tRNA (1 ⁇ g/ ⁇ l final) 0.625 ⁇ l 1 M HEPES, pH 7.0 (25 mM final, pass through 0.22 ⁇ filter prior to use to remove particulates) 0.75 ⁇ l 10 % SDS (0.3 % final) and DEPC- water to 25 ⁇ l final volume.
  • the labelled cDNA target samples were filtered in Millipore 0.22 ⁇ filter spin column (Ultrafree-MC, Millipore, USA) according to the manufacturer's instmctions, followed by incubation of the reaction mixture at 100 °C for 2-5 minutes.
  • the cDNA probes were cooled at room temp for 2-5 minutes by spinning at maximum speed in a microcentrifuge.
  • a LifterSlip (Erie Scientific Company, USA) was carefully placed on top of the SSA4 microarrays spotted on lmmobilizerTM MicroArray Slide, and the hybridization mixture was applied to the array from the side.
  • the slides were washed sequentially by plunging gently in 2 x SSC/0.1% SDS at room temperature until the cover slip falls of into the washing solution, then in lx SSC pH 7.0 (150 mM NaCl, 15 mM Sodium Citrate) at room temperature for 1 minute, then in 0.2 x SSC, pH 7.0 (30 mM NaCl, 3 mM Sodium Citrate) at room temperature for 1 minute, and finally in 0.05 x SSC (7.5 mM NaCl, 0.75 mM Sodium Citrate) for 5 seconds, followed by drying of the slides by spinning at 1000 xg for 2 minutes.
  • the slides were stored in a slide box in the dark until scanning.
  • Microarray Data Analysis The slides were scanned in a ScanArray 4000XL confocal laser scanner (Packard Instruments, USA). The hybridization data were analysed using the GenePix Pro 4.01 microarray analysis software (Axon, USA).
  • Example 11 Interpretation of Splice Array Data Using LNA Discriminating Probes.
  • the eukaryotic pre-mRNA is the subject of Splicing and Alternative Splicing, hence sequences refer to RNA sequences, Original sequence refers to pre-mRNA, and splice forms refer to mRNA sequences.
  • the splicing is conducted by a cellular machinery named the spliceosome.
  • exons and introns can be used to refer to regions of pre-RNA sequences (or more specifically a single splice form). It is noted that a part of the corresponding DNA/pre-mRNA sequence that is an exon (not excised) in one splice form can potentially be absent in another splice form (e.g., partly absent in exon truncation and completely absent in exon skipping).
  • the terms "constant regions” and "variable regions” are useful for characterizing the process of identifying different splice forms.
  • Splicing can be defined as the production of a new sequence via the excision of part(s) of an original sequence ( Figure 40).
  • Alternative splicing can be defined as the production of more than one novel sequence via the excision of different parts of the original sequence.
  • Alternative splicing can be categorized in terms of (i) whether or not the variable region is flanked by a single constant region or surrounded by two constant regions, (ii) the size of the variable region (e.g., exon skipping/intron retention vs. extension and truncation) [(intron/exon) 5' and 3'], and (iii) the number of variable regions (and hence the number of splice forms).
  • Capture Probe design can be divided into 3 distinct types according to their position: Merged Probes (MP) or Junction Probes, Unique Internal Probes (UIP), and Shared Internal Probes (SIP) (Figure 42). Considering the case of a single variable region surrounded by constant regions, there are several different possible capture probe positions for each type ( Figure 43). Data Interpretation
  • the aim of the analyses can be to determine (i) whether a given original sequence is subject to alternative splicing (i.e., whether there is more than one splice form present), and (ii) whether there is a difference in alternative splicing of the original sequence between two biological samples (i.e., whether the proportions between the two splice forms differ between biological samples).
  • the analysis can also be used for data validation.
