Methods For Using Mutant RNA Polymerases Wtih Reduced Discrimination Between Non-Canonical Nucleoside Triphosphates

Abstract

A method for synthesizing a nucleic acid molecule comprising at least one non-canonical nucleoside triphosphate using a mutant polymerase having a reduced discrimination between canonical and non-canonical substrates is disclosed. The method comprises incubating a template nucleic acid in a reaction mixture comprising the mutant nucleic acid polymerase and the appropriate canonical and non-canonical nucleoside triphosphates which are desired substrates for the mutant nucleic acid polymerase. The present invention is also a method of determining the sequence of a nucleic acid molecule using the mutant polymerase to create a nucleic acid molecule comprising at least one non-canonical nucleoside triphosphate.

1 . A method for synthesizing a nucleic acid molecule comprising at least one non-canonical nucleotide, comprising the steps of:
 a) incubating a template nucleic acid in a reaction mixture under nucleic acid synthesis conditions containing
 (i) a mutant RNA polymerase exhibiting an amino acid selected from the group consisting of phenylalanine, methionine and leucine in place of tyrosine (Y) in the conserved wild-type motif having a consensus sequence consisting of
 . . . K-(7 amino acids)-YG . . . 
 
 wherein the mutant RNA polymerase has a reduced discrimination between canonical and non-canonical nucleoside triphosphates, and 
 (ii) at least one non-canonical nucleoside triphosphate, and 
   b) obtaining the synthesis of a nucleic acid molecule comprising at least one non-canonical nucleotide.   
     
     
         2 . The method of  claim 1  wherein the template nucleic acid is DNA. 
     
     
         3 . The method of  claim 1  wherein the template nucleic acid is RNA. 
     
     
         4 . The method of  claim 1  wherein the nucleic acid molecule comprising the at least one non-canonical nucleotide is synthesized by extension of a primer molecule, at least part of which is sufficiently complementary to a portion of the template to hybridize therewith. 
     
     
         5 . The method of  claim 1  wherein the nucleic acid molecule comprising the at least one non-canonical nucleotide is synthesized de novo without using a primer molecule. 
     
     
         6 . The method of  claim 1  wherein the at least one non-canonical nucleoside triphosphate is selected from the group consisting of a 2′-deoxy nucleoside triphosphate, a 2′,3′-dideoxy nucleoside triphosphate and a nucleoside triphosphate that has a fluorine or an amino group on the 2′-position of the sugar. 
     
     
         7 . The method of  claim 1  wherein the synthesized nucleic acid molecule is selected from the group consisting of a ribozyme, a nucleic acid molecule for gene therapy, a nucleic acid molecule for use in a vaccine, a nucleic acid molecule for use as an antiviral, a nucleic acid molecule for use as an antimicrobial, a nucleic acid molecule for use as an antisense composition for regulating gene expression, a nucleic acid molecule for use in a composition for hybridization to a complementary nucleic acid and a nucleic acid molecule for use as primer or a probe for detection of a complementary nucleic acid. 
     
     
         8 . The method of  claim 1  wherein the synthesized nucleic acid molecule is single-stranded. 
     
     
         9 . The method of  claim 1  wherein the method is part of an amplification reaction selected from the group consisting of NASBA, 3SR and TMA. 
     
     
         10 . A kit for performing the method of  claim 1  comprising:
 (i) the mutant RNA polymerase exhibiting an amino acid selected from the group consisting of phenylalanine, methionine, and leucine in place of tyrosine (Y) in the conserved wild-type motif having a consensus sequence consisting of
 . . . K-(7 amino acids)-YG . . . 
   wherein the mutant RNA polymerase has a reduced discrimination between canonical and non-canonical nucleoside triphosphate substrates; and   (ii) data or information describing conditions under which the method of  claim 1  may be performed.   
     
     
         11 - 40 . (canceled)