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WO2000070093A1 - Jeu d'echantillons comprenant des compositions d'oligonucleotides et de polynucleotides modifies - Google Patents

Jeu d'echantillons comprenant des compositions d'oligonucleotides et de polynucleotides modifies Download PDF

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Publication number
WO2000070093A1
WO2000070093A1 PCT/US2000/013185 US0013185W WO0070093A1 WO 2000070093 A1 WO2000070093 A1 WO 2000070093A1 US 0013185 W US0013185 W US 0013185W WO 0070093 A1 WO0070093 A1 WO 0070093A1
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Prior art keywords
oligonucleotide
ohgonucleotides
array
modified
arrays
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PCT/US2000/013185
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English (en)
Inventor
Roderic M. K. Dale
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Oligos Etc. Inc.
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Priority to AU52696/00A priority Critical patent/AU5269600A/en
Publication of WO2000070093A1 publication Critical patent/WO2000070093A1/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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the field of this invention is arra ⁇ s having associated ohgonucleotides and/or polynucleotides. methods of producing such arrays, and uses thereof
  • arrays of binding agents such as ohgonucleotides and polynucleotides
  • binding agents such as ohgonucleotides and polynucleotides
  • One important use of arrays is in the analysis of differential gene expression, where the expression of genes in different cells, normally a cell of interest and a control, is compared and any discrepancies in expression are identified In such assays, the presence of discrepancies indicates a difference in the classes of genes expressed m the cells being compared
  • arrays find use by serving as a substrate with associated binding fragments such as ohgonucleotides Nucleic acid sequences are obtamed from analogous cells, tissues or organs of a healthy and diseased organism, and hybridized to
  • the present mvention provides arrays having associated o gonucleotide and/or polynucleotides with modified structures (e g , V, 2', 3', 5' and/or modifying the ⁇ bose oxygen), methods of making such arrays, assays for using such arrays, and kits containing such arrays
  • modified structures e g , V, 2', 3', 5' and/or modifying the ⁇ bose oxygen
  • the modifications described herein provide numerous advantages, including a higher binding affinity for complementary nucleic acids, acid resistance and/or nuclease resistance
  • the mvention comprises an array device comprised of a support surface and polymer molecules bound to the support surface
  • the polymer molecules are not naturally occurring ohgonucleotides or polynucleotides, but rather modified backbones with bases attached m the desired sequential positioning and the desired spacmg between the bases
  • the backbone is preferabh modified to obtain improved results compared to natural o
  • the modified associated ohgonucleotides and/or polynucleotides of the mvention provide additional binding with respect to corresponding, unmodified ohgonucleotides having the same sequence
  • the binding affinity is preferably mcreased by a modification at the 2' site of the sugar group e g a 2'-F or a 2'-OR modification such as 2'-0-methyl or 2'-0-methoxyethoxy Alternatively or in combmation.
  • the bmding affimty can be mcreased by modification m the 3' linkage group, e g phosphoramidate linkages, or a modification replacmg the oxygen with a carbon
  • the modified associated ohgonucleotides and/or polynucleotides of the array exhibit substantial acid resistance, allow mg the arrays to be treated with low pH solutions This allows the array to be exposed to low pH in order to remove any bound nucleic acids that are not modified, e g , bound test nucleic acids
  • the modified associated ohgonucleotides and/or polynucleotides of the array exhibit substantial resistance to nuclease degradation
  • These molecules preferably have an end-blocking group that confers nuclease resistance to the molecule, e g , a butanol or butyl group
  • Nucleases can be used to digest the test substrate binding agent, freeing the associated binding agents for further use
  • the chemical modification may be on the 5' end for ohgonucleotides and/or polynucleotides attached to a substrate at the 3' end, or alternatively the chemical modification may be on the 3' end for ohgonucleotides and/or polynucleotides attached to a substrate at the 5' end
  • arrays also offer the significant advantage that the individual chip can be tested for efficacy and/or quality p ⁇ or to use with a test sample which is particularly helpful if the amount of test sample is limited or if the array is being used as a medical device and must comply with FDA quality control requirements
  • the present invention further pro ⁇ ides an assay usmg the arrays of the mvention to determine physiological responses such as gene expression, where the response is determmed by the hybridization pattern of the array after exposure to test samples
  • the test samples may be mRNA, cDNA, whole cell extracts, and the like
  • modified ohgonucleotides and/or polynucleotides of the arrays of the mvention that the chemical modifications enhance the chemical binding mteractions. e g , mcrease bmdmg affinity over standard Watson-Crick base pai ⁇ ng with complementary ohgonucleotides and/or polynucleotides, particularly when binding to mRNA
  • the modified ohgonucleotides and or polynucleotides of the array may be synthesized to have approximately the same T m , by varying the length of the nucleic acids m each composition
  • modified ohgonucleotides and/or polynucleotides of the mvention hyb ⁇ dize more tightly with complementary RNA sequences than natural DNA ohgonucleotides, allowing the use of shorter binding fragments (e g one or more ohgonucleotides in lieu of a complete cDNA)
  • the ohgonucleotides and or polynucleotides can be used m a variety of array applications, such as identification of new genes, determination of expression levels, diagnosis of disease, and the like
  • FIGS. 1 -7 illustrate the chemical structure of exemplary modifications that result in acid stability
  • FIGS 8-9 illustrate the chemical structure of end-blocked, acid stable molecules used in the invention
  • Figure 10 illustrates other potential modifications that may be used m the present invention
  • nucleic acid and “nucleic acid molecule” as used interchangeably herem, refer to a molecule compnsed of one or more nucleotides, l e , nbonucleotides, deoxynbonucleotides, or both
  • m cludes monomers and polymers of nbonucleotides and deoxynbonucleotides, with the nbonucleotides and/or deoxynbonucleotides bemg connected together, m the case of the polymers, via 5' to 3' linkages
  • linkages may mclude any of the linkages known m the nucleic acid synthesis art mcludmg, for example, nucleic acids compnsmg 5' to 2' linkages
  • the nucleotides used in the nucleic acid molecule may be naturally occu ing or may be synthetically produced analogues that are capable of forming base-pair relationships with naturally occurring base pairs.
  • non-naturally occurring bases that are capable of forming base-pairing relationships include, but are not limited to, aza and deaza pyrimidine analogues, aza and deaza purine analogues, and other heterocyclic base analogues, wherein one or more of the carbon and nitrogen atoms of the purine and pyrimidine rings have been substituted by heteroatoms, e.g., oxygen, sulfur, selenium, phosphorus, and the like.
  • oligonucleotide refers to a nucleic acid molecule comprising from about 2 to about 300 nucleotides.
  • Ohgonucleotides for use in the present invention are preferably from 80-200, more preferably from 100-150 in length.
  • polynucleotide refers to nucleic acid molecules comprising a plurality of nucleotide monomers including but not limited to nucleic acid molecules comprising over 200 nucleotides.