  • biases in the microarray platform include (a) noise in terms of non-specific binding and subsequent false signal, (b) differences in dye labeling efficiency, (c) differences in capture probe affinity, (d) differences in sample conditions (e.g., number of cells, and amount of RNA), and (e) differences in reverse transcriptase efficiency of different splice forms. Biases can be corrected for by various means of normalization and/or standardization. Data Analysis
  • 0 denotes the relation between the proportions of the signals (capture probes a and b) between the labeled extracts from biological samples (labeled with Cy5 & Cy3). That is,
  • Example 12 Exemplary Microarrays
  • the nucleic acid arrays of the invention can be generated by standard methods for either synthesis of nucleic acid probes that are then bonded to a solid support or synthesis of the nucleic acid probes on a solid support (e.g., by sequential addition of nucleotides to a reactive group on the solid support).
  • photogenerated acids are produced in light-irradiate sites of the chip and used to deprotect the 5'-OH group of nucleic acid monomers and oligomers (e.g., to remove an acid-labile protecting group such as 5'-O-DMT) to which a nucleotide is to be added (Gao et al., Nucleic Acid Research 29:4744-4750, 2001).
  • Standard methods can also be used to label the nucleic acids in a test sample with, e.g., a fluorescent label, incubate the labeled nucleic acid sample with the array, and remove any unbound or weakly bound test nucleic acids from the array.
  • capture probes were immobilized using AQ technology with a HEG5 linker (U.S.P.N. 6,033,784) onto an lmmobilizerTM slide.
  • An exemplary chip consists of 288 spots in four replicates (i.e., 1152 spots) with a pitch of 250 ⁇ m, and an exemplary hybridization buffer is 5xSSCT (i.e., 750 mM NaCl, 75 mM Sodium Citrate, pH 7.2, 0.05% Tween) and 10 mM MgCl 2.
  • An exemplary target is a 45-mer oligonucleotide with Cy5 at the 5' end and with a final concentration in the hybridization solution of 1 ⁇ M.
  • Hybridization was performed with 200 ⁇ L hybridization solution in a hybridization chamber created by attaching a CoverWellTM gasket to the lmmobilizer slide. The incubation was conducted overnight at 4°C. After hybridization, the hybridization solution was removed, and the chamber was flushed with 3 x 1.0 mL hybridization buffer described above without any target nucleic acid. A cover WellTM chamber was then filled with 200 ⁇ L hybridization solution without target. The slide was observed with a Zeiss Axioplan 2 epifluorescence microscope with a 5x Fluar objective and a Cy5 filterset from OMEGA. The temperature of the microscope stage was controlled with a Peltier element. Thirty-five images at each temperature were acquired automatically with a Photometries camera, automated shutter, and motorized microscope stage. The images were acquired, stitched together, calibrated and stored in stack by the software package "MetaNue"
  • Arrays can be generated using capture probes of any desired length (e.g., arrays of pentamers, hexamers, or heptamers.)
  • 1, 2, 3, 4, 5, 6, 7, 8, or more nucleotides of the probes are L ⁇ A nucleotides.
  • at least 1, 2, 3, 5, 7, 9, or all of the A and T nucleotides in the probes are L ⁇ A A and L ⁇ A T nucleotides.
  • L ⁇ A nucleotides can be placed in any position of the capture probe, such as at the 5' terminus, between the 5' and 3' termini, or at the 3' terminus.
  • L ⁇ A nucleotides may be consecutive or may be separated by one or more other nucleotides.
  • the microarrays can be used to analyze target nucleic acids of any "AT” or "GC” content, and are especially useful for analyzing nucleic acids with high "AT” content because of the increased affinity of the microarrays of the present invention for such nucleic acids compared to traditional microarrays.
  • the array has at least 100, 200, 300, 400, 500, 600, 800, 1000, 2000, 5000, 8000, 10000, 15000, 20000, or more different probes.
  • nucleotides with a universal base can be included in the capture probes to increase the T m of the capture probes (e.g., capture probes of less than 7, 6, 5, or 4 nucleotides).
  • non-discriminatory nucleotides include inosine, random nucleotides, 5 nitro-indole, LNA, inosine, and LNA 2-aminopurine.
  • 1, 2, 3, 4, 5, or more nucleotides with a universal base are located at the 5' and/or 3' termini of the capture probes.
