CN-122029426-A - Novel analytical method for biomolecular interactions

Abstract

The present disclosure provides a method of designing a structure useful for the formation of an interactive complex, the structure produced, and uses thereof. In one aspect, the present disclosure provides for the design and manufacture of structures described in this specification. Since the unit molecule is an object of observation or use, the structural body can be designed to be suitable for the unit molecule. In one embodiment, a structure for forming a complex between unit molecules may be designed. The structure described in the present specification has a function of promoting the formation of a complex between unit molecules.

Inventors

• FUJITA DAISHI
• YOSHIMURA MASAHIKO
• MATSUDA FUYUKI
• IKEDA YUKIO

Assignees

• 国立大学法人京都大学

Dates

Publication Date

20260512

Application Date

20241010

Priority Date

20231010

Claims (20)

1. 1. A method of manufacturing a structure for forming an interactive complex, characterized in that, The structure comprises a substrate comprising a first connector anchor point and a second connector anchor point, The first linker immobilization point and the second linker immobilization point being capable of binding or having bound thereto a first linker and a second linker, respectively, the first linker and the second linker being capable of binding or having bound thereto a first unit molecule and a second unit molecule, respectively, The relative positional relationship of the first connector sub-fixing point and the second connector sub-fixing point is fixed, The first unit molecule and the second unit molecule are capable of forming together an interactive complex, The method includes the step of designing the structure based on the interaction complex.
2. 2. The method of claim 1, wherein the step of designing the structure includes designing the structure with a structure such that a crossing index of the first unit molecule and the second unit molecule satisfies-5D-15.
3. 3. The method of claim 1, wherein the step of designing the structure comprises designing the structure based on the dimensions of the first unit molecule and the second unit molecule.
4. 4. The method of claim 1, wherein the step of designing the structure comprises designing the structure based on a predicted value of a binding dissociation constant between the first unit molecule and the second unit molecule.
5. 5. The method of claim 1, wherein the step of designing the structure includes determining the lengths of the first linker and the second linker based on at least one of the dimensions of the first unit molecule and the second unit molecule, and a binding dissociation constant between the first unit molecule and the second unit molecule.
6. 6. The method of claim 1, wherein the step of designing the structure includes determining a distance between the first linker fixing point and the second linker fixing point based on at least one of a size of the first unit molecule and the second unit molecule, and a binding dissociation constant between the first unit molecule and the second unit molecule.
7. 7. The method of claim 1, wherein the step of designing the structure comprises selecting the substrate from a plurality of options based on the size of the first unit molecule and the second unit molecule.
8. 8. The method of claim 1, wherein the step of designing the structure comprises: selecting the substrate from a plurality of options; determining a distance between the first connector anchor point and the second connector anchor point, and Determining the length of the first connector and the second connector.
9. 9. A method of analyzing an interaction complex, comprising: A step of manufacturing the structure according to the method of claim 1; A step of preparing a structure including the first unit molecule bound to the first linker fixing point via the first linker and the second unit molecule bound to the second linker fixing point via the second linker, and A step of analyzing the interaction complex.
10. 10. The method of claim 9, wherein a binding dissociation constant between the first unit molecule and the second unit molecule is determined.
11. 11. The method of claim 10, wherein the binding dissociation constant (K d ) is in the range of 10 -2 ～10 -6 M.
12. 12. The method of claim 10, wherein the binding dissociation constant is determined based on a standard curve made from results for reference unit molecules for which binding dissociation constants are known.
13. 13. The method of claim 9, wherein the structure of the interaction complex is determined.
14. 14. A method of performing an immobilized enzyme reaction, comprising: A step of manufacturing the structure according to the method of claim 1; and Contacting the structure with a substrate for an enzymatic reaction, wherein the interactive complex is an enzyme.
15. 15. A structure for forming an interactive complex, characterized in that, The structure comprises a substrate comprising a first connector anchor point and a second connector anchor point, The first linker immobilization point and the second linker immobilization point being capable of binding or having bound thereto a first linker and a second linker, respectively, the first linker and the second linker being capable of binding or having bound thereto a first unit molecule and a second unit molecule, respectively, The relative positional relationship of the first connector sub-fixing point and the second connector sub-fixing point is fixed, The first unit molecule and the second unit molecule are capable of forming together an interactive complex.
16. 16. The structure according to claim 15, wherein the cross index of the first unit molecule and the second unit molecule is equal to or less than-5 and equal to or less than D and equal to or less than 15.
17. 17. The structure of claim 15, manufactured by the method of claim 1.
18. 18. A method of resolving an interaction complex, comprising: (A) Providing a structure for forming an interactive complex at a predetermined location, the structure comprising a substrate comprising a first linker fixing point and a second linker fixing point, a first linker and a second linker bound to the substrate via the first linker fixing point and the second linker fixing point, respectively, and a first unit molecule and a second unit molecule bound to the first linker and the second linker, respectively, wherein the relative positional relationship of the first linker fixing point and the second linker fixing point is fixed, the first unit molecule and the second unit molecule being capable of forming together an interactive complex, the predetermined location being defined as a relative position with respect to the substrate, the interactive complex being formed at the predetermined location; (B) A step of forming the interaction complex, and (C) A step of resolving the interaction complex by resolving data derived from the interaction complex at the predetermined location.
19. 19. The method of claim 18, further comprising the step of irradiating a particle beam or electromagnetic wave to the predetermined location.
20. 20. The method of claim 18, further comprising the step of collecting a particle beam or electromagnetic wave originating from the interactive complex.

