Method for Detecting and/or Characterizing the Interaction Between Proteins and Small Molecules

• US 20200132678A1
• Filed: 06/21/2018
• Published: 04/30/2020
• Est. Priority Date: 06/21/2017
• Status: Active Grant

• Current Assignee: Dynamic Biosensors GmbH
• Original Assignee: Dynamic Biosensors GmbH
• Inventors: Beike, Hanna, Daub, Herwin, Fjolla, Ismajli, Langer, Andreas, Rant, Ulrich, Strasser, Ralf
• Granted Patent: US 11,549,939 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92

Abstract

A method for characterizing the interaction between a protein and a small molecule by detecting a change in fluorescence emitted by a fluorescent dye and a nucleic acid structure which can be used in said method.

Claims

1. A method for detecting and/or characterizing the interaction between a protein and a small molecule, comprising the steps of:

a) providing a solid surface to which at least one linker is bound, wherein the protein, at least one fluorescent dye and a competitor compound are attached to the at least one linker and wherein the competitor compound binds to the protein;

b) contacting the solid surface with the small molecule under conditions that allow the small molecule to bind to the protein and to release the competitor compound from the protein; and

c) detecting a change in the fluorescence emitted by the fluorescent dye.

2. The method of claim 1, wherein the protein is attached to a first linker and one fluorescent dye and the competitor compound are attached to a second linker.

3. The method of claim 1, wherein the protein, one fluorescent dye and the competitor compound are all attached to the same linker.

4. The method of claim 3, wherein the linker has two branches, wherein the protein and one fluorescent dye are attached to the first branch and the competitor compound is attached to the second branch.

5. The method of claim 1, wherein the solid surface is a metal surface.

6. The method of claim 1, wherein the linker is a nucleic acid linker.

7. The method of claim 6, wherein the nucleic acid linker is double-stranded.

8. The method of claim 1, wherein by detecting the change in fluorescence emitted by the fluorescent dye a time course of association and/or dissociation of the small molecule with the protein is measured.

9. The method of claim 1, wherein the change in fluorescence is detected continuously after the solid surface has been contacted with the small molecule.

10. The method of claim 1, wherein the competitor compound is able to quench the fluorescence of the fluorescent dye.

11. The method according to claim 10, wherein the competitor compound quenches the fluorescence of the fluorescent dye after the competitor compound has been released from the protein.

12. The method of claim 1, wherein additionally a quencher is attached to the linker.

13. A nucleic acid structure comprising:

(i) a first and a second nucleic acid molecule which form a double strand with each other over a part of the first and a part of the second nucleic acid molecule;

(ii) a third nucleic acid molecule which forms a double strand with a part of the first nucleic acid molecule which does not form a double strand with the second nucleic acid molecule; and

(iii) a fourth nucleic acid molecule which forms a double strand with a part of the second nucleic acid molecule which does not form a double strand with the first nucleic acid molecule, wherein;

a fluorescent dye is attached to the first nucleic acid molecule;

a quencher is attached to the second nucleic acid molecule;

a protein is attached to the third nucleic acid molecule, and

a competitor compound is attached to the fourth nucleic acid molecule.

14. A method for characterizing the interaction between a protein and a small molecule, comprising contacting the small molecule with the nucleic acid structure of claim 13 under conditions that allow the small molecule to bind to the protein and to release the competitor compound from the protein, and detecting a change in the fluorescence emitted by the fluorescent dye.

15. The nucleic acid structure of claim 13, wherein the nucleic acid structure is attached to a solid surface.