Fine-tuned ultraspecific nucleic acid hybridization probes

Abstract

Compositions and methods for highly specific nucleic acid probes and primers are provided. The probe system comprises a complement strand and a protector stand that form a partially double-stranded probe. The reaction standard free energy of hybridization between the probe and target nucleic acid as determined by Expression 1 (ΔG°rxn=ΔG°t-TC−ΔG°nh-PC+(ΔG°v-TC−ΔG°h-PC)) is from about −4 kcal/mol to about +4 kcal/mol. Alternatively, the reaction standard free energy of hybridization between the probe and target nucleic acid is determined by Expression 1 to be within 5 kcal/mol of the standard free energy as determined by Expression 2 (−Rτln(([P]0−[C]0)/[C]0)]), where the [P]0 term of Expression 2 equals the concentration of the protector strand and the [C]0 term of Expression 2 equals the concentration of the complement strand. In addition, a method for on-the-fly fine tuning of a reaction using the present probe is provided.

What is claimed is: 
     
         1 . A composition for selective interaction with a target nucleic acid molecule, the composition comprising:
 a first concentration of a first nucleic acid strand comprising a first region, second region and third region;   a second concentration of a second nucleic acid strand comprising a fourth region and fifth region, wherein the target nucleic acid molecule comprises a sixth region and a seventh region;   wherein the first and second concentrations are such that an interaction between the composition and the target nucleic acid molecule possesses a standard free energy as determined by Expression 1 within 5 kcal/mol of a standard free energy as determined by Expression 2, where the [P] 0  term of Expression 2 equals the second concentration and the [C] 0  term of Expression 2 equals the first concentration; and   wherein the ΔG° t-TC  term of Expression 1 represents the standard free energy of hybridization between the sixth region and the first region, wherein the ΔG° nh-PC  term of Expression 1 represents the standard free energy of hybridization between the fifth region and the third region, wherein the ΔG° v-TC  term of Expression 1 represents the standard free energy of hybridization between the seventh region and the second region, and wherein the ΔG° h-PC  term of Expression 1 represents the standard free energy of hybridization between the fourth region and the second region.   
     
     
         2 . The composition of  claim 1  further comprising a label conjugated to the first nucleic acid strand and wherein the label is selected from the group consisting of organic fluorophores, haptens, nanoparticles, and radioisotopes. 
     
     
         3 . The composition of  claim 2  further comprising a label conjugated to the second nucleic acid strand and wherein the label is selected from the group consisting of organic fluorophores, haptens, nanoparticles, and radioisotopes, and wherein Expression 3 is substituted for Expression 1. 
     
     
         4 . The composition of  claim 1  wherein the values for ΔG° t-TC  and ΔG° nh-PC  differ by more than 10%, or differ by more than 1 kcal/mol. 
     
     
         5 . The composition of  claim 1  wherein the value of ΔG° t-TC −ΔG° nh-PC  is not between −1 kcal/mol and +1 kcal/mol. 
     
     
         6 . The composition of  claim 1  wherein ΔG° t-TC  is from about −2 kcal/mol to about −16 kcal/mol. 
     
     
         7 . The composition of  claim 1  wherein the first nucleic acid strand and the second nucleic acid strand form a partially double-stranded nucleic acid probe or primer. 
     
     
         8 . The composition of claim  27  further comprising an excess of the second nucleic acid strand. 
     
     
         9 . The composition of  claim 1  wherein fewer than 50% of the nucleotides in the first region are in a double-stranded state in the evaluated minimum free energy structure, as computed in the operational temperature and salinity conditions. 
     
     
         10 . A process comprising the steps of:
 selecting a target nucleotide sequence in a nucleic acid molecule, the target nucleotide sequence comprising a sixth nucleotide subsequence, a seventh nucleotide subsequence, and an eighth nucleotide subsequence;   selecting a first nucleotide sequence comprising a first nucleotide subsequence, a second nucleotide subsequence, and a third nucleotide subsequence;   selecting a second nucleotide sequence comprising a fourth nucleotide subsequence and a fifth nucleotide subsequence;   calculating a standard free energy using Expression 1, wherein the ΔG° t-TC  term of Expression 1 represents the standard free energy of hybridization between the sixth region and the first region, wherein the ΔG° nh-PC  term of Expression 1 represents the free energy of hybridization between the fifth region and the third region, wherein the ΔG° v-TC  term of Expression 1 represents the standard free energy of hybridization between the seventh region and the second region, and wherein the ΔG° h-PC  term of Expression 1 represents the standard free energy of hybridization between the fourth region and the second region; and   determining if the standard free energy calculated using Expression 1 meets a predetermined condition; and   synthesizing a first nucleic acid strand comprising the first nucleotide sequence and a second nucleic acid strand comprising the second nucleotide sequence if the predetermined condition is met.   
     