Description

Novel analytical method for biomolecular interactions Technical Field The present disclosure relates to a method of designing a structure useful for the formation of an interactive complex, a structure produced thereby, and uses thereof. The present disclosure also relates to a novel method for complex analysis using the structure, and in particular, to drug discovery and development. Background Many molecules, such as biomolecules, function by interacting with other molecules, and when interactions occur between molecules, complexes are formed, regardless of the length of time. Therefore, in order for a molecule to function, the formation of a complex is important, but a technique for observing or utilizing the formation of a complex is still in progress. For example, as a method for determining the complex structure, elemental analysis, mass spectrometry, nuclear Magnetic Resonance (NMR), powder X-ray analysis, or the like is used, but information obtained by each method is different in emphasis. For example, patent document 1 describes that complexes between IgE antibodies and target molecules thereof are analyzed by microscopic observation. The interactive complex is composed of a plurality of molecules, and thus the size becomes large, and analysis becomes difficult. In addition, many drugs act by intervening intermolecular interactions (complex formation). Prior art literature Patent literature Patent document 1 European patent application publication No. 18215047 Disclosure of Invention The inventors of the present invention developed methods for designing and manufacturing novel structures for forming interactive complexes and found that the structures are useful in a variety of applications. In particular, the use of the structure to form a complex at a predetermined position enables analysis of the complex by measuring the predetermined position, and is useful for drug discovery and development. The present disclosure provides, for example, the following items: (item A1) A method of manufacturing a structure for forming an interactive complex, wherein the structure comprises a substrate comprising a first linker fixing point and a second linker fixing point, the first linker fixing point and the second linker fixing point being capable of binding or being bound to a first linker and a second linker, respectively, the first linker and the second linker being capable of binding or being bound to a first unit molecule and a second unit molecule, respectively, the relative positional relationship of the first linker fixing point and the second linker fixing point being fixed, the first unit molecule and the second unit molecule being capable of forming together an interactive complex, the method comprising the step of designing the structure based on the interactive complex. (Item A2) The method according to any one of the above items A, wherein the step of designing the structure includes designing the structure having a structure such that a crossing index of the first unit molecule and the second unit molecule satisfies-5≤D≤15. (Item A3) The method according to any one of the above item a, wherein the step of designing the structure includes designing the structure based on the sizes of the first unit molecule and the second unit molecule. (Item A4) The method according to any one of the above items a, wherein the step of designing the structure includes designing the structure based on a predicted value of a binding dissociation constant between the first unit molecule and the second unit molecule. (Item A5) The method according to any one of the above item a, wherein the step of designing the structure includes determining the lengths of the first linker and the second linker based on at least one of the sizes of the first unit molecule and the second unit molecule, and the binding dissociation constant between the first unit molecule and the second unit molecule. (Item A6) The method according to any one of the above item a, wherein the step of designing the structure includes determining a distance between the first linker fixing point and the second linker fixing point based on at least one of a size of the first unit molecule and the second unit molecule, and a binding dissociation constant between the first unit molecule and the second unit molecule. (Item A7) The method of any one of the above items a, wherein the step of designing the structure includes selecting the substrate from a plurality of options based on the sizes of the first unit molecules and the second unit molecules. (Item A8) The method of any one of the above items a, wherein the step of designing the structure comprises selecting the substrate from a plurality of options, determining a distance between the first connector anchor point and the second connector anchor point, and determining lengths of the first connector and the second connector. (Item A9) A method for analyzing an interaction complex, comprising the steps o