Method for sequencing and characterizing polymeric biomolecules using aptamers and a method for producing aptamers

Abstract

The present invention relates to methods for sequencing a polymeric biomolecule and methods for structurally characterizing the same comprising using aptamers. In a preferred embodiment of this invention, these methods relate to using the single polymeric biomolecule. The invention also relates to a method for selecting aptamers useful for sequencing nucleic acids and aptamers generated by the method. The invention also provides aptamers that recognize and bind to AMP, dAMP, GMP, dGMP, CMP and dCMP.

We claim:  
     
         1 . A method for sequencing a polymeric biomolecule comprising the steps of separating a terminal monomer from the polymeric biomolecule and detecting the separated terminal monomer using an aptamer.  
     
     
         2 . A method for structurally characterizing a polymeric biomolecule comprising the step of contacting the polymeric biomolecule with an aptamer that specifically detects a monomer of the polymeric biomolecule.  
     
     
         3 . The method according to  claim 1 , wherein a single polymeric biomolecule is sequenced.  
     
     
         4 . The method according to  claim 1  or  2 , wherein the polymeric biomolecule is selected from the group consisting of a polynucleotide, a polysaccharide or a polypeptide.  
     
     
         5 . The method according to  claim 1  or  2 , wherein the aptamer is a single-stranded DNA molecule.  
     
     
         6 . The method according to  claim 1 , wherein the detection step is carried out at a low temperature.  
     
     
         7 . The method according to  claim 2 , wherein the step of contacting the polymeric biomolecule with the monomer is carried out a low temperature.  
     
     
         8 . The method according to  claim 2 , wherein the low temperature is approximately a temperature between less than 10° C. to above freezing point.  
     
     
         9 . The method according to  claim 1 , wherein the sequencing is automated.  
     
     
         10 . The method according to  claim 1  or  2 , wherein a label is attached to the aptamer.  
     
     
         11 . The method according to  claim 1  or  2 , wherein the method further comprises the contacting the aptamer with a second factor which is labeled.  
     
     
         12 . The method according to  claim 10  or  11 , wherein the label is an optically detectable species.  
     
     
         13 . The method according to  claim 1 , wherein the polymeric biomolecule is a deoxyribose nucleic acid and the separation step comprises the use of an exonuclease.  
     
     
         14 . The method according to  claim 1 , wherein the polymeric biomolecule is a polysaccharide and the separation step comprises the use of a mixture of exoglycosidases.  
     
     
         15 . The method according to  claim 1 , wherein the polymeric biomolecule is a polypeptide and the separation step comprises the use of a carboxy exopeptidase.  
     
     
         16 . The method according to  claim 1 , wherein the separated terminal monomer is deposited onto a surface.  
     
     
         17 . The method according to  claim 16 , wherein the surface is passivated against non-specific adsorption of the recognition molecules.  
     
     
         18 . The method according to  claim 16 , wherein the surface is patterned into regions of differing hydrophilicity to restrict area onto which the terminal monomer is deposited.  
     
     
         19 . A method for producing an aptamer for recognizing a target monomer comprising the steps of (1) separating the aptamer from a mixture of aptamers by subjecting the mixture of aptamers to an affinity system comprising the target monomer at low temperature, (2) amplifying the aptamer that bound to the affinity system, and (3) repeating the separation and amplification steps until the aptamer having the desired affinity and selectivity for the target monomer is obtained.  
     
     
         20 . The method according to  claim 19 , wherein the low temperature is approximately a temperature between less than 10° C. to above freezing point.  
     
     
         21 . The method according to  claim 19  and  20 , wherein the target monomer is a ribonucleotide or deoxyribonucleotide.  
     
     
         22 . A method for producing an aptamer for recognizing a target nucleotide or a target nucleoside comprising the steps of separating the aptamer from a mixture of aptamers using an affinity system, wherein the affinity system comprises the target nucleotide attached to a solid support through the 5′-carbon of the sugar ring of the target nucleotide and amplifying the aptamers bound to the target by polymerase chain reaction (PCR).  
     
