Fine-tuned ultraspecific nucleic acid hybridization probes

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.

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.