     
         11 . The process of  claim 10  further comprising the step of calculating a standard free energy using Expression 2, wherein the predetermined condition comprises being within 5 kcal/mol of the standard free energy calculated using Expression 2, and wherein the terms [C] 0  and [P] 0  of Expression 2 represent a predetermined concentration of the first nucleic acid strand and the second nucleic acid strand, respectively. 
     
     
         12 . The process of  claim 11  wherein in the instance the standard free energy as determined by Expression 1 is not within 5 kcal/mol of the standard free energy as determined by Expression 2, then the process is repeated at least in part until the standard free energy as determined by Expression 1 is within 5 kcal/mol of the standard free energy as determined by Expression 2 such that the process further comprises at least one of the following steps:
 (1) selecting a new set of nucleotide sequences and subsequences; and 
 (2) modifying the predetermined concentration of at least one of the first nucleic acid strand or the second nucleic acid strand. 
 
     
     
         13 . The process of  claim 10  wherein the predetermined condition comprises the standard free energy calculated by Expression 1 being from about −4 kcal/mol to about +4 kcal/mol. 
     
     
         14 . A composition for selective interaction with a target nucleic acid molecule, the composition comprising:
 a first nucleic acid strand comprising a first region, a second region, and a third region, wherein said first region possesses a nucleotide sequence that is complementary to a nucleotide sequence of a sixth region of the target nucleic acid molecule, wherein the second region possesses a nucleotide sequence that is complementary to a nucleotide sequence of a seventh region of the target nucleic acid molecule; and   a second nucleic acid strand comprising a fourth region and a fifth region, wherein the fourth region possesses a nucleotide sequence that is complementary to the nucleotide sequence of the second region, and wherein the fifth region possesses a nucleotide sequence that is complementary to the nucleotide sequence of the third region;   wherein the composition possesses a standard free energy of hybridization with the target nucleic acid molecule from about −4 kcal/mol to about +4 kcal/mol, wherein the standard free energy of hybridization is determined by Expression 1, wherein the ΔG° t-TC  term of Expression 1 represents the standard free energy of hybridization between the first region and the sixth region, wherein the ΔG° nh-PC  term of Expression 1 represents the free energy of hybridization between the third region and the fifth region, wherein the ΔG° v-TC  term of Expression 1 represents the standard free energy of hybridization between the seventh region and the second region, and wherein the ΔG° h-PC  term of Expression 1 represents the standard free energy of hybridization between the fourth region and the second region.   
     
     
         15 . The composition of  claim 14  comprising a first concentration of the first nucleic acid strand and a second concentration of the second nucleic acid strand, wherein the standard free energy of hybridization of the composition with respect to the target nucleic acid molecule as determined by Expression 1 is not from about −4 kcal/mol to about +4 kcal/mol, but instead is within 3 kcal/mol of a standard free energy of hybridization as determined by Expression 2, wherein the terms [C] 0  and [P] 0  of Expression 2 represent the first concentration and the second concentration, respectively. 
     
     
         16 . The composition of  claim 14  wherein ΔG° t-TC  is from about −5 kcal/mol to about −15 kcal/mol. 
     
     
         17 . The composition of  claim 14  wherein the first nucleic acid strand and the second nucleic acid strand form a partially double-stranded nucleic acid molecule, and wherein the first region possesses no secondary structure. 
     
     
         18 . The composition of  claim 14  wherein the sum of the standard free energy of hybridization between the first region and the sixth region and between the second region and the seventh region (ΔG° t-TC +ΔG° v-TC ) is more negative than −15 kcal/mol. 
     
     
         19 . The composition of  claim 14  wherein the sum of the standard free energy of hybridization between the third region and the fifth region and between the between the fourth region and the second region (ΔG° nh-PC +ΔG° h-PC ) is more negative than −15 kcal/mol. 
     
     
         20 . A method comprising the steps of:
 applying a first composition to a sample to determine the presence or quantity of a first target nucleic acid molecule at an operating condition, wherein the first composition is the composition of  claim 1 ; and   wherein the operating condition comprises a temperature between about 4° C. and about 75° C. to permit rapid hybridization of the first composition to the first target nucleic acid molecule.   
     
     
         21 . The method of  claim 20  further comprising the step of applying denaturants or crowding agents to the sample, wherein denaturants or crowding agents are selected form the group consisting of formamide, ethanol, methanol, Tween, Triton, sodium dodecasulfate, DMSO, polyethylene glycol, and dextran. 
     
     
         22 . The method of  claim 20  further comprising applying a second composition to the sample to determine the presence or quantity of a second target nucleic acid molecule, wherein the second composition is the composition of  claim 1 , and wherein the first and second target nucleic acid sequences overlap by at least five nucleotides thereby providing an overlapping region, wherein within the overlapping region, the first target nucleic acid sequences differ from the second target nucleic acid sequence by one or two nucleotides. 
     
     
         23 . The method of  claim 22  wherein the operation condition is sufficient to amplify the first and second target nucleic acid sequences. 
     
     
         24 . The method of  claim 22  wherein the operation conditions is sufficient for polymerase chain reaction.