Compositions of toehold primer duplexes and methods of use

Abstract

Provided herein are primers and primer systems having improved specificity and kinetics over existing primers, and methods of use thereof.

1 . A partially double-stranded primer comprised of first and second nucleic acid strands arranged into
 (1) one double-stranded target-non-specific region,   (2) one double-stranded target-specific region, and   (3) one single-stranded target-specific region contributed to by the first nucleic acid strand,   wherein the double-stranded target-non-specific region has a standard free energy approximately equal to the standard free energy for the single-stranded target-specific region bound to a target nucleic acid.   
     
     
         2 . The partially double-stranded primer of  claim 1 , wherein the second nucleic acid strand comprises a non-extendable nucleotide at its 3′ end and/or the first nucleic acid strand comprises a non-natural nucleotide at or near the 3′ end of its target-non-specific region. 
     
     
         3 . The partially double-stranded primer of  claim 2 , wherein the non-extendable nucleotide is a non-natural nucleotide or a dideoxy nucleotide, optionally wherein the non-natural nucleotide is iso-C, iso-G or deoxyuridine. 
     
     
         4 . (canceled) 
     
     
         5 . The partially double-stranded primer of  claim 1 , wherein the double-stranded target non-specific region is about 4-21 nucleotides in length and/or the single-stranded target-specific region is about 4-20 nucleotides in length. 
     
     
         6 . (canceled) 
     
     
         7 . The partially double-stranded primer of  claim 1 , wherein the first and second nucleic acid strands are comprised of DNA or RNA. 
     
     
         8 . A system comprising
 a nucleic acid target,   a polymerase, and   a partially double-stranded primer comprising a first and a second nucleic acid strand arranged into
 (1) one double-stranded target-non-specific region, 
 (2) one double-stranded target-specific region, and 
 (3) one single-stranded target-specific region contributed to by the first nucleic acid strand. 
   
     
     
         9 . The system of  claim 8 , wherein the nucleic acid target is a single-stranded. 
     
     
         10 . The system of  claim 8 , wherein the nucleic acid target is DNA or RNA. 
     
     
         11 . The system of  claim 8 , wherein the system comprises a plurality of different partially double-stranded primers. 
     
     
         12 . The system of  claim 8 , wherein the system comprises at least two partially double-stranded primers which together can be used to amplify a region of the nucleic acid target. 
     
     
         13 . The system of  claim 8 , wherein the nucleic acid target comprises repetitive sequence, secondary structure and/or high GC content. 
     
     
         14 . The system of  claim 8 , wherein the nucleic acid target is present in a plurality of different nucleic acid targets, optionally wherein the nucleic acid target is present as a single copy or in low copy in a plurality of different nucleic acid targets. 
     
     
         15 . (canceled) 
     
     
         16 . A method comprising
 contacting a partially double-stranded primer of  claim 1  to a sample, and detecting hybridization of the primer to a target in the sample.   
     
     
         17 . The method of  claim 16 , wherein the partially double-stranded primer is labeled with a detectable moiety, optionally wherein the detectable moiety comprises a fluorophore or a radioisotope. 
     
     
         18 . (canceled) 
     
     
         19 . The method of  claim 16 , wherein the target is present as a single copy in the sample. 
     
     
         20 . A method comprising
 hybridizing a single-stranded target-specific region of a first strand of a partially double-stranded primer of  claim 1  to a nucleic acid target, thereby dissociating the first strand of the primer from a second strand of the primer, and   extending the first strand at its 3′ end, in a target-complementary manner, in the presence of a polymerase.   
     
     
         21 . A method comprising
 performing a nucleic acid synthesis reaction in the presence of a nucleic acid target, a polymerase, and one or more partially double-stranded primers of  claim 1 .   
     
     
         22 . The method of  claim 21 , wherein the nucleic acid synthesis reaction is a nucleic acid amplification reaction, optionally a polymerase chain reaction (PCR), or wherein the nucleic acid synthesis reaction is a transcription reaction, optionally a reverse transcription reaction. 
     
     
         23 .- 26 . (canceled) 
     
     
         27 . A kit comprising
 one or more partially double-stranded primers of  claim 1 , and   one or more nucleic acid synthesis reagents.   
     
     
         28 .- 29 . (canceled) 
     
     
         30 . A kit comprising
 a first single-stranded nucleic acid in a first container, and   a second single-stranded nucleic acid that is complementary to a region of the first single-stranded nucleic acid, in a second container,   wherein, when the first and second single-stranded nucleic acids are hybridized to each other, a partially double-stranded nucleic acid is formed that comprises
 (1) a double-stranded target-non-specific region, 
 (2) a double-stranded target-specific region, and 
 (3) a single-stranded target-specific region contributed to by the first nucleic acid, 
   wherein the first single-stranded nucleic acid comprises a non-natural nucleotide and/or the second single-stranded nucleic acid comprises a non-extendable nucleotide at its 3′ end.   
     
     
         31 .- 33 . (canceled) 
     
     
         34 . A single stranded primer that partially self-hybridizes to form
 (1) one or more double-stranded target-non-specific region,   (2) one or more double-stranded target-specific region,   (3) one or more single-stranded target-specific region, and   (4) one or more hairpin loop regions,   wherein the one or more double-stranded target-non-specific region has a concentration-adjusted standard free energy approximately equal to the concentration-adjusted standard free energy for the one or more single-stranded target-specific region bound to a target nucleic acid.   
     
     
         35 . A method of performing a multiplexed nucleic acid amplification reaction comprising amplifying multiple unique nucleic acid molecules using a primer of  claim 1 .