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Modified nucleic acid probesRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidModified nucleic acid probes description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060110727, Modified nucleic acid probes. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATION [0001] This application is a continuation-in-part of U.S. Ser. No. 08/440,742 filed May 10, 1995, which is a continuation-in-part of PCT application (designating the United States) SN PCT/US94/12305 filed Oct. 26, 1994, which is a continuation-in-part of U.S. Ser. No. 08/284,064 filed Aug. 2, 1994, which is a continuation-in-part of U.S. Ser. No. 08/143,312 filed Oct. 26, 1993, each of which is incorporated herein by reference in its entirety for all purposes. FIELD OF THE INVENTION [0002] The present invention provides probes comprised of nucleotide analogues immobilized in arrays on solid substrates for analyzing molecular interactions of biological interest, and target nucleic acids comprised of nucleotide analogues. The invention therefore relates to the molecular interaction of polymers immobilized on solid substrates including related chemistry, biology, and medical diagnostic uses. BACKGROUND OF THE INVENTION [0003] The development of very large scale immobilized polymer synthesis (VLSIPS.TM.) technology provides pioneering methods for arranging large numbers of oligonucleotide probes in very small arrays. See, U.S. application Ser. No. 07/805,727 and PCT patent publication Nos. WO 90/15070 and 92/10092, each of which is incorporated herein by reference for all purposes. U.S. patent application Ser. No. 07/082,937, filed Jun. 25, 1993, and incorporated herein for all purposes, describes methods for making arrays of oligonucleotide probes that are used, e.g., to determine the complete sequence of a target nucleic acid and/or to detect the presence of a nucleic acid with a specified sequence. [0004] VLSIPS.TM. technology provides an efficient means for large scale production of miniaturized oligonucleotide arrays for sequencing by hybridization (SBH), diagnostic testing for inherited or somatically acquired genetic diseases, and forensic analysis. Other applications include determination of sequence specificity of nucleic acids, protein-nucleic acid complexes and other polymer-polymer interactions. SUMMARY OF THE INVENTION [0005] The present invention provides arrays of oligonucleotide analogues attached to solid substrates. Oligonucleotide analogues have different hybridization properties than oligonucleotides based upon naturally occurring nucleotides. By incorporating oligonucleotide analogues into the arrays of the invention, hybridization to a target nucleic acid is optimized. [0006] The oligonucleotide analogue arrays have virtually any number of different members, determined largely by the number or variety of compounds to be screened against the array in a given application. In one group of embodiments, the array has from 10 up to 100 oligonucleotide analogue members. In other groups of embodiments, the arrays have between 100 and 10,000 members, and in yet other embodiments the arrays have between 10,000 and 1,000,0000 members. In preferred embodiments, the array will have a density of more than 100 members at known locations per cm.sup.2, or more preferably, more than 1000 members per cm.sup.2. In some embodiments, the arrays have a density of more than 10,000 members per cm.sup.2. [0007] The solid substrate upon which the array is constructed includes any material upon which oligonucleotide analogues are attached in a defined relationship to one another, such as beads, arrays, and slides. Especially preferred oligonucleotide analogues of the array are between about 5 and about 20 nucleotides, nucleotide analogues or a mixture thereof in length. [0008] In one group of embodiments, nucleoside analogues incorporated into the oligonucleotide analogues of the array will have the chemical formula: wherein R.sup.1 and R.sup.2 are independently selected from the group consisting of hydrogen, methyl, hydroxy, alkoxy (e.g., methoxy, ethoxy, propoxy, allyloxy, and propargyloxy), alkylthio, halogen (Fluorine, Chlorine, and Bromine), cyano, and azido, and wherein Y is a heterocyclic moiety, e.g., a base selected from the group consisting of purines, purine analogues, pyrimidines, pyrimidine analogues, universal bases (e.g., 5-nitroindole) or other groups or ring systems capable of forming one or more hydrogen bonds with corresponding moieties on alternate strands within a double- or triple-stranded nucleic acid or nucleic acid analogue, or other groups or ring systems capable of forming nearest-neighbor base-stacking