  • modified oligonucleotide and “modified polynucleotide” as used herein refers to ohgonucleotides and or polynucleotides with one or more chemical modifications at the molecular level of the natural molecular structures of all or any of the bases, sugar moieties, internucleoside phosphate linkages, as well as to molecules having added substituents, such as diamines, cholesterol or other lipophilic groups, or a combination of modifications at these sites.
  • the internucleoside phosphate linkages can be phosphodiester, phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate and/or sulfone internucleotide linkages, or 3'-3', 5'-2' or 5'-5' linkages, and combinations of such similar linkages (to produce mixed backbone modified ohgonucleotides).
  • the modifications can be internal (single or repeated) or at the end(s) of the oligonucleotide molecule, and can include additions to the molecule of the internucleoside phosphate linkages, such as cholesteryl, diamine compounds with varying numbers of carbon residues between amino groups and te ⁇ ninal ribose, and deoxyribose and phosphate modifications which cleave or cross-link to the opposite chains or to associated enzymes or other proteins.
  • Electrophilic groups such as ribose-dialdehyde could covalently link with an epsilon amino group of the lysyl-residue of such a protein.
  • modified ohgonucleotides and “modified polynucleotides” also include ohgonucleotides and/or polynucleotides comprising modifications to the sugar moieties (e.g., 2'- substituted ribonucleotides or deoxyribonucleotide monomers), any of which are connected together via 5' to 3' linkages.
  • Modified ohgonucleotides may also be comprised of PNA or morpholino modified backbones where target specificity of the sequence is maintained.
  • a modified oligonucleotide of the invention (1) does not have the structure of a naturally occumng oligonucleotide and (2) will hybridize to a natural oligonucleotide mRNA or cDNA Further the modification preferably provides (3) higher binding affinity, (4) greater acid resistance, and (5) better stabilits agamst digestion with enzymes as compared to a natural oligonucleotide
  • oligonucleotide backbone refers to the structure of the chemical moiety linking nucleotides m a molecule
  • the invention preferably comprises a backbone which is different from a naturally occurring backbone and is further characterized b ⁇ (1) holding bases m correct sequential order and (2) holdmg bases a correct distance between each other to allow a natural oligonucleotide to hybndize to it This ma
  • end-blocked refers to an oligonucleotide with a chemical modification at the molecular level that prevents the degradation of selected nucleotides, e g , by nuclease action This chemical modification is positioned such that it protects the integral portion of the oligonucleotide, for example the region of the oligonucleotide that is targeted for hybndization (I e , the test sequence of the oligonucleotide)
  • An end block may be a 3' end block or a 5' end block
  • a 3' end block may be at the 3 '-most position of the molecule, or it may be internal to the 3' ends, provided it is 3' of the mtegral sequences of the oligonucleotide
  • substantially nuclease resistant refers to ohgonucleotides that are resistant to nuclease degradation as compared to naturally occurring or unmodified ohgonucleotides
  • Modified ohgonucleotides of the mvention are at least 1 25 times more resistant to nuclease degradation than their unmodified counterpart, more preferably at least 2 times more resistant, even more preferably at least 5 times more resistant, and most preferably at least 10 times more resistant than their unmodified counterpart
  • Such substantially nuclease resistant ohgonucleotides mclude, but are not limited to, ohgonucleotides with modified backbones such as phosphorothioates, methylphosphonates, ethylphosphotnesters, 2'-0-methylphosphoroth ⁇ oates, 2'-0-methyl-p-ethoxy nbonucleotides, 2'-0- alkyls, 2'-0-alkyl-n(0-alkyl), 3'--
  • substantially acid resistant refers to ohgonucleotides that are resistant to acid degradation as compared to unmodified ohgonucleotides
  • the relative acid resistance of an oligonucleotide will be measured by companng the percent degradation of a resistant oligonucleotide with the percent degradation of its unmodified counterpart (1 e , a correspondmg oligonucleotide with "normar backbone bases and phosphodiester linkages)
  • An oligonucleotide that is acid resistant is preferably at least 1 5 times more resistant to acid degradation, at least 2 times more resistant, even more preferably at least 5 times more resistant, and most preferably at least 10 times more resistant than their unmodified counterpart
  • alkyl refers to a branched or unbranched saturated hyrdrocarbon chain containing 1-6 carbon atoms, such as methyl, ethyl, propyl, tert-butyl, n-hexyl and the like
  • array type refers to the type of gene represented on the array by the associated test ohgonucleotides, where the type of gene that is represented on the anay is dependent on the intended purpose of the array, e g , to monitor expression of key human genes, to monitor expression of known oncogenes, etc I e , the use for which the anay is designed As such, all of the test ohgonucleotides on a given array conespond to the same type or category or group of genes Genes are considered to be of the same type if they share some common linking characteristics, such as species of ongm, e g , human, mouse, rat, etc , tissue or cell type of ongm, e g , muscle, neural, dermal, organ, etc , disease state, e g , cancer, functions, e g , protem kinases, tumor supressors and the like, participation m the same normal biological process, e g , apoptos
  • one anay type is a "cancer array” in which each of the "unique” associated test ohgonucleotides conespond to a gene associated with a cancer disease state
  • a "human array” may be an array of test ohgonucleotides correspondmg to umque tightly regulated human genes
  • an "apoptosis array” may be an array type in which the associated test ohgonucleotides conespond to umque genes associated with apoptosis
  • association oligonucleotide refers to the oligonucleotide or polynucleotide composition that makes up each of the samples associated to the array
  • associated oligonucleotide mcludes oligonucleotide compositions of umque sequences and/or control or calibrating sequences (e g , ohgonucleotides correspondmg to housekeepmg genes)
  • the oligonucleotide and/or polynucleotide compositions are preferably compnsed of smgle stranded nucleic acid, where all of the nucleic acids m a sample composition may be identical to each other Alternatively, there may be nucleic acids having two or more sequences m each composition, for example two different ohgonucleotides that are separate but complementary to each other THE INVENTION IN GENERAL Arrays having
  • the modified ohgonucleotides and or polynucleotides of the array may be synthesized to have approximately the same T m . by varying the length of the nucleic acids m each composition Thus, an oligonucleotide with an A-T rich sequence would be designed to be longer than an oligonucleotide with a G-C nch sequence to provide approximate!, the same T m
  • the T m of each of the compositions on an array can be held relatively constant by providing lengths of ohgonucleotides and polynucleotides based on the bmdmg affimty of the base sequence
  • Acid stable associated ohgonucleotides and/or polynucleotides of the mvention are stable when exposed to a pH of 1-2, while their bmdmg partners are not
