# US 2023/0054300 A1 · Double-stranded dna molecule for the detecting and characterizing molecular interactions

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- Number: US 2023/0054300 A1
- Document ID: US20230054300A1
- Status: Published application
- Kind: A1
- Application number: 16/955,596
- Filed: 2018-12-21
- Published: 2023-02-23
- Earliest claimed: 2017-12-21
- Assignees: Paris Sciences Et Lettres—Quartier Latin; Centre National de la Recherche Scientifique (CNRS); Ecole Normale Superieure; Universite De Paris
- Applicants: Paris Sciences Et Lettres - Quartier Latin; Centre National De La Recherche Scientifique (Cnrs); Ecole Normale Superieure; Université De Paris; Institut National De La Sante Et De La Recherche Medicale (Inserm)
- Inventors: Térence STRICK; Charlie GOSSE; Dorota KOSTRZ; Jinglong WANG; Marc NADAL
- Web page: https://patentgrep.com/patent/US20230054300A1

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- G01N 33/54373: {involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings}
- C12Q 1/6825: Nucleic acid detection involving sensors
- G01N 33/6803: {General methods of protein analysis not limited to specific proteins or families of proteins}
- G01Q 60/24: AFM [Atomic Force Microscopy] or apparatus therefor, e.g. AFM probes
- 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
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- G01Q: SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]

## Abstract

The present application relates to a double-stranded DNA molecule comprising a first double-stranded DNA molecule (1) connected to a second double-stranded DNA molecule (2) by at least one covalent bond which is not a phosphodiester, phosphorothioate, phosphoramidate or phosphorodiamidate bond, preferably by a tether, said tether preferably being a double-stranded DNA molecule.

## Claims

### Claim 1

A double-stranded DNA molecule comprising a first double-stranded DNA molecule (1) connected to a second double-stranded DNA molecule (2) by a tether comprising double-stranded DNA, wherein the tether is attached by
(i) at least one covalent bond to a nucleotide of the first double-stranded DNA molecule (1), and by
(ii) at least one covalent bond to a nucleotide of the second double-stranded DNA molecule (2),
wherein
the at least one covalent bond of (i) and the at least one covalent bond of (ii) are not phosphodiester bonds, phosphorothioate bonds, phosphoramidate bonds or phosphorodiamidate bonds, and
the nucleotide of (i) and the nucleotide of (ii) are not the ultimate nucleotides of double-stranded DNA molecules (1) and (2).

### Claim 2 (depends on 1)

The double-stranded DNA molecule according to claim 1, wherein the tether is a double-stranded DNA molecule.

### Claim 3 (depends on 1)

The double-stranded DNA molecule according to claim 1, wherein the tether is attached to
the first double-stranded DNA molecule (1) by a first covalent bond between a first extremity of the tether and an intermediate region of the first double-stranded DNA molecule (1),
and to
the second double-stranded DNA molecule (2) by a second covalent bond between a second extremity of the tether and an intermediate region of the second double-stranded DNA molecule (2).

### Claim 4 (depends on 1)

The double-stranded DNA molecule according to claim 1, wherein
a first test molecule is linked to a first extremity of the first double-stranded DNA molecule (1) and
a second test molecule is linked to a first extremity of the second double-stranded DNA molecule (2).

### Claim 5 (depends on 4)

The double-stranded DNA molecule according to claim 4, wherein
the second extremity of the first double-stranded DNA molecule (1) is linked to a first support and
the second extremity of the second double-stranded DNA molecule (2) is linked to a second support.

### Claim 6 (depends on 4)

The double-stranded DNA molecule according to claim 4, wherein:
the first double-stranded DNA molecule (1) and/or the second double-stranded DNA molecule (2) has a length of 300 to 5000 base pairs;
the first extremity of the first double-stranded DNA molecule (1) and/or the first extremity of the second double-stranded DNA molecule (2) has a length of 10 to 150 base pairs; and/or
the tether has a length of about 300 to about 50,000 base pairs.

### Claim 7 (depends on 4)

The double-stranded DNA molecule according to claim 4, wherein the first and/or second test molecule is selected from the group consisting of the following molecules: polymers, amino acids, peptides, polypeptides, proteins, nucleosides, nucleotides, polynucleotides, oligonucleotides, sugars, polysaccharides, small molecules, drugs, aptamers, antigens, antibodies, lipids, lectins, hormones, vitamins, viruses, virus fragments, nanoparticles, cell surface molecules, and transcription factors.

### Claim 8

(canceled)

### Claim 9 (depends on 1)

A device comprising the double-stranded DNA molecule according to claim 1 with its supports.

