Stoichiometric tuning of nucleic acid hybridization probes by auxiliary oligonucleotide species

Abstract

This invention describes a method of controlling the hybridization yield of nucleic acid probes by adjusting the relative concentrations of auxiliary oligonucleotides to the probes and the targets. The auxiliary oligonucleotide is partially or fully complementary to either the probe or the target, and is released upon hybridization of the probe to the target.

What is claimed is: 
     
         1 . A method for providing a nucleic acid probe for selective capture or enrichment of a nucleic acid molecule bearing a target nucleic acid sequence with a desired yield, comprising in order:
 contacting a first sample containing the nucleic acid molecule bearing the target nucleic acid sequence with a test solution comprising the nucleic acid probe at a temperature and a buffer condition conducive to hybridization of the target nucleic acid sequence to the nucleic acid probe, wherein the nucleic acid probe comprises a first concentration of a first nucleic acid molecule and a second concentration of a second nucleic acid molecule, wherein the second concentration is greater than the first concentration, wherein the first nucleic acid molecule comprises a first probe subsequence and a second probe subsequence, wherein the first probe subsequence is complementary to a first target subsequence of the nucleic acid molecule and the second probe subsequence is complementary to a second target subsequence of the nucleic acid molecule, wherein at least a portion of the target nucleic acid sequence is contained within the first target subsequence, and wherein the second nucleic acid molecule comprises a third probe subsequence that is complementary to at least a subsequence of the first probe subsequence;   determining an experimental yield, wherein the experimental yield is the proportion of the target nucleic acid sequence in the first sample that is hybridized to the first nucleic acid molecule;   determining a third concentration of the second nucleic acid molecule, wherein the third concentration [P]′ 0  is determined by Equation 1, wherein [P] 0  is the second concentration, wherein χ 1  is the experimental yield, and wherein χ 2  is the desired yield, wherein the desired yield is the desired proportion of the target nucleic acid sequence that is hybridized to the first nucleic acid molecule; and   providing instructions to use the third concentration of the second nucleic acid molecule for preparation of the nucleic acid probe or preparing the nucleic acid probe with the second nucleic acid molecule at the third concentration.   
     
     
         2 . The method of  claim 1 , wherein the first nucleic acid molecule further comprises a fourth probe subsequence that is not complementary to the target nucleic acid sequence nor is complementary to any sequence on the nucleic acid molecule within 30 nucleotides of the target nucleic acid sequence, and the second nucleic acid molecule further comprises a fifth probe subsequence that is at least 80% complementary to the fourth subsequence. 
     
     
         3 . The method of  claim 1 , wherein the first nucleic acid molecule is a DNA oligonucleotide. 
     
     
         4 . The method of  claim 1 , wherein the first nucleic acid molecule comprises deoxyuridines, RNA nucleotides, or a photocleavable linker moiety. 
     
     
         5 . The method of  claim 1 , wherein the second nucleic acid molecule is a DNA oligonucleotide. 
     
     
         6 . The method of  claim 1 , wherein the second concentration is between 1.1 and 10,000 times the first concentration. 
     
     
         7 . The method of  claim 1 , wherein the first nucleic acid molecule further comprises a functional moiety capable of interacting with a binding partner, wherein the step of determining the experimental yield is performed by capturing the first nucleic acid molecule through interaction of the functional moiety and binding partner. 
     
     
         8 . The method of  claim 1  wherein prior to determining the experimental yield, capturing nucleic acid molecules hybridized to the first nucleic acid molecule through solid-phase separation. 
     
     
         9 . The method of  claim 8  wherein prior to determining the experimental yield, selectively degrading the first nucleic acid molecule after capturing through solid-phase separation. 
     
     
         10 . The method of  claim 9 , wherein the selective degradation of the first nucleic acid molecule is through a nuclease. 
     
     
         11 . The method of  claim 9  wherein the first nucleic acid molecule comprises a photocleavable linker moiety. 
     
     
         12 . The method of  claim 11 , wherein the selective degradation of the first nucleic acid molecule is through illumination by light of a wavelength sufficient to cleave the photocleavable linker moiety. 
     
     
         13 . A method for selective capture or enrichment of a nucleic acid molecule bearing a target nucleic acid sequence with a desired yield comprising contacting a sample containing the nucleic acid molecule bearing the target nucleic acid sequence with the nucleic acid probe of  claim 1  or  2 , wherein the second nucleic acid molecule of the nucleic acid probe of  claim 1  or  2  is at the third concentration. 
     
     
         14 . A nucleic acid probe composition for selectively capturing or enriching a nucleic acid molecule bearing a target nucleic acid sequence comprising a first concentration of a first nucleic acid molecule and a second concentration of a second nucleic acid molecule, wherein the first nucleic acid molecule comprises a first probe subsequence and a second probe subsequence, wherein the first probe subsequence is complementary to a first target subsequence of the nucleic acid molecule and the second probe subsequence is complementary to a second target subsequence of the nucleic acid molecule, wherein at least a portion of the target nucleic acid sequence is included in the first target subsequence, and wherein the second nucleic acid molecule comprises a third probe subsequence and a fourth probe subsequence, wherein the third probe subsequence is complementary to the first target subsequence or a subsequence contained within the first target subsequence, and the fourth probe subsequence is complementary to a third target subsequence, wherein the third target subsequence is separate from the first and second target subsequences, but is within 30 nucleotides of the first or second target subsequences. 
     
     
         15 . The composition of  claim 14 , wherein the first nucleic acid molecule further comprises a functional moiety capable of interacting with a binding partner 
     
     
         16 . The composition of  claim 14 , wherein the second concentration is between 0.001 and 1,000 times the first concentration. 
     
     
         17 . The composition of  claim 14 , wherein the first nucleic acid molecule is a DNA oligonucleotide. 
     
     
         18 . The composition of  claim 14 , wherein the first nucleic acid molecule comprises deoxyuridines, RNA nucleotides, or a photocleavable linker moiety. 
     
     
         19 . The composition of  claim 14 , wherein the second nucleic acid molecule is a DNA oligonucleotide.