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WO2010058189A1 - Oligonucléotides capables de distinguer des séquences d’acide nucléique qui comprennent une séquence conservée - Google Patents

Oligonucléotides capables de distinguer des séquences d’acide nucléique qui comprennent une séquence conservée Download PDF

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Publication number
WO2010058189A1
WO2010058189A1 PCT/GB2009/002748 GB2009002748W WO2010058189A1 WO 2010058189 A1 WO2010058189 A1 WO 2010058189A1 GB 2009002748 W GB2009002748 W GB 2009002748W WO 2010058189 A1 WO2010058189 A1 WO 2010058189A1
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Prior art keywords
nucleic acid
oligonucleotide
target nucleic
oligonucleotides
sequence
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PCT/GB2009/002748
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English (en)
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Andrew Thompson
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Trillion Genomics Limited
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Application filed by Trillion Genomics Limited filed Critical Trillion Genomics Limited
Priority to US13/130,946 priority Critical patent/US20110319283A1/en
Publication of WO2010058189A1 publication Critical patent/WO2010058189A1/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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

  • the present invention relates to the field of molecular biology and in particular to oligonucleotides for use in nucleic acid hybridisation, and to methods and reagents for distinguishing between nucleic acid sequences that comprise a conserved sequence.
  • the reliability of the hybridisation process depends inter alia upon specific recognition of the target sequence by the probe sequence.
  • a probe strand binds to a target strand having similar but not identical complementary sequence, the affinity of the two strands for one another is reduced but cross-hybridisation is still possible. If two targets with similar sequences are present in a biological sample, it may be difficult to discriminate between them where both targets bind to the same probe.
  • splicing or cis-splicing
  • pre-mRNA also known as heterogeneous nuclear RNA [hnRNA] or immature RNA
  • hnRNA heterogeneous nuclear RNA
  • trans-splicing involves ligation of two or more exons from different genes.
  • Differential splicing by extending or skipping of one or more exons, or retaining of one or more introns, creates alternative mRNA splice variants of the same gene.
  • Many genes which are subject to splicing have a modular structure in which domains or "blocks" of a gene sequence can be recombined in different ways to give alternative mRNA splice variants. The presence of such conserved or shared domains makes it difficult to distinguish different splice variants in hybridisation-dependent gene expression profiling assays.
  • chromosomal translocations or deletions may result in two "parent" genes being fused to form a "chimeric" gene (also referred to as a "gene fusion") and a mutant chimeric gene product may be expressed.
  • the chimeric gene comprises part of the sequence of one parent gene and part of the sequence of another parent gene. Chimeric genes are found in certain cancers such as leukemia, where the presence of particular translocations or deletions is associated with the severity of the disease.
  • One characteristic fusion found in leukemia is a BCR-ABL gene fusion ("B2A2"; see Bohlander, 2000, Cytogenet Cell Genet. 91 (1-4):52-6).
  • the best mode of treatment by targeting and killing cancer cells which express chimeric genes may be determined following identification of the presence of these fused sequences. It would be useful to be able to reliably detect a chimeric gene (or its mRNA) in a simple assay.
  • boundary-spanning oligonucleotide probes complementary to sequences flanking juxtaposed domain sequences can be used to detect splice variants or gene fusion mRNAs (Kane et al., 2000, Nucleic Acids Res. 28: 4552-4557).
  • the experimental results for that approach using 50-mer oligonucleotides showed significant cross-hybridisation of different splice variants to the boundary-spanning probes. This may not be unexpected since splice variants that share one exon spanned by a boundary- spanning probe can hybridise to part of the sequence of the probe.
  • T m melting temperature
  • the problem of detecting a splice variant or a chimeric gene mRNA is compounded when attempting to perform in situ hybridisation, as corresponding chromosomal gene sequence(s) and pre-mRNA may additionally be present. Also, amplification of the target may be difficult or impossible, so any probe used should be highly sensitive to detect. Furthermore, generally speaking it is necessary to have high affinity probes for in situ hybridisation to assist binding in the presence of extensive secondary structure and auxiliary proteins bound to RNA and also to ensure that the probe remains bound to the target after extensive wash steps to remove unbound probe. The use of short probes may thus not be a suitable option for improving detection of alternative splice variants or chimeric genes in situ.
  • oligonucleotide arrays that allows the simultaneous detection of multiple sequences present in a sample.
  • nucleic acids in a sample are labelled with a fluorescent dye and the labelled material is hybridised to oligonucleotide probes on an array.
  • the array may be designed such that individual probes are present at discrete locations on the array, allowing determination of the presence of a particular target sequence by measuring the amount of fluorescence that is emitted from each probe location on the array.
  • Various techniques have been developed for labelling of large populations of nucleic acids in a single reaction, particularly techniques for labelling populations of poly-adenylated messenger RNA molecules.
  • Antisense oligonucleotides have been proposed as a method for inhibiting the expression of particular genes (Crooke, 1998, Antisense Nucleic Acid Drug Dev. 8(2): 133-4) or for changing patterns of alternative splicing (Wilton & Fletcher, 2005, Gene Ther. 5(5): 467- 83). For the purposes of detecting splice variants or chimeric gene mRNAs, highly specific antisense reagents are required.
  • the present invention provides inter alia an oligonucleotide (also referred to herein as a "probe”) able to discriminate between a nucleic acid target and a nucleic acid variant thereof (for example, mRNA splice variants, or chimeric gene and corresponding parent gene mRNAs) in which the target and variant that share at least one domain of conserved or identical sequence.
  • a nucleic acid target for example, mRNA splice variants, or chimeric gene and corresponding parent gene mRNAs
  • a nucleic acid variant thereof for example, mRNA splice variants, or chimeric gene and corresponding parent gene mRNAs
  • the present invention also provides an array of two or more oligonucleotides able to discriminate between a nucleic acid target and a variant thereof that shares at least one domain of conserved or identical sequence.
  • an antisense oligonucleotide able to specifically hybridise with a target sequence comprising a domain structure, for example, a target sequence which shares at least one domain of conserved or identical sequence with a variant of the target sequence.
  • oligonucleotides of the invention are particularly suitable for in situ hybridisation, in vivo applications like antisense therapy, and also for other applications like microarray detection of alternative splice variants or chromosomal translocations in cancer.
  • an oligonucleotide (such as an oligonucleotide probe, also referred to herein as a "probe") which is hybridisable with greater affinity to a target nucleic acid than to a nucleic acid variant of the target nucleic acid, the target nucleic acid comprising a first domain and a second domain flanking a domain junction, the first domain having a sequence which is conserved with a first sequence in the nucleic acid variant, wherein the oligonucleotide has a first portion and a second portion flanking a portion junction, the first portion being complementary in part to the first domain and the second portion being complementary in part to the second domain, but wherein the first portion comprises at least a first discontinuity relative to the first domain and the second portion comprises at least a second discontinuity relative to the second domain, each discontinuity comprising or consisting of a sequence mismatch and/or a non-nucleotide spacer.
  • oligonucleotides utilise discontinuities in their sequence to enhance specificity (also referred to herein as "discrimination"). Oligonucleotide probes having discontinuities are known in the prior art, but not with the structure and uses of the presently described oligonucleotides.
  • the oligonucleotide of the invention comprising at least two discontinuities will typically bind to a target nucleic acid (also referred to herein as a "target” or “target sequence”) with a lower affinity and consequently with a lower T m than a corresponding oligonucleotide that is fully complementary to the same target.
  • a target nucleic acid also referred to herein as a "target” or “target sequence”
  • target sequence also referred to herein as a "target” or “target sequence”
  • the oligonucleotide of the present invention will be slightly less sensitive than a corresponding fully complementary oligonucleotide of the same length.
  • the difference between the T m of a duplex formed by the oligonucleotide of the invention and its target compared to the T m of the duplex formed by the oligonucleotide of the invention and a nucleic acid variant comprising only one domain of conserved or identical sequence will be larger than that for a corresponding fully complementary oligonucleotide which lacks a discontinuity.
  • This feature of the oligonucleotide of the invention allows enhanced detection of the target vs variant nucleic acids.
  • tethered oligonucleotide probes comprising two sequences linked by a single non-nucleic acid linker can have enhanced specificity for mRNA targets with extensive secondary structure, compared to single sequences of the same length (Cload & Schepartz, 1994, J. Am. Chem. Soc. 116: 437 - 442).
  • tethered probes do not have the structure or uses of the presently described oligonucleotides.
  • the oligonucleotide of the present invention may have first and second portions of substantially equal length, for example lengths which do not differ by more than 0-10% or
  • the first and second portions may be structurally bilaterally symmetrical about the portion junction.
  • first and second portions may have the same number of monomers on each side of the portion junction, although the sequence of the first and second portions will usually not be the same.
  • the first and second portions of the oligonucleotide may have substantially the same T m .
  • substantially the same is meant that the T m of the first portion is identical with, or up to 10% different from, for example up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% different from, the T m of the second portion.
  • discrimination of the oligonucleotide between the target nucleic acid and its target nucleic acid variant(s) may be enhanced by using such an oligonucleotide.
  • the T m of the first and second portions may be calculated theoretically, for example using the Nearest Neighbour method (see Breslauer et al., 1986, PNAS USA 83(11): 3746-50;
  • first and second portions have substantially the same T m , these portions will typically, but not necessarily, be of unequal length.
  • Each of the first and second discontinuities may be positioned adjacent nucleotide 2 to 20 (i.e. adjacent nucleotide 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20), for example adjacent nucleotide 3 or nucleotide 4 or adjacent nucleotide 8, 9, 10, 11 or 12, of the oligonucleotide, relative to the portion junction.
  • Each discontinuity may be of a length equivalent to 1 to 5 nucleotides, for example 1 , 2, 3, 4, or 5 nucleotides, such as 1 or 2 nucleotides.
  • Each sequence mismatch may comprise a natural nucleotide which is non-complementary to a base at a corresponding position in the target nucleic acid.
  • the sequence mismatch may comprise an artificial mismatch.
  • the oligonucleotide may comprise a mixture of one or more natural nucleotide mismatches and one or more artificial mismatches.
  • the artificial mismatch may comprise a universal base analogue or an abasic mismatch.
  • Each discontinuity may be a non-nucleotide spacer, for example polyethylene glycol, a phosphoramidite spacer such as a C3 phosphoramidite spacer, or an amino acid.
  • the oligonucleotide may comprise a natural nucleotide or a nucleotide analogue, for example a 2-O-methyl analogue, a bridged nucleic acid monomer (such as a locked nucleic acid [LNA] monomer), a peptide nucleic acid (PNA) monomer, a universal nucleoside, or a combination of any of these.
  • a nucleotide analogue for example a 2-O-methyl analogue, a bridged nucleic acid monomer (such as a locked nucleic acid [LNA] monomer), a peptide nucleic acid (PNA) monomer, a universal nucleoside, or a combination of any of these.
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • Nucleotide analogues suitable for use in the oligonucleotide may have enhanced binding affinity compared with their native DNA or RNA counterparts.
  • the analogue may have an enhanced affinity for RNA such as a 2'-modified RNA analogue (for example, 2-O-Methyl RNA, 2' Fluoro-RNA and 2-0-ethyl RNA).
  • Bridged nucleic acid monomers comprise a linkage from the 2' position in the ribose ring to the 4'-position.
  • Bridged nucleic acid monomers (for example, LNA monomers) are suitable analogues for oligonucleotides intended for binding and recognition of DNA targets. Oligonucleotides comprising both one or more 2'-modified RNA analogues and one or more bridged nucleic acid monomers are also encompassed.
  • An oligonucleotide comprising PNA, and a chimeric oligonucleotide comprising PNA and DNA are suitable for binding to a DNA target. Due to lower solubility than other analogues, PNA oligonucleotides longer than 20 bases are currently difficult to manufacture so PNA probes may be shorter and the discontinuities may be position closer to the portion junction (for example, 3 to 4 bases rather than 8 to 12 bases, as described further below). Chimeric DNA-PNA oligonucleotides may be longer.
  • the oligonucleotide may comprise a label, for example a fluorescent tag, a mass tag, biotin, an enzyme, and/or a nanoparticle.
  • the label may include a sequence which is non- complementary to the target nucleic acid.
  • the oligonucleotide may be in the form of a molecular beacon.
  • the term "molecular beacon” refers to a single-stranded oligonucleotide that form a stem-and-loop (also known as "hairpin") structure under suitable conditions known in the art.
  • the loop contains a probe sequence that is complementary to a target nucleic acid sequence, and the stem is formed by annealing - in the absence of target - of complementary arm sequences located at either side of the probe sequence.
  • a fluorescent tag is linked (for example, covalently linked) at or near the end of one arm and a corresponding non- fluorescent quencher is linked (for example, covalently linked) to the end of the other arm.
  • fluorescein as a fluorophore and DABCYL or Methyl Red as the quencher
  • ROX as the fluorophore
  • DABCYL Methyl Red
  • a molecular beacon does not fluoresce when it is free in solution because the stem places the fluorophore sufficiently close to the non-fluorescent quencher that they transiently share electrons, eliminating the ability of the fluorophore to fluoresce.
  • the molecular beacon hybridises to a nucleic acid strand containing the target nucleic acid sequence, it undergoes a spontaneous conformational reorganisation that forces the stem to dissociate and the fluorophore and the quencher to move away from each other, thus allowing the molecular beacon to fluorescence (see Tyagi & Kramer, 1996, Nat. Biotech. 14: 303-308; Tyagi et al., 1998, Nat. Biotech.
  • Molecular beacons may comprise nucleotide analogues such as LNA or 2'-O-methyl RNA (Wang et al., 2005, J Am Chem Soc. 127(45): 15664-15665; Tsourkas et al., 2003, Nucleic Acids Res. 31(6): 5168-5174).
  • the oligonucleotide may have a 5' to 3' structure comprising the first portion, the portion junction and the second portion, where the first portion has a 5' to 3' structure: 1 to 45 nucleotides (for example, 5 to 12 nucleotides), a discontinuity, and 2 to 12 nucleotides, and the second portion has a 5 1 to 3' structure: 2 to 12 nucleotides, a discontinuity, and 1 to 45 nucleotides (for example, 5 to 12 nucleotides).
  • the oligonucleotide may be up to about 100 nucleotides in length, for example about 40 to 70 nucleotides, less than 40 nucleotides, about 30 nucleotides, less than 20 nucleotides, or at least about 16 nucleotides in length.
  • the target nucleic acid of the invention may be an mRNA molecule.
  • the mRNA molecule may be a splice variant of a gene, the nucleic acid variant being an alternative splice variant of the same gene.
  • the portion junction of the oligonucleotide may correspond in position with the domain junction in the target nucleic acid.
  • the domain junction corresponds in position with a splice boundary.
  • the target nucleic acid may be a chimeric gene or its expressed mRNA.
  • the first domain of the target nucleic acid has a sequence which is conserved with the first sequence in the nucleic acid variant, the first domain and second domain of the target nucleic acid in combination provide a unique sequence which may be detected specifically by an oligonucleotide of the present invention.