  • microarrays of the invention may also be used as a general tool to analyze the PCR products generated by amplification of a test sample with PCR primers for one or more nucleic acids of interest.
  • PCR primers can be used to amplify nucleic acids with a particular exon or exon-exon combination, and then the PCR products can be identified and/or quantified using a microarray of the invention.
  • PCR primers to specific exons can be used to amplify nucleic acids that are then applied to a microarray for detection and/or quantification as described herein.
  • species-specific PCR primers e.g., primers specific for an exon whose sequence differs among species
  • the hybridization pattern of the PCR products to the array can be used to distinguish between different bacteria, viruses, or yeast and even between different strains of the same pathogenic species.
  • the array is used to determine whether a patient sample contains a bacteria strain that is known to be resistant or susceptible to particular antibiotics or contains a viras or yeast strain known to be resistant or susceptible to certain drugs. Changes in product composition or raw material origin can also be detected using a microarray. The arrays can also be used to determine the composition of mRNA cocktails.
  • Exemplary environmental microbiology applications of these arrays include identification of major rRNA types in contaminated soil samples and classification of microbial isolates. These rRNA amplificates are formed from rRNA by rtPCR or from the rDNA gene by conventional PCR. Numerous general and selective primers for different groups of organisms have been published. Most frequently an almost full length amplificate of the 16S rDNA gene is used (e.g., the primers 26F and 1492R). For purifying rRNA from a soil sample, standard methods such as one or more commercial extraction kits from companies such as QIAGEN ("Rneasy", Q-biogene "RNA PLUS,” or “Total RNA safe" can be used.
  • Example 14 Methods for Minimizing the Variance in Melting Temperatures in Nucleic Acid Populations of the Invention Any simultaneous use of more than one primer or probe is made difficult because the involved primers or probes must work under the same conditions. An indication of whether or not two or more primers or probes will work under the same conditions is the relative T ms at which the hybridized oligonucleotides dissociate.
  • the ⁇ T m is of importance.
  • ⁇ T m expresses the difference between T m of the match and the T m of the mismatch hybridizations. Generally, the larger ⁇ T m obtained, the more specific detection of the sequence of interest.
  • a large ⁇ T m facilitates more probes to be used simultaneously and in this way a higher degree of multiplexity can be applied.
  • High affinity nucleotide analogs such a LNA can be also be used universally to equalize the melting properties of oligonucleotides with different AT and CG content.
  • the increased affinity of LNA adenosine and LNA thymidine corresponds approximately to the normal affinity of DNA guanine and DNA cytosine.
  • An overall substitution of all DNA-A and DNA-T with LNA-A and LNA-T results in melting properties that are nearly sequence independent but only depend on the length of the oligonucleotide. This may be important for design of oligonucleotide probes used in large multiplex analysis.
  • LNA can be mixed with DNA during standard oligonucleotide synthesis
  • LNA can be placed at optimal positions in probes in order to adjust T m .
  • the specificity of PCR may also be enhanced by the use of LNA in primers, or probes, and this facilitates a higher degree of multiplexity.
  • the T m of the primers or probes can be adjusted to work at the same temperature. Amplification or hybridization is more specific when LNA is included in primers or probes. This is due to the LNA increased ⁇ T m , which relates to higher specificity. Once ⁇ T m of the primers or probes is high, more primers or probes can potentially be brought to work together.
  • Prediction ofT m LNA can be used to enhance any experiment that is based on hybridization.
  • the series of algorithms described herein have been developed to predict the optimal use of LNA. Melting properties of 129 different LNA substituted capture probes hybridized described herein to their corresponding DNA targets were measured in solution using UV- spectrophotometry. The data set was divided into a training set with 90 oligonucleotides and a test set with 39 oligonucleotides. The training set was used for training of both linear regression models and neural networks. Neural networks trained with nearest neighbour information, length, and DNA/LNA neighbour effect are efficient for prediction of T m with the given set of data.
  • All assays in which DNA/RNA hybridization is conducted may benefit from the use of LNA in terms of increased specificity and quality.
