Double-stranded dna molecule for the detecting and characterizing molecular interactions

Patent metadata

Field	Value
Publication number	US-2023054300-A1
Application number	US-201816955596-A
Country	US
Kind code	A1
Filing date	Dec 21, 2018
Priority date	Dec 21, 2017
Publication date	Feb 23, 2023
Grant date	—

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.

First 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).

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

3 . 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).

4 . 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).

5 . 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.

6 . 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.

7 . 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.

8 . (canceled)

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

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.

11 . 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).

12 . (canceled)

13 . 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, F LF , to the double-stranded DNA molecule, which allows the test molecules to associate; b) applying a high physical force, F HF , 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 z LF 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 z HF-A and z HF-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
z HF-A is the extension when the test molecules are associated and
z HF-D is the extension when the test molecules are dissociated; and

comparing z LF , z HF-A , and z HF-D extensions, as a function of time t.

14 . 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 z LF-A and z LF-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
z LF-A is the extension when the test molecules are associated and
z LF-D is the extension when the test molecules are dissociated;

determining z HF-A and z HF-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
z HF-A is the extension when the test molecules are associated and
z HF-D is the extension when the test molecules are dissociated; and

comparing z LF-A , z LF-D , z HF-A , and z HF-D extensions, as a function of time t.

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

16 . 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.

17 . 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 F CF , 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 t CF-A , and/or
determining spontaneous association after time t CF-D .

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

19 . 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.

20 . The double-stranded DNA molecule according to claim 5 , wherein at least one of the two suppor

Assignees

• Paris Sciences Lettres Quartier Latin
• Centre Nat Rech Scient
• Ecole Normale Superieure
• Univ Paris
• Inst Nat Sante Rech Med

Inventors

• Strick Térence
• Gosse Charlie
• Kostrz Dorota
• Wang Jinglong
• Nadal Marc

Classifications

• G01N33/6803

  General methods of protein analysis not limited to specific proteins or families of proteins · CPC title

• G01Q60/24

  AFM [Atomic Force Microscopy] or apparatus therefor, e.g. AFM probes · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• G01N33/54373 Primary

  involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings · CPC title

• C12Q1/6825

  Nucleic acid detection involving sensors · CPC title