  • the first domain sequence and the first sequence in the nucleic acid variant may be a shared exon or a shared gene sequence or part thereof.
  • the second domain adjacent the first domain renders the combined sequence formed in the target nucleic acid unique.
  • the second domain may be a sequence (for example, an intron) which is not conserved between the target nucleic acid and its variant.
  • sequence of the second domain may itself be conserved between the target nucleic acid and its variant, but here the target nucleic acid has a unique sequence due to the positioning of the first domain adjacent the second domain in the target nucleic acid; a conserved second domain is positioned elsewhere in the variant.
  • the target nucleic acid in another aspect of the invention is a chromosome or any portion thereof.
  • the oligonucleotide may in one aspect be for use in detection of the target nucleic acid, for example by in situ hybridisation.
  • the oligonucleotide may additionally or alternatively be for use in an array.
  • the oligonucleotide of the invention is also referred to herein as a "probe” or an "oligonucleotide probe".
  • the oligonucleotide may be an antisense oligonucleotide.
  • the antisense oligonucleotide may comprise nucleotides with enhanced nuclease resistance and enhanced binding affinity such as 2'-O-methyl analogues, 2'-O- methoxyethoxy (2'-MOE) analogues or 2'-deoxy-2'-fluoro-D-arabinose analogues.
  • the antisense oligonucleotide may comprise, in addition to the first and second portions, an RNase H-recruiting portion that can recruit RNase H to a duplex of the antisense oligonucleotide with RNA.
  • the RNase H-recruiting portion the antisense oligonucleotide may for example have a length of between 5 and 7 nucleotides.
  • the antisense oligonucleotide may additionally comprise stretches of nuclease resistant nucleotides with enhanced binding affinity flanking the RNase H-recruiting portion, for example on both the 3' and 5 1 sides of the RNase H-recruiting portion.
  • a set of oligonucleotides comprising two or more oligonucleotides, in which a first oligonucleotide is as defined herein and a second or further oligonucleotide is as defined herein except that the second or further oligonucleotide is hybridisable with greater affinity to one or more nucleic acid variants of the target nucleic acid.
  • the set of oligonucleotides may be for use in simultaneous or sequential detection of the target nucleic acid and the one or more nucleic acid variants.
  • the set of oligonucleotides of the invention may be attached to a solid surface such as an array (or microarray) or a magnetic bead.
  • an array comprising an oligonucleotide or a set of oligonucleotides as defined herein.
  • a method for detecting the presence or absence of a target nucleic acid in a sample comprising the steps of:
  • step (ii) contacted nucleic acids labelled in step (i) with an oligonucleotide of the invention as defined herein under conditions which allow hybridisation of the oligonucleotide to the target nucleic acid to form a duplex molecule;
  • a method for detecting the presence or absence of a target nucleic acid and one or more nucleic acid variants of the target nucleic acid in a sample comprising the steps of:
  • step (i) labelling nucleic acids in the sample;
  • step (i) contacted nucleic acids labelled in step (i) with a set of oligonucleotides of the invention as defined herein under conditions which allow hybridisation of the oligonucleotides to form duplex molecules with the target nucleic acid and the nucleic acid variants;
  • the or each oligonucleotide may be immobilised on a solid surface (for example a matrix, a planar surface, a bead such as a magnetic bead or a fluorescently encoded microparticle).
  • a solid surface for example a matrix, a planar surface, a bead such as a magnetic bead or a fluorescently encoded microparticle.
  • the invention may allow the simultaneous or sequential detection of multiple target nucleic acids and their variants.
  • multiplexed detection of multiple targets using the oligonucleotide probes of this invention is provided.
  • step (ii) contacting the sample with the oligonucleotide labelled in step (i) under conditions which allow hybridisation of the oligonucleotide to the target nucleic acid to form a duplex molecule;
  • a method for detecting the presence or absence of a target nucleic acid and one or more nucleic acid variants of the target nucleic acid in a sample comprising the steps of:
  • step (ii) contacting the sample with oligonucleotides labelled in step (i) under conditions which allow hybridisation of the oligonucleotides to form duplex molecules with the target nucleic acid and the nucleic acid variants;
  • the methods defined above may be in situ hybridisation methods, in which case the or each oligonucleotide may be labelled with a fluorescent tag, a mass tag, biotin, an enzyme, and/or a nanoparticle.
  • the method of this invention may further comprise the step of quantifying the amount of target nucleic acid or nucleic acid variant by measuring the amount of bound label.
  • the optional washing steps of the methods of the invention serve to destabilise and remove incorrectly hybridised duplexes or non-specifically adsorbed labelled nucleic acid.
  • detection methods such as surface-sensitive methods that can discriminate between the presence and absence of a duplex formed between the oligonucleotide and target may be used.
  • Suitable detection methods that do not require a wash step after hybridisation include surface plasmon resonance and evanescent wave fluorescence detection.
  • the nucleic acids in the sample may be amplified prior to hybridisation. Suitable methods of amplification include PCR, Strand Displacement Amplification (SDA) and Rolling Circle Amplification (RCA). Methods for amplification of mRNA, such as in vitro transcription (IVT), may be used.
  • SDA Strand Displacement Amplification
  • RCA Rolling Circle Amplification
  • Conditions in step (ii) of the methods of the invention may be optimised for salt concentration and/or temperature such that the or each oligonucleotide binds with enhanced specificity to its target nucleic acid.
  • appropriate selection of the salt concentration and/or specific temperature or range of temperatures for formation of duplex molecules can allow enhanced discrimination of the oligonucleotide between a target nucleic acid and one or more target nucleic acid variants.
  • hybridisation between the or each oligonucleotide and its target nucleic acid may be simulated using appropriate software to determine optimal salt concentration and/or temperature to enhance specificity. For example, software such as
  • DINAMeIt (Markham & Zuker, 2005, Nucleic Acids Res. 33(Web Server issue): W577-81) may be used.
  • DINAMeIt is a web application that exploits the open source software tool, UNAFoId (Markham, N. R. & Zuker, M. (2008) In Keith, J. M., editor, Bioinformatics,
  • kits comprising one or more oligonucleotides of the invention as defined herein or a set of oligonucleotides of the invention as defined herein.
  • the oligonucleotides of the kit may for example be provided in an unbound form (for example, in solution or as a lyophilised powder) for use in the hybridisation to targets in solution or to tissue sections or to cellular samples immobilised on a surface such as a glass slide.
  • the oligonucleotides of the kit may be provided as an array of immobilised oligonucleotides in which, for example, the oligonucleotides are immobilised at pre-determined discrete locations on an array surface.
  • an oligonucleotide (such as an oligonucleotide probe) which is hybridisable with greater affinity to a target nucleic acid than to a nucleic acid variant of the target nucleic acid, the target nucleic acid comprising a first domain and a second domain flanking a domain junction, the first domain having a sequence which is conserved with a first sequence in the nucleic acid variant, wherein the oligonucleotide has a first portion and a second portion flanking a portion junction, the first portion being complementary or complementary in part to the first domain and the second portion being complementary or complementary in part to the second domain, and wherein the first portion and the second portion have substantially the same T m .
  • the T m of the first portion is identical with, or up to 10% different from, for example up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1 % different from, the T m of the second portion.
  • first and/or second portions of the oligonucleotide may be completely complementary to the first and second domains, respectively.
  • first portion may comprise at least a first discontinuity relative to the first domain and/or the second portion may comprise at least a second discontinuity relative to the second domain, each discontinuity comprising or consisting of a sequence mismatch and/or a non-nucleotide spacer.
  • discrimination of the oligonucleotide between the target nucleic acid and its target nucleic acid variant(s) may be enhanced by using an oligonucleotide where the first and second portions have substantially the same T m , irrespective of whether or not the oligonucleotide comprises one or more discontinuities relative to the target nucleic acid.
  • Figs 1a-c illustrate three forms of oligonucleotides according to the invention, employing nucleic acid analogues and comprising abasic spacer discontinuities.
  • Fig. 1a shows an oligonucleotide comprising 2-O-methyl modified nucleotides
  • Fig. 1b shows an oligonucleotide comprising 2-O-methyl modified nucleotides interspersed with LNA nucleotides
  • Fig. 1c shows an oligonucleotide comprising DNA nucleotides interspersed with LNA nucleotides;
  • Figs 2a-b illustrate two further forms of oligonucleotides, where each discontinuity comprises a universal nucleotide.
  • Fig. 2a shows an oligonucleotide comprising 2-O-methyl modified nucleotides
  • Fig. 2b shows an oligonucleotide comprising 2-O-methyl modified nucleotides interspersed with LNA nucleotides;
  • Figs 3a-c illustrate another three forms of oligonucleotides according to the invention, employing nucleic acid analogues and comprising simple linker discontinuities.
  • Fig. 3a shows an oligonucleotide comprising 2-O-methyl modified nucleotides and with a discontinuity that comprises a 3-carbon spacer (C3 spacer)
  • Fig. 3b shows an oligonucleotide comprising 2-O-methyl modified nucleotides interspersed with LNA nucleotides and with a discontinuity that comprises a 12-atom ethylene glycol linker
  • Fig. 3c shows an oligonucleotide comprising DNA nucleotides interspersed with LNA nucleotides and with a discontinuity comprising an 18-atom ethylene glycol linker;
  • Fig. 4 illustrates a PNA oligonucleotide probe design with a discontinuity comprising an alpha amino acid linker
  • Figs 5(i)-(viii) illustrate different base options suitable for introduction into oligonucleotides of the invention
  • Fig. 6 is a schematic representation of a synthesis method for the preparation of singly labelled oligonucleotides
  • Fig. 7 (in two parts) is a schematic representation of a synthesis method for the preparation of multiply labelled oligonucleotides
  • Fig. 8 is a schematic representation of an alternative synthesis method (compared to that shown in Fig. 7) for the preparation of multiply labelled oligonucleotides;
  • Figs 9a-b show a series of isobaric mass tag sets.
  • Fig. 9a illustrates a mass tagged oligonucleotide probe based on the peptide (N) - Acetate - Valine - Piperazin-1-ylacetic acid - Alanine - Beta-alanine - (C), while
  • Fig. 9b illustrates a set of 9 isobaric tags constructed by combinatorial rearrangement of the mass modifier amino acids, beta- alanine, alanine and valine, and isotopes of acetic acid in the tags ;
  • Fig. 10 is a graph showing melting curves of exemplar Probes 1 and 2 against a target nucleic acid B2A2 and variant nucleic acids ABL and BCR.
  • the x-axis represents temperature in 0 C while the y-axis represents mole fraction;
  • Fig. 11 is a graph showing melting curves of exemplar Probes 3 and 4 against a target nucleic acid B2A2 and variant nucleic acids ABL and BCR.
  • the x-axis represents temperature in 0 C while the y-axis represents mole fraction;
  • Fig. 12 is a graph showing melting curves of exemplar Probes 5 and 6 against a target nucleic acid B2A2 and variant nucleic acids ABL and BCR.
  • the x-axis represents temperature in 0 C while the y-axis represents mole fraction;
  • Fig. 13 is a graph showing melting curves of exemplar Probes 7 and 8 against a target nucleic acid B2A2 and variant nucleic acids ABL and BCR using 50 mM salt.
  • the x-axis represents temperature in 0 C while the y-axis represents mole fraction;
  • Fig. 14 is a graph showing melting curves of exemplar Probes 7 and 8 against a target nucleic acid B2A2 and variant nucleic acids ABL and BCR using 1 M salt.
  • the x-axis represents temperature in °C while the y-axis represents mole fraction;
  • Fig. 15 is a graph showing melting curves of exemplar Probes 7 and 8 against a target nucleic acid B2A2 and variant nucleic acids ABL and BCR using 1 M salt but where the probes, target and variant nucleic acids are RNA rather than DNA.
  • the x-axis represents temperature in 0 C while the y-axis represents mole fraction;
  • Fig. 16 is a graph showing a mole fraction difference curve (dM/dT curve) that corresponds to the melting curves for PROBE1 shown in Fig. 10.
  • the x-axis represents temperature in 0 C while the y-axis represents mole fraction difference (dM/dT);
  • Fig. 17 is a graph showing a typical set of fluorescence difference curves (dF/dT curves) for the hybridization of PROBE9 to its intended target B2A2_FRET and corresponding hybridizations to ABL_FRET and BCR_FRET targets.
  • the x-axis represents temperature in 0 C while the y-axis represents fluorescence difference (dF/dT).
  • nucleic acid refers to polymers composed of naturally occurring nucleotides, polymers composed of synthetic or modified nucleotides (i.e. nucleotide analogues), or a combination of natural, synthetic and/or modified nucleotides. Furthermore, these terms encompass polymers including non-nucleotide structures such as linkers.
  • a polynucleotide that is a RNA or DNA may include naturally occurring moieties such as the naturally occurring bases and ribose or deoxyribose rings, or they may be composed of synthetic or modified moieties as described elsewhere herein.
  • the linkage between nucleotides is commonly the 3'-5' phosphate linkage, which may be a natural phosphodiester linkage, a phosphothioester linkage, and other synthetic linkages.
  • modified backbones include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates.
  • Additional linkages include phosphotriester, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate and sulfone internucleotide linkages.
  • Other polymeric linkages include 2'-5' linked analogs of these (see for example US6,503,754 and US6,506,735).
  • the monosaccharide may be modified by being, for example, a pentose or a hexose other than a ribose or a deoxyribose.
  • the monosaccharide may also be modified by substituting hydryoxyl groups with hydro or amino groups, by esterifying additional hydroxyl groups, and so on. Further backbone modifications suitable for use in the invention are described below.
  • nucleic acid and nucleotide are synonymous and interchangeable.
  • a target may be a nucleic acid target such as a chromosome or any portion thereof, an expressed nucleic acid, particularly mRNA, a recombinant nucleic acid molecule such as a plasmid or oligonucleotide, or another nucleic acid fragment.
  • a nucleic acid target may be naturally occurring or synthetic or a combination of the two.
  • the target may be of any length provided that it is able to complement a probe of this invention.
  • the nucleic acid target may be DNA or RNA.
  • the target is DNA, it is understood that the DNA is hybridisable in the methods of the invention.
  • the DNA may be provided for use in the methods in a denatured or single-stranded form capable of hybridising to a single-stranded oligonucleotide probe.
  • the term “complementary” refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule.
  • the term “complementary” and similar words relate to the ability of a first nucleic acid base in one strand of a nucleic acid or oligonucleotide to interact specifically only with a particular second nucleic acid base in a second strand of a nucleic acid or oligonucleotide.
  • a and T or U interact with each other
  • G and C interact with each other.
  • hybridise refers to a process of forming a nucleic acid or oligonucleotide duplex by causing strands with complementary sequences to interact with each other. The interaction occurs by virtue of complementary bases on each of the strands specifically interacting to form a pair. The ability of strands to hybridise to each other depends on a variety of conditions, as known in the art. Nucleic acid or oligonucleotide strands may be stated to hybridise with each other when a sufficient number of corresponding positions in each strand are occupied by nucleotides that can interact with each other. For reasons described herein, the sequences of strands forming a duplex need not be 100% complementary (or identical) to each other to be specifically hybridisable.