  • Direct companson of two different samples of bmdmg partners usmg a smgle array has the advantage of limiting potential experimental vanation present when comparmg multiple arrays Performing the experiment with the same sample on the same array allows a confirmation of the results obtained m the first instance, thus effectively confirming results without havmg vanation m the array composition
  • associated end-blocked ohgonucleotides and/or polynucleotides display a resistance to nucleases, allowing the arrays to be exposed to DNA nucleases to free the array from a sample of binding partners
  • An array of the invention having nuclease resistant associated ohgonucleotides can be treated with an approp ⁇ ate nuclease and reused with a different or the same sample
  • the arrays of the present invention encompass associated ohgonucleotides chemically modified to be acid stable from a pH of 0 01 to 7 0. and more preferably acid stable in a pH of 1 0 to 4 0, allowing such molecules to retain their structural mtegnties in acidic environments
  • any 2'-mod ⁇ fied oligonucleotide may be used in the present invention
  • the ohgonucleotides of the invention are and 2'-0-alkyl-n(0-alkyl) ohgonucleotides which, unlike unsubstituted phosphodiester or phosphorothioate DNA or RNA, exhibit significant acid resistance m solutions with pH as low as 0-1 even at 37 °C Acid stability of this first component coupled with the mtroduction of 3' and/or 5' acid stable, exonuclease resistant ends, confers several unique properties on 2'-0-alkyl and 2'-0-alk5 l-n(O-alky
  • the relative nuclease resistance of a oligonucleotide can be measured by comparmg the percent digestion of a resistant oligonucleotide with the percent digestion of its unmodified counterpart (1 e , a correspondmg oligonucleotide with "normal" backbone, bases, and phosphodiester linkage) Percent degradation may be determined by usmg analytical HPLC to assess the loss of full length ohgonucleotides, or by any other suitable methods (e g , by visualizing the products on a sequencing gel using staining, autoradiography fluorescence, etc , or measuring a shift in optical density) Degradation is generally measured as a function of time
  • Comparison between unmodified and modified ohgonucleotides can be made by ratiomg the percentage of mtact modified oligonucleotide to the percentage of mtact unmodified oligonucleotide For example, if, after 15 minutes of exposure to a nuclease, 25% (I e , 75% degraded) of an unmodified oligonucleotide is mtact, and 50% (I e , 50% degraded) of a modified oligonucleotide is mtact, the modified oligonucleotide is said to be 2 times (50% divided by 25%) more resistant to nuclease degradation than is the unmodified oligonucleotide Generally, a substantially nuclease resistant oligonucleotide will be at least about 1 25 times more resistant to nuclease degradation than an unmodified oligonucleotide with a correspondmg sequence, typically at least about 1 5 times more resistant,
  • Percent acid degradation may be determmed by usmg analytical HPLC to assess the loss of full length ohgonucleotides, or by any other suitable methods (e g , by visualizing the products on a sequencing gel usmg staining, autoradiography, fluorescence, etc , or measuring a shift m optical density) Degradation is generally measured as a function of time Comparison between unmodified and modified ohgonucleotides can be made by ratioing the percentage of mtact modified oligonucleotide to the percentage of intact unmodified oligonucleotide For example, if.
  • substantially "acid resistant" ohgonucleotides will be at least about 1 25 times more resistant to acid degradation than an unmodified oligonucleotide with a conespondmg sequence, typically at least about 1 5 times more resistant, preferably about 1 75 more resistant, more preferably at least 5 times more resistant and even more preferably at least about 10 times more resistant after 30 mmutes of exposure at 37° C to a pH of about 1 5 to about 4 5
  • the end-blocked ohgonucleotides of the compositions and methods of the invention are substantially nuclease resistant, substantially acid resistant, and preferably, both substantially nuclease resistant and substantially acid resistant
  • This embodiment mcludes ohgonucleotides completely or partially denvatized by one or more linkages from the group compnsed of phosphorothioate linkages, 2'-0-methyl-phosphod ⁇ esters, 2'-0-alkyl, 2'-0-ethyl, 2'-0-propyl, 2'-0- butyl, 2'-0-alkyl-n(0-alkyl).
  • This embodiment also mcludes other modifications that render the ohgonucleotides and/or polynucleotides substantially resistant to nuclease activity
  • Methods of rendering an oligonucleotide nuclease resistant mclude, but are not limited to, covalently modifying the purme or pynmidme bases that compnse the oligonucleotide
  • bases may be methylated, hydroxymethylated, or otherwise substituted (e g , glycosylated) such that the ohgonucleotides compnsmg the modified bases are rendered substantially nuclease resistant
  • the oligonucleotide and/or polynucleotide will have a backbone substantially resistant to acid degradation, exonuclease digestion, and endonuclease digestion
  • an oligonucleotide is uniformly modified with 2'-0-alkyl or 2'-0-alkyl- n(O-al
  • the associated ohgonucleotides and/or polynucleotides of the current invention are used for diagnostic purposes
  • ohgonucleotides of the current invention may be used to detect complementary ohgonucleotides by contacting an oligonucleotide of the invention with an oligonucleotide sample under conditions that allow for the hybridization of the oligonucleotide of the invention to any complementary oligonucleotide present in the sample, and detecting such hybndization
  • a preferred embodiment of the present invention is an end-blocked oligonucleotide with the chemical backbone structure RNA-butanol-3 ' or 2'-0-alkyl-0-alkyl
  • a particularly preferred embodiment of the present mvention is an oligonucleotide with the chemical backbone structure of 5'-butanol-2'-0-methyl RNA-butanol-3', 5'-butanol-2'-0-alkyl-0-alkyl RNA-butanol-3' or 2'-0-alkyl-0-alkyl RNA
  • the end-blocking group on one end of the oligonucleotide may not be needed, dependmg on the manner of association with the substrate, as will be apparent to one skilled in the art upon readmg the present disclosure
  • Each associated oligonucleotide and/or polynucleotide composition of the pattern present on the surface of the substrate is preferably made up of a set of unique nucleic acids, and preferably a umque oligonucleotide composition
  • umque composition is meant a collection or population of smgle stranded ohgonucleotides capable of participating in a hybridization event under appropnate hybndization conditions, where each of the individual ohgonucleotides may be the same — have the same nucleotide sequence ⁇ or different sequences, for example the oligonucleotide composition may consist of two different ohgonucleotides that are complementary to each other (I e , the two different ohgonucleotides are complementary but physically separated so as to be smgle stranded, l e , not hybridized to each other)
  • the oligonucleotide compositions will compn
  • compositions having umque ohgonucleotides the sequence of the ohgonucleotides are chosen in view of the type and the mtended use of the array on which they are present.
  • the umque ohgonucleotides are preferably chosen so that each distmct umque oligonucleotide does not cross- hybridize with any other distinct umque oligonucleotide on the array. 1 e .
