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Biomolecule affinity constant determination method based on DNA (Deoxyribonucleic Acid) origami

A measurement method and biomolecular technology, applied in the field of detection, can solve problems such as large errors, cumbersome steps, and complexity, and achieve the effects of high specificity, good repeatability, and less sample consumption

Inactive Publication Date: 2013-03-06
SHANGHAI INST OF APPLIED PHYSICS - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] Therefore, the technical problem to be solved by the present invention is that the existing biomolecular affinity constant determination method cannot be measured at the single molecule level, and there are many conditions set in the experiment, the steps are cumbersome and complicated, and the errors introduced are also large. For a relatively large problem, DNA origami has the advantages of precise and controllable reaction sites and the advantage that atomic force microscopy (AFM) can directly observe single antigen-antibody molecular complexes on the reaction interface, providing a DNA origami-based biomolecule Affinity Constant Determination Method

Method used

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  • Biomolecule affinity constant determination method based on DNA (Deoxyribonucleic Acid) origami
  • Biomolecule affinity constant determination method based on DNA (Deoxyribonucleic Acid) origami
  • Biomolecule affinity constant determination method based on DNA (Deoxyribonucleic Acid) origami

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Embodiment 1

[0038] Example 1 Determination of digoxin and digoxin antibody affinity constant

[0039] 1. Preparation of digoxin-modified rectangular DNA origami

[0040] The DNA origami designed in this example utilizes M13mp18DNA and staple single-strand self-assembly to form a rectangular pattern with a length and width of 100 nm and 70 nm, respectively. Such as figure 1 Shown, at two positions on the DNA origami, the ends of the staple strands are modified with digoxigenin for binding to the digoxigenin antibody.

[0041] Mix M13mp18 DNA single strands and staple single strands (including digoxin-modified staple single strands) at a molar concentration of 1:10, and place in 1×TAE / Mg 2+ Buffer system (Tris 40mM, acetic acid 20mM, EDTA 2mM, MgCl21 2 .5mM, pH value 8), the total volume is 61μL, and then placed on the PCR instrument to anneal from 95°C to 20°C at an annealing speed of 0.1°C / 10s. After the reaction is completed, excess staples are removed by ultrafiltration Short chains...

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Abstract

The invention discloses a biomolecule affinity constant determination method based on DNA (Deoxyribonucleic Acid) origami. The biomolecule comprises a reactant I and a reactant II which are integrated together and dissociated. The method comprises the following steps of: (1) connecting the reactant I to the tail of a staple chain of the DNA origami, and then, executing self-assembly together with a scaffold chain to form the DNA origami; (2) adding the reactant II, after reaching equilibrium in reaction, collecting data by executing scanning imaging with an atomic force microscope, calculating the concentration [AbAg] of a compound of the reactant I and the reactant II, the concentration[Ag] of the free reactant I and the concentration [Ab] of the free reactant II; and (3) substituting the formula K=[AbAg] / [Ag][Ab] with the obtained data to calculate the affinity constant of the reactant I and the reactant II. The method can be used for accurately calculating the affinity constant of the biomolecule at the molecular level, thereby having the advantages of high efficiency, excellent repeatability and less usage of samples.

Description

technical field [0001] The invention belongs to the field of detection methods, in particular to a method for measuring the affinity constant of biomolecules based on DNA origami. Background technique [0002] Antibody affinity (affinity) refers to the firmness of the combination of antigen and antibody. The greater the affinity, the faster the binding reaction of the antibody, and the less likely the antigen-antibody complex is to dissociate. Antibody affinity is the sum of non-covalent attraction and repulsion between the binding site of the antibody and the corresponding antigenic determinant, and it is an important indicator for evaluating the quality of the antibody. The magnitude of antibody affinity is expressed by affinity constant, and the affinity constant (K) is the equilibrium constant of the antigen / antibody binding reaction. [0003] At present, there are many methods for determining the affinity constant, such as: thiocyanate elution method, urea elution met...

Claims

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Application Information

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IPC IPC(8): G01Q60/34
Inventor 樊友杰李宾吴娜胡钧
Owner SHANGHAI INST OF APPLIED PHYSICS - CHINESE ACAD OF SCI
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