Ultraspecific Nucleic Acid Sensors for Low-Cost Liquid Biopsies

Abstract

Ultraspecific, programmable nucleic acid sensors capable of detecting and preferentially amplifying target DNA molecules comprising a particular SNP or mutation are provided. In some cases, the ultraspecific programmable nucleic acid sensors are useful for detecting SNP-containing DNA molecules indicative of cancer such as cell-free DNA circulating in the blood or indicative of organ transplant rejection Also provided are methods for construction of such ultraspecific nucleic acid sensors and methods for preferential amplification of target DNA molecules containing a mutation of interest, as well as testing systems for early cancer screening and routine monitoring of circulating cancer DNA using liquid biological samples such as serum, plasma, or saliva.

1 . A method for detecting a single nucleotide polymorphism (SNP)-containing DNA molecule in a sample, the method comprising:
 (a) contacting an ultraspecific riboregulator to a sample, wherein the riboregulator is a synthetic nucleic acid molecule comprising a docking site, a toehold domain, and a hairpin domain, the hairpin domain comprising a fully or partially double-stranded stem domain and a loop domain, wherein the stem domain is complementary to a SNP-containing nucleic acid molecule, whereby, if present in the sample, the SNP-containing nucleic acid molecule will bind to and form a complex with the ultraspecific riboregulator, leaving a 3′ region of the riboregulator in position to produce a reporter protein; and   (b) performing an isothermal amplification to selectively amplify the SNP-containing nucleic acid molecule using a DNA polymerase and monomers.   
     
     
         2 . The method of  claim 1 , wherein the SNP-containing nucleic acid molecule is amplified using a SNP-specific forward primer, a SNP-specific blocking strand, a conventional reverse primer, a DNA polymerase, and monomers. 
     
     
         3 . The method of  claim 1 , wherein the sample is a biological sample. 
     
     
         4 . The method of  claim 3 , wherein the biological sample is a liquid sample. 
     
     
         5 . The method of  claim 1 , wherein detecting said SNP-containing nucleic acid molecule is a positive or negative indicator of a disease-associated SNP in said sample. 
     
     
         6 . The method of  claim 1 , wherein the toehold domain is 2-6 nucleotides in length. 
     
     
         7 . A method for preferential amplification of a target DNA molecule comprising a mutation, wherein the method comprises
 (a) contacting an oligonucleotide primer molecule and an oligonucleotide displacer molecule to a sample comprising a mutant DNA template, wherein the oligonucleotide primer molecule comprises a short forward toehold domain and a branch migration region at its 3′ end, wherein the oligonucleotide displacer molecule comprises a short reverse toehold domain and a branch migration region at its 5′ end, whereby the oligonucleotide primer and oligonucleotide displacer molecules associate with the mutant DNA template; and   (b) contacting a DNA polymerase and nucleotide monomers to the contacted sample of (a),   wherein, in the presence of the mutant DNA template, the oligonucleotide primer completes a branch migration reaction to permit extension by the DNA polymerase and amplification of the mutant DNA template, and the oligonucleotide displacer completes a branch migration reaction that prevents extension by the DNA polymerase and amplification of a wild-type, non-mutated DNA template.   
     
     
         8 . The method of  claim 7 , further comprising (c) repeating steps (a) and (b) along with a conventional amplification primer in the reverse direction until a desired degree of amplification of the target DNA molecule comprising the mutation is achieved. 
     
     
         9 . The method of  claim 7 , wherein the oligonucleotide primer molecule further comprises a docking site at its 5′ end, and wherein the oligonucleotide displacer molecule comprises a docking site at its 3′ end. 
     
     
         10 . The method of  claim 7 , wherein the sample is a biological sample. 
     
     
         11 . The method of  claim 10 , wherein the biological sample is a liquid sample. 
     
     
         12 . The method of  claim 7 , wherein detecting said target DNA molecule comprising a mutation is a positive or negative indicator of a disease-associated mutation in said sample. 
     
     
         13 .- 27 . (canceled) 
     
     
         28 . A method for preferential amplification of a target DNA molecule comprising a SNP mutation, wherein the method comprises:
 (a) contacting a SNP-specific intramolecular competitive primer to a sample comprising a target DNA molecule, wherein the SNP-specific intramolecular competitive primer comprises (i) a 5′ wild-type targeting hairpin comprising a fully or partially double-stranded stem domain, a loop domain, and a short toehold domain which is three to four nucleotides in length, and (ii) a 3′ mutant targeting hairpin comprising a fully or partially double-stranded stem domain complementary to a target DNA molecule comprising a SNP mutation, a loop domain, and a short toehold domain, wherein the wild-type targeting hairpin and the mutant targeting hairpin are separated by a spacer domain, and whereby the SNP-specific intramolecular competitive primer associates with the target DNA molecule; and   (b) extending the mutant targeting hairpin of the SNP-specific intramolecular competitive primer along the target DNA molecule with a DNA polymerase molecule and monomers to be incorporated into the extended mutant targeting hairpin, using the target DNA molecule as a template,   wherein, in the presence of the target DNA molecule comprising the SNP mutation, the mutant targeting hairpin binds to the DNA template, leaving the 3′ end in position to prime polymerization by the DNA polymerase molecule, and   wherein, in the presence of the target DNA molecule not containing the SNP mutation, the wild-type targeting hairpin binds to the DNA template and prevents primer polymerization by blocking its 3′ end from binding to the DNA template.   
     
     
         29 . The method of  claim 28 , further comprising (c) repeating steps (a) and (b) along with a conventional amplification primer in the reverse direction until a desired degree of amplification of the target DNA molecule comprising a mutation is achieved. 
     
     
         30 . The method of  claim 28 , wherein the short toehold domain is 3 nucleotides in length. 
     
     
         31 . The method of  claim 28 , wherein the short toehold domain is 4 nucleotides in length. 
     
     
         32 . The method of  claim 28 , wherein the sample is a biological sample. 
     
     
         33 . The method of  claim 32 , wherein the biological sample is a liquid sample. 
     
     
         34 . The method of  claim 28 , wherein detecting said target DNA molecule comprising a mutation is a positive or negative indicator of a disease-associated mutation in said sample.