     
         23 . The method according to  claim 22 , wherein the aptamers are amplified using primers that are labeled.  
     
     
         24 . The method according to  claim 22 , wherein the aptamers are labeled with fluorescent dye.  
     
     
         25 . The method according to  claim 22 , wherein the target nucleotide is attached to the solid support through the Hoogsteen on the 5′ carbon on the sugar ring.  
     
     
         26 . The method according to  claim 22 , wherein the separation step is conducted at a low temperature.  
     
     
         27 . The method according to  claim 26 , wherein the low temperature is approximately a temperature between less than 10° C. to above freezing point.  
     
     
         28 . An aptamer produced according to the method of  claim 19  or  22 .  
     
     
         29 . A single-stranded nucleic acid molecule comprising a DNA sequence 
 5′-CGGRGGAGGNACGGRGGAG-3′ (SEQ ID NO: 1),    wherein R is G or A and N is any one of G, A, T or C.    
     
     
         30 . The single-stranded nucleic acid molecule according to  claim 29 , comprising a DNA sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36.  
     
     
         31 . A single-stranded nucleic acid molecule comprising a DNA sequence 
 5′-GGGAGGGTN 1 N 2 N 3 GGN 4 G-3′ (SEQ ID NO: 2),    wherein N 1 , N 2 , N 3 , and N 4  is any monomer selected from the group consisting of A, C, G and T.    
     
     
         32 . The single-stranded nucleic acid molecule according to  claim 31 , comprising a DNA sequence selected from the group consisting of SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 67; SEQ ID NO: 75 and SEQ ID NO: 76.  
     
     
         33 . The single-stranded nucleic acid molecule according to  claim 31 , wherein N 4  is T or C.  
     
     
         34 . A single-stranded nucleic acid molecule comprising a DNA sequence 
 5′-GGT N 1 N 2 N 3 GGN 4 G-3′ (SEQ ID NO: 3)    wherein N 1 , N 2 , N 3 , and N 4  is any monomer selected from the group consisting of A, C, G and T.    
     
     
         35 . The single-stranded nucleic acid molecule according to  claim 34 , comprising a DNA sequence selected from the group consisting of SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72; SEQ ID NO: 73 and SEQ ID NO: 74.  
     
     
         36 . A single-stranded nucleic acid molecule comprising a DNA sequence 
 5′-TGGGN 1 TGGGN 2 N 3 TGGGN 4 AGGGT-3′ (SEQ ID NO: 4 or SEQ ID NO: 90),    wherein N 1 , N 2 , and N 4  is any monomer selected from the group consisting of A, C, G and T and N 3  is no momomer or any monomer selected from the group consisting of A, C, G and T.    
     
     
         37 . The single-stranded nucleic acid molecule according to  claim 36 , comprising a DNA sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 42; SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57.  
     
     
         38 . A single-stranded nucleic acid molecule comprising a DNA sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45; SEQ ID NO: 48; SEQ ID NO: 50, SEQ ID NO: 51, SEQ IS NO: 52, SEQ ID NO: 53, SEQ ID NO: 58, SEQ ID NO: 65, SEQ ID NO: 68, SEQ ID NO: 69.  
     
     
         39 . The nucleic acid molecule according to claims  29 - 38  that is not more than 120 nucleotides in length.  
     
     
         40 . The nucleic acid molecule according to  claim 39  that is not more than 50 nucleotides in length.  
     
     
         41 . The nucleic acid molecule according to  claim 29 , wherein upstream of the DNA sequence is a 5′ flanking region comprising the DNA sequence 5′-CCTACT-3′ and downstream of the DNA sequence is a 3′ flanking region comprising the DNA sequence 5′-AGTAGG-3′.  
     
     
         42 . The nucleic acid molecule according to  claim 29 , wherein upstream of the DNA sequence is a 5′ flanking region comprising the DNA sequence 5′-AGATG-3′ and downstream of the DNA sequence is a 3′ flanking region comprising the DNA sequence 5′-CATCG-3′.  
     
