Methods for detecting molecular interactions

Patent metadata

Field	Value
Publication number	US-9164090-B2
Application number	US-67188108-A
Country	US
Kind code	B2
Filing date	Aug 14, 2008
Priority date	Aug 17, 2007
Publication date	Oct 20, 2015
Grant date	Oct 20, 2015

Abstract

The present invention relates to methods for dynamically detecting the interactions between various materials including bioactive molecules and for detecting target molecules. More specifically, the present invention relates to a method for dynamically detecting bait-prey interactions and a method for easily detecting target molecules which blocks or activates the interactions, the method comprising: allowing a material capable of forming a nano-assembly matrix, a bait and a prey to interact with each other, and analyzing whether a nano-assembly matrix is formed by the interaction between the bait and the prey in vitro or in vivo; or allowing a material capable of forming a nano-assembly matrix, a bait and a prey to interact with each other, inducing the formation of a nano-assembly matrix by a mediator (regulator) material in vitro or in vivo, and then analyzing whether the prey and the bait co-localizes on the nano-assembly matrix.

First claim

What is claimed is:

1. A method for detecting molecular interactions within living cells, comprising the steps of:
(i) providing into said living cells ferritin of same types or different types, having a bait bound thereto and other ferritin of same types or different types, having a prey bound thereto to the same field or system,
wherein the ferritin is a protein having a plurality of the same or different binding moieties and can be assembled by the self-assembly between them; wherein the ferritin having a bait bound thereto and the ferritin having a prey bound thereto are the same or different types;
(ii) forming a nano-assembly matrix by the interaction between the bait and the prey; and
(iii) examining whether the nano-assembly matrix was formed, thus detecting the bait-prey interaction.

2. The method for detecting molecular interactions according to claim 1 , wherein the interaction between the bait and the prey occurs directly or indirectly in step (i).

3. The method for detecting molecular interactions according to claim 1 , wherein a material capable of mediating or regulating the interaction between the bait and the prey is additionally added in step (i).

4. The method for detecting molecular interactions according to claim 1 , wherein each of the bait and the prey is provided in a state in which a label is bound thereto.

5. The method for detecting molecular interactions according to claim 3 , wherein the material capable of mediating or regulating the interaction between the bait and the prey is provided in a state in which a label is bound thereto.

6. A method for detecting molecular interactions within living cells, comprising the steps of:
(i) providing into said living cells ferritin of same types or different types, having a bait and mediator (regulator) materials bound thereto, to the same field or system,
wherein the ferritin is a protein having a plurality of the same or different binding moieties and can be assembled by the self-assembly between them,
wherein the ferritin form a nano-assembly matrix by the interaction between the mediator materials;
(ii) providing a prey to the same field or system; and
(iii) examining whether the prey co-localizes with the formed nano-assembly matrix by interacting with the bait present on the formed nano-assembly matrix, thus detecting the bait-prey interaction.

7. A method for detecting molecular interactions within living cells, comprising the steps of:
(i) providing into said living cells ferritin of same types or different types, having first mediator (regulator) materials bound thereto, and a bait having second mediator (regulator) molecules bound thereto, to the same field or system,
wherein the ferritin is a protein having a plurality of the same or different binding moieties and can be assembled by the self-assembly between them,
wherein the ferritin form a nano-assembly matrix-first mediator (regulator) materials complex by the interactions between the first mediator (regulator) materials, and the bait having second mediator molecules is bound to the formed nano-assembly matrix-first mediator (regulator) materials complex by the interaction between the second mediator materials and the first mediator material of the formed nano-assembly matrix;
(ii) providing a prey to the same field or system; and
(iii) examining whether the prey co-localizes with the formed nano-assembly matrix by interacting with the bait bound to the formed nano-assembly matrix, thus detecting the bait-prey interaction.

8. The method for detecting molecular interactions according to claim 6 or 7 , wherein a material mediating or regulating the interactions between the mediator (regulator) materials is additionally added in step (i).