  • Exemplary uses include sequencing, 5 primer extension assays, PCR amplification, such as multiplex PCR, allele specific PR amplification, molecular beacons, (e.g., nucleic acids be multiplexed with one colour based on multiple T m 's), Taq-man probes, in situ hybridization probes (e.g., chromosomal and bacterial 16S rRNA probes), capture probes to the mRNA poly- A tail, capture probes for microarray detection of SNPs, capture probes for expression microarrays (sensitivity 10 increased 5-8 times), and capture probes for assessment of alternative mRNA splicing.
  • sequencing 5 primer extension assays
  • PCR amplification such as multiplex PCR
  • allele specific PR amplification e.g., allele specific PR amplification
  • molecular beacons e.g., nucle
  • Example 15 Exemplary Methods for the Prediction of Melting Temperatures for Nucleic Acid Populations of the Invention
  • LNA units have different melting properties than DNA and RNA nucleotides.
  • thermodynamical models for melting temperature prediction have existed for DNA and RNA only, but not for LNA.
  • T m prediction model for LNA/DNA mixed oligonucleotides has been developed.
  • the T m prediction tool is available on-line at the Exiqon website (www.LNA-Tm.com and http://www.exiqon.com/Poster/Tmpred-ET- view.pdf).
  • RNA to hybridize in a temperature dependent manner e.g. the microarray techniques, PCR reactions and blotting techniques.
  • the melting properties of nucleic acid duplexes, in particular the melting temperature T m are crucial for optimal design of such experiments.
  • T m is usually computed using a two-state thermodynamical model (Breslauer, Meth. EnzymoL,
  • the model described herein predicts the T m of duplexes of mixed LNA/DNA oligonucleotides hybridized to their complementary DNA strands.
  • DNA monomers are
  • LNA monomers are denoted with uppercase letters, e.g., there are eight types of monomers in the mixed strand: a, c, g, t, A, C, G and T.
  • the model is based on the formula (SantaLucia, 1998, supra; Allawi et al, Biochemistry 36:10581-10594, 1997).
  • the LNA model differs from SantaLucia' s DNA model in the way the changes in enthalpy lHand entropy ⁇ S are calculated. As in SantaLucia' s model, they depend on nearest neighbor sequence information and special contributions for the terminal base-pairs in the two ends of the duplex. However, with eight types of monomers (LNA and DNA) the increased number of nearest neighbor combinations requires more model parameters to be determined and hence more data. Parameter Reduction
  • zlHand ⁇ S are calculated as a sum of contributions from all nearest neighbor pairs in the sequence.
  • LNA doubles the number of monomer types and quadruples the number of possible nearest neighbor pairs.
  • Parameter reduction strategies are used for matching the model complexity to limited data sets.
  • a strategy for reducing model complexity is to sum AH from single base-pair contributions, which do not take the influence of adjacent nucleotides into account.
  • nearest neighbor contributions are added as a correction term to the single base-pair contributions.
  • Another strategy is to use hierarchically reduced monomer alphabets.
  • similar monomers are identified with the same letter.
  • the smallest alphabet, ⁇ D,L ⁇ simply identifies the monomer type: DNA or LNA.
  • the sequence GcTAAcTt can be written as SsWWWsWw or as LDLLLDLD.
  • the principle is to split AH and AS into contributions that depend on different levels of detail of the sequence.
  • the fine levels of detail require many parameters to be determined, while the coarse levels need fewer parameters.
  • the more detailed contributions can then be treated as minor corrections, thus effectively reducing the total number of model parameters.
  • Model parameters were determined using data from melting experiments on hundreds of oligonucleotides.
  • the oligonucleotides were random sequences with lengths between 8 and 20 and a percentage of LNA between 20 and 70. Melting curves were obtained using a Perkin-Elmer UV ⁇ -40 spectrophotometer, but only the T m values were used for modeling. Model parameters were adjusted using a gradient descent algorithm that minimizes the error function p ⁇ _ "1 ___ t pred _ iexp
  • thermodynamic quantities The aim of this work has been to estimate T m values as accurately as possible.