  • an oligonucleotide sequence which is "conserved" with another oligonucleotide sequence indicates that the sequences may share at least 60% sequence identity with each other over a given domain or portion or region of the sequences, for example up to 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99 or 100% sequence identity.
  • Sequence identity between oligonucleotide sequences can be determined by comparing an alignment of the sequences. When an equivalent position in the compared sequences is occupied by the same base, then the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical bases at positions shared by the compared sequences.
  • comparing sequences in one aspect no gaps are introduced during the determination of sequence identity. In another aspect, optimal alignments may require gaps to be introduced into one or more of the sequences to take into consideration possible insertions and deletions in the sequences.
  • Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. Calculation of maximum percent identity involves the production of an optimal alignment, taking into consideration gap penalties.
  • Suitable computer programs for carrying out sequence comparisons are widely available in the commercial and public sector. Examples include the MatGat program (Campanella et al., 2003, BMC Bioinformatics 4: 29), the Gap program (Needleman & Wunsch, 1970, J.
  • the FASTA program can alternatively be accessed publically from the European Bioinformatics Institute (http://www.ebi.ac.uk/fasta) and the University of Virginia (http://fasta.biotech.virginia.edu/fasta_www /cgi). FASTA may be used to search a sequence database with a given sequence or to compare two given sequences (see http://fasta.bioch. Virginia. edu/fasta_www/cgi/search_frm2. cgi). Typically, default parameters set by the computer programs may be used when comparing sequences. The default parameters may change depending on the type and length of sequences being compared.
  • oligonucleotide sequences which are conserved with each other may hybridise under high stringency conditions.
  • high stringency conditions may involve a wash in a 0.0165-0.0330 M NaCI buffer solution at a temperature of about 5-10 0 C below the calculated or actual T m of the probe (for example, about 65°C).
  • the buffer solution may, for example, be SSC buffer (0.15M NaCI and 0.015M tri-sodium citrate), with the high stringency wash taking place in 0.1 x SSC buffer. Steps involved in hybridisation of nucleic acid sequences have been described for example in Sambrook et al. (1989; Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor).
  • splice junction refers to a site in mRNA where splicing to join an intron and an exon (or to join two exons in the case of trans-splicing) has taken place.
  • the oligonucleotide need not correspond to the full length of the target.
  • the oligonucleotide may include sequences which are not generally complementary to the target.
  • a "discontinuity" is herein defined as an interruption in the chemical sequence of nucleotides with one or more non-base-pairing nucleotides ("mismatches") and/or non-nucleotide "spacers”.
  • Oligonucleotides of the invention may be produced by chemical synthesis using standard oligonucleotide synthesis methods known in the art. Methods may be purely synthetic, for example, the cyanoethyl phosphoramidite method (Beaucage & Caruthers, 1981, Tetrahedron Lett. 22: 1859-1862; McBride & Caruthers, Tetrahedron Lett. 24: 245-248). Synthetic methods useful for making oligonucleotides are also described by lkuta et al. (1984, Ann. Rev. Biochem. 53: 323-356) (phosphotriester and phosphite-triester methods), and Narang et al. (1980, Methods Enzymol. 65: 610-620) (phosphotriester method). PNA molecules may be made using known methods such as those described by Nielsen et al. (1994, Bioconjug. Chem. 5: 3-7).
  • H-phosphonate method for oligonucleotide synthesis may also be used. This method was first reported by Hale et al. (1957, J. Chem. Soc. 3291) and revisited later by Sekine and Hata (1975, Tetrahedron Lett. 16: 1711), Sekine et al. (1979, Tetrahedron Lett. 20: 1145), Garegg et al. (1985, Chemica Scripta 25: 280), and Garegg et al. (1986, Chemica Scripta 26: 59).
  • the H-phosphonate method involves condensing the 5' hydroxyl group of the nascent oligonucleotide with a nucleoside having a 3' phosphonate moiety. Once the entire chain is constructed, the phosphite diester linkages are oxidized with t-butyl hydroperoxide or iodine to yield the corresponding phosphotriester. See, for example, Froehler (1993, "Oligodeoxynucleotide Synthesis", Methods MoI. Biol. Vol. 20, Protocols for Oligonucleotides and Analogs, p. 63-80, S.
  • the H-phosphonate method allows the production of modified backbones such as a phosphorothioate backbone (Stawinski & Str ⁇ mberg, 2005, Methods MoI Biol. 288: 81-100) which is suitable for the present invention.
  • oligonucleotides of this invention comprise different portions or regions, smaller subsequences of the oligonucleotides may be synthesised and then assembled by ligation (Borodina et al., 2003, Anal Biochem. 318(2): 309-313).
  • Artificial mismatches may comprise natural nucleotides that are known to be non- complementary to the base at the appropriate position in the target sequence.
  • universal nucleoside analogues such as 1-(2'-Deoxy-.beta.-D- ribofuranosyl)-3-nitropyrrole, which maximizes stacking interactions while minimizing hydrogen-bonding interactions without sterically disrupting a DNA duplex (Nichols et al., 1994, Nature 369: 492; and Bergstrom et al., 1995, J.A.C.S. 117: 1201).
  • analogues 1-(2'-Deoxy-beta-D-ribofuranosyl)-5-nitroindole and 1-(2'-deoxy-beta-D- ribofuranosyl)-4-nitroimidazole may be used.
  • nitropyrrole and nitroindole analogues are reported to be most favorable as universal nucleotides as they show the least discrimination for base pairing with natural nucleotides (Bergstrom et al., 1997, Nucleic Acids Res. 25(10): 1935-1942).
  • Abasic spacers may also be used such as the "D-spacer" in which deoxyribose residues without a base are introduced into the sequence of the probe oligonucleotide (see for example Takeshita et al., 1987, J Biol Chem. 262(21): 10171-10179).
  • the abasic spacer and the universal nucleotides are available as deoxyribose nucleotides but other sugar modifications, in particular 2'-modified sugars, are also envisaged.
  • a 3-carbon spacer may be introduced into oligonucleotides as a discontinuity by employing a 'C3 phosphoramidite spacer'.
  • a C3 spacer will separate a pair of phosphodiester groups by approximately the same distance as ribose in terms of the backbone configuration.
  • Longer discontinuities may be introduced, for example as 9-atom, 12-atom and 18-atom spacers which are commercially available as phosphoramidites (for example from Glen Research Corporation, Sterling, Virginia, USA).
  • FMOC amino acids may be used.
  • Preferred spacers include glycine, alanine, beta-alanine, serine and lysine.
  • Two alpha amino acids together will have approximately the same intemucleotide distance as a single PNA nucleotide. Longer spacings can be introduced using more alpha amino acids or by introducing longer amino acids such as amino hexanoic acid.
  • the oligonucleotides of the invention may incorporate "artificial mismatches", so the overall binding affinity of the oligonucleotides is reduced compared to unmodified probes. It may therefore be useful to use nucleic acid analogues with enhanced binding affinity compared to natural phosphodiester deoxyribosenucleic acids. It is known that RNA analogues with certain modifications at the 2' position of the ribose ring show enhanced binding affinity for RNA targets compared to corresponding DNA/RNA hybrids (see Cummins et al., 1995, Nucleic Acids Res. 23(11): 2019-24).
  • RNA analogues also show reduced binding affinity for DNA compared to DNA/DNA hybrids (Tsourkas et al., 2003, Nucleic Acids Res. 31 (6): 5168-74).
  • the ability to bind preferentially to RNA over DNA with enhanced melting temperature makes 2'-modified analogues particularly useful for in situ hybridisation applications for detection of alternatively spliced RNA in a background of genomic DNA.
  • 2'-O-methyl analogues in particular are readily available as phosphoramidite monomers for automated synthesis and are suitable for use with this invention. Additionally or alternatively, 2'-fluoro-modified analogues may be used.
  • nucleic acid analogues for use with this invention are 'bridged' analogues such as locked nucleic acids ("LNA”; Thomsen et al., 2005, RNA 11 (11): 1745-8) and 2'-4'- BNA(NC) (Rahman et al., 2008, J Am Chem Soc. 130(14): 4886-96).
  • Bridged nucleic acid analogues show enhanced binding affinity for RNA compared with their natural nucleic acid counterparts, and are thus suitable for in situ hybridisation applications.
  • Bridged analogues also show enhanced binding affinity for DNA compared with their natural nucleic acid counterparts, and are therefore useful for detection of chromosomal targets such as chromosomal translocations and for the detection of labelled cDNAs.
  • LNA monomers may be introduced into 2'-O- methyl oligonucleotide sequences to enhance binding affinity of the resultant oligonucleotide (Kierzek et al., 2005, Nucleic Acids Res. 33(16): 5082-93).
  • the LNA monomers may be positioned every second base but in one embodiment not at the 5' end of an oligonucleotide.
  • PNA is another analogue for use with this invention (Nielsen et al., 1994, Bioconjug Chem. 5(1): 3-7). PNA has enhanced binding affinity for both DNA and RNA targets compared to DNA oligonucleotides. PNA is less soluble than other DNA analogues and it is currently difficult to produce usable PNA oligonucleotides with a length greater than 20 bases. Hence PNA oligonucleotides may be shorter than oligonucleotides made with sugar/phosphate backbones.
  • the invention encompasses oligonucleotides comprising lengths of PNA and DNA (see Uhlmann, 1998, Biol Chem. 379(8-9): 1045-52). These "mixed" oligonucleotides may be longer than PNA-only probes. Linkages between PNA and DNA can be useful points for introducing discontinuities in such mixed oligonucleotides.
  • T m For several applications of the oligonucleotides, it may be useful to determine the T m . Higher T m values correspond to more stable duplexes.
  • the stability of DNA duplexes can be calculated using known methods for prediction of melting temperatures (Breslauer et al., 1986, PNAS USA 83(11): 3746-3750; Lesnick & Freier, 1995, Biochemistry 34: 10807- 10815, 1995; McGraw et al., 1990, Biotechniques 8: 674-678, 1990; and Rychlik et al., 1990, Nucleic Acids Res. 18: 6409-6412).
  • Exemplar oligonucleotides designs Exemplar oligonucleotides designs
  • Figs 1a-c illustrate three different forms of oligonucleotides according to the invention.
  • Fig. 1a shows an oligonucleotide comprising 2-O-methyl modified nucleotides.
  • the oligonucleotide comprises two adjacent portions complementary in part to adjacent target nucleic acid domains.
  • the two oligonucleotides portions are symmetrical about a portion junction ("PJ"; shown as a dashed line), which separates the portions in the oligonucleotide.
  • the portion junction thus corresponds with a domain junction (for example, a splice junction or a gene fusion junction) in the target nucleic acid.
  • Each of the oligonucleotide portions has a sequence of 21 bases.
  • the oligonucleotide sequence then comprises a discontinuity (at a position corresponding to base 11 of the target sequence downstream from the domain junction) formed from an abasic deoxyribose sugar residue.
  • ribose may be used instead of deoxyribose.
  • Further alternatives are an abasic 2'-O-methylribose sugar or an abasic bridged sugar.
  • the discontinuity in the oligonucleotide shown in Fig. 1a is at a position corresponding to base 11 downstream from the domain junction in the target sequence but in this oligonucleotide and in other embodiments of the invention (for example, as described herein), the discontinuity could be located closer to the portion junction if desired.
  • the overall length of the oligonucleotide can also be varied extensively while retaining enhanced specificity. In addition, two or more spacers could be introduced to provide a longer discontinuity. The flanking regions of complementary sequence would then start at a correspondingly increased distance from the domain junction.
  • Fig. 1b shows an oligonucleotide comprising 2-O-methyl modified nucleotides interspersed with LNA nucleotides.
  • the oligonucleotide differs in structure from that shown in Fig. 1a in that the LNA nucleotides in Fig. 1 b are positioned every second base, avoiding the 5' end.
  • oligonucleotide in Fig. 1b is symmetrical about the portion junction and as such the alternating pattern of LNA insertions is not continued across the junction (which is flanked instead of two adjacent 2-O-methyl nucleotides).
  • continuous runs of alternating 2'-O-methyl and LNA nucleotides may be used, for example an oligonucleotide having the sequence: 5'-(OMe-LNA) 5 -dSpacer-(OMe-LNA) 10 -dSpacer- (OMe-LNA) 5 -3 ⁇
  • Fig. 1c shows an oligonucleotide comprising DNA nucleotides interspersed with LNA nucleotides.
  • the oligonucleotide differs in structure to that shown in Fig. 1b in that LNA nucleotides are positioned every third base in Fig. 1c.
  • LNA nucleotides may be positioned differently in the oligonucleotide sequence.
  • An alternative oligonucleotide design may for example have the following structure: 5'-LNA-(DNA-DNA-LNA) 3 -dSpacer-(DNA-DNA- LNA) 6 -dSpacer-(LNA-DNA-DNA) 3 -LNA-3'.
  • Figs 2a-b illustrate two further forms of oligonucleotides according to the invention.
  • Fig. 2a shows an oligonucleotide comprising 2-O-methyl modified nucleotides.
  • the oligonucleotide differs in structure from that shown in Fig. 1a in that the discontinuities in the oligonucleotide of Fig. 2a are artificial mismatches formed by the universal base nitropyrrole.
  • universal nucleotides such as nitroindole or nitroimidazole could be used to create artificial mismatches.
  • Fig. 2b shows an oligonucleotide comprising 2-O-methyl modified nucleotides interspersed with LNA nucleotides.
  • the oligonucleotide differs in structure from that shown in Fig. 1b in that the discontinuities in Fig. 2b comprise artificial mismatches using the universal base nitroindole.
  • universal nucleotides such as nitropyrrole or nitroimidazole could be used to create artificial mismatches.
  • Figs 3a-c illustrate three further forms of oligonucleotides according to the invention.
  • Fig. 3a shows an oligonucleotide comprising 2-O-methyl modified nucleotides.
  • the oligonucleotide differs in structure from that shown in Fig. 1a in that the discontinuity as shown in Fig. 3a comprises a 3-carbon spacer (C3 spacer).
  • Fig. 3b shows an oligonucleotide comprising 2-O-methyl modified nucleotides interspersed with LNA nucleotides.
  • the oligonucleotide differs in structure from that shown in Fig. 1 b in that the discontinuity as shown in Fig. 3b comprises a 12-atom ethylene glycol linker.
  • the 12-atom ethylene glycol linker is fairly flexible, so the second ten bases of sequence in each portion of the oligonucleotide could be complementary to target domains further from the domain junction than in the Fig. 1b oligonucleotide.
  • base 12 of the oligonucleotide shown in Fig. 3b may be complementary to base 13 or 14 from the domain junction in the target sequence.
  • more than one spacer may be used if a larger interruption in the continuity of the oligonucleotide sequence with respect to the target sequence is required.
  • Other spacers such as a C3 spacer may additionally be incorporated.
  • the LNA nucleotides in the Fig. 3b oligonucleotide are inserted every second base, avoiding the 5' terminus.
