Compositions and methods for enhanced sensitivity and specificity of nucleic acid synthesis

Abstract

The present invention relates to nucleic acid inhibitors, compositions and method for enhancing synthesis of nucleic acid molecules. In a preferred aspect, the invention relates to inhibition or control of nucleic acid synthesis, sequencing or amplification. Specifically, the present invention discloses nucleic acids having affinity for polypeptides with polymerase activity for use in such synthesis, amplification or sequencing reactions. The nucleic acid inhibitors are capable of inhibiting nonspecific nucleic acid synthesis under certain conditions (e.g., at ambient temperatures). Thus, in a preferred aspect, the invention relates to “hot start” synthesis of nucleic acid molecules. Accordingly, the invention prevents, reduces or substantially reduces nonspecific nucleic acid synthesis. The invention also relates to kits for synthesizing, amplifying, reverse transcribing or sequencing nucleic acid molecules comprising one or more of the nucleic acid inhibitors or compositions of the invention. The invention also relates to using the inhibitors of the invention to prevent viral replication or treat viral infections in a subject. Thus, the invention relates to therapeutic methods and pharmaceutical compositions using the inhibitors of the invention. The invention thus may be used for in vivo and in vitro inhibition of nucleic acid synthesis and/or inhibition of polymerase activity.

1 - 55 . (canceled) 
     
     
         56 . A method of inhibiting one or more reverse transcriptases comprising: contacting a sample or a cell with one or more nucleic acid molecules which binds or has affinity for one or more reverse transcriptases causing said nucleic acid molecule to inhibit the polymerase activity of said reverse transcriptases. 
     
     
         57 . The method of  claim 56 , wherein said nucleic acid molecule comprises one or more modifications to inhibit or prevent degradation or digestion of said nucleic acid molecule. 
     
     
         58 - 59 . (canceled) 
     
     
         60 . The method of  claim 56 , wherein said reverse transcriptase is selected from the group consisting of M-MLV reverse transcriptase, AMV reverse transcriptase, RSV reverse transcriptase, RAV reverse transcriptase, MAV reverse transcriptase and HIV reverse transcriptase. 
     
     
         61 . The method of  claim 56 , wherein said reverse transcriptase has reduced, substantially reduced or eliminated RNase H activity. 
     
     
         62 . The method of  claim 56 , wherein said nucleic acid molecule comprises one or more modifications so as to be non-extendable. 
     
     
         63 . The method of  claim 62 , wherein said modification is to the 3′-most nucleotide. 
     
     
         64 . The method of  claim 63 , wherein said modification is phosphorylation of the 3′-hydroxyl of the nucleotide. 
     
     
         65 . The method of  claim 56 , wherein said nucleic acid molecule is linear, circular, supercoiled, double stranded with one or more single stranded portions, hairpin structure, or complexed with other molecules such as peptides or proteins. 
     
     
         66 . The method of  claim 56 , wherein said nucleic acid molecule comprises DNA, RNA, DNR/RNA hybrids, double stranded DNA, double stranded RNA or DNA/RNA double stranded molecules. 
     
     
         67 . The method of  claim 56 , wherein said nucleic acid molecule is double-stranded. 
     
     
         68 . The method of  claim 56 , wherein said nucleic acid molecule is a single stranded nucleic acid molecule capable of folding into double stranded form. 
     
     
         69 . The method of  claim 67  or  68 , wherein said nucleic acid molecule comprises one or more internal, 5′ and/or 3′ single stranded portions. 
     
     
         70 . The method of  claim 57 , wherein said modification a phosphorothioate. 
     
     
         71 . The method of  claim 57 , wherein said modification is methylation or alkylation of one or more hydroxyl groups. 
     
     
         72 . The method of  claim 56 , wherein said nucleic acid molecule comprises a 5′- and a 3′-portion, wherein said 3′-portion comprises one or more deoxyribonucleotides or derivatives thereof and said 5′-portion comprises one or more ribonucleotides or derivatives thereof and wherein all or a portion of said 3′-portion is capable of base pairing to all or a portion of said 5′-portion. 
     
     
         73 . The method of  claim 72 , wherein said 5′-portion comprising one or more ribonucleotides forms a 5′-overhang. 
     
     
         74 . The method of  claim 65 , wherein said hairpin structure comprises a series of contiguous ribonucleotides base paired or hybridized with a series of contiguous deoxyribonucoleotides. 
     
     
         75 . The method of  claim 56 , wherein said nucleic acid molecule prevents binding or interaction of said reverse transcriptase with a primer/template. 
     
     
         76 . The method of  claim 56 , wherein said inhibiting is reduced, substantially reduced, inhibited, or eliminated when reaction conditions are changed. 
     
     
         77 . The method of  claim 56 , wherein said binding or affinity of said nucleic acid molecule to said reverse trancriptase is reduced, substantially reduced, inhibited, or eliminated under conditions for nucleic acid synthesis, amplification or sequencing.