  • the oligonucleotide will not cross-hybridize to any other oligonucleotide compositions that correspond to a different gene falling within the broad category or type of genes represented on the anay under appropnate conditions
  • the nucleotide sequence of each umque oligonucleotide of a composition will have less than 90% homology, usually less than 85 % homology and more usually less than 80% o homology with any other different associated oligonucleotide composition of the array, where homolog-s is determmed by sequence analysis comparison using the FASTA program using default settings
  • the sequence of umque associated ohgonucleotides m the compositions are not conserved sequences found m a number of different genes (at least two), where a conserved sequence is defined as a stretch of from about 4 to about 80 nucleotides which have at least about 90% sequence identity, where sequence identity is measured as above
  • the associated oligonucleotide will generally have a length of from
  • associated modified oligonucleotide composition will not cross-hybndize with any other associated ohgonucleotides on the anay under standard hybndization conditions
  • associated ohgonucleotides and hybridization conditions can be altered to allow bmdmg to multiple associated oligonucleotide compositions
  • the oligonucleotide sequences may be more similar and/or less stringent hybndization conditions may be used
  • ohgonucleotides and/or polynucleotides of the mvention may contain any modification that confers on the molecules greater bmdmg with other nucleic acids, that mcreases the acid stability and or mcreases the nuclease stability of the molecule This mcludes ohgonucleotides and/or polynucleotides completely denvatized phosphorothioate linkages, 2'-0-methylphosphod ⁇ esters, 2'- O-alkyl, 2'-0-alkyl-n(0-alkyl), 2'-fluoro.
  • nucleotides m each oligonucleotide and/or polynucleotide may each contain the same modifications, may contain combmations of these modifications, or may combme these modifications with phosphodiester linkages Additional methods of rendering ohgonucleotides and/or polynucleotides nuclea
  • the ring structure of the nbose group of the nucleotides in the modified oligonucleotide and/or polynucleotide may also have an oxygen in the ring structure substituted with N-H, N-R, S and/or methylene
  • 2'-0-alkyl substituted ohgonucleotides and/or polynucleotides exhibit marked acid stability and endonuclease resistance, are sensitive to 3' exonucleases
  • the 3' or 5' and 3' ends of the nboohgonucleotide sequence are preferably attached to an exonuclease blocking function
  • one or more phosphorothioate nucleotides can be placed at either end of the oligonbonucleotide
  • one or more inverted bases can be placed on either end of the ohgoribonucleotide, or one or more alkyls.
  • a prefened embodiment of the present mvention is an oligonucleotide compnsmg a oligonucleotide havmg the following structure
  • A-B-C wherem "B” is a 2'-0-alkyl or 2'-0-alkyl-n(0-alkyl) oligonbonucleotide between about 2 and about 300 bases m length, and "A” and “C” are respective 5' and 3' end blocking groups (e g , one or more phosphorothioate nucleotides (but typically fewer than six), inverted base linkages, or alkyl, alkenyl, or alkynl groups or substituted nucleotides or 2'-0-alkyl-n(0-alkyl))
  • a partial list of blockmg groups mcludes inverted bases, dideoxynucleotides.
  • methylphosphates alkyl groups, aryl groups, cordycepm, cytosine arabanoside, 2'-methoxy, ethoxy nucleotides, phosphoramidates, a peptide linkage, dinitrophenyl group, 2'- or 3'-0-methyl bases with phosphorothioate linkages, 3'-0-methyl bases, fluorescent, cholesterol, biotin, acndine.
  • An enzyme-resistant butanol preferably has the structure OH- CH 2 CH 2 CH 2 CH 2 (4-hydroxybutyl) which is also referred to as a C4 spacer
  • Ohgonucleotides can be synthesized on commercially purchased DNA synthesizers from ⁇ luM to >lmM scales usmg standard phosphoramidite chemistry and methods that are well known in the art, such as, for example, those disclosed in Stec et al , J Am Chem Soc 106 6077-6089 (1984), Stec et al , J. Org Chem 50(20) 3908-3913 (1985), Stec et al , J Chromatog 326 263-280 (1985), LaPlanche et al , Nuc Acid Res 14(22) 9081-9093 (1986), and Fasman, Practical Handbook of Biochemistry and Molecular Biology.
  • Ohgonucleotides can be deprotected following phosphoramidite manufacturer's protocols Unpu ⁇ fied ohgonucleotides may be dried down under vacuum or precipitated and then dried Sodium salts of ohgonucleotides can be prepared usmg the commercially available DNA-Mate (Barkosigan Inc ) reagents or conventional techniques such as a commercially available exchange resin, e g , Dowex, or by addition of sodium salts follow ed by precipitation, diafiltration, or gel filtration, etc Ohgonucleotides to be purified can be chromatographed on commercially available reverse phase or ion exchange media, e g , Waters Protein Pak Pharmacia's Source Q, etc Peak fractions can be combined and the samples desalted and concentrated by means of reverse phase chromatography on poly(styrene-divinylbenzene) based columns like Hamilton's PRP.
  • ohgonucleotides may be electrophoretically punfied usmg polyacrylamide gels
  • Lyophilized or dried-down preparations of ohgonucleotides can be dissolved in pyrogen-free, stenle, physiological saline (I e , 0 85 %> salme) sterile Sigma water, and filtered through a 0 45 micron Ge nan filter (or a stenle 0 2 micron pyrogen-free filter)
  • the descnbed ohgonucleotides may be partially or fully substituted with any of a broad vanety of chemical groups or linkages mcludmg, but not limited to phosphoramidates, phosphorothioates, alkyl phosphorates, 2'-0-methyls, 2'-mod ⁇ fied RNAs, morphohno groups, phosphate esters, propyne groups, or chimencs of any combmation of the above groups or other linkages (or analogs thereof)
  • a vanety of standard methods can be used to punfy the presently descnbed ohgonucleotides
  • the ohgonucleotides of the present mvention can be punfied by chromatography on commercially available reverse phase (for example, see the RAININ Instrument Co , Inc instruction manual for the DYNAMAX®-300A, Pure-DNA reverse-phase columns, 1989, or current updates thereof, herein incorporated by reference) or ion exchange media such as Waters' Protem Pak or
  • the modified polynucleotides and ohgonucleotides that are associated on the array may also be produced used established techniques such as polymerase cham reaction (PCR) and reverse transc ⁇ ption (RT) These methods are similar to those currently known m the art (see e g , PCR Strategies Michael A Innis (Editor), et al (1995) and PCR Introduction to Biotechmques Series, C R Newton, A Graham (1997)), and preferabh the enzymes used to produce the polynucleotides or ohgonucleotides are optimized for incorporation of modified nucleotide monomers Methods of identifying which enzymes are best suited for incorporation of nucleotide monomers with specific modifications (e g , which enzymes will best incorporate 2'-mod ⁇ fied dNTPs) are well known in the art, and thus one skilled m the art would be able to identify enzymes for use with the present invention based upon this disclosure For example, the process directed evolution can be used to unveil mechanisms of
  • Genomic sequencing programs may also reveal conse ⁇ ed regions m the enzyme structure and regions of vanabi ty between enzymes from closely related species, thus identifying regions of an enzyme that may be altered without affecting the desired activity It would be well withm the skill of one m the art to use such techniques to identify an enzyme ith optimal performance for producing the modified polynucleotides and ohgonucleotides of the invention Techmques for identification of specific enzymes for production of polynucleotides for association on the arrays of the invention are descnbed m Schmidt-Dannert C, et al , Trends Bwtechnol 17 135-6 (1999), Moreno-Hagelsieb G, et al , Bwl Res 29 127-40 (1996), Colacmo F, et al , Bwtechnol Genet
  • oligonucleotide or polynucleotide is considered pure when it has been isolated so as to be substantially free of, inter aha, incomplete products produced dunng the synthesis of the desired oligonucleotide or polynucleotide Preferabh .