  • continuous runs of alternating 2'-O-methyl and LNA nucleotides may also be used, for example an oligonucleotide having the sequence: 5'-(OMe-LNA) 5 -Linker-(OMe-LNA) 10 - Linker-(OMe-LNA) 5 -3 ⁇
  • Fig. 3c shows an oligonucleotide comprising DNA nucleotides interspersed with LNA nucleotides.
  • the oligonucleotide differs in structure from the oligonucleotide shown in Fig. 1c in that the discontinuity as shown in Fig. 3c is an 18-atom ethylene glycol linker.
  • the second ten bases of sequence in each portion of the oligonucleotide could be complementary to target sequences further from the portion junction than in the Fig. 1c oligonucleotide, if a greater interruption in the continuity of the oligonucleotide sequence with respect to the target sequence is required.
  • the alternative design described with respect to Fig. 1c is also suitable when using an 18-atom ethylene glycol linker to produce a mismatch.
  • Fig. 4 illustrates a PNA-containing oligonucleotide according to the invention.
  • the PNA probe comprises two portions of sequence complementary to adjacent target nucleic acid domains (such as domains adjacent a splice junction or a gene fusion junction). The two portions are shown on either side of a dashed line marking the portion junction which separates the junctions in the oligonucleotide. The dashed line accordingly corresponds with the domain junction which separates domains in the target.
  • Each of the junction sequences consist of eight bases of sequence.
  • oligonucleotides in Figs 1-4 are shown without labels. However, labels may be incorporated into each of the oligonucleotide, for example at locations as discussed elsewhere.
  • Figs 5(i)-(viii) illustrate various bases that can be incorporated into the oligonucleotides of the invention.
  • Figs 5(i) to 5(iv) illustrate the standard "Watson-Crick" bases adenine, thymine, cytosine and guanine, respectively.
  • Several modifications may be made to these standard bases. Such modifications may normalise or increase base pairing energies.
  • Non-limiting examples of modified bases are shown in Figs 5(v) - 5(viii).
  • Fig. 5(v) shows a cytosine analogue 5-propynyl cytosine which pairs more stably with guanine than standard cytosine (Wagner et al., 1993, Science. 260(5113): 1510-3).
  • Fig. 5(vi) shows a thymine analogue 5-propynyI uracil which pairs more stably with adenine than standard thymine.
  • Fig. 5(vii) shows an adenine analogue 2,6-diaminopurine which forms three hydrogen bonds with thymine, rather than two formed with standard adenine.
  • the analogue therefore pairs more stably with thymine than standard adenine (Gaffney et al., 1984, Tetrahedron 40: 3-13).
  • Fig. 5(viii) shows a cytosine analogue 5-methyl cytosine which pairs more stably with guanine than standard cytosine.
  • 5-methyl cytosine base substitutions when combined with 2'-O-methoxyethyl sugar modifications are particularly stable (see US6, 503,754 and US6,506,735).
  • modified bases for use in the invention include 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2- thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioaIkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7
  • Further modified bases include tricyclic pyrimidines such as phenoxazine cytidine (1 H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1-pyrimido[5,4-b][1 ,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g.
  • Modified bases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2- pyridone.
  • Oligonucleotides may be readily modified in the phosphate moiety. Under certain conditions, such as low salt concentration, analogues such as methylphosphonates, (Miller et al., 1983, Nucleic Acids Res. 11(18): 6225-42), triesters (Miller et al., 1974, Biochemistry 13(24): 4887-96) and phosphoramidates have been shown to increase duplex stability.
  • analogues such as methylphosphonates, (Miller et al., 1983, Nucleic Acids Res. 11(18): 6225-42), triesters (Miller et al., 1974, Biochemistry 13(24): 4887-96) and phosphoramidates have been shown to increase duplex stability.
  • a known method for synthesis of methylphosphonates produces a mixture of stereoisomers with the methylated phosphate groups acting as chiral centres.
  • N3'->P5' phosphoramidates are also advantageous as they have a neutral backbone and are simple to produce in a specific stereochemical conformation (Gryaznov et al., 1995, PNAS USA 92(13): 5798-802).
  • phosphate modifications include phosphorothioates (Efimov et al., 1995, Nucleic Acids Res. 23(20): 4029-33), phosphorodithioates (Capaldi et al., 2000, Nucleic Acids Res. 28(9): E40) and boranophosphates (Shimizu et al., 2006, J Org Chem. 71 (11): 4262- 9), each of which increase the resistance of oligonucleotides to exonuclease degradation, lsosteric replacement of phosphorus by sulphur gives nuclease resistant oligonucleotides (Huang & Benner, 2002, J Org Chem. 67(12): 3996-4013). Replacement by carbon at either phosphorus or linking oxygen is also a further possibility (Milligan et al., 1993, J. Med. Chem. 36(14): 1923 - 1937).
  • mass tags can be used with this invention.
  • suitable mass tags are disclosed in WO 97/27327, WO 97/27325, WO 97/27331 and WO 03/025576.
  • These publications disclose tags that comprise polyamide compounds, essentially peptides or peptide-like tags, which means that these tags can be prepared using a number of peptide synthesis methods that are well known in the art (see for example Jones, 1991 , "The chemical synthesis of peptides", Oxford University Press; Fields & Noble, 1990, lnt J Pept Protein Res 35(3): 161-214; Albericio, 2000, Biopolymers 55(2): 123-139).
  • peptide and peptide-like tags enable coupling of these tags to oligonucleotides using a variety of peptide conjugation techniques that are known in the art.
  • Methods for coupling peptides to oligonucleotides "on column” via 5' amine functionalities are disclosed in Zaramella et al. (2004, J Am Chem Soc 126(43): 14029 - 14035).
  • Methods for conjugating peptides to oligonucleotides via thiol groups at the termini of the oligonucleotides are disclosed in Arar et al. (1995, Bioconjug Chem. 6(5): 573-577).
  • Oligonucleotides can be coupled to peptides with terminal cysteine residues as disclosed in Wei et al. (1994, Bioconjug Chem. 5(5): 468-74).
  • peptide tags can be synthesised on Controlled Pore Glass (CPG) beads of the kind used for DNA synthesis.
  • CPG Controlled Pore Glass
  • an oligonucleotide can be synthesized directly on the peptide (Haralambidis et al., 1990, Nucleic Acids Res. 18(3): 493-499).
  • the peptide should be linked to the resin with a suitable linker that will be cleaved under conditions normally used for cleaving and deprotecting oligonucleotides.
  • HMBA 4-Hydroxymethylbenzoic acid linker
  • tagged oligonucleotide conjugates according to the invention are prepared by initially synthesizing the peptide tag component of the conjugate on a CPG resin that is compatible with automated oligonucleotide synthesis.
  • step (1) of the method in Fig. 6 aminopropyl CPG is initially coupled to the aforementioned HMBA linker. Peptide synthesis is initiated from this linker.
  • step (2) an O-trityl protected FMOC serine residue is coupled to the HMBA linker.
  • step (3) multiple cycles of standard FMOC synthesis takes place, i.e. the desired peptide tag is synthesised using commercially available FMOC- amino acids.
  • an FMOC protected photocleavable linker (4-[4-(1-(FMOC- amino)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid available from IRIS Biotech GmbH, Tiredwitz, Germany) is introduced followed by FMOC-alanine, followed by 1-Fmoc- piperidin-4-ylacetic acid (Sigma Aldrich, UK), followed again by FMOC-alanine, which is finally acetylated to block the terminal amino group so that it does not interfere in subsequent oligonucleotide synthesis.
  • the CPG resin is transferred from the automated peptide synthesizer reaction vessel or column into a column or vessel suitable for use in an automated DNA synthesizer.
  • step (4) the trityl protection group is removed using standard deprotection conditions in the DNA synthesizer, typically trichloroacetic acid or dichloroacetic acid in dichloromethane.
  • step (5) an optional Dimethoxtrityl (DMTr) protected linker (or “spacer”) is introduced to reduce steric hindrance from the solid support and the peptide (Spacer Phosphoramidite 9 from Glen Research, Stirling, VA, USA).
  • DMTr Dimethoxtrityl
  • step (6) multiple cycles of standard automated phosphoramidite DNA synthesis are carried out to generate the desired DNA sequence of the oligonucleotide, linked to the peptide.
  • step (7) the side chain deprotection groups are cleaved along with the HMBA linker releasing the deprotected conjugate into solution (also referred to as an NH 3 /H 2 O step).
  • the released conjugate is then purified by high performance liquid chromatography (HPLC), gel filtration, gel electrophoresis or other standard purification techniques known in the art.
  • HPLC high performance liquid chromatography
  • Fig. 6 introduces the peptide tag at the 3 J end of the oligonucleotide.
  • reverse' synthesis (5' to 3') is also possible (see Claeboe et al., 2003, Nucleic Acids Res. 31(19): 5685-91).
  • Reverse synthesis phosphoramidites used with the method in Fig. 6 (and also the methods in Figs 7 and 8) would yield oligonucleotides modified at the 5' terminus.
  • Phosphoramidite monomers for 5' to 3' synthesis are commercially available, for example from Glen Research Corporation (Sterling, Virginia, USA).
  • FMOC derivatives Many amino acids are known in the art and not all are available as FMOC derivatives. It is, however, possible for one of ordinary skill in the art to prepare FMOC protected derivatives of unprotected amino acids for use with the present invention using standard methods (for example, Fields & Noble, 1990, lnt J Pept Protein Res. 35(3): 161-214).
  • PNA peptide conjugates can be readily synthesized as both PNA and peptides can be synthesized by FMOC chemistry. Examples of such oligonucleotides are disclosed in Thompson et al. (2007, Nucleic Acids Res. 35(4): e28).
  • mass tags can be incorporated into the oligonucleotide through conjugation to thymidine analogues, for example, as disclosed in Brown et al. (2001 , Tetrahedron Lett. 42: 2587-2592). In that publication, a thymidine analogue is described with a linker coupled to the purine ring of the thymidine.
  • This thymidine analogue has a hydroxyl group protected with an FMOC group on the end of the linker that can be made available after the nucleotide has been coupled into an oligonucleotide during automated oligonucleotide synthesis to allow a phosphoramidite modified tag to be incorporated into an oligonucleotide. Since this analogue can be incorporated within the chain, multiple linkers and hence tags can be couple to the oligonucleotide. Alternatively, branched peptides can be synthesized incorporating multiple tag peptide sequences. The branched peptides can be synthesized on CPG, with subsequent synthesis of the oligonucleotide (see Haralambidis et al., 1990, Nucleic Acids Res. 18(3): 501-505).
  • tag oligonucleotide conjugates according to this invention are prepared by initially synthesizing the peptide tag component of the conjugate on a controlled pore glass (CPG) resin that is compatible with automated oligonucleotide synthesis.
  • CPG controlled pore glass
  • step (1) of Fig. 7 aminopropyl CPG is initially coupled to the aforementioned HMBA linker.
  • a spacer could be introduced between the aminopropyl glass and the linker of desired or a CPG resin that has a longer linker could be employed.
  • Peptide synthesis is initiated from the HMBA linker.
  • an N-alpha-Fmoc-N- epsilon-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl-L-lysine ( ⁇ -FMOC- ⁇ -Dde-Lys) residue is coupled to the HMBA linker.
  • One or more spacers such as beta-alanine could be introduced between the lysine and the HMBA linker if desired to reduce potential steric interference from the resin.
  • the Dde protection group is removable under 2% hydrazine in Dimethylformamide (DMF) (Bycroft et al., 1993, J. Chem. So ⁇ , Chem. Commun.
  • step (3) a further ⁇ - FMOC- ⁇ -Dde-Lys residue is coupled, followed by an ⁇ -FMOC-O-trityl protected serine residue.
  • the final FMOC group is removed and the serine amino group is acetylated to prevent further reaction.
  • step (4) the Dde protection groups are removed.
  • step (5) multiple cycles of FMOC peptide synthesis are conducted, i.e. the desired peptide tag is synthesized from both of the free epsilon amino groups exposed by the removal of the Dde groups using commercially available FMOC-amino acids.
  • an FMOC protected photocleavable linker (4-[4-(1-(FMOCamino)ethyl)-2-methoxy-5- nitrophenoxy)butanoic acid available from IRIS Biotech GmbH, Tiredwitz, Germany) is introduced followed by FMOC-alanine, followed by 1-Fmoc-piperidin-4-ylacetic acid (Sigma Aldrich, UK), followed again by FMOC-alanine, which is finally acetylated to block the terminal amino group so that it does not interfere in subsequent oligonucleotide synthesis.
  • step (5) and step (6) the CPG resin is transferred from the automated peptide synthesizer reaction vessel or column into a column or vessel suitable for use in an automated DNA synthesizer.
  • step (6) of Fig. 7 the trityl protection group is removed using standard deprotection conditions in the DNA synthesizer, typically trichloroacetic acid or dichloroacetic acid in dichloromethane.
  • step (7) an optional Dimethoxtrityl (DMTr) protected linker (or "spacer”) is introduced to reduce steric hindrance from the solid support and the peptide (here, Spacer Phosphoramidite 9 from Glen Research, Stirling, VA, USA).
  • DMTr Dimethoxtrityl
  • spacer is introduced to reduce steric hindrance from the solid support and the peptide (here, Spacer Phosphoramidite 9 from Glen Research, Stirling, VA, USA).
  • step (8) multiple cycles of standard automated DNA synthesis are carried out to generate the desired DNA sequence linked to the peptide.
  • step 9 the side chain deprotection groups are cleaved along with the HMBA linker releasing the deprotected conjugate into solution (also referred to as an NH 3 /H 2 O step).
  • the released conjugate would then be purified by high performance liquid chromatography, gel filtration, gel electrophoresis or other standard techniques known in the art.
  • the number of peptides linked to a single oligonucleotide can be varied by varying the number of ⁇ -FMOC- ⁇ -Dde-Lysine residues introduced into the peptide in the first steps of the synthesis shown in Fig. 7. If only a single tag is desired then only one ⁇ -FMOC- ⁇ -Dde- Lysine residue need be coupled, but if 5 tags were desired this can be achieved by coupling 5 ⁇ -FMOC- ⁇ -Dde-Lysine residues.
  • step (7) of Fig. 7 could take place prior to removal of the Dde groups. This may be desirable if many peptides or if large peptides are to be coupled to a single oligonucleotide causing steric hindrance that might reduce the efficiency of the introduction the linker.
  • ⁇ -Dde- ⁇ -FMOC-Lysine can be employed in place of ⁇ -FMOC- ⁇ -Dde-Lysine.
  • the O-trityl serine needed to initiate oligonucleotide synthesis can be coupled at the epsilon amino while the peptide tag is synthesized at the alpha position after removal of the Dde group.
  • the alpha- Dde group can be removed prior to the epsilon-FMOC group, and the O-trityl serine can be coupled at this position.
  • One or more optional spacers, such as beta-alanine residues can be introduced before coupling of the O-trityl protected serine, if desired.
  • tag oligonucleotide conjugates are prepared by initially synthesizing the peptide tag component of the conjugate on a CPG resin that is compatible with automated oligonucleotide synthesis.
