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

Abstract

Molecular constructs, populations thereof, arrays, compositions, methods and kits for differentiating a target molecule from an off-target molecule are provided.

What is claimed is: 
     
         1 . A molecular construct 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, said 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 with 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.   
     
     
         2 . The molecular construct of  claim 1 , wherein said off-target domain is generally homologous with respect to said target domain. 
     
     
         3 . The molecular construct of  claim 1 , wherein said off-target domain differs from said target domain either chemically by at least one functional group or conformationally or both. 
     
     
         4 . 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.   
     
     
         5 . The population of  claim 4 , wherein said off-target domain is generally homologous with respect to said target domain. 
     
     
         6 . The population of  claim 4 , wherein said off-target domain differs from said target domain either chemically by at least one functional group or conformationally or both. 
     
     
         7 . The population of  claim 4 , wherein said population comprises:
 a) a first subpopulation of at least one said molecular construct with respect to a first target domain, and   b) a second subpopulation of at least one said molecular construct with respect to a second target domain,   wherein said first target domain differs from said second target domain.   
     
     
         8 . The population of  claim 7 , wherein said first target domain and said second target domain are disposed on the same target molecule. 
     
     
         9 . The population of  claim 7 , wherein said first target domain and said second target domain are disposed on different target molecules. 
     
     
         10 . The population of  claim 7 , wherein said first target domain is an off-target domain with respect to a second target molecule. 
     
     
         11 . The population of  claim 4 , wherein said population comprises:
 a) a first subpopulation comprising at least one said molecular construct with respect to a first off-target domain and   b) a second subpopulation comprising at least one said molecular construct with respect to a second off-target domain, and   wherein said first off-target domain differs from said second off-target domain.   
     
     
         12 . The population of  claim 11 , wherein said first off-target domain and said second off-target domain are disposed on the same off-target molecule. 
     
     
         13 . The population of  claim 11 , wherein said first off-target domain and said second off-target domain are disposed on different off-target molecules. 
     
     
         14 . The population of  claim 11 , wherein said first off-target domain is a target domain with respect to a second off-target molecule. 
     
     
         15 . The population of  claim 4 , wherein
 a) the second domain of said molecular constructs of a first portion of said population comprises a first subdomain that is at least partially complementary to the first domain and a second subdomain that is not complementary to the first domain; and   b) the second domain of said molecular constructs of a second portion of said population comprises a first subdomain that is at least partially complementary to the first domain and a second subdomain that is not complementary to the first domain;   wherein the second subdomain of the molecular constructs of the first portion differs from the second subdomain of the molecular constructs of the second portion and modulates the binding of the first domains to the second domains of the first portion with respect to the binding of the first domains to the second domains of the second portions.   
     
     
         16 . An array comprising more than one biosensor element on a solid support, wherein each biosensor element comprises a molecular construct in accordance with  claim 1 . 
     
     
         17 . A kit comprising at least one molecular construct of  claim 1 . 
     
     
         18 . A method for differentiating a target molecule from an off-target molecule, said method comprising contacting a molecular construct with an unknown molecule. 
     
     
         19 . 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.   
     
     
         20 . The method of  claim 19 , wherein said competitor strand comprises two hybridization domains separated by a domain which does not hybridize with said probe strand. 
     
     
         21 . The method of  claim 19 , wherein said hybridization domains are the complement of said probe strand. 
     
     
         22 . The method of  claim 19 , wherein the nucleic acid molecules of the sample are detectably labeled. 
     
     
         23 . The method of  claim 19 , wherein said population of probe complexes are in solution. 
     
     
         24 . The method of  claim 19 , wherein said population of probe complexes is spatially constrained. 
     
     
         25 . The method of  claim 22 , wherein said population of probe complexes is linked to a solid support. 
     
     
         26 . The method of  claim 19 , wherein step a) is performed with increasing concentrations of sample nucleic acid molecules. 
     