     
         44 . The nucleic acid molecule according to  claim 42 , wherein the 5′ flanking region is 5′-GCCTCATGTCGAACCTACTGGA-3′ (SEQ ID NO: 77) and the 3′ flanking region is 5′-GGAAGTAGGTGAGGGAG-3′ (SEQ ID NO: 78).  
     
     
         45 . The nucleic acid molecule according to  claim 31 , wherein upstream of the DNA sequence is a 5′ flanking region comprising the DNA sequence 5′-TCATGTCGAAGGGGCGTATGGGCTTTG-3′ (SEQ ID NO: 79) and downstream of the DNA sequence is a 3′ flanking region comprising the DNA sequence 5′-ACATGT-3′.  
     
     
         46 . The nucleic acid molecule according to  claim 31 , wherein upstream of the DNA sequence is a 5′ flanking region comprising the DNA sequence TGATCCGCGGCAGTGC-3′ (SEQ ID NO: 80) and downstream of the DNA sequence is a 3′ flanking region comprising the DNA sequence 5′-TGCTTGGAGCAATGGCGATGACGGATC-3′ (SEQ ID NO: 81).  
     
     
         47 . The nucleic acid molecule according to  claim 36 , wherein upstream of the DNA sequence is a 5′ flanking region comprising the DNA sequence 5′-AGTGACACCAC-3′ (SEQ ID NO: 82) and downstream of the DNA sequence is a 3′ flanking region comprising the DNA sequence 5′-TGTGGAATCAC-3′ (SEQ ID NO: 83).  
     
     
         48 . The nucleic acid molecule according to  claim 36 , wherein upstream of the DNA sequence is a 5′ flanking region comprising the DNA sequence 5′-AGATCGCCATAAG-3′ (SEQ ID NO: 84) and downstream of the DNA sequence is a 3′ flanking region comprising the DNA sequence 5′-GGAGCAATGGCGAT-3′ (SEQ ID NO: 85).  
     
     
         49 . The nucleic acid molecule according to claims  29 ,  31 ,  34  and  36 , wherein one or more of the phosphodiester linkages between the nucleotides have been replaced with a linkage that increases the stability of the nucleic acid molecule.  
     
     
         50 . The nucleic acid molecule according to  claim 29  that recognizes and binds to a nucleotide selected from the group consisting of an AMP or a dAMP.  
     
     
         51 . The nucleic acid molecule according to  claim 31  or  34  that recognizes and binds to a nucleotide selected from the group consisting of an CMP or a dCMP.  
     
     
         53 . The nucleic acid molecule according to  claim 36  that recognizes and binds to a nucleotide selected from the group consisting of a GMP or a dGMP.  
     
     
         54 . The nucleic acid molecule according to  claim 38 , wherein the DNA sequence is SEQ ID NO: 17 and wherein the nucleic acid molecule recognizes and binds to a nucleotide selected from the group consisting of an AMP or dAMP.  
     
     
         55 . The nucleic acid molecule according to  claim 38 , wherein the DNA sequence is selected from the group consisting of SEQ ID NO: 65, SEQ ID NO: 68 and SEQ ID NO: 69 and wherein the nucleic acid molecule recognizes and binds to a nucleotide selected from the group consisting of an CMP or dCMP.  
     
     
         56 . The nucleic acid molecule according to  claim 38 , wherein the DNA sequence is selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45; SEQ ID NO: 48; SEQ ID NO: 50, SEQ ID NO: 51, SEQ IS NO: 52, SEQ ID NO: 53 and SEQ ID NO: 58, and wherein the nucleic acid molecule recognizes and binds to a nucleotide selected from the group consisting of an GMP or dGMP.  
     
     
         57 . The nucleic acid molecule according to any one of claims  48 - 56 , wherein equilibrium dissociation constant of the binding of the nucleic acid molecule to the nucleotide is one hundred micromolar to submicromolar.  
     
     
         58 . The nucleic acid molecule according to  claim 57 , wherein the equilibrium dissociation constant of the binding of the nucleic acid molecule to the nucleotide is less than 3 μM.