  • aminopropyl CPG is initially coupled to an HMBA linker.
  • Peptide synthesis is initiated from this linker.
  • an ⁇ -FMOC-O-trityl protected serine residue is coupled to the HMBA linker.
  • One or more spacers such as beta-alanine could be introduced between the serine residue and the HMBA linker if desired to reduce potential steric interference from the resin.
  • step (3) an ⁇ -FMOC- ⁇ -FMOC-protected Lysine residue is introduced and coupled to the serine residue.
  • step (4) multiple cycles of standard FMOC peptide synthesis are conducted, i.e. the desired peptide tag is synthesized from both of the free amino groups exposed by the removal of the FMOC groups on the lysine amino groups.
  • the peptide tags are synthesized using commercially available FMOC-amino acids.
  • an FMOC protected photocleavable linker (4-[4-(1-(FMOC-amino)ethyl)-2-methoxy-5- nitrophenoxy)butanoic acid available from IRIS Biotech GmbH, Tiredwitz, Germany) is introduced followed by FMOC-alanine, followed by 1-Fmoc-piperidin-4-ylacetic acid (Sigma Aldrich, UK), followed again by FMOC-alanine, which is finally acetylated to block the terminal amino group so that it does not interfere in subsequent oligonucleotide synthesis.
  • step (4) and step (5) the CPG resin is transferred from the automated peptide synthesizer reaction vessel or column into a column or vessel suitable for use in an automated DNA synthesizer.
  • step (5) the trityl protection group is removed using standard deprotection conditions in the DNA synthesizer, typically trichloroacetic acid or dichloroacetic acid in dichloromethane.
  • step (6) an optional Dimethoxtrityl protected linker (or "spacer") is introduced to reduce steric hindrance from the solid support and the peptide (Spacer Phosphoramidite 9 from Glen Research, Stirling, VA, USA). A wide variety of similar spacers is known in the art and could be substituted for the linker shown, if desired.
  • step (7) multiple cycles of standard automated DNA synthesis are carried out to generate the desired DNA sequence linked to the peptide.
  • step (8) the side chain deprotection groups are cleaved along with the HMBA linker releasing the deprotected conjugate into solution (also referred to as an NH 3 /H 2 O step).
  • the released conjugate would then be purified by HPLC, gel filtration, gel electrophoresis or other standard purification techniques known in the art.
  • the number of peptides linked to a single oligonucleotide can be varied by varying the number of ⁇ -
  • FMOC- ⁇ -FMOC-Lysine residues introduced into the peptide in the first steps of the synthesis. After the introduction of the first ⁇ -FMOC- ⁇ -FMOC-Lysine, removal of the
  • FMOC groups will expose two amino groups. This means in the next cycle of synthesis 2 further ⁇ -FMOC- ⁇ -FMOC-Lysine groups could be introduced and four in the next cycle if desired. Three cycles of coupling of ⁇ -FMOC- ⁇ -FMOC-Lysine would make 8 amino groups available for the synthesis of peptide tags.
  • the FMOC protected linker 4-[4-(1-(FMOCamino)ethyl)-2-methoxy-5- nitrophenoxy)butanoic acid is commercially available from Iris Biotech GmbH (Marktredwitz, Germany) and from Advanced ChemTech, lnc (Kentucky, USA). This can be readily incorporated into peptides during conventional FMOC synthesis.
  • Branched peptides (or other polyamides) with one or more attached oligonucleotides, and comprising a plurality of cleavable branches, form another aspect of the invention.
  • Non-natural amino acids may be used in this invention, including FMOC-Piperazin-1- ylacetate, 1-Fmoc-piperidin-4-ylacetic acid, N,N-Dimethyl glycine, ⁇ -Dimethylamino-DL- alanine (all commercially available from Sigma Aldrich, UK).
  • exemplar peptide sequences of the invention include:
  • Cysteic Acid - Alanine - Aspartic acid - Alanine [SEQ ID NO: 5] Cysteic Acid - Alanine - Aspartic acid - Valine [SEQ ID NO: 6] Cysteic Acid - Valine - Aspartic acid - Alanine [SEQ ID NO: 7] Cysteic Acid - Valine - Aspartic acid - Valine [SEQ ID NO: 8].
  • the collision cleavable linker (Piperazin-1 - ylacetic acid) is also a charge-carrying group due to the presence of the tertiary amino group in the piperazine ring.
  • the charge-carrying groups are separate entities, i.e. N,N-Dimethyl glycine and cysteic acid, respectively.
  • the charge-carrying groups could act as the mass modifier as well, allowing the adjacent mass modifier amino acid group to be removed resulting in a smaller tag if that were desirable.
  • the proline residue can be substituted with piperidin-4-ylacetic acid.
  • the N,N-Dimethyl glycine can be replaced with any easily protonated positive charge-carrying group including the natural amino acids lysine and histidine, tertiary amino group containing molecules such as ⁇ -Dimethylamino-alanine. Secondary amino containing groups may also be introduced such as nipecotic acid. Pyridine containing compounds such as nicotinic acid are also appropriate.
  • Fig. 9a illustrates a mass tagged oligonucleotide probe based on the peptide (N) - Acetate - Valine - Piperazin-1-ylacetic acid - Alanine - Beta-alanine - (C) and its tag product following photocleavage ("P").
  • This structure is another variant of the series-1 peptides shown above.
  • Fig. 9b illustrates a set of 9 isobaric tags constructed by combinatorial rearrangement of the mass modifier amino acids, beta-alanine, alanine and valine, and isotopes of acetic acid in the tags. All of the isotope-substituted reagents are commercially available (for example, from Cambridge Isotope Laboratories, Inc; Andover, MA, USA).
  • tags can be constructed by changing the amino acids and other entities used as mass modifiers.
  • the acetate group blocking the N-terminal amino group of the peptide tags could be replaced with any carboxylic acid, e.g. propionic or butyric acids, which can be obtained with isotope substitutions (for example, from
  • the mass modifiers comprise individual amino acids of valine or alanine or their isotopes.
  • other amino acids can be used for this purpose.
  • more than one amino acid can be inserted into the sequence to act as mass modifiers.
  • beta-amino acids can also be used to act as mass modifiers as these do not readily support formation of oxazolone structures and thus do not cleave as easily as alpha amino acids at any given collision energy.
  • Fig. 9b the combination of alanine and beta-alanine is used to act as a mass modifier.
  • the combination of valine and acetate is also used to act as a single mass modifier in Fig. 9b.
  • an acid cleavable linker can be used. Since most MALDI matrix materials are acidic, addition of the matrix will effect cleavage of the mass tags.
  • a simple method for introducing an acid labile group is to include a P3'-N5' phosphoramidate at the 5' terminus of the oligonucleotide adjacent to the mass tag (Shchepinov et al., 2001 , Nucleic Acids Res. 29(18): 3864-72).
  • the entire probe label complex can be desorbed, and cleavage of the tags can take place by collision using Post Source Decay in a Time-Of-Flight mass spectrometer or in the mass analyzer of an ion trap instrument or in a collision cell in alternative geometries that are used with MALDI, such as the Q-TOF geometry.
  • Suitable PNA probes with a collision cleavable linker between the tag peptide and a PNA probe have been described previously (see Thompson et al., 2007, Nucleic Acids Res. 35(4): e28).
  • fluorescent tags suitable for labelling the oligonucleotides of the invention are known in the art. Fluorescein is widely used as a fluorescent reagent for oligonucleotide labelling. In addition, related compounds including rhodamine and its derivatives, eosin and its derivatives are all useful. Methods for introducing these kinds of dye into oligonucleotides are well known (see Urdea et al., 1998, Nucleic Acids Res. 16(11): 4937- 56; Giusti & Adriano, 1993, PCR Methods Appl. 2(3): 223-7).
  • Cyanine dyes are also suitable labels for oligonucleotides of the present invention (Anderson et al., 1993, "Synthesis of a Carbocyanine Phosphoramidite and its use in Oligonucleotide Labelling", International Conference on Nucleic Acid Medical Applications; Southwick et al., 1990, Cytometry 11: 418-430).
  • Alexa dyes are another suitable class of dyes for labelling of oligonucleotides (see Panchuk-Voloshina et al., 1999, J Histochem Cytochem. 47(9): 1179-88).
  • Binding of unlabelled oligonucleotides to unlabelled targets may be detected using intercalating dyes.
  • exemplary dyes include SYBR Green, Thiazole Orange, TOTO (a dimer of Thiazole Orange), Oxazole Yellow (YO and its dimer YOYO) (Rye et al., 1992, Nucleic Acids Res. 20(11): 2803-12; Schneeberger et al., 1995, PCR Methods Appl. 4(4): 234-8.
  • Suitable methods for immobilization of oligonucleotides to solid-phase supports are well known in the art.
  • suitable attachment methods are described by Pease et al. (1994, PNAS USA 91(11): 5022-5026) and Khrapko et al. (1991 , MoI Biol (Mosk) (USSR) 25: 718-730).
  • a method for immobilization of 3'-amine oligonucleotides on casein-coated slides is described by Stimpson et al. (1995, PNAS USA 92: 6379-6383).
  • Particularly useful methods of attaching oligonucleotides to solid-state substrates are described by Maskos and Southern (1992, Nucleic Acids Res. 20(7): 1679-1684) and Guo et al. (1994, Nucleic Acids Res. 22: 5456-5465).
  • modified oligonucleotides are provided as arrays of oligonucleotides on a planar substrate, where each distinct oligonucleotide is present at a distinct location on the planar array. These arrays may also be referred to as microarrays.
  • in situ synthesis and robotic spotting of pre-synthesised oligonucleotides (for examples, see Yershov et al., 1996, PNAS USA 93: 4913-4918).
  • In situ synthesis has the advantage, in principle, of low cost as the array is assembled directly from small quantities of the 4- nucleotide monomers. The manufacturing process is highly automated and precise.
  • a disadvantage of the in situ approach is that it is not possible to purify the oligonucleotide located in each spot on the array.
  • the first in situ synthesised arrays were produced in the laboratories of Ed Southern using a glass "mask" etched with microfabricated fluidic channels linked to a conventional oligonucleotide synthesiser.
  • the mask plate is placed on top of a suitably derivatised glass array surface, such as a microscope slide.
  • the synthesiser then delivers reagents to each channel in the required order to perform conventional oligonucleotide synthesis on the microscope slide surface. In this way, narrow strips of oligonucleotides can be synthesised on a microscope slide.
  • a second technology uses a circular delivery device linked to an oligonucleotide synthesiser.
  • the delivery device is clamped to the surface of prepared microarray slide.
  • the desired nucleotide monomer is delivered to the array surface.
  • the delivery device is then detached and moved along the surface of the slide so that it still largely overlaps the area to which the first monomer has been delivered, and then the second desired monomer is delivered. This extends the first monomer into a dinucleotide in the region that overlaps with the first delivery area, while adding a single monomer to the area that has not yet been treated.
  • the delivery device is then shifted to a third partially overlapping location where the third monomer is delivered. The process is repeated until the entire desired sequence of a gene is delivered to the surface of the slide.
  • a further approach to in situ manufacture of short oligonucleotides employs photolithography.
  • Specialist phosphoramidate monomers with photo-cleavable protective groups are required for this technology and specialist mask illumination equipment.
  • the photolithographic method for in situ array synthesis starts with a reactive surface that has been protected with the photo-labile masking groups. Regions of the surface designated to be reacted with a particular nucleotide monomer are activated by exposing that region with light to remove the photo-labile protective group. In practice, this is effected by shining light through a "mask" so that only desired regions of the array surface are exposed to light.
  • the cleaved protected groups are washed away and the desired protected nucleotide monomer is coupled to the exposed surface.
  • the coupling reaction effectively blocks all the available reactive groups with protected nucleotide monomer.
  • the next designated base can then be added to the array by exposing the required areas with light. If a region that has already been coupled to a nucleotide is exposed then the protective groups are removed from the protected nucleotide making it available for chain extension. Addition of the next protected nucleotide will then either be coupled to the exposed surface of the array or to unprotected nucleotide. Thus after the second cycle of deprotection and coupling the surface will comprise unreacted regions, regions with a single nucleotide attached and regions with dinucleotides attached.
  • the process can be continued until the surface of the array is derivatised at each location with a specific desired nucleotide.
  • To produce an array of N-mers should require no more than 4xN deprotection and coupling steps, and in general it takes fewer steps (for reference see Pease et al., 1994, PNAS USA 91 : 5022-5026; Nuwaysir et al., 2002, Genome Res. 12(11): 1749-55).
  • arrays employing the above photolithographic methods require spacer and universal nucleotide analogues that have photocleavable protective groups.
  • a number of other approaches to in situ photolithographic synthesis of oligonucleotide arrays have also been developed that do not require specialist phosphoramidates, and would thus allow arrays of oligonucleotides according to this invention to be constructed using photolithographic methods and conventional nucleotides (see McGaII et al., 1996, PNAS USA 93(24): 13555-60; Serafinowski et al., 2003, J Am Chem Soc. 125(4): 962-5).
  • a further suitable method of in situ oligonucleotide synthesis uses inkjet dispensing to deliver standard phosphoramidates to specific locations on a microarray slide in the required order to produce the desired sequences (Hughes et al., 2001 , Nat Biotechnol. 19: 342-347). That method uses conventional oligonucleotide synthesis reagents and protocols, relying on accurate dispensing to deliver the desired reagents to the correct locations on the array.
  • arrays of in situ synthesized modified oligonucleotides according to this invention may be constructed using any of the above in situ oligonucleotide synthesis methods.
  • robotic spotting uses widely available robotic equipment.
  • pins or "quills" are dipped by a robotic arm or translation stage, into microtitre plates with different oligonucleotides in each well and delivered to desired locations on a microscope slide. All that is required are microtitre plates with the desired oligonucleotides.
  • An advantage of robotically spotted oligonucleotides is the pre-synthesis of oligonucleotides. This allows purification of the oligonucleotides and validation of the sequence before use, which is useful for the present invention particularly when oligonucleotides of 50 nucleotides or more in length are used.
  • Coupling of oligonucleotides to an array surface may involve the synthesis of amine- or thiol- modified oligonucleotides. These nucleophilic functional groups can covalently react with the derivatised surface of a microscope slide glass support. Various reactive functionalities have been used to derivatise array surfaces, including isocyanates, N- hydroxysuccinimide esters and epoxides. The last are a popular choice due to their relative stability, particularly in aqueous environments, which means that they remain active even after periods of storage.
  • oligonucleotides are held tethered in an aqueous environment so that the physics of their hybridisation is much more like the behaviour of untethered oligonucleotides in solution. This is advantageous from the point of view of oligonucleotide design in the present invention as most design algorithms use hybridisation models based on the parameters of solution phase hybridisation.
  • Arrays or microarrays of oligonucleotides according to the invention have a number of applications.
  • the microarrays allow reliable analysis of different mRNA splice variants. Attempts have been made by various groups to produce microarrays that can resolve different alternative splice variants (see Kane et al., 2000, Nucleic Acids Res. 28: 4552-4557) but these prior art approaches have high levels of cross-hybridisation between different splice variants.