### Claim 10

A double-stranded DNA molecule comprising a first double-stranded DNA molecule (A) and a second double-stranded DNA molecule (B), wherein
the first double-stranded DNA molecule (A) comprises a cleavage site which is present only in the first double-stranded DNA molecule (A),
the first double-stranded DNA molecule (A) is connected to the second double-stranded DNA molecule (B) by two covalent bonds which are not phosphodiester bonds, phosphorothioate bonds, phosphoramidate bonds or phosphorodiamidate bonds, and
one of the two covalent bonds is located on each side of the cleavage site.

### Claim 11 (depends on 1)

A process for manufacturing a double-stranded DNA molecule according to claim 1, comprising a step of:
a) cleaving a precursor molecule of the first and second double-stranded DNA molecules (1) and (2) at a cleavage site that is present only in the precursor molecule, thereby generating a double-stranded DNA molecule comprising a first double-stranded DNA molecule (1) and a second double-stranded DNA molecule (2).

### Claim 12

(canceled)

### Claim 13 (depends on 1)

A method of characterizing an interaction between at least two test molecules linked to a double-stranded DNA molecule according to claim 1, comprising:
a) applying a low physical force, FLF, to the double-stranded DNA molecule, which allows the test molecules to associate;
b) applying a high physical force, FHF, to the double-stranded DNA molecule, which makes it possible to determine whether the test molecules are associated or dissociated; and
c) detecting a change in conformation of the DNA molecule comprising:
determining zLF extension between a second extremity of the first double-stranded DNA molecule (1) and a second extremity of the second double-stranded DNA molecule (2) in step a);
determining zHF-A and zHF-D extensions between the second extremity of the first double-stranded DNA molecule (1) and the second extremity of the second double-stranded DNA molecule (2), in step b), wherein
zHF-A is the extension when the test molecules are associated and
zHF-D is the extension when the test molecules are dissociated; and
comparing zLF, zHF-A, and zHF-D extensions, as a function of time t.

### Claim 14 (depends on 13)

The method according to claim 13, wherein the method further comprises the following additional step:
d) detecting a change in conformation of the DNA molecule comprising:
determining zLF-A and zLF-D extensions between the second extremity of the first double-stranded DNA molecule (1) and the second extremity of the second double-stranded DNA molecule (2) in step a), wherein
zLF-A is the extension when the test molecules are associated and
zLF-D is the extension when the test molecules are dissociated;
determining zHF-A and zHF-D extensions between the second extremity of the first double-stranded DNA molecule (1) and the second extremity of the second double-stranded DNA molecule (2), in step b), wherein
zHF-A is the extension when the test molecules are associated and
zHF-D is the extension when the test molecules are dissociated; and
comparing zLF-A, zLF-D, zHF-A, and zHF-D extensions, as a function of time t.

### Claim 15 (depends on 14)

A method according to claim 14, wherein the tether of the double-stranded DNA molecule has a length of at least 700 bp.

### Claim 16 (depends on 13)

A method according to claim 13, wherein the physical force in step a) is from 0.01 pN to 0.4 pN and/or wherein the physical force in step b) is from 0.5 to 70 pN.

### Claim 17 (depends on 1)

A method of characterizing an interaction between at least two test molecules linked to a double-stranded DNA molecule according to claim 1, comprising:
a) applying a constant force FCF, to the double-stranded DNA molecule, which allows the test molecules to associate and dissociate; and
b) detecting a change in conformation of the DNA molecule comprising:
determining spontaneous dissociation of the test molecules after time tCF-A, and/or
determining spontaneous association after time tCF-D.

### Claim 18 (depends on 17)

The method according to claim 17, wherein the constant force FCF of step a) is at least 0.03 pN.

### Claim 19 (depends on 13)

The method according to claim 13, wherein the characterization of the interaction comprises determining at least one of the following: characteristic association time, characteristic dissociation time, dissociation rate constant, dissociation activation energy, distance separating the transition state from the complex during dissociation, and equilibrium dissociation constant.

### Claim 20 (depends on 5)

The double-stranded DNA molecule according to claim 5, wherein at least one of the two supports is a movable support.

### Claim 21 (depends on 6)

The double-stranded DNA molecule according to claim 6, wherein the first double-stranded DNA molecule (1) and/or the second double-stranded DNA molecule (2) has a length of 650 to 1500 base pairs

### Claim 22 (depends on 6)

The double-stranded DNA molecule according to claim 6, wherein the first extremity of the first double-stranded DNA molecule (1) and/or the first extremity of the second double-stranded DNA molecule (2) has a length of 30 to 50 base pairs.