  • a machine learning approach has been adopted in which the prediction of the physical lHand AS quantities is less important.
  • the parameters of this model may be inaccurate as thermodynamic quantities.
  • the gradient descent algorithm produces a broad ensemble of models in which the AH and AS parameters can vary substantially, while maintaining an accuracy in the predicted T m .
  • the thermodynamic meaning of AH and ⁇ S is based on a two-state assumption, which may not be realistic in every case. Even short oligonucleotides can form different secondary stmctures or melt through multiple-state transitions (T ⁇ stesen et ⁇ l., J. Phys. Chem. B.
  • the T m prediction model has been tested on two data sets that were not used during the training process.
  • One set consisted of pure DNA oligonucleotides without LNA monomers and had a standard deviation of the residuals (SEP) of 1.57 degrees.
  • the other set consisted of mixed oligonucleotides with both LNA and DNA and had a SEP of 5.25 degrees.
  • SEP standard deviation of the residuals
  • the difference in prediction accuracy between the two types of oligonucleotides suggests that T m prediction of mixed strands is a more complex task than Tm prediction of pure DNA. This is possibly due to irregularities in the duplex helical stracture induced by the LNA monomers (Nielsen et al, Bioconjug. Chem. 11:228-238, 2000).
  • Example 16A Algorithm to Optimize the Substitution Pattern of Nucleic Acids of the Invention
  • High affinity nucleotides such as LNA and other nucleotides that are conformationally restricted to prefer the C3'-endo conformation or nucleotides with a modified backbone and/or nucleobase stabilize a double helix configuration.
  • the most stable duplex between a high affinity capture oligonucleotide and an unmodified target oligonucleotide should generally arise when all nucleotides in the capture probe or primer are replaced by their high affinity analogue.
  • the most stable duplex should thus be formed between a fully modified LNA capture probe and the corresponding DNA/RNA target molecule.
  • Such a fully modified capture probe should be more efficient in capturing target molecules, and the resulting duplex is more thermally stable.
  • a fully modified capture probe may thus form duplexes with itself, or if it is long enough, internal hairpins that are even more stable than duplexes with the desired target molecule.
  • Probes with even a small inverse repeat segment where all constituent positions are substituted with high affinity nucleotides may bind to itself and be unable to bind the target.
  • a sequence dependent substitution pattern is desirably used to avoid substitutions in positions that may form self- complementary base-pairs.
  • a computer algorithm can be used to automatically determine the optimal substitution pattern for any given capture probe sequence according to the following two criteria.
  • the capture probe should contain as many substitutions as possible in order to bind as much target as possible at any given temperature and to increase the thermal stability of the formed duplex.
  • the second criterion is substituted with the following alternative criterion to obtain capture probes with similar thermal stability. The number and position of capture probe substitutions should be adjusted so that all the duplexes between capture probes and targets have a similar thermal stability (i.e., T m equalization).
  • the second criterion for increasing thermal stability is more desirable that the alternative second criterion for T m equalization.
  • the second alternative criterion is desirably used since T m equalization is desirable for these probes and primers.
  • An exemplary algorithm works as follows. For each nucleotide sequence in an array of length n, all possible substitution patterns, i.e., 2" different sequences are evaluated. Each evaluation consist of estimating the energetic stability of the duplex between the substituted capture sequence and a perfect match unmodified target ("target duplex") and the energetic stability of the most stable duplex that can be formed between two substituted capture probes themselves (“self duplex”). The energetic stability estimate for a duplex may be calculated, e.g., using a Smith-
  • Gap initiation penalty -8
  • Gap continuation penalty -50 a c g t A C G T a -2 c -2 -2 g -2 3 -2 t 2 -2 1 -2 A -3 -3 -3 4 -3 C -3 -3 6 -3 -3 -3 -3
  • This scoring matrix was partly based on the best parameter fit to a large (over 1000) number of melting curves of different DNA and LNA containing duplexes and partly by visual scoring of test capture probe efficiency. If desired, this scoring matrix may be optimized by optimizing the parameter fit as well as increasing or optimizing the dataset used to obtain these parameters.
