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 . A composition for inhibiting nucleic acid synthesis, comprising a nucleic acid inhibitor that is capable of binding or has affinity to an enzyme with polymerase activity.  
     
     
         2 . The composition of  claim 1 , wherein said nucleic acid inhibitor forms a hairpin or comprises a double stranded nucleic acid molecule.  
     
     
         3 . The composition of  claim 1 , wherein said binding or affinity of said nucleic acid inhibitor to said enzyme is inhibited, reduced, substantially reduced, or eliminated under conditions for nucleic acid synthesis, amplification or sequencing.  
     
     
         4 . The composition of  claim 1 , wherein said nucleic acid inhibitor is capable of forming a complex with said enzyme.  
     
     
         5 . The composition of  claim 1 , further comprising one or more enzymes having polymerase activity.  
     
     
         6 . The composition of  claim 5 , wherein said enzyme is thermophilic.  
     
     
         7 . The composition of  claim 1 , wherein said nucleic acid inhibitor is denatured or has reduced capacity to inhibit under conditions for nucleic acid synthesis, amplification or sequencing.  
     
     
         8 . The composition of  claim 5 , wherein said enzyme having nucleic acid polymerase activity is selected from the group consisting of a DNA polymerase, an RNA polymerase and a reverse transcriptase.  
     
     
         9 . The composition of  claim 8 , wherein said DNA polymerase is selected from the group consisting of Taq, Tne, Tma, Pfu, VENT™, DEEPVENT™, KOD, Tfl, and Tth DNA polymerases, and mutants, variants and derivatives thereof.  
     
     
         10 . The composition of  claim 8 , wherein said reverse transcriptase is selected from the group consisting of M-MLV reverse transcriptase, RSV reverse transcriptase, AMV reverse transcriptase, RAV reverse transcriptase, MAV reverse transcriptase and HIV reverse transcriptase, and mutants, variants and derivatives thereof.  
     
     
         11 . The composition of  claim 8 , wherein said reverse transcriptase is substantially reduced in RNase H activity.  
     
     
         12 - 29 . (canceled)  
     
     
         30 . A kit for use in synthesis, amplification or sequencing of a nucleic acid molecule, said kit comprising one or more of the nucleic acid inhibitors of  claim 1 .  
     
     
         31 . The kit of  claim 30 , further comprising one or more components selected from the group consisting of one or more nucleotides, one or more DNA polymerases, one of more reverse transcriptases, one or more suitable buffers, one or more primers and one or more terminating agents.  
     
     
         32 - 34 . (canceled).  
     
     
         35 . A method for inhibiting or preventing nucleic acid synthesis, amplification or sequencing comprising: 
 mixing one or more nucleic acid inhibitors of  claim 1  with one or more enzymes having polymerase activity; and    incubating said mixture under conditions sufficient to inhibit or prevent nucleic acid synthesis, amplification and/or sequencing.    
     
     
         36 . An oligonucleotide comprising a 5′- and a 3′-portion, wherein said 3′-portion comprises a series of contiguous deoxyribonucleotides or derivatives thereof and said 5′-portion comprises a series of contiguous 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.  
     
     
         37 . The oligonucleotide according to  claim 36 , wherein said 5′-portion comprising ribonucleotides forms a 5′-overhang.  
     
     
         38 . The oligonucleotide according to  claim 36 , wherein said the 3′-most nucleotide comprises one or more modifications so as to be non-extendable.  
     
     
         39 . The oligonucleotide according to  claim 38 , wherein said modification is phosphorylation of the 3′-hydroxyl of the nucleotide.  
     
     
         40 . The oligonucleotide according to  claim 36 , wherein said, comprising one or more modifications so as to be resistant to one or more nucleases.  
     
     
         41 . The oligonucleotide according to  claim 40 , wherein said modification is a phosphorothioate.  
     
     
         42 . The oligonucleotide according to  claim 40 , wherein said modification is a methylation of a hydroxyl group.  
     
     
         43 . A method of inhibiting a polymerase enzyme within a cell, comprising: 
 introducing into a cell an oligonucleotide, said oligonucleotide comprising a 5′- and a 3′-portion, wherein said 3′-portion comprises a series of contiguous deoxyribonucleotides or derivatives thereof and said 5′-portion comprises a series of contiguous ribonucleotides or derivatives thereof and wherein all or a portion of said 3′-portoin is capable of base pairing to all or a portion of said 5′-portion; and    causing the inhibition of the polymerase with said oligonucleotide.    
     
     
         44 . The method according to  claim 43 , wherein said 5′-portion of said oligonucleotide comprising ribonucleotides forms a 5′-overhang.  
     
     
         45 . The method according to  claim 43 , wherein said polymerase is a reverse transcriptase.  
     
     
         46 . The method according to  claim 45 , wherein said polymerase is HIV reverse transcriptase.  
     
     
         47 . A method of inhibiting replication of a virus, comprising: 
 providing a virus, said virus comprising a reverse transcriptase and requiring activity of the reverse transcriptase for replication;    contacting said reverse transcriptase with an oligonucleotide that inhibits activity of said reverse transcriptase thereby inhibiting replication of said virus.    
     
     
         48 . The method according to  claim 47 , wherein said oligonucleotide comprises a 5′- and a 3′-portion, wherein said 3′-portion comprises a series of contiguous deoxyribonucleotides or derivatives thereof and said 5′-portion comprises a series of contiguous 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.  
     
     
         49 . The method according to  claim 48 , wherein said 5′-portion comprising ribonucleotides forms a 5′-overhang.  
     
     
         50 . The method according to  claim 47 , wherein said virus is a HIV.  
     
     
         51 . The method according to  claim 47 , wherein said contacting comprises introducing said oligonucleotide into a cell.  
     
     
         52 . A method of treating a viral infection in a subject, comprising: 
 administering to said subject a composition comprising an oligonucleotide comprising a 5′- and a 3′-portion, wherein said 3′-portion comprises a series of contiguous deoxyribonucleotides or derivatives thereof and said 5′-portion comprises a series of contiguous 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.    
     
     
         53 . An oligonucleotide which binds or has affinity for one or more reverse transcriptases.  
     
     
         54 . The oligonucleotide of  claim 53 , which comprises one or more ribonucleotides or derivatives thereof and one or more deoxyribonucleotides or derivatives thereof.  
     
     
         55 . The oligonucleotide of  claim 53 , wherein said oligonucleotide is resistant to degradation or digestion.  
     
     
         56 . A method of inhibiting one or more reverse transcriptases comprising: 
 contacting a sample or a cell with one or more oligonucleotides which binds or has affinity for one or more reverse transcriptases causing said oligonucleotides to inhibit the polymerase activity of said reverse transcriptases.    
     
     
         57 . The method of  claim 56 , wherein said oligonucleotide comprises one or more modifications to inhibit or prevent degradation or digestion of said oligonucleotide.  
     
     
         58 . A method of treating a viral infection in a subject comprising: 
 administering to said subject an effective amount of the oligonucleotide of  claim 53;  and    causing said oligonucleotide to inhibit or prevent said viral infection in said subject.    
     
     
         59 . A pharmaceutical composition comprising the oligonucleotide of  claim 53.