9. The method for detecting molecular interactions according to claim 6 or 7 , wherein a material which mediating or regulating the interaction between the bait and the prey is additionally added in step (iii).

10. The method for detecting molecular interactions according to claim 6 or 7 , wherein each of the bait, the prey and the mediator (regulator) material is provided in a state in which a label is bound thereto.

11. The method for detecting molecular interactions according to any one claim among claims 1 , 6 and 7 , wherein the bait, the prey and/or the mediator (regulator) material is a bioactive molecule.

12. The method for detecting molecular interactions according to claim 11 , wherein the bioactive molecule is selected from the group consisting of nucleic acids, nucleotides, proteins, peptides, amino acids, saccharides, lipids, vitamins and chemical compounds.

13. The method for detecting molecular interactions according to claim 4 or 5 , wherein the label is a radioactive label, a fluorescent material or a luminescent material.

14. The method for detecting molecular interactions according to claim 13 , wherein the fluorescent material is a fluorescent dye, a tetracystein motif, a fluorescent protein, or a fluorescent nanoparticle.

15. The method for detecting molecular interactions according to claim 10 , wherein the label is a radioactive label, a fluorescent material or a luminescent material.

16. The method for detecting molecular interactions according to claim 15 , wherein the fluorescent material is a fluorescent dye, a tetracystein motif, a fluorescent protein, or a fluorescent nanoparticle.

17. The method for detecting molecular interactions according to any one claim among claims 1 , 6 and 7 , wherein the living cells are selected from the group consisting of Zebra fish, C. elegans , yeast, fly or frog, mammal (except human) and plant.

18. The method for detecting molecular interactions according to any one claim among claims 1 , 6 and 7 , wherein the step of providing ferritin into the living cells is carried out by the use of any one selected from the group consisting of transducible peptide (or fusogenic peptide), lipid (or liposome) gene transporter or the binding complex thereof; electroporation and magnetofection.

19. The method for detecting molecular interactions according to any one claim among claims 1 , 6 and 7 , wherein the formation of nano-assembly matrix is examined using any one selected from the group consisting of an optical means, a scanner, a radioactive label detecting device, a fluorescence polarization reader (FP reader), a spectrophotometer, MRI (magnetic resonance imaging), SQUID, MR relaxometer, a fluorescence detector, a luminescence detector.

20. The method for detecting molecular interactions according to any one claim among claims 1 , 6 and 7 , wherein the binding location of the prey by interaction with the bait is measured using any one selected from the group consisting of an optical means, a scanner, a radioactive label detecting device, a fluorescence polarization reader (FP reader), a spectrophotometer, MRI (magnetic resonance imaging), SQUID, MR relaxometer, a fluorescence detector, a luminescence detector.

21. A method for detecting a prey as a target, interacting with a bait within living cells, the method comprising the steps of:
(i) providing ferritin having a bait bound thereto and a library of prey bound to other ferritin, to the same field or system,
wherein the ferritin is a protein having a plurality of the same or different binding moieties and can be assembled by the self-assembly between them;
(ii) forming nano-assembly matrices by the interactions between the bait and a prey;
(iii) measuring whether the nano-assembly matrices were

Assignees

• Kim Tae Kook
• Lee Seung Goo
• Lee Sang Kyu
• Lee Kyoung Hu
• Ha Jae-Seok
• Korea Advanced Inst Sci & Tech
• Korea Res Inst Of Bioscience

Inventors

• Kim Tae Kook
• Lee Seung Goo
• Lee Sang Kyu
• Lee Kyoung Hu
• Ha Jae-Seok

Classifications

• G01N33/6845

  Methods of identifying protein-protein interactions in protein mixtures · CPC title

• G01N33/54326 Primary

  Magnetic particles · CPC title

• G01N33/54346

  Nanoparticles · CPC title

• G01N33/5008

  for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics · CPC title

• G01N33/536

  with immune complex formed in liquid phase · CPC title