Molecular Constructs for Differentiating a Target Molecule from an Off-Target Molecule

A population of molecular constructs for differentiating a target molecule from an off-target molecule, said target molecule having a target domain and said off-target molecule having an off-target domain that differs from said target domain, each molecular construct comprising

• a) a first domain that is capable of binding to said target domain, and
• b) a second domain that is at least partially hybridizable to said first domain,
• wherein said first domain is capable of binding to said off-target domain;
• wherein a hybrid of said first domain with said second domain is in a state of predetermined stable equilibrium in the absence of said target domain and in a state of predetermined metastable equilibrium in the presence of said target domain;
• and
• wherein the free energy of displacement of said second domain by said target domain from said hybrid of said first domain with said second domain is energetically more favored than the free energy of displacement of said second domain by said off-target domain from said hybrid of said first domain with said second domain.

A method for detecting the presence of a nucleic acid sequence of interest, said method comprising:

• a) contacting a population of probe complexes with a sample, wherein said population of probe complexes comprises at least
• i) a first subpopulation of probe complexes comprising a probe strand and a competitor strand, wherein said competitor strand comprises at least one hybridization domain and at least one domain which does not hybridize with said probe strand, wherein said hybridization domain is at least partially complementary to said probe strand, and
• ii) a second subpopulation of probe complexes comprising a probe strand and a competitor strand, wherein said competitor strand comprises at least one hybridization domain and at least one domain which does not hybridize with said probe strand, wherein said hybridization domain is at least partially complementary of said probe strand, wherein the non-hybridization domain of the second subpopulation differs from the non-hybridization domain of the first subpopulation;
• and
• b) detecting the formation of complexes between the probe strand and a nucleic acid molecule from the sample, wherein the presence of such complexes is indicative of the presence of the nucleic acid sequence of interest.

A method for detecting the presence of a nucleic acid sequence of interest, said method comprising:

• a) contacting a population of molecular constructs with a sample may comprise said nucleic acid sequence, wherein said population of molecular constructs comprises at least
• i) a first subpopulation of molecular constructs, each molecular construct comprising a probe strand and a competitor strand, wherein said probe strand comprises at least one hybridization domain and at least one other domain which does not hybridize with said competitor strand, wherein said at least one hybridization domain is at least partially complementary to said competitor strand, and
• ii) a second subpopulation of molecular constructs, each molecular construct comprising a probe strand and a competitor strand, wherein said probe strand comprises at least one hybridization domain and at least one other domain which does not hybridize with said competitor strand, wherein said at least one hybridization domain is at least partially complementary to said competitor strand, wherein the non-hybridization domain of the second subpopulation differs from the non-hybridization domain of the first subpopulation;
• and
• b) detecting the formation of complexes between the probe strand and a nucleic acid sequence from the sample, wherein the presence of such complexes is indicative of the presence of the nucleic acid sequence of interest.