  • Arrays comprising the oligonucleotides of this invention have reduced levels of cross-hybridisation compared with the prior art methods.
  • oligonucleotides probes may be coupled to magnetic beads.
  • Avidin labelled magnetic microparticles are available from a number of sources, for example Avidin Dynabeads® (Invitrogen, Inc., San Diego, CA, USA) or Sera- Mag® Streptavidin beads (Seradyn, Inc., Indianapolis, IN, USA).
  • Biotinylated oligonucleotide probes according to the invention can be readily coupled to avidinated beads.
  • Labelled samples can be contacted with probe-coupled magnetic particles under hybridising conditions.
  • the magnetic beads then permit facile separation of hybridised from unhybridised probes by using a magnet to retain beads in the reaction vessel while removing the solution phase, unbound probes.
  • Non-specifically bound probes can then be washed and the wash buffer can be removed while magnetically retaining the probe/particle complexes.
  • the wash process can be repeated multiple times.
  • the captured labelled sample material can then be eluted for detection or it can detected in situ depending on the mode of detection used.
  • fluorescently encoded microparticles may be used, for example the xMAP system from Luminex, lnc (Austin, TX, USA).
  • the xMAP system provides an array of up to 100 independently resolvable fluorescent beads that can be detected in a flow sorting device.
  • This technology enables multiple hybridisation probes to be labelled with distinct encoded fluorescent beads for multiplex nucleic acid detection applications (see Dunbar, 2006, Clin Chim Acta. 363(1-2): 71-82; Flagella et al., 2006, Anal Biochem. 352(1): 50-60). This technology is very well suited to multiplexed detection of multiple targets using the probes of this invention.
  • genomic DNA and mRNA are common targets and non- limiting techniques for labelling these molecules in particular are discussed below.
  • Genomic DNA may be amplified by cloning genomic DNA into bacterial plasmids, which can then be amplified in a bacterial vector.
  • Whole genome amplification can also be achieved using Multiple Displacement Amplification (MDA), which is a non-specific amplification approach that allows large amounts of genomic DNA to be produced in a single reaction with generic primers (Hosono et al., 2003, Genome Res. 13(5): 954-64).
  • MDA Multiple Displacement Amplification
  • PCR can be used to introduce labels into the amplified genomic DNA by linking labels to the primers used in the amplification reaction.
  • Amplification of mRNA may be achieved by a number of methods known in the art. Suitable methods include in vitro transcription (IVT). There are various methods of IVT RNA amplification and labelling (see for example: Moll et al., 2004, Anal Biochem. 334(1): 164-174). In such methods, RNA is labelled by incorporation of tagged nucleotides during the IVT reaction.
  • IVTT in vitro transcription
  • PCR-based methods of mRNA amplification are known in the art (see for example Herrler et al. 2000, J MoI Med. 78(7): B23; Petalidis et al., 2003, Nucleic Acids Res. 31(22): e142) and may be used in the present invention.
  • labelled oligonucleotides may have the following applications.
  • Leukemia is characterised by a number of chromosomal translocations, and specific translocations have prognostic implications.
  • FISH Fluorescence In Situ Hybridisation
  • intact chromosomes are contacted with labelled probes that bind to specific sequences on the chromosomes allowing the translocations to be identified and the leukemia to be characterized (see for example: Dohner et al., 1997, Leukemia 11 (S2): S19-24).
  • the oligonucleotides of the invention would allow enhanced discrimination of the FISH method.
  • the oligonucleotides of the invention may be molecular beacons.
  • Molecular beacons are fluorescent probes that fluoresce only in the presence of their target.
  • Molecular beacons can be used in RT-PCR for detection of the amplification products (Tyagi and Kramer, 1996, Nat Biotechnol. 14(3): 303-308).
  • molecular beacon microarrays can be constructed. These would have the advantage that the target nucleic acid would not have to be labelled and only oligonucleotide-bound material would be detected, in contrast to conventional microarrays where non-specifically adsorbed labelled target is also detected.
  • the oligonucleotides of the invention may be used as antisense compounds with enhanced specificity.
  • Antisense compounds operate by various modes of action.
  • One mode of action of particular interest here is the use of antisense oligonucleotides to modulate alternative splicing (Sazani & KoIe, 2003, J Clin Invest. 112(4): 481-6).
  • Antisense sequences complementary to exon/intron boundaries of unspliced mRNA may prevent binding of splicing factors thus inhibiting formation of the spliceasome. This can direct splicing to alternative splice sites. With two antisense oligonucleotides targeting both exon/intron boundaries for a single exon, an exon can be skipped in its entirety.
  • Exemplary genes that are good targets for splice modulation are transmembrane receptors (such as the TNF-alpha receptor gene) that can be produced in a soluble form by alternative splicing to remove a transmembrane domain.
  • Other targets include genes that regulate apoptosis such as bcl-x.
  • Bcl-xL and bcl-xS are generated by alternative splicing of bcl-x intron 2, via the use of a common 3 1 splice site and two alternative 5 J splice sites.
  • Bcl- xL the longer splice variant, is anti-apoptotic and high levels of bcl-xL have been correlated with resistance to chemotherapy (Liu et al., 1999, Am J Pathol. 155(6): 1861-7).
  • Oligonucleotides targeted against the bcl-xL 5 J splice site were shown to shift splicing towards production of bcl-xS in various cancer cell lines (Taylor et al., 1999, Nat Biotechnol. 17(11): 1097-100; Mercatante et al., 2001 , J Biol Chem. 276(19): 16411-7).
  • Oligonucleotides of the invention may be used to mediate RNase H based gene inhibition allowing selective suppression of specific splice variants of a given target gene.
  • RNase H specifically degrades the RNA portion of DNA/RNA duplexes.
  • RNase H appears to be a ubiquitous intracellular and extracellular nuclease that is postulated to provide an anti-viral defense mechanism for cells. The presence of this enzyme can be exploited to selectively degrade target ribonucleic acids in cells by designing single-stranded DNA oligonucleotides complementary to specific RNA sequences in cells.
  • Antisense oligonucleotides of the invention can be designed to specifically to degrade particular splice variants of a gene with greatly reduced affinity for alternative splice variants that may comprise one the blocks of sequence in the target. This may be used to investigate the function of splice variants and as a therapeutic approach.
  • RNase H however only recognizes certain types of modified nucleic acids, so only certain analogues can be used in antisense molecules intended for an RNase H dependent effect. Phophorothioate backbones do recruit RNase to phosphorothioate oligonucleotide/RNA duplexes. However, certain sugar modifications prevent recruitment of RNase H to oligonucleotide/RNA duplexes.
  • 2-O-methyl and larger modifications inhibit RNase H recruitment even if the native phosphodiester or phosphorothioate backbones are present.
  • 2 J -deoxy-2'-fluoro-beta-D-arabinonucleic acid is a 2' modification that does support RNase H activity and is discussed below. Since many modifications, that are otherwise desirable for the enhancement of binding affinity or exonuclease stability, inhibit RNase H activity, a different approach to oligonucleotide design is necessary.
  • Chimeric oligonucleotides provide a window of sequence that supports RNase H activity flanked at the 3' and 5' ends by other nucleotides that introduce other desirable properties into the sequence (Monia et al., 1993, J Biol Chem. 268(19): 14514-22; Lok et al., 2002, Biochemistry 41(10): 3457-67).
  • the RNase H recruiting region of the antisense oligonucleotide preferably comprise a length of between 5 and 7 nucleotides.
  • a suitable nucleic acid analogue for antisense applications is 2'-O-methoxyethoxy (2 1 - MOE), a 2' sugar modification.
  • 2'-MOE oligonucleotides are also RNA analogues with enhanced affinity for RNA binding. They have good toxicity profiles in animal studies (Prakash et al., 2008, J Med Chem. 51 (9): 2766-76).
  • 2'-MOE oligonucleotides with a phosphorothioate backbone have shown promise as orally deliverable antisense agents (Tillman et al., 2008, J Pharm Sci. 97(1): 225-36).
  • a further suitable nucleic acid analogue for antisense applications is 2'-deoxy-2'-fluoro- beta-D-arabinonucleic acid (2'-F-ANA).
  • This analogue has been shown to have enhanced RNA affinity, like other 2'-modified nucleotide analogues.
  • 2'-F-ANA can also mediate RNase H cleavage of RNA in 2'-F-ANA/RNA duplexes.
  • 2'-F-ANA show significant resistance to exonuclease degradation (Ferrari et al., 2006, Ann N Y Acad Sci. 1082: 91-102).
  • 2'-F-ANA analogues can also mediate siRNA (Dowler et al., 2006, Nucleic Acids Res. 34(6): 1669-75).
  • dsRNA-dependent protein kinase PKR which is normally activated in virally infected cells to induce cell death
  • antisense RNA antisense RNA oligonucleotides of sufficient length to activate PKR complementary to chimeric mRNA fragments expressed by chimeric genes resulting from chromosomal translocations can be used to kill cancer cells that express mutated genes (Shir & Levitzki, 2002, Nat Biotechnol. 20(9): 895-900).
  • Antisense oligonucleotides with the mismatch modifications of this invention designed to flank deletions or translocations in mutant genes to produce a dsRNA molecule are likely to be more specific than unmodified oligonucleotides for the purposes of inducing cell death in cancerous cells.
  • This PKR-mediated killing strategy may be useful in treating many cancers that express a unique RNA species.
  • Oligonucleotide designs can be using evaluated using various software modelling tools. Examples of predicted melting curves generated using the open source software tool DINAMeIt (Markham & Zuker, supra; DINAMeIt version used: UNAFoId 3.6) are shown in Figs 10 to 15 and are discussed below.
  • DINAMeIt is a software tool that models the interaction between two nucleic acids. Unlike simple melting temperature models, DINAMeIt can simulate the entire annealing process between two complementary oligonucleotides over the entire temperature range of aqueous solutions, i.e. from 0 to 100 0 C. In addition, DINAMeIt can model the interaction of pairs of sequences that are not perfectly complementary, i.e. an oligonucleotide comprising a mismatch compared with its target sequence. DINAMeIt also accounts for self-interactions of individual sequences both in terms of intra-molecular interactions and inter-molecular interactions. This allows evaluation of oligonucleotide probes such as molecular beacons that are designed to form stable structures in the absence of target.
  • DINAMeIt is well suited to assess oligonucleotide probe designs according to the invention, which is a useful exercise prior to synthesis of such oligonucleotides.
  • DINAMeIt was used to show the effect of various oligonucleotide designs using the BCR-ABL gene fusion as a model system.
  • a characteristic B2A2 fusion between the BCR and ABL genes found in numerous leukemias (Bohlander 2000, supra) was selected as a model target to asses distinguish from the parent BCR and ABL genes.
  • Sequences of the B2A2 mRNA domains extending 25 bases flanking the B2A2 fusion junction (i.e. domain junction), corresponding parent BCR and ABL sequences, and exemplar oligonucleotide probe designs are shown in Table 1.
  • PROBE1 TACTGGCCGCTGAAGGGCTTCTTCCTTATTGATGGTCAGC
  • PROBE2 TACTGGCCGCCGAAGGGCTTCTTCCTTATCGATGGTCAGC
  • DINAMeIt has been used to calculate the melting curves for the interaction of the oligonucleotides (PROBE1 to PROBE8) listed in Table 1 with the three targets (B2A2, BCR and ABL).
  • the oligonucleotides are all designed to bind to B2A2 but can cross-hybridise with ABL and BCR because of the presence of shared domain sequence.
  • PROBE sequences parts of the oligonucleotides that can bind to ABL are shown underlined while the portions of the oligonucleotides that can bind to BCR are shown in bold. Discontinuities in the oligonucleotide sequences are shown in italics.
  • DINAMeIt calculates melting curves for the interaction of two sequences only using a relatively small number of user-defined parameters: the sequence of each oligonucleotide in the pair; oligonucleotide concentration; type of oligonucleotide (DNA or RNA); and salt concentration. Duplex simulations were carried out using equal concentrations of oligonucleotide and target. The concentration of oligonucleotides or target was set to be 10 "5 molar. DINAMeIt can simulate DNA duplex hybridisations with different concentrations of sodium and magnesium ions. In the DINAMeIt version we used, for RNA hybridisations the parameters only support simulations in 1M sodium with no correction available for magnesium.
  • salt (NaCI) concentration was set to be 50 mM and oligonucleotide probes and target were assumed to be DNA.
  • salt (NaCI) was set to 1M to evaluate the effect of salt on DNA hybridisation and to evaluate the hybridisation of RNA, respectively.
  • concentration of MgCI 2 was set to zero.
  • oligonucleotide design was employed to allow discrimination of the B2A2 target from the parent cross-hybridising sequences.
  • a 40- mer probe was designed so that 20 bases of the oligonucleotide match the sequence from BCL and 20 bases of the oligonucleotide match the sequence from ABL.
  • This is referred to as a "symmetric length” or “symmetric length probe” design, in that portions of the oligonucleotide probe are the same length on either side of the portion junction, and is used in the prior art to design so-called "junction probes" for microarrays (Su et al., 2008, BMC Genomics. 9: 273; Johnson et al., 2003, Science. 302(5653): 2141-2144; Kane et al., 2000, Nucleic Acids Res. 28: 4552-4557).
  • PROBE 1 as shown in Table 1 was designed to be perfectly complementary to the target fusion sequence B2A2. This also means that 20 bases of PROBE 1 are complementary to the BCR gene and 20 bases are complementary to the ABL gene.
  • PROBE2 as shown in Table 1 is also a 40-mer designed to bind to the B2A2 sequence at the junction between the BCR and ABL donor-sequences. PROBE2, however, has two mismatches introduced into the sequence at the 10 th position downstream from the junction between the BCR and ABL sequences in the B2A2 sequence. Thus, only 19 bases of PROBE2 are complementary to the BCR sequence and only 19 bases of PROBE2 are complementary to the ABL sequence. In Table 1 , the PROBE2 mismatches are marked in italics. By comparison with PROBE1 , it can be seen that two T residues have been changed to C residues in the mismatched PROBE2.
  • the melting curve predicted by DINAMeIt for the hybridisation of PROBE1 to its intended target B2A2 is shown in Fig. 10 as a dense solid line and the melting curves for the hybridisation of PROBE1 to ABL and BCR under the same hybridisation conditions are shown in Fig. 10 as dense dashed lines.
  • the graph shows the mole fraction that is present as a duplex on the y-axis, i.e. the fraction of the starting quantities of oligonucleotide and target that are duplexed for a given temperature on the x-axis.
  • the melting curve for the hybridisation of PROBE2 to its intended target B2A2 is shown as a light solid line in Fig. 10 and the melting curves for hybridisation of PROBE2 to ABL and BCR are shown in Fig. 10 as light dashed lines to enable comparison with the non- mismatched probe design.