  • a punfied oligonucleotide or polynucleotide will also be substantially free of contaminants which may hinder or otherwise mask the bmdmg activity of the molecule
  • the arrays of the subject mvention have a plurality of associated modified ohgonucleotides and/or polynucleotides stably associated with a surface of a solid support, e g , covalently attached to the surface with or without a linker molecule
  • Each associated sample on the array comprises a modified oligonucleotide composition, of known identity, usually of known sequence, as described m greater detail below Any conceivable substrate may be employed m the invention
  • the modified oligonucleotide compositions are stably associated with the surface of a solid support, where the support may be a flexible or ngid solid support
  • stably associated is meant that the sample of associated modified ohgonucleotides and/or polynucleotides maintain their position relative to the solid support under hvbndization and washing conditions
  • the samples can be non-covalently or covalently stably associated with the support surface
  • Examples of non-covalent association include non-specific adsorption, bmdmg based on electrostatic mteractions (e g , ion pair mteractions), hydrophobic interactions, hydrogen bondmg mteractions, specific bmdmg through a specific binding pair member covalently attached to the support surface, and the like
  • covalent bindmg m include covalent bonds formed between the ohgonucleotides and a functional group present on the surface of the rigid support
  • the array is present on either a flexible or ngid substrate
  • a flexible substrate is capable of being bent, folded or similarly manipulated without breakage
  • rigid is meant that the support is solid and does not readily bend, l e , the support is not flexible
  • the rigid substrates of the subject arrays are sufficient to provide physical support and structure to the associated ohgonucleotides and/or polynucleotides present thereon under the assay conditions in which the array is employed, particularly under high throughput handling conditions
  • the rigid supports of the subject mvention are bent, they are prone to breakage
  • the substrate may be biological, nonbiological, organic, inorganic, or a combmation of any of these, existing as particles, strands, precipitates, gels, sheets, tubing, spheres, containers, capillanes, pads, slices, films, plates
  • the substrate is preferably flat but may take on a vanety of alternative surface configurations
  • the substrate may contam raised or depressed regions on which the synthesis takes place
  • the substrate and its surface preferably form a ngid support on which to carry out the reactions descnbed herem
  • the substrate and its surface are also chosen to provide appropnate light-absorbing characteristics
  • the substrate may be a polymenzed Langmuir Blodgett film, functiona zed glass, Si, Ge, GaAs, GaP, S ⁇ 0 2 , SPN 4 , modified silicon, or any one of a wide vanety of gels or polymers such as (poly)tetrafluoroethvlene, (poly)v ⁇ nyl ⁇ dened ⁇ fluonde, polystyrene, polycarbonate or combinations thereof
  • Other substrate matenals will be readily apparent to those of skill m the art upon review of this disclosure
  • the substrate is flat glass or single-crystal silicon with surface relief features of less than 10 angstroms
  • the surface of the substrate is etched using well known techniques to provide for desired surface features
  • the synthesis regions may be more closely placed within the focus point of impinging light, be provided with reflective "mirror" structures for maximization of light collection from fluorescent sources, etc
  • the surface may be composed of any of a wide variety of matenals, for example, polymers, plastics, resms polvsacchandes, silica or silica-based matenals, carbon, metals, morgamc glasses, membranes or any of the above-listed substrate matenals
  • the surface may provide for the use of caged binding members which are attached firmly to the surface of the substrate
  • the surface will contain reactive groups, which could be carboxyl, ammo, hydroxyl, or the like
  • the surface will be optically transparent and will have surface S ⁇ --OH functionalities, such as are found on silica surfaces
  • the surface of the substrate is preferably provided with a layer of linker molecules, although it will be understood that the linker molecules are not required elements of the mvention
  • the linker molecules are preferably of sufficient length to permit modified ohgonucleotides and/or polynucleotides of the mvention and on a substrate to hybridize to natural nucleic acid molecules and to interact freely with molecules exposed to the substrate
  • the linker molecules should be 6-50 atoms long to provide sufficient exposure
  • the linker molecules may also be, for example, aryl acetylene, ethyiene glycol ohgomers containing 2-10 monomer umts, diamines, diacids. amino acids, or combmations thereof
  • Other linker molecules which can bmd to modified ohgonucleotides of the invention may be used in light of this disclosure
  • the linker molecules can be attached to the substrate via carbon-carbon bonds usmg, for example, (poly)tnf_uorochloroethylene surfaces, or preferably, by siloxane bonds (usmg, for example, glass or silicon oxide surfaces) Siloxane bonds with the surface of the substrate may be formed m one embodiment via reactions of linker molecules bearing tnchlorosilyl groups
  • the linker molecules may optionally be attached m an ordered array, 1 e , as parts of the head groups m a polymenzed Langmuir Blodgett film
  • the linker molecules are adsorbed to the surface of the substrate
  • the linker molecules and modified nucleotides used herem are provided with a functional group to which is bound a protective group
  • the protective group is on the distal or terminal end of the linker molecule opposite the substrate
  • the protective group may be either a negative protective group (1 e the protective group renders the linker molecules less reactive with a monomer upon exposure) or a positive protective group (1 e , the protective group renders the linker molecules more reactive with a monomer upon exposure)
  • negative protective groups an additional step of reactivation will be required In some embodiments, this will be done by heating
  • the protective group on the linker molecules may be selected from a wide variety of positive hght-reactive groups preferably mcludmg mtro aromatic compounds such as o-nitrobenzyl derivatives or benzylsulfonyl
  • the substrate, the region for attachment of an individual oligonucleotide group could be of any size or shape For example, squares, ellipsoids rectangles triangles, circles, or portions thereof, along with irregular geometric shapes, may be utilized
  • Duplicate synthesis regions may also be applied to a smgle substrate for purposes of redundancy
  • the regions on the substrate can have a surface area of between about 1 cm 2 and 10 10 cm 2 Preferabh. the regions have areas of less than about 10 x to 10 7 cm 2 , more preferably less than 10 3 to 10 6 cm 2 , and even more preferably less than 10 5 cm 2
  • a smgle substrate supports more than about 10 different oligonucleotide and/or polynucleotide compositions and preferably more than about 100 different oligonucleotide and or polynucleotide compositions, although m some embodiments more than about 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , or 10 s different compositions are provided on a substrate
  • the modified nucleotides be substantially pure In preferred embodiments regions of the substrate contam ohgonucleotides or polynucleotides which are at least about 50%.