  • the heptamer sequence ATGCAGA in which each position can be either an LNA or a DNA nucleotide is used.
  • the target duplex formed between a fully modified capture probes with this sequence and its unmodified target receive a score of 34 as illustrated below.
  • Capture sequence A-T-G-C-A-G-A
  • the most stable self duplex that can be formed between two modified capture probes has an almost equivalent energetic stability with a score of 30 as illustrated below.
  • Target sequence A-G-A-C-G-T-A
  • the capture probe efficiency of a fully modified probe is likely reduced by its propensity to form a stable duplex with itself.
  • ATGcaGA in which capital letters represent LNA nucleotides
  • the stability of the target duplex is reduced slightly from 34 to 29.
  • Target sequence t-a-c-g-t-c-t
  • Target sequence A-G-a-c-G-T-A
  • the difference between the stability of the desired target duplex and the undesired self duplex can be further increased by using the capture sequence AtgcaGA where the target duplex has a score of 24.
  • Capture sequence A-t-g-c-a-G-A
  • Capture sequence A-t-g-c-a-G-A
  • ATCcaGA is the substitution pattern with the highest degree of substitution for which the stability of the target duplex is adequately more stable than the stability of the best self duplex (e.g., above 25%).
  • This algorithm can be used to determine desirable substitution patterns for any size capture probe or any given probe sequence.
  • the following simple design rules may also be applied for probe design, especially for short probes.
  • the best self alignment for the corresponding DNA capture probe in the sequence is determined using a simple Smith- Waterman scoring matrix of:
  • Example 16B Computer code for a preferred software program of the invention.
  • the oligod program takes a gene sequence as input and returns sequences for
  • # # # -maxhits n # n is the maximal number of alignments to show for each oligo # # -maxoligo n # n is the maximal number of oligos to suggest for each sequence # # -min_score n # n is the minimal score required for a hit to be included in the scoring of a oligo
  • hybridisation matrix # -matrix hybridisationmatrix #
  • the format of the hybridisation matrix is that used by the fasta
  • # freq is the frequence of LNA default is 4 # # -blastdb "dbl db2 db3" #
  • $glb-> ⁇ bin ⁇ "/usr/bin”
  • $glb-> ⁇ formatdb ⁇ "$glb-> ⁇ bin ⁇ /formatdb”
  • $glb-> ⁇ blastall ⁇ "$glb-> ⁇ bin ⁇ /blastall"
  • $glb-> ⁇ ssearch ⁇ "$glb-> ⁇ bin ⁇ /ssearch"
  • $glb-> ⁇ cookiefn ⁇ "$glb-> ⁇ dir ⁇ /lwpcookies.txt";

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Abstract

L'invention concerne des acides nucléiques améliorés et des procédés destinés au profilage d'expression des ARNm, pour identifier et profiler des variants épissés déterminés d'ARNm et pour détecter les mutations, les délétions ou les duplications d'exons déterminés ou d'autres variants épissés, p.ex., des altérations associées avec des maladies telles que le cancer dans un échantillon d'acide nucléique, p.ex. dans un échantillon biologique ou un échantillons de patient.