     
         27 . The method of  claim 19 , wherein the non-hybridization domain comprises a loop domain. 
     
     
         28 . An array of a plurality of populations in accordance with  claim 4  for detecting a plurality of different target molecules in a sample, wherein the free energy of displacement of each of those populations differs with respect to each other of those populations so as to be capable of identifying different target molecules in said sample. 
     
     
         29 . The array of  claim 28 , wherein the number of different targets is from about 2 to 10. 
     
     
         30 . The array of  claim 28 , wherein the number of different targets is from 100 to 1000. 
     
     
         31 . The array of  claim 28 , wherein the number of different targets is greater than 1000. 
     
     
         32 . The array of  claim 28 , wherein the ratio of the free energy of displacement of one population to another population is about 1.1. 
     
     
         33 . The array of  claim 28 , wherein the ratio of the free energy of displacement of one population to another population is from 1.1 to 1.2. 
     
     
         34 . The array of  claim 28 , wherein the ratio of the free energy of displacement of one population to another population is from 1.2 to 1.5. 
     
     
         35 . The array of  claim 28 , wherein the ratio of the free energy of displacement of one population to another population is from 1.5 to 2.0. 
     
     
         36 . The array of  claim 28 , wherein the ratio of the free energy of displacement of one population to another population is greater than 2. 
     
     
         37 . An array of a plurality of populations in accordance with  claim 4  for determining the relative number of target molecules in a sample, wherein the free energy of displacement of each of those populations differs with respect to each other of those populations so as to be capable of determining the relative number of target molecules of a population from another population in said sample. 
     
     
         38 . The array of  claim 37 , wherein the number of different targets is from about 2 to 10. 
     
     
         39 . The array of  claim 37 , wherein the number of different targets is from 100 to 1000. 
     
     
         40 . The array of  claim 37 , wherein the number of different targets is greater than 1000. 
     
     
         41 . The array of  claim 37 , wherein the ratio of the free energy of displacement of one population to another population is about 1.1. 
     
     
         42 . The array of  claim 37 , wherein the ratio of the free energy of displacement of one population to another population is from 1.1 to 1.2. 
     
     
         43 . The array of  claim 37 , wherein the ratio of the free energy of displacement of one population to another population is from 1.2 to 1.5. 
     
     
         44 . The array of  claim 37 , wherein the ratio of the free energy of displacement of one population to another population is from 1.5 to 2.0. 
     
     
         45 . The array of  claim 37 , wherein the ratio of the free energy of displacement of one population to another population is greater than 2. 
     
     
         46 . An array of a plurality of populations in accordance with  claim 4  for identifying a plurality of different target molecules in a sample and determining the relative number of each of those different target molecules in the sample, wherein
 a) the free energy of displacement of each of those populations differs with respect to each other of those populations so as to be capable of identifying each different target molecule in said sample and 
 b) the free energy of displacement of each of those populations differs with respect to each other of those populations so as to be capable of determining the relative number of each of those different target molecules of a population from another population in said sample. 
 
     
     
         47 . The array of  claim 46 , wherein the number of different targets is from about 2 to 10. 
     
     
         48 . The array of  claim 46 , wherein the number of different targets is from 100 to 1000. 
     
     
         49 . The array of  claim 46 , wherein the number of different targets is greater than 1000. 
     
     
         50 . The array of  claim 46 , wherein the ratio of the free energy of displacement of one population to another population is about 1.1. 
     
     
         51 . The array of  claim 46 , wherein the ratio of the free energy of displacement of one population to another population is from 1.1 to 1.2. 
     
     
         52 . The array of  claim 46 , wherein the ratio of the free energy of displacement of one population to another population is from 1.2 to 1.5. 
     
     
         53 . The array of  claim 46 , wherein the ratio of the free energy of displacement of one population to another population is from 1.5 to 2.0. 
     
     
         54 . The array of  claim 46 , wherein the ratio of the free energy of displacement of one population to another population is greater than 2. 
     
     
         55 . 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.