  • each region will contam a substantially pure modified oligonucleotide or polynucleotide composition havmg a smgle sequence
  • the substrates of the arrays of the mvention compnse at least one surface on which the pattern of associated ohgonucleotides and/or polynucleotides is present, where the surface may be smooth, substantially planar, or have irregulanties.
  • Modification la ⁇ ers of mterest include inorganic and organic layers such as metals, metal oxides, polymers, small organic molecules and the like
  • each composition will be sufficient to provide for adequate hybridization and detection of nucleic acids during the assay m which the array is employed Generally, the amount of oligonucleotide or polynucleotide in each composition will be at least about 0 1 ng usually at least about 0 5 ng and more usually at least about 1 ng, where the amount may be as high as 1000 ng or higher, but will usually not exceed about 20 ng and more usually will not exceed about 10 ng.
  • the copy number of each oligonucleotide or polynucleotide m a composition will be sufficient to provide enough hybndization sites to yield a detectable signal, and will generally range from about 0 01 frnol to 50 fmol, usually from about 0 05 fmol to 20 frnol and more usually from about 0 1 frnol to 5 frnol Where the composition has an overall circular dimension, the diameter of the sample will generally
  • type or land can refer to a plurality of different charactenzing features, where such features mclude species specific genes, where specific species of mterest include eukaryotic species, such as mice, rats, rabbits, pigs, primates, humans, etc , function specific genes, where such genes mclude oncogenes, apoptosis genes, cytokines, receptors, protem kinases, etc , genes specific for or involved m a particular biological process, such as apoptosis, differentiation, cell cycle regulation, cancer, aging, proliferation, etc .
  • features mclude species specific genes, where specific species of mterest include eukaryotic species, such as mice, rats, rabbits, pigs, primates, humans, etc , function specific genes, where such genes mclude oncogenes, apoptosis genes, cytokines, receptors, protem kinases, etc , genes specific for or involved m a particular biological process, such as apopto
  • locations m include organs, such as heart, liver, prostate, lung etc , tissue, such as nerve, muscle, connective, etc , cellular, such as axonal, lymphocytic, etc , or subcellular locations, e g , nucleus, endoplasmic reticulum, Golgi complex, endosome, lyosome, peroxisome mitochondna cytoplasm, cytoskeleton. plasma membrane, extracellular space specific genes that change expression level over time, e g .
  • ohgonucleotides preferably from 80-300 nt in length, more preferably from 100-200 nt m length, are used on the arrays These are especially useful m place of cDNAs for determining the presence of mRNA m a sample, as the modified ohgonucleotides have the advantage of rapid synthesis and punfication and analysis prior to attachments to the substrate surface
  • ohgonucleotides with 2 1 modified sugar groups show mcreased bmdmg affimty with RNA, and these ohgonucleotides are particularly advantageous m identifying mRNA in a sample exposed to an array
  • modified ohgonucleotides allow the compositions to bind with the same affinity as a much longer unmodified nucleic acid, e g an unmodified cDNA
  • multiple ohgonucleotides may be used Multiple ohgonucleotides directed at a particular gene or RNA molecule may be mterspersed in a single region or the different ohgonucleotides may each be m a discrete region, e g to determine presence or absence of related molecules m a sample
  • the arrays of the present mvention typically compnse one or more additional associated oligonucleotide composition which does not correspond to the array type, I e , the type or land of gene represented on the arra
  • the array may comprise one or more compositions that are made of non "umque" ohgonucleotides, e g , ohgonucleotides correspondmg to commonly expressed genes
  • compositions compnsmg ohgonucleotides that bmd to plasmid and bactenophage ohgonucleotides.
  • compositions compnsmg housekeepmg genes and other control genes from the same or another species may be present, e g , to serve in the normalization of mRNA abundance and standardization of hybndization signal intensity m the sample assayed with the anay
  • Patents and patent applications descnbmg arrays of ohgonucleotides and methods for their fabncation mclude 5,242,974, 5,384,261. 5,405,783, 5,412,087, 5,424,186. 5,429,807, 5,436,327, 5,445,934, 5,472,672, 5,527,681, 5,529.756, 5,545,531, 5,554,501, 5,556,752, 5,561,071, 5,599,895, 5,624,711, 5,639,603, 5,658.734, 5,700,637, 5,744,305, 5,837,832, 5,843,655, 5,861,242, 5,874.974, 5,885,837, WO 93/17126, WO 95/11995, WO 95/35505, EP 742 287, and EP 799 897 Patents and patent applications descnbmg methods of using arrays m vanous applications mclude 5,143,854, 5,288,644, 5,324,633 5,432,049,
  • the modified ohgonucleotides for use with the present invention are synthesized pnor to attachment onto the substrate. This affords the advantages that (1) ohgonucleotides of known composition and sequence can be produced, (2) ohgonucleotides can be analyzed and punfied prior to attachment, which eliminates "shortmers," I e , ohgonucleotides with insufficient length and/or incorrect sequence, (3) the methods used to produce ohgonucleotides are less prone to error than current methods for production of cDNA, e g PCR with Taq polymerase, and (4) attachment to the substrate may be momtored or assayed without destroymg the array
  • modified ohgonucleotides can be attached usmg the techmques of, for example U S Patent No 5,807,522, which is mcorporated herein by reference for teaching methods of polymer attachment Other similar methods may be used, as will be apparent to one skilled m the art upon reading the present technology
  • Oligonucleotide and/or polynucleotide arrays provide a high throughput technique that can assay a large number of polynucleotides m a sample
  • a vanety of different array formats have been developed and are known to those of skill m the art
  • the arrays of the subject mvention find use m a vanety of applications, mcludmg gene expression analysis, drug screening, mutation analysis and the like.
  • Arrays can be used, for example, to examine differential expression of genes and can be used to determine gene function
  • anays can be used to detect differential expression of a polynucleotide between a test cell and control cell (e g , cancer cells and normal cells)
  • a test cell and control cell e g , cancer cells and normal cells
  • high expression of a particular message in a cancer cell which is not observed in a conesponding normal cell, can indicate a cancer specific gene product.
  • Exemplary uses of arrays are further described in, for example. Pappalarado et al , Sem. Radiation Oncol. 8:217 (1998), and Ramsay, Nature Biotechnol. 16:40 (1998). Methods for analyzing the data collected from hybridization to arrays are well known in the art.
  • data analysis can include the steps of determining fluorescent intensity as a function of substrate position from the data collected, removing outliers, i.e., data deviating from a predetermined statistical distribution, and calculating the relative binding affinity of the test nucleic acids from the remaining data.
  • the resulting data can be displayed as an image with the intensity in each region varying according to the binding affinity between associated ohgonucleotides and or polynucleotides and the test nucleic acids.
  • Ohgonucleotides having a sequence unique to that gene are preferably used in the present invention. Different methods may be employed to choose the specific region of the gene to be targeted. A rational design approach may also be employed to choose the optimal oligonucleotide sequence for the hybridization array. Preferably, the region of the gene that is selected is chosen based on the following criteria. First, the sequence that is chosen should yield an oligonucleotide composition that preferably does not cross-hybridize with any other oligonucleotide composition present on the array.