PCT/DK2003/000715 2002-10-21 2003-10-21 Oligonucleotides utiles dans la detection d'acides nucleiques d'interet WO2004035819A2 (fr)

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WO2006076025A3 (fr) * 2004-05-14 2006-12-21 Amaox Inc Biocapteurs de cellule immune et procede d'utilisation de ceux-ci
WO2006085984A3 (fr) * 2004-07-09 2007-02-22 Amaox Inc Biocapteurs de cellules immunes et procedes d'utilisation
WO2008111908A1 (fr) * 2007-03-15 2008-09-18 Jyoti Chattopadhyaya Ribothymidines carbocycliques 2', 4' à conformation verrouillée à 5 et 6 éléments destinées à traiter des infections et le cancer
US8232055B2 (en) 2002-12-23 2012-07-31 Agilent Technologies, Inc. Comparative genomic hybridization assays using immobilized oligonucleotide features and compositions for practicing the same
US8273235B2 (en) 2010-11-05 2012-09-25 Roshan V Chapaneri Dark colored chromium based electrodeposits
US8321138B2 (en) 2005-07-29 2012-11-27 Agilent Technologies, Inc. Method of characterizing quality of hybridized CGH arrays
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US9771583B2 (en) 2007-09-19 2017-09-26 Applied Biosystems, Llc siRNA sequence-independent modification formats for reducing off-target phenotypic effects in RNAI, and stabilized forms thereof
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AU2001257239A1 (en) * 2000-04-25 2001-11-07 Affymetrix, Inc. Methods for monitoring the expression of alternatively spliced genes
WO2002097398A2 (fr) * 2000-10-25 2002-12-05 Exiqon A/S Supports de substrats fermes appropries pour l'analyse de biomolecules

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US8232055B2 (en) 2002-12-23 2012-07-31 Agilent Technologies, Inc. Comparative genomic hybridization assays using immobilized oligonucleotide features and compositions for practicing the same
WO2006076025A3 (fr) * 2004-05-14 2006-12-21 Amaox Inc Biocapteurs de cellule immune et procede d'utilisation de ceux-ci
WO2006085984A3 (fr) * 2004-07-09 2007-02-22 Amaox Inc Biocapteurs de cellules immunes et procedes d'utilisation
JP2008505649A (ja) * 2004-07-09 2008-02-28 アモークス リミテッド 免疫細胞バイオセンサーおよびその使用方法
US8321138B2 (en) 2005-07-29 2012-11-27 Agilent Technologies, Inc. Method of characterizing quality of hybridized CGH arrays
WO2008111908A1 (fr) * 2007-03-15 2008-09-18 Jyoti Chattopadhyaya Ribothymidines carbocycliques 2', 4' à conformation verrouillée à 5 et 6 éléments destinées à traiter des infections et le cancer
US8461124B2 (en) 2007-03-15 2013-06-11 Jyoti Chattopadhyaya Five- and six-membered conformationally locked 2′,4′-carbocyclic ribo-thymidines for the treatment of infections and cancer
US10900038B2 (en) 2007-09-19 2021-01-26 Applied Biosystems, Llc siRNA sequence-independent modification formats for reducing off-target phenotypic effects in RNAI, and stabilized forms thereof
US9771583B2 (en) 2007-09-19 2017-09-26 Applied Biosystems, Llc siRNA sequence-independent modification formats for reducing off-target phenotypic effects in RNAI, and stabilized forms thereof
US10329564B2 (en) 2007-09-19 2019-06-25 Applied Biosystems, Llc siRNA sequence-independent modification formats for reducing off-target phenotypic effects in RNAi, and stabilized forms thereof
US11142800B2 (en) 2010-10-07 2021-10-12 The General Hospital Corporation Biomarkers of cancer
US8273235B2 (en) 2010-11-05 2012-09-25 Roshan V Chapaneri Dark colored chromium based electrodeposits
WO2015200697A1 (fr) * 2014-06-25 2015-12-30 The General Hospital Corporation Ciblage de hsatii (human satellite ii)
US10301624B2 (en) 2014-06-25 2019-05-28 The General Hospital Corporation Targeting human satellite II (HSATII)
US12152241B2 (en) 2014-06-25 2024-11-26 The General Hospital Corporation Targeting human satellite II (HSATII)
EP3587432A4 (fr) * 2017-02-21 2021-01-06 Osaka University Composé d'acide nucléique et oligonucléotide
US11286275B2 (en) 2017-02-21 2022-03-29 Osaka University Nucleic acid compound and oligonucleotide
US11905308B2 (en) 2017-02-21 2024-02-20 Osaka University Nucleic acid compound and oligonucleotide
US11578327B2 (en) 2018-02-14 2023-02-14 Deep Genomics Incorporated Oligonucleotide therapy for Wilson disease

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