  • Various features of the invention are illustrated by the melting curves shown in Fig. 10. It can be seen by comparison of the melting curves of PROBE1 and PROBE2 that the introduction of the mismatches into PROBE2 reduces the overall binding affinity of PROBE2 compared with PROBE1 as the PROBE2 curves are shifted to the left, i.e. to a lower temperature profile. Similarly, it can be seen that there is significant binding of PROBE1 to both the BCR and ABL sequences. In particular, there is very significant binding of PROBE1 to the ABL sequence. Although the overall binding affinity of PROBE2 is reduced compared to PROBE1 , the difference in binding affinity between PROBE2 bound to its intended B2A2 target and PROBE2 bound to either the ABL or BCR sequence is increased compared to PROBE1.
  • Fig. 10 can be quantitatively compared in various ways.
  • hybridising PROBE1 at about 74.5 0 C with its intended target in the presence of the ABL and BCR targets yields a mole fraction of PROBE1 that binds to ABL of about 0.5 x 10 ⁇ 6 while the mole fraction of PROBE1 binding to its intended target is about 2.9 x 10 '6 .
  • the ratio of PROBE1 binding with B2A2 to PROBE1 binding with ABL is thus 5.8:1.
  • the mole fraction binding to BCR would be negligible at this temperature.
  • Example 2 Symmetric T m probes
  • an alternative oligonucleotide probe design is employed to enable higher levels of discrimination of the B2A2 target from the parent targets, ABL and BCR.
  • a 40-mer oligonucleotide probe was designed so that the T m (calculated using the Nearest Neighbour method) of the oligonucleotide portion that hybridises to the sequence from BCL is as similar as possible to the portion of the oligonucleotide that hybridises to the sequence from ABL in the probe.
  • T m calculated using the Nearest Neighbour method
  • the lengths of the portions will typically differ in order to make the portion T m s symmetrical, hence the probes are typically or often asymmetric with regard to the relative lengths of the two oligonucleotide portions.
  • PROBE3 shown in Table 1 was designed to be perfectly complementary to its target. In addition, a 24 base portion of PROBE3 is complementary to the BCR gene while a 16 base portion of PROBE3 is complementary the ABL gene. The T m s of these probe portions were designed to be as similar as possible.
  • PROBE4 shown in Table 1 is also a 40-mer oligonucleotide designed to bind to the B2A2 sequence at the junction between the BCR and ABL sequences. PROBE4, however, has two mismatches introduced into the sequence at position 7 from the 5'-end of the oligonucleotide and at position 30 from the 5'-end of the oligonucleotide. These mismatches are shown in italics.
  • the melting curve for the hybridisation of PROBE3 to its intended target B2A2 is shown as a dense solid line and the melting curves for hybridisation of PROBE3 to ABL and BCR are shown as dense dashed lines.
  • the melting curve for the hybridisation of PROBE4 to its intended target B2A2 is shown as a light solid line and the melting curves for hybridisation of PROBE4 to ABL and BCR are shown as light dashed lines.
  • Example 1 Various features of the improved oligonucleotide probe design are illustrated by the melting curves shown in Fig. 11. As with the symmetric length design in Example 1 , it can be seen by comparison of the melting curves of PROBE3 and PROBE4 that the introduction of the mismatches into PROBE4 reduces the overall binding affinity of PROBE4 compared with PROBE3 as the PROBE4 curve is shifted to the left, i.e. to a lower temperature profile. Similarly, by comparison with Example 1 it can be seen that there is still significant binding of PROBE3 to both the BCR and ABL sequences. However, there is considerably less binding of PROBE3 to the ABL sequence compared with the binding of PROBE1 with the ABL sequence.
  • binding of PROBE3 to the BCR sequence has increased so that it shows a pattern of binding that is very similar to that of PROBE3 binding to the ABL sequence.
  • the symmetric T m probe design has reduced the differences in the amount of cross-hybridisation between PROBE3 and the BCR and ABL targets when compared with PROBE1 but increased the average discrimination against both "off-target" hybridisations.
  • Fig. 10 it could be seen that for a given mole fraction of non-specific binding, i.e. for a mole fraction of binding to either BCR or ABL of 0.5 x 10-6, the specific binding of the probes to B2A2 was relatively low, 2.9 x 10 "6 and 4.9 x 10 "6 for PROBE1 and PROBE2 respectively.
  • Fig. 11 it can be seen that the overall increase in discrimination enabled by the improved symmetric T m probe design means that the mole fraction of specific probe binding to B2A2 for a given mole fraction of non-specific binding under the same conditions, e.g.
  • 0.5 x 10 "6 is now greater than 9.0 x 10 "6 over a range of hybridisation temperatures, shown as the shaded area under the PROBE3 curve. However, for PROBE3 it can be seen that the range where specific binding is greater than 9.0 x 10 '6 and non-specific binding is less than 0.5 x 10 '6 is from 69.5 0 C to 70.5 0 C, or about 1 0 C.
  • PROBE4 means that the mole fraction of specific oligonucleotide probe binding to B2A2 for a given mole fraction of non-specific binding, e.g. 0.5 x 10 "6 , is now greater than 9.0 x
  • PROBE4 curve In contrast to PROBE3, it can be seen for PROBE4 that the range where specific binding is greater than 9.0 x 10 "6 and non-specific binding is less than 0.5 x 10 '6 is from 59.5 0 C to 63.5 0 C, or about 4 0 C.
  • both PROBE3 and PROBE4 show a significant improvement in specificity for target sequence compared with PROBE1 , which clearly demonstrates the advantage of the symmetric T m design.
  • the greater range of temperature at which high specificity can be achieved with PROBE4 compared with PROBE3 illustrates that the improved design also increases the "robustness" of the oligonucleotide probe.
  • This difference between PROBE3 and PROBE4 clearly illustrates that there is still a further advantage gained by introducing mismatches into symmetric T m -designed oligonucleotides to enhance discrimination.
  • a symmetric length design is employed again but using a shorter oligonucleotide probe compared to Example 1 to determine if shorter oligonucleotides show higher levels of discrimination of the B2A2 target from the parent targets, ABL and BCR.
  • PROBE5 as shown in Table 1 was designed to be perfectly complementary to its target. Thus, 15 bases of PROBE5 are complementary to the BCR gene while 15 bases of PROBE5 are complementary the ABL gene.
  • PROBE6 shown in Table 1 has two mismatches introduced into the sequence at position 8 and at position 23 from the 5'-end of the oligonucleotide probe. The mismatches are shown in italics in Table 1. By comparison with PROBE5, it can be seen that two A residues have been changed to two T residues in the mismatched probe. PROBE6 thus has 14 bases complementary to the BCR sequence and 15 bases complementary to the ABL sequence.
  • the melting curve for the hybridisation of PROBE5 to its intended target B2A2 is shown as a dense solid line and the melting curves for the hybridisation of PROBE5 to ABL and BCR are shown as dense dashed lines.
  • the melting curve for the hybridisation of PROBE6 to its intended target B2A2 is shown as a light solid line and the melting curves for hybridisation of PROBE4 to ABL and BCR are shown as light dashed lines.
  • Various features of the shortened symmetric length probe design are illustrated by the melting curves shown in Figure 12.
  • Example 1 As with the longer symmetric length design in Example 1 , it can be seen by comparison of the melting curves of PROBE5 and PROBE6 that the introduction of the mismatches into PROBE6 reduces the overall binding affinity of PROBE6 compared with PROBE5 as the PROBE6 curve is shifted to the left, i.e. to a lower temperature profile. Similarly, it can be seen that there is still significant binding of PROBE5 to both the BCR and ABL sequences. In particular, there is significant binding of PROBE5 to the ABL sequence although relatively less compared with the binding of PROBE1 with the ABL sequence. As with PROBE1 , binding of PROBE5 to the BCR sequence is considerably less than the binding of PROBE5 to the ABL sequence. Thus, the reduced length of PROBE5 has enhanced discrimination against the cross-hybridising targets, BCR and ABL, when compared with PROBE1.
  • the ratio of PROBE5 binding with B2A2 to PROBE5 binding with ABL is thus 8.4:1. This is a significant improvement compared with PROBE 1 and clearly demonstrates the advantage of a shortened probe. The mole fraction binding to BCR would still be negligible at this T m though.
  • the symmetric T m design was employed to enable higher levels of discrimination of the B2A2 target from the parent targets, ABL and BCR.
  • a 34-mer oligonucleotide probe was designed so that the T m of the oligonucleotide portion that hybridises to the sequence from BCL is as similar as possible to the portion that hybridises to the sequence from ABL.
  • Tm is calculated using the Nearest Neighbour method.
  • the oligonucleotides are shown in Table 1 as PROBE7 and PROBE8.
  • PROBE7 was designed to be perfectly complementary to its target. In addition, 20 bases of PROBE7 are complementary to the BCR gene while 14 bases of PROBE7 are complementary the ABL gene. The T m s of these two oligonucleotide probe portions are designed to be as similar as possible.
  • PROBE8 in Table 1 is also a 34-mer designed to bind to the B2A2 sequence at the boundary between the BCR and ABL sequences but has two mismatches introduced into the sequence at position 7 and at position 26 from the 5'- end of the oligonucleotide probe. In Table 1 , these mismatches are shown in italics. By comparison with PROBE7, it can be seen that two A residues have been changed to T residues in PROBE8.
  • the mismatches have been introduced so that the melting temperatures of the subsequences on either side of the mismatch within each oligonucleotide probe portion are as close to each other as possible. This has the effect of minimizing the T m s of the subsequences on either side of the mismatch.
  • the melting curve for the hybridisation of PROBE7 to its intended target B2A2 is shown as a dense solid line and melting curves for the hybridisation of PROBE7 to ABL and BCR are shown as dense dashed lines.
  • the melting curve for the hybridisation of PROBE8 to its intended target B2A2 is shown as a light solid line and the melting curve for hybridisation of PROBE8 to ABL and BCR are shown as light dashed lines.
  • Various features of the shortened symmetric T m design are illustrated by the melting curves shown in Fig. 13. As with the symmetric length design, it can be seen by comparison of the melting curves of PROBE7 and PROBE8 that the introduction of the mismatches into PROBE4 reduces the overall binding affinity of PROBE8 compared with PROBE7 as the PROBE8 curve is shifted to the left, i.e. to a lower temperature profile. Similarly, it can be seen that there is still significant binding of PROBE7 to both the BCR and ABL sequences. However, there is considerably less binding of PROBE7 to the ABL sequence compared with the binding of PROBE5 with the ABL sequence.
  • Fig. 12 for Example 3 it could be seen that for a given mole fraction of non-specific binding, i.e. for a mole fraction of binding to either BCR or ABL of 0.5 x 10 ⁇ 6 , the specific binding of the oligonucleotide probes to B2A2 was relatively low, 4.2 x 10 "6 and 6 x 10 "6 for PROBE5 and PROBE6, respectively.
  • Fig. 13 it can be seen that the overall increase in discrimination enabled by the improved symmetric T m probe design means that the mole fraction of specific probe binding of PROBE7 to B2A2 for a given mole fraction of non-specific binding under the same conditions, e.g.
  • 0.5 x 10 "6 is now greater than 9.0 x 10 "6 over a range of hybridisation temperatures, shown as the shaded area under the PROBE7 curve. However, for PROBE7 it can be seen that the range where specific binding is greater than 9.0 x 10 "6 and non-specific binding is less than 0.5 x 10 "6 is from just over 64 0 C to just over 65 0 C, or about 1 0 C.
  • 0.5 x 10 "6 is now greater than 9.0 x 10 "6 over a range of hybridisation temperatures shown as the shaded area under the PROBE4 curve.
  • the range where specific binding is greater than 9.0 x 10 "6 and non-specific binding is less than 0.5 x 10 "6 is from 56.5 0 C to just over 59 0 C, or a little over 2.5°C.
  • both PROBE7 and PROBE8 show a significant improvement in specificity compared with PROBE5, which clearly demonstrates the advantage of the symmetric T m design.
  • Example 5 Effect of increased salt concentration on symmetric T m 34-mer probes:
  • the effect of increasing salt in hybridisation reactions was evaluated using the 34-mer symmetric T m designed PROBE7 and PROBE8.
  • ABL and BCR was evaluated using 1M salt rather than 50 mM salt as used in Example 4.
  • Example 6 Effect of RNA hybridisation on symmetric T n , 34-mer probes
  • the effect of switching to RNA hybridisation was evaluated using the 34- mer symmetric T m design from Examples 4 and 5, i.e. the binding of PROBE7 and PROBE8 were evaluated as if they and their targets were RNA.
  • the hybridisation of PROBE7 and PROBE8 to B2A2 was evaluated using 1M salt rather than 50 mM salt that was used in Example 4.
  • one aspect of the invention is to employ nucleic acid analogues with enhanced binding affinity compared to natural phosphodiester deoxyribosenucleic acids, particularly RNA analogues such as 2'-O-methyl RNA or LNA. It is expected that these analogues will have a similar effect on probe binding as the switch to RNA shown in Example 6, i.e. there will be a general increase in the T m of the oligonucleotide probe compared to the corresponding DNA oligonucleotide probe along with an increase in specificity when used against RNA targets. FRET studies of probe hybridization
  • the probes in Table 2 are designed to bind to B2A2 but can cross-hybridize with ABL and BCR because of the presence of shared portions of sequence.
  • the portion of sequence shared by B2A2 and ABL is shown underlined, while the portion of sequence shared by B2A2 and BCR is shown in bold.
  • the design of probes to maximize the discrimination of the B2A2 probe for its target over the two parent sequences is discussed below.
  • the T m s of the portions of the sequence corresponding to each target are discussed below although only the perfect match probe sequences have been calculated.
  • Probes 17 to 20 contain LNA nucleotides. In these sequences, LNA nucleotides are marked in upper case while normal DNA nucleotides are in lower case.
  • Probes and target oligonucleotides were made up in stock solutions of 50 pmol/ ⁇ l.
  • Hybridization reactions were made up as follows: 1 ⁇ l of Probe 1 ⁇ l of Target 5 ⁇ l of 10 x Hybridization Buffer
  • Mg Magnesium-containing Hybridization Buffer (Mg) comprising 100 mM MgCI 2 ; 250 mM NaCI; 200 mM TRIS HCI (pH 7.5) in water.
  • Fig. 16 shows a mole fraction difference curve (dlWdT curve) that corresponds to the melting curves for PROBE1 shown in Fig. 10. Again, the first derivatives of the melt curves in Fig. 10 are shown in Fig. 16.
  • Fig. 17 illustrates a typical set of dF/dT curves for the hybridization of PROBE9 to its intended target, B2A2_FRET (solid line), and the corresponding hybridizations to the ABL-FRET target (Dot-Dashed line) and BCR_FRET target (Dashed line), which share half their sequences with B2A2_FRET.
  • the maximum points on the dF/dT curves can be regarded as the melting temperatures of the corresponding probe/target duplex.
  • DdT Discriminating Difference in melt Temperature
  • Table 3 below lists the probes and the T m determined from the dF/dT curves for the hybridization of each probe with the targets B2A2_FRET, ABL_FRET and BCR-FRET.
  • the symmetric length design approach is applied again, with the design of 40-mer probe such that 20 bases of the probe matches the sequence from BCL and 20 bases matches the sequence from ABL in the probe.