  • sequences that are avoided include those found in: highly expressed gene products, structural RNAs, repeated sequences found in the sample to be tested with the array and sequences found in vectors.
  • a further consideration is to select ohgonucleotides with sequences that provide for minimal or no secondary structure, structure which allows for optimal hybridization but low non-specific binding, equal or similar thermal stabilities, and optimal hybridization characteristics.
  • array type refers to the nature of the oligonucleotide and/or polynucleotide compositions present on the array and the types of genes to which the associated compositions correspond. These array types include, but are not limited to: human array; mouse anay; developmental array; cancer array; apoptosis array; oncogene and tumor suppressor array; cell cycle gene array; cytokine and cytokine receptor array; growth factor and growth factor receptor array; neuroarrays; and the like.
  • the human ana ⁇ human genes that mav be represented by the composition on the arrays include those for (a) oncogenes and tumor suppressors, (b) cell cycle regulators, (c) stress response proteins, (d) ion channel and transport protems (e) intracellular signal transduction modulators and effectors, (f) apoptosis-related proteins (g) DNA synthesis, repair and recombination proteins, (h) transcription factors and general DNA binding proteins, (1) growth factor and chemokme receptors, (j) interleul ⁇ n and interferon receptors, (k) hormone receptors, (1) neurotransmitter receptors, (m) cell surface antigens and cell adhesion protems, (n) growth factors, cytokines and chemokmes, (o) mterleukins and mterferons, (p) hormones, (q) extracellular matrix protems, (r) cytoskeleton and motihty protems (s) RNA processmg and turnover
  • the arrays of the invention can be used m among other applications differential gene expression assays
  • anays are useful m the differential expression analysis of (a) diseased and normal tissue, e g , neoplastic and normal tissue (b) different tissue or tissue types, (c) developmental stage, (d) response to external or internal stimulus, (e) response to treatment and the like
  • the arrays are also useful m broad scale expression screening for drug discovery and research, such as the effect of a particular active agent on the expression pattern of genes m a particular cell, where such information can be used to reveal drug toxicity, carcinogenicity, etc , environmental momtonng, disease research and
  • test sample is contacted with the array under hybndization conditions, where such conditions can be adjusted, as desired, to provide for an optimum level of specificity m view of the particular assay being performed
  • hybndization conditions where such conditions can be adjusted, as desired, to provide for an optimum level of specificity m view of the particular assay being performed
  • each population of labeled test samples are separately contacted to identical arrays or together to the same array under conditions of hybridization, preferably under stnngent hybndization conditions (for example, at 50 °C or higher and 0 1X SSC (15 mM sodium chlonde/01 5 ⁇ iM sodium citrate)), such that test nucleic acids hybndize to complementary ohgonucleotides and/or polynucleotides on the substrate surface
  • test nucleic acids have the same label
  • different arrays can be employed for each physiological source
  • the same array can be employed sequentially for each physiological source, with test samples removed from the array as described below
  • the labels of the test nucleic acids are different and distinguishable for each of the different physiological sources being assayed
  • distinguishable labels include two or more different emission wavelength fluorescent dyes, like C ⁇ 3 and Cy5, two or more isotopes with different energies of emission, like 32 P and 33 P, labels which generate signals under different treatment conditions, like temperature pH treatment by additional chemical agents, etc , or generate signals at different time points after treatment
  • Using one or more enzymes for signal generation allows for the use of an even greater ⁇ anet ⁇ of distinguishable labels, based on different substrate specificity of enzymes (e g , alkaline phosphatase/peroxidase)
  • non-hybndized labeled nucleic acid is removed from the support surface, convemently by washing, generating a pattern of hybndized oligonucleotide and/or polynucleotide on the substrate surface
  • a vanety of wash solutions are known to those of skill m the art and may be used
  • the resultant hybndization patterns of labeled, hybridized ohgonucleotides and/or polynucleotides may be visualized or detected m a vanety of ways, with the particular manner of detection being chosen based on the particular label of the test nucleic acid, where representative detection means include scintillation countmg autoradiography, fluorescence measurement, colonmetnc measurement, light emission measurement and the like
  • the hybndization patterns may be compared to identify differences between the patterns Where arrays in which each of the different ohgonucleotides and/or polynucleotides corresponds to a known gene are employed, any discrepancies can be related to a differential expression of a particular gene m the physiological sources bemg compared
  • the array may be treated to remove the bound test nucleic acids
  • the associated nucleic acid compositions remam mtact following treatment, allowing reuse of the treated arra>
  • the array of the mvention substantially retams its bmdmg capabilities, and any differences m bmdmg ability may be determmed usmg control sequences associated on the array
  • the array of the mvention retams at least 75% of its bmdmg capabilities, more preferably the array retams at least 85% of its bmdmg capabilities, and even more preferably the array of the mvention retams at least 95% of its bmdmg capabilities
  • Arrays with associated modified oligonucleotide and/or polynucleotide compositions can be exposed to a low pH environment, e g . pH from 0 5 -4 5, which results m the degradation of non- modified nucleic acids
  • the arrays of the mvention are rinsed to remove any unwanted test nucleic acid fragments, residual label and the like, and the arrays are prepared for reuse
  • the array may be regenerated by removal and/or degradation of the test sample
  • a two hour incubation of the sample-bound array m an acid solution at pH 1 5, 39 °C results m complete loss of a full-length unmodified 14-mer oligonucleotide Under these conditions the bound arra ⁇ ohgonucleotides of the mvention maintain full length structural integrity Following the acid incubation a variety of wash conditions may be used to clear the test sample from the probe array
  • Exemplary clearmg conditions for use with the arrays of the invention are (1) Incubation of the bound array with pH 1-2 acid solution, 8 hours at 39 °C followed with three rmses at 39 °C with strmgent wash buffer. 0 1 X SSC pH 7 0, and two rmses with hybndization buffer, pH approximately 7 0 These two solutions are for removal of degraded sample and the regeneration of the substrate array and hence do not require a low pH Array may then be reused
  • the associated acid stable ohgonucleotides of the array remain 1) associated to the substrate surface, 2) structuralh mtact, and 3) capable of bmding with another test bmdmg partner
  • nuclease resistant may be treated with a nuclease to remove bound test nucleic acids and label
  • the nuclease used can be chosen depending on the nature of the binding between the associated oligonucleotide and/or polynucleotide and the molecules of the test sample and the attachment of the oligonucleotide and/or polynucleotide to the arra ⁇ For example, if the associated ohgonucleotides are end-blocked ohgonucleotides.
