# US 2019/0169682 A1 · Molecular Constructs for Differentiating a Target Molecule from an Off-Target Molecule

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- Number: US 2019/0169682 A1
- Document ID: US20190169682A1
- Status: Published application
- Kind: A1
- Application number: 16/160,426
- Filed: 2018-10-15
- Published: 2019-06-06
- Earliest claimed: 2011-05-06
- Assignees: Rutgers, The State University of New Jersey
- Inventors: Kenneth J. BRESLAUER; William H. BRAUNLIN; Leslie C Beadling; Jens VOLKER
- Web page: https://patentgrep.com/patent/US20190169682A1

## CPC

- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- C12Q 1/6811: Selection methods for production or design of target specific oligonucleotides or binding molecules
- C12Q 1/6825: Nucleic acid detection involving sensors
- C12Q 1/6832: Enhancement of hybridisation reaction
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

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

## Claims

### Claim 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.

### Claim 2 (depends on 1)

The molecular construct of claim 1, wherein said off-target domain is generally homologous with respect to said target domain.

### Claim 3 (depends on 1)

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.

### Claim 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.

### Claim 5 (depends on 4)

The population of claim 4, wherein said off-target domain is generally homologous with respect to said target domain.

### Claim 6 (depends on 4)

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.

### Claim 7 (depends on 4)

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.

### Claim 8 (depends on 7)

The population of claim 7, wherein said first target domain and said second target domain are disposed on the same target molecule.

### Claim 9 (depends on 7)

The population of claim 7, wherein said first target domain and said second target domain are disposed on different target molecules.

### Claim 10 (depends on 7)

The population of claim 7, wherein said first target domain is an off-target domain with respect to a second target molecule.

### Claim 11 (depends on 4)

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.

### Claim 12 (depends on 11)

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.

### Claim 13 (depends on 11)

The population of claim 11, wherein said first off-target domain and said second off-target domain are disposed on different off-target molecules.

### Claim 14 (depends on 11)

The population of claim 11, wherein said first off-target domain is a target domain with respect to a second off-target molecule.

### Claim 15 (depends on 4)

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.

### Claim 16 (depends on 1)

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.

### Claim 17 (depends on 1)

A kit comprising at least one molecular construct of claim 1.

### Claim 18

A method for differentiating a target molecule from an off-target molecule, said method comprising contacting a molecular construct with an unknown molecule.

### Claim 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.

### Claim 20 (depends on 19)

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.

### Claim 21 (depends on 19)

The method of claim 19, wherein said hybridization domains are the complement of said probe strand.

### Claim 22 (depends on 19)

The method of claim 19, wherein the nucleic acid molecules of the sample are detectably labeled.

### Claim 23 (depends on 19)

The method of claim 19, wherein said population of probe complexes are in solution.

### Claim 24 (depends on 19)

The method of claim 19, wherein said population of probe complexes is spatially constrained.

### Claim 25 (depends on 22)

The method of claim 22, wherein said population of probe complexes is linked to a solid support.

### Claim 26 (depends on 19)

The method of claim 19, wherein step a) is performed with increasing concentrations of sample nucleic acid molecules.

### Claim 27 (depends on 19)

The method of claim 19, wherein the non-hybridization domain comprises a loop domain.

### Claim 28 (depends on 4)

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.

### Claim 29 (depends on 28)

The array of claim 28, wherein the number of different targets is from about 2 to 10.

### Claim 30 (depends on 28)

The array of claim 28, wherein the number of different targets is from 100 to 1000.

### Claim 31 (depends on 28)

The array of claim 28, wherein the number of different targets is greater than 1000.

### Claim 32 (depends on 28)

The array of claim 28, wherein the ratio of the free energy of displacement of one population to another population is about 1.1.

### Claim 33 (depends on 28)

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.

### Claim 34 (depends on 28)

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.

### Claim 35 (depends on 28)

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.

### Claim 36 (depends on 28)

The array of claim 28, wherein the ratio of the free energy of displacement of one population to another population is greater than 2.

### Claim 37 (depends on 4)

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.

### Claim 38 (depends on 37)

The array of claim 37, wherein the number of different targets is from about 2 to 10.

### Claim 39 (depends on 37)

The array of claim 37, wherein the number of different targets is from 100 to 1000.

### Claim 40 (depends on 37)

The array of claim 37, wherein the number of different targets is greater than 1000.

### Claim 41 (depends on 37)

The array of claim 37, wherein the ratio of the free energy of displacement of one population to another population is about 1.1.

### Claim 42 (depends on 37)

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.

### Claim 43 (depends on 37)

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.

### Claim 44 (depends on 37)

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.

### Claim 45 (depends on 37)

The array of claim 37, wherein the ratio of the free energy of displacement of one population to another population is greater than 2.

### Claim 46 (depends on 4)

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.

### Claim 47 (depends on 46)

The array of claim 46, wherein the number of different targets is from about 2 to 10.

### Claim 48 (depends on 46)

The array of claim 46, wherein the number of different targets is from 100 to 1000.

### Claim 49 (depends on 46)

The array of claim 46, wherein the number of different targets is greater than 1000.

### Claim 50 (depends on 46)

The array of claim 46, wherein the ratio of the free energy of displacement of one population to another population is about 1.1.

### Claim 51 (depends on 46)

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.

### Claim 52 (depends on 46)

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.

### Claim 53 (depends on 46)

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.

### Claim 54 (depends on 46)

The array of claim 46, wherein the ratio of the free energy of displacement of one population to another population is greater than 2.

### Claim 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.