interactions within a double- or triple-stranded complex. In other embodiments, the oligonucleotide analogues are not constructed from nucleosides, but are capable of binding to nucleic acids in solution due to structural similarities between the oligonucleotide analogue and a naturally occurring nucleic acid. An example of such an oligonucleotide analogue is a peptide nucleic acid or polyamide nucleic acid in which bases which hydrogen bond to a nucleic acid are attached to a polyamide backbone. [0009] The present invention also provides target nucleic acids hybridized to oligonucleotide arrays. In the target nucleic acids of the invention, nucleotide analogues are incorporated into the target nucleic acid, altering the hybridization properties of the target nucleic acid to an array of oligonucleotide probes. Typically, the oligonucleotide probe arrays also comprise nucleotide analogues. [0010] The target nucleic acids are typically synthesized by providing a nucleotide analogue as a reagent during the enzymatic copying of a nucleic acid. For instance, nucleotide analogues are incorporated into polynucleic acid analogues using taq polymerase in a PCR reaction. Thus, a nucleic acid containing a sequence to be analyzed is typically amplified in a PCR or RNA amplification procedure with nucleotide analogues, and the resulting target nucleic acid analogue amplicon is hybridized to a nucleic acid analogue array. [0011] Oligonucleotide analogue arrays and target nucleic acids are optionally composed of oligonucleotide analogues which are resistant to hydrolysis or degradation by nuclease enzymes such as RNAase A. This has the advantage of providing the array or target nucleic acid with greater longevity by rendering it resistant to enzymatic degradation. For example, analogues comprising 2'-O-methyloligoribonucleotides are resistant to RNAase A. [0012] Oligonucleotide analogue arrays are optionally arranged into libraries for screening compounds for desired characteristics, such as the ability to bind a specified oligonucleotide analogue, or oligonucleotide analogue-containing structure. The libraries also include oligonucleotide analogue members which form conformationally-restricted probes, such as unimolecular double-stranded probes or unimolecular double-stranded probes which present a third chemical structure of interest. For instance, the array of oligonucleotide analogues optionally include a plurality of different members, each member having the formula: Y-L.sup.1-X.sup.1-L.sup.2-X.sup.2, wherein Y is a solid substrate, X.sup.1 and X.sup.2 are complementary oligonucleotides containing at least one nucleotide analogue, L.sup.1 is a spacer, and L.sup.2 is a linking group having sufficient length such that X.sup.1 and X.sup.2 form a double-stranded oligonucleotide. An array of such members comprise a library of unimolecular double-stranded oligonucleotide analogues. In another embodiment, the members of the array of oligonucleotide are arranged to present a moiety of interest within the oligonucleotide analogue probes of the array. For instance, the arrays are optionally conformationally restricted, having the formula --X.sup.11-Z-X.sup.12, wherein X.sup.11 and X.sup.12 are complementary oligonucleotides or oligonucleotide analogues and Z is a chemical structure comprising the binding site of interest. [0013] Oligonucleotide analogue arrays are synthesized on a solid substrate by a variety of methods, including light-directed chemical coupling, and selectively flowing synthetic reagents over portions of the solid substrate. The solid substrate is prepared for synthesis or attachment of oligonucleotides by treatment with suitable reagents. For example, glass is prepared by treatment with silane reagents. [0014] The present invention provides methods for determining whether a molecule of interest binds members of the oligonucleotide analogue array. For instance, in one embodiment, a target molecule is hybridized to the array and the resulting hybridization pattern is determined. The target molecule includes genomic DNA, cDNA, unspliced RNA, mRNA, and rRNA, nucleic acid analogues, proteins and chemical polymers. The target molecules are optionally amplified prior to being hybridized to the array, e.g., by PCR, LCR, or cloning methods. [0015] The oligonucleotide analogue members of the array used in the above methods are synthesized by any described method for creating arrays. In one embodiment, the oligonucleotide analogue members are attached to the solid substrate, or synthesized on the solid substrate by light-directed very large scale immobilized polymer synthesis, e.g., using photo-removable protecting groups during synthesis. In another