  • the appropnate nuclease would be one that recognizes RNA-DNA hybrids, e g , Ribonuclease H
  • the appropnate nuclease would be one that recognizes double stranded DNA complexes, e g , Deoxy ⁇ bonuclease I or II, and Exodeoxynbonuclease III or V
  • the appropnate nuclease is one that recognizes RNA-RNA hybnds, such as micrococcal nuclease
  • nucleases that are 5' or 3' specific may be
  • kits for performing analyte bmdmg assays usmg the arrays of the present mvention
  • Such kits accordmg to the subject mvention will at least compnse the arrays of the mvention having associated modified ohgonucleotides and/or polynucleotides
  • Kits also preferably compnse an agent for removal of test bmdmg agents, e g , a solution with low pH and or with nuclease activity
  • the kits may further compnse one or more additional reagents employed m the vanous methods, such as 1) primers for generatmg test nucleic acids.
  • dNTPs and/or rNTPs either premixed or separate, optionally with one or more uniquely labeled dNTPs and/or rNTPs (e g , biotinylated or Cy3 or Cy5 tagged dNTPs) 3) post synthesis labeling reagents, such as chemically act ⁇ e derivatives of fluorescent dyes, 4) enzymes, such as reverse transcnptases DNA polymerases, and the like, 5) various buffer mediums, e g hybridization and washmg buffers, 6) labeled probe purification reagents and components like spin columns etc , and 7) signal generation and detection reagents, e g streptavidm- alkahne phosphatase conjugate chemifluorescent or chemiluminescent substrate, and the like
  • Ohgonucleotides were synthesized using commercial phosphoramidites on commercially purchased DNA synthesizers from ⁇ 1 uM to >lmM scales usmg standard phosphoramidite chemistry and methods that are well known m the art, such as, for example, those disclosed in Stec et al , J Am Chem Soc 106 6077-6089 (1984), Stec et al J Org Chem 50(20) 3908-3913 (1985), Stec et al , J Chromatog 326 263-280 (1985), LaPlanche et al , Nuc Acid Res 14(22) 9081-9093 (1986), and Fasman, Practical Handbook of Biochemistry and Molecular Biology, 1989. CRC Press, Boca Raton, FL, herem incorporated by reference
  • ohgonucleotides were deprotected following phosphoramidite manufacturer's protocols Unpunfied ohgonucleotides were either dned down under vacuum or precipitated and then dned Sodium salts of ohgonucleotides were prepared usmg the commercially available DNA-Mate (Barkosigan Inc ) reagents or conventional techmques such as commercially available exchange resm, e g , Dowex, or by addition of sodium salts followed by precipitation, diafiltration, or gel filtration, etc A vanety of standard methods were used to punfy and produce the presently descnbed ohgonucleotides In bnef, ohgonucleotides were purified by chromatography on commercially available reverse phase (for example, see the RAININ Instrument Co , Inc instruction manual for the DYNAMAX®-300A, Pure-DNA reverse-phase columns, 1989, or current updates thereof, herem incorporated by reference) or
  • duplexes with the 2'-0-methyl substitutions display a significantly mcreased T m compared to RNA or DNA with a 2' H or 2' OH, respectively RNA or DNA with propyl or fluoro substitutions at the 2' position display an even higher T m than does the 2'-0-methyl
  • a 14 mer heteropolymer was synthesized as a regular phosphodiester DNA (O), a phosphorothioate DNA (S), an unblocked 2 -O-methyl RNA (2'om), a 2 -O-methyl RNA with 3' and 5' butanol blocked ends (B2'om), and a phosphorothioate chimera havmg four 2'-0-methyl phosphorothioate bases on either side of 6 mtenor phosphorothioate DNA bases (SD) They were punfied, desalted, lyophilized, and dissolved at 300 A 260 per ml m sterile water Samples were removed and diluted 1 to 4 with 0 1 N HCI to give a final pH of approximately 1 5, and placed m a heat block at 39 °C Aliquots were taken at the times mdicated and diluted 1 20 mto a solution of 0 025 M NaOH and 0 03 M NaCl, and were run on an analytical
  • the 2'-0-methyl ohgonucleotides are far more stable than the correspondmg phosphodiester, phosphorothioate. or a mixed 2'-0-methyl phosphorothioate structure that Agrawal et al recommended to mcrease bioavailabihty

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Abstract

L'invention concerne des jeux d'échantillons comprenant des oligonucléotides modifiés associés, des procédés permettant de préparer ces jeux d'échantillons, des tests faisant appel à de tels jeu d'échantillons et des assortiments de matériel contenant de tels jeu d'échantillons. Dans une forme d'exécution, ces jeux d'échantillons présentent une affinité de liaison accrue avec des acides nucléiques complémentaires, en particulière avec l'ARN complémentaire. Dans une autre forme d'exécution, les acides nucléiques associés composant le jeu d'échantillon présentent une résistance importante à l'acide ce qui permet de traiter ces jeu d'échantillons avec des solutions à faible pH. Dans une forme d'exécution encore différente, les acides nucléiques associés modifiés du jeu d'échantillons présentent une résistance élevée à la dégradation par les nucléases.
PCT/US2000/013185 1999-05-13 2000-05-11 Jeu d'echantillons comprenant des compositions d'oligonucleotides et de polynucleotides modifies WO2000070093A1 (fr)

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WO2005040419A1 (fr) * 2003-10-14 2005-05-06 Novartis Ag Jeu ordonne de microechantillons oligonucleotidiques
GB2409454B (en) * 2002-10-01 2007-05-23 Nimblegen Systems Inc Microarrays having multiple oligonucleotides in single array features
JP2007521011A (ja) * 2003-07-02 2007-08-02 パーキンエルマー ラス インコーポレイテッド マイクロｒｎａ配列や小さな干渉ｒｎａ配列の標識と検出のための分析方法
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US8183361B2 (en) 2001-07-10 2012-05-22 Lakewood-Amedex, Inc. Oligonucleotide-containing pharmacological compositions and their use
US8188259B2 (en) 2001-07-10 2012-05-29 Lakewood-Amedex, Inc. Oligonucleotide-containing pharmacological compositions and their use
US8916529B2 (en) 2001-07-10 2014-12-23 Lakewood-Amedex, Inc. Oligonucleotide-containing pharmacological compositions and their use
US9567584B2 (en) 2001-07-10 2017-02-14 Lakewood Amedex, Inc. Oligonucleotide—containing pharmacological compositions and their use
GB2409454B (en) * 2002-10-01 2007-05-23 Nimblegen Systems Inc Microarrays having multiple oligonucleotides in single array features
JP2007521011A (ja) * 2003-07-02 2007-08-02 パーキンエルマー ラス インコーポレイテッド マイクロｒｎａ配列や小さな干渉ｒｎａ配列の標識と検出のための分析方法
EP1644533A4 (fr) * 2003-07-02 2007-11-14 Perkinelmer Las Inc Analyse et procede de marquage et de detection de microsequences d'arn et de petites sequences de l'arn d'interference
WO2005040419A1 (fr) * 2003-10-14 2005-05-06 Novartis Ag Jeu ordonne de microechantillons oligonucleotidiques
JP2007512805A (ja) * 2003-10-14 2007-05-24 ノバルティス　アクチエンゲゼルシャフト オリゴヌクレオチドマイクロアレイ

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