  • MELTING Le Novere N, 2001 , Bioinformatics. 17(12): 1226-7
  • the 5' segment complementary to ABL has a T 171 of 62.3 0 C
  • the 3' segment complementary to BCR has a T m of 52.1 0 C.
  • the dF/dT curves measured for the hybridization of PROBE9 to its intended target B2A2_FRET is shown in Fig. 17 as the dense solid line and the dF/dT curves for the hybridization of PROBE1 to ABL and BCR under the same hybridization conditions are shown in Fig. 17 as the dense dashed lines.
  • the graph shows the change in fluorescence intensity (dF/dT) on the y-axis.
  • the curves for the hybridization of the probe with the three targets are overlaid on the same graph to give an idea of the amount of cross hybridization to expect for a given set of hybridization conditions if the B2A2 target and cross-hybridizing
  • Table 3 List of probes and T m (in 0 C) determined from the dF/dT curves for the hybridization of each probe with the targets B2A2_FRET, ABL_FRET and BCR-FRET
  • PROBE9 is designed to be perfectly complementary to its target (B2A2_FRET). Similarly, 20-bases of PROBE9 will be complementary to either the BCR gene or the ABL gene.
  • PROBE10 in Table 2 is also a 40-mer designed to bind to the B2A2 sequence at the boundary between the BCR and ABL sequences so that 20 bases of PROBE10 binds to the BCR sequence and 20 bases of PROBE10 binds to the ABL sequence.
  • PROBE10 has two mismatches introduced into the sequence at the 10 th position from the boundary between the BCR and ABL sequences on either side of the boundary.
  • the first mismatch is marked as the bold C nucleotide in the underlined portion of the probe that binds to the ABL sequence while the second mismatch is also a C nucleotide that is not shown in bold, in the bold portion of the probe sequence that bind to the BCR sequence in the B2A2 target.
  • PROBE9 it can be seen that two T residues have been changed to C residues in the mismatched probe.
  • PROBE9 has a T m of 7O 0 C for hybridization with B2A2_FRET and a DdT value of 3 0 C in the sodium only (Na) buffer.
  • PROBE9 has a T m of 76.5 0 C for hybridization with B2A2_FRET and a DdT value of only 1 0 C in the buffer containing magnesium ions (Mg).
  • PROBE10 has a T m of 64.5 0 C for hybridization with B2A2_FRET and a DdT value of 4.5 0 C in the Na buffer.
  • PROBE10 has a T m of 71.5 for hybridization with B2A2_FRET and DdT value of 2.5 0 C in the Mg buffer.
  • T m 71.5 for hybridization with B2A2_FRET
  • DdT 2.5 0 C in the Mg buffer.
  • Example 8 - Symmetric T n , probes As for Example 2, the improved probe design according to this invention is employed to demonstrate higher levels of discrimination of the B2A2 target from the parent targets, ABL and BCR. Again, the 40-mer probe is designed using the symmetric T m design and PROBE11 and PROBE12 in Table 2 have the same sequences as PROBE3 and PROBE4 in Table 1 respectively.
  • the lengths of the probe portions differ, hence the probes may be asymmetric with regard to the relative lengths of the two probe portions.
  • PROBE11 has a T m of 71 0 C for hybridization with B2A2_FRET and a DdT value of 8 0 C in the Na buffer.
  • PROBE9 has a T m of 77.5 0 C for hybridization with B2A2_FRET and a DdT value of 5 0 C ih the Mg buffer.
  • PROBE12 has a T m of 63 0 C for hybridization with B2A2_FRET and a DdT value of 9.5 0 C in the Na buffer.
  • PROBE12 has a T m of 70 for hybridization with B2A2_FRET and DdT value of 7 0 C in the Mg buffer.
  • both PROBE11 and PROBE12 show an improvement in specificity compared with PROBE9, which clearly demonstrates an advantage of the symmetric T m design.
  • the increase of DdT of PROBE12 compared with PROBE11 illustrates that there is still a further advantage gained by introducing mismatches into probes designed with a symmetric T m to enhance probe discrimination and that the combined introduction of artificial mismatches and symmetric T m probe designs have an additive effect on probe specificity
  • Example 3 used DINAmelt to explore the specificity of a shorter probe compared to Example 1 to see if shorter probes show higher levels of discrimination of the B2A2 target from the parent targets, ABL and BCR.
  • PROBE13 and PROBE14 have the same sequences as PROBE5 and PROBE6 from Example 3.
  • the 5' segment complementary to ABL has a T m of 56.2 0 C
  • the 3' segment complementary to BCR has a T m of 40.0 0 C.
  • PROBE13 has a T m of .64.5 0 C for hybridization with B2A2_FRET and a DdT value of 4.5 0 C in the Na buffer.
  • PROBE13 has a T m of 72 for hybridization with B2A2_FRET and a DdT value of only 2 0 C in the Mg buffer.
  • PROBE 14 has a T m of 57 0 C for hybridization with B2A2_FRET and a DdT value of 5.0 0 C in the Na buffer.
  • PROBE14 has a T m of 66.5 for hybridization with B2A2_FRET and DdT value of 4.5 0 C in the Mg buffer.
  • T m 66.5 for hybridization with B2A2_FRET
  • DdT 4.5 0 C in the Mg buffer.
  • the introduction of the mismatches into PROBE14 reduces the overall T m of PROBE10 compared with PROBE9 in both the Na and Mg buffer.
  • DdT of PROBE14 in comparison to PROBE13 of about 0.5 and 2.5 0 C for the Na and Mg hybridization buffers respectively.
  • This difference between PROBE13 and PROBE14 clearly illustrates the advantage of introducing mismatches into shorter probes to enhance probe discrimination.
  • the shortened probes have a lower T m than the longer probes.
  • the shorter probes appear to be slightly more specific, i.e. the 30-mer probes have higher DdT values than the corresponding 40-mer probes.
  • PROBE15 and PROBE16 have the same sequences as PROBE7 and PROBE8 from Example 4, to test the improved symmetric T m probe design according to the invention to see if it will deliver higher levels of discrimination of the B2A2 target from the parent targets, ABL and BCR.
  • the T m s of the probe portions are as similar as possible (5' ABL-segment is 52.O 0 C and the 3' BCR-segment is 52.1 0 C as determined by MELTING using the same conditions as Example 7), the lengths of the probe portions will typically differ, hence the probes are often asymmetric with regard to the relative lengths of the two probe portions.
  • PROBE 15 has a T m of 66.5 0 C for hybridization with B2A2_FRET and a DdT value of 1O 0 C in the Na buffer.
  • PROBE15 has a T m of 73.5 0 C for hybridization with B2A2_FRET and a DdT value of 7 0 C in the Mg buffer.
  • PROBE16 has a T m of 58 0 C for hybridization with B2A2_FRET and a DdT value of 10.5 0 C in the Na buffer.
  • PROBE16 has a T m of 66.5 for hybridization with B2A2_FRET and DdT value of 1O 0 C in the Mg buffer.
  • DdT value of PROBE 15 with PROBE 13
  • the corresponding DdT values of PROBE16 with PROBE14 there is a significant increase in DdT values for the symmetric T m probe design according to this invention, although the lengths of these symmetric T m probes is slightly greater (34 bases) than for the symmetric length probes (30 bases).
  • the introduction of the mismatches into PROBE16 reduces the overall T m of PROBE16 compared with PROBE15 in both the Na and Mg buffer.
  • DdT of PROBE16 in comparison to PROBE15 of about 0.5 and 3 0 C for the Na and Mg buffers respectively, again showing the benefit of the mismatch for enhancing discrimination.
  • the improved symmetric T m design of the 34-mer probes also shows a small increase in DdT over the longer symmetric T m 40-mer probe design in Example 8 by the measures used here.
  • probes designed according to this invention can benefit from 2' sugar modifications, which increase binding affinity but also increase sensitivity of the probes to structural deformation of probe/target duplexes.
  • One preferred modification is Locked Nucleic Acid (Nielsen et al., 1999, J Biomol Struct Dyn. 17(2):175-91; Koshkin and Wengel, 1998, J. Org. Chem. 63: 2778-2781 , 1998; Koshkin et al., 1998, J. Am. Chem. Soc. 120:13252-13253; Kvasrn ⁇ and Wengel, 1999, Chem. Commun. 657-658; Jensen et al., 2001 , J. Chem.
  • PROBE17 and PROBE18 use the same symmetric length probe design as Examples 3 and 9, with a number of LNA nucleotides introduced into the sequence.
  • LNA nucleotides and oligonucleotides are supplied by Exiqon A/S (Vedbaek, Denmark). Melting Temperatures for LNA/DNA oligonucleotide duplexes can be calculated using a tool on the Exiqon website (http://www.exiqon.com/oligo-tools). The Exiqon model does not take into account salt or oligonucleotide concentration and so the prediction is approximate. According to the Exiqon T m model, the 5 ! segment of PROBE17 complementary to ABL has a T m of 76°C, while the 3' segment complementary to BCR has a T m of 6O 0 C. These clearly differ quite substantially.
  • PROBE17 has a T m of 76 0 C for hybridization with B2A2_FRET and a DdT value of 8 0 C in the Na buffer.
  • PROBE17 has a T m of 86 for hybridization with B2A2_FRET and a DdT value of only 4.5 0 C in the Mg buffer.
  • PROBE18 has a T m of 68 0 C for hybridization with B2A2_FRET and a DdT value of 9.5 0 C in the Na buffer.
  • PROBE18 has a T m of 79 0 C for hybridization with B2A2_FRET and DdT value of 5.5 0 C in the Mg buffer.
  • Example 12 Effect of 2'-modification on the hybridization of symmetric T m 34-mer probes:
  • PROBE19 and PROBE20 use the same symmetric T m probe design as Examples 4 and 10, with a number of LNA nucleotides introduced into the sequence.
  • the 5' segment of PROBE19 complementary to ABL has a T m of 71 0 C
  • the 3' segment complementary to BCR has a T m of 72 0 C.
  • PROBE19 has a T m of 80.5 0 C for hybridization with B2A2_FRET and a DdT value of 12.5 0 C in the Na buffer.
  • PROBE19 has a T m of 88.5 0 C for hybridization with B2A2_FRET and a DdT value of 9.5 0 C in the Mg buffer.
  • PROBE20 has a T m of 72.5 0 C for hybridization with B2A2_FRET and a DdT value of 17 0 C in the Na buffer.
  • PROBE20 has a T m of 81.5 for hybridization with B2A2_FRET and DdT value of 12 0 C in the Mg buffer.
  • nucleic acid analogues with enhanced binding affinity compared to natural phosphodiester deoxyribosenucleic acids, particularly RNA analogues such as 2'-O-methyI RNA or 2'- FIuoro RNA. It is expected that these analogues will have a similar effect on probe binding as the switch to LNA shown in Examples 11 and 12, i.e. there will be a general increase in the T m of the probe compared to the corresponding DNA probe along with an increase in specificity when used against RNA targets.

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Abstract

La présente invention concerne, entre autres, un oligonucléotide (ou « sonde ») capable de distinguer un acide nucléique cible et un acide nucléique variant de celui-ci (par exemple, des variants d’épissage d’ARNm, ou un gène chimérique et des ARNm du gène parent correspondant) où la cible et le variant partagent au moins un domaine de séquence conservée ou identique. Dans un aspect, l’oligonucléotide comporte une première portion et une deuxième portion flanquant une jonction de portions, où la première portion comprend au moins une première discontinuité par rapport au premier domaine d’une séquence cible et la deuxième portion comprend au moins une deuxième discontinuité par rapport à un deuxième domaine d’une séquence cible, chaque discontinuité comprenant ou étant constituée d’un mésappariement de séquence et/ou un espaceur non nucléotidique.
PCT/GB2009/002748 2008-11-24 2009-11-24 Oligonucléotides capables de distinguer des séquences d’acide nucléique qui comprennent une séquence conservée WO2010058189A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011070333A2 (fr) 2009-12-10 2011-06-16 Trillion Genomics Limited Sondes
WO2012126639A1 (fr) * 2011-03-24 2012-09-27 Qiagen Gmbh Sondes d'hybridation modifiées
US20230086246A1 (en) * 2012-02-03 2023-03-23 California Institute Of Technology Signal encoding and decoding in multiplexed biochemical assays

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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US9758834B2 (en) * 2011-03-28 2017-09-12 Nanostring Technologies, Inc. Compositions and methods for diagnosing cancer
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US10837014B2 (en) 2012-05-16 2020-11-17 Translate Bio Ma, Inc. Compositions and methods for modulating SMN gene family expression
US10174323B2 (en) 2012-05-16 2019-01-08 The General Hospital Corporation Compositions and methods for modulating ATP2A2 expression
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AU2013262709A1 (en) 2012-05-16 2015-01-22 Rana Therapeutics, Inc. Compositions and methods for modulating MECP2 expression
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US10364455B2 (en) 2012-09-27 2019-07-30 Bioo Scientific Corporation Methods and compositions for improving removal of ribosomal RNA from biological samples
US20140350235A1 (en) * 2013-05-23 2014-11-27 The Board Of Trustees Of The Leland Stanford Junior University In situ oligonucleotide synthesis on a paramagnetic support
MA41795A (fr) * 2015-03-18 2018-01-23 Sarepta Therapeutics Inc Exclusion d'un exon induite par des composés antisens dans la myostatine
EP4522744A1 (fr) * 2022-05-12 2025-03-19 Wave Life Sciences Ltd. Compositions d'oligonucléotides et procédés associés

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994002500A1 (fr) * 1992-07-17 1994-02-03 Aprogenex, Inc. Sondes et amorces d'oligonucleotides servant a detecter la translocation chromosomique
WO1996012803A1 (fr) * 1994-10-24 1996-05-02 Genset Methode de traitement ex vivo de cellules tumorales de patients atteints de lmc
WO1997046711A1 (fr) * 1996-06-06 1997-12-11 Wisconsin Alumni Research Foundation Hybridation par mesappariement artificiel

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994002500A1 (fr) * 1992-07-17 1994-02-03 Aprogenex, Inc. Sondes et amorces d'oligonucleotides servant a detecter la translocation chromosomique
WO1996012803A1 (fr) * 1994-10-24 1996-05-02 Genset Methode de traitement ex vivo de cellules tumorales de patients atteints de lmc
WO1997046711A1 (fr) * 1996-06-06 1997-12-11 Wisconsin Alumni Research Foundation Hybridation par mesappariement artificiel

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011070333A2 (fr) 2009-12-10 2011-06-16 Trillion Genomics Limited Sondes
WO2012126639A1 (fr) * 2011-03-24 2012-09-27 Qiagen Gmbh Sondes d'hybridation modifiées
US20230086246A1 (en) * 2012-02-03 2023-03-23 California Institute Of Technology Signal encoding and decoding in multiplexed biochemical assays
US20240182954A1 (en) * 2012-02-03 2024-06-06 California Institute Of Technology Signal encoding and decoding in multiplexed biochemical assays

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