embodiment, the oligonucleotide members are attached to the solid substrate by forming a plurality of channels adjacent to the surface of said substrate, placing selected monomers in said channels to synthesize oligonucleotide analogues at predetermined portions of selected regions, wherein the portion of the selected regions comprise oligonucleotide analogues different from oligonucleotide analogues in at least one other of the selected regions, and repeating the steps with the channels formed along a second portion of the selected regions. The solid substrate is any suitable material as described above, including beads, slides, and arrays, each of which is constructed from, e.g., silica, polymers and glass. Definitions [0016] An "Oligonucleotide" is a nucleic acid sequence composed of two or more nucleotides. An oligonucleotide is optionally derived from natural sources, but is often synthesized chemically. It is of any size. An "oligonucleotide analogue" refers to a polymer with two or more monomeric subunits, wherein the subunits have some structural features in common with a naturally occurring oligonucleotide which allow it to hybridize with a naturally occurring oligonucleotide in solution. For instance, structural groups are optionally added to the ribose or base of a nucleoside for incorporation into an oligonucleotide, such as a methyl or allyl group at the 2'-O position on the ribose, or a fluoro group which substitutes for the 2'-O group, or a bromo group on the ribonucleoside base. The phosphodiester linkage, or "sugar-phosphate backbone" of the oligonucleotide analogue is substituted or modified, for instance with methyl phosphonates or O-methyl phosphates. Another example of an oligonucleotide analogue for purposes of this disclosure includes "peptide nucleic acids" in which native or modified nucleic acid bases are attached to a polyamide backbone. Oligonucleotide analogues optionally comprise a mixture of naturally occurring nucleotides and nucleotide analogues. However, an oligonucleotide which is made entirely of naturally occurring nucleotides (i.e., those comprising DNA or RNA), with the exception of a protecting group on the end of the oligonucleotide, such as a protecting group used during standard nucleic acid synthesis is not considered an oligonucleotide analogue for purposes of this invention. [0017] A "nucleoside" is a pentose glycoside in which the aglycone is a heterocyclic base; upon the addition of a phosphate group the compound becomes a nucleotide. The major biological nucleosides are .beta.-glycoside derivatives of D-ribose or D-2-deoxyribose. Nucleotides are phosphate esters of nucleosides which are generally acidic in solution due to the hydroxy groups on the phosphate. The nucleosides of DNA and RNA are connected together via phosphate units attached to the 3' position of one pentose and the 5' position of the-next pentose. Nucleotide analogues and/or nucleoside analogues are molecules with structural similarities to the naturally occurring nucleotides or nucleosides as discussed above in the context of oligonucleotide analogues. [0018] A "nucleic acid reagent" utilized in standard automated oligonucleotide synthesis typically caries a protected phosphate on the 3' hydroxyl of the ribose. Thus, nucleic acid reagents are referred to as nucleotides, nucleotide reagents, nucleoside reagents, nucleoside phosphates, nucleoside-3'-phosphates, nucleoside phosphoramidites, phosphoramidites, nucleoside phosphonates, phosphonates and the like. It is generally understood that nucleotide reagents carry a reactive, or activatible, phosphoryl or phosphonyl moiety in order to form a phosphodiester linkage. [0019] A "protecting group" as used herein, refers to any of the groups which are designed to block one reactive site in a molecule while a chemical reaction is carried out at another reactive site. More particularly, the protecting groups used herein are optionally any of those groups described in Greene, et al., Protective Groups In Organic Chemistry, 2nd Ed., John Wiley & Sons, New York, N.Y., 1991, which is incorporated herein by reference. The proper selection of protecting groups for a particular synthesis is governed by the overall methods employed in the synthesis. For example, in "light-directed" synthesis, discussed herein, the protecting groups are photolabile protecting groups such as NVOC, MeNPoc, and those disclosed in co-pending Application PCT/US93/10162 (filed Oct. 22, 1993), incorporated herein by reference. In other methods, protecting groups are removed by chemical methods and include groups such as FMOC, DMT and others known to those of skill in the art. 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