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Single-molecule mechanical method for measuring inhibition of small-molecule drug on protein and nucleic acid interaction

A technology for inhibiting proteins and small molecules, which is applied in measuring devices, scientific instruments, and analytical materials, etc., and can solve the problems of single-molecule mechanics measurement of small-molecule drug inhibitory protein-nucleic acid interaction methods that have not been reported.

Pending Publication Date: 2021-01-22
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, so far, no method has been reported based on single-molecule mechanics to measure small-molecule drug inhibitory protein-nucleic acid interactions.

Method used

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  • Single-molecule mechanical method for measuring inhibition of small-molecule drug on protein and nucleic acid interaction
  • Single-molecule mechanical method for measuring inhibition of small-molecule drug on protein and nucleic acid interaction
  • Single-molecule mechanical method for measuring inhibition of small-molecule drug on protein and nucleic acid interaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Constructing a DNA structure-based unimolecular reactor for CpG motifs

[0048] Table 1 Artificial oligonucleotide DNA name and sequence information

[0049]

[0050]

[0051]DNA hairpin construct handle preparation. The handle of the DNA hairpin structure contains biotin and digoxin modification, and is prepared by polymerase chain reaction, and the process is as follows. Add 1 µl of biotin / digoxigenin handle upstream primer (Table 1, concentration 10 µM), 1 µl biotin / digoxigenin handle downstream primer (Table 1, concentration 10 µM) to an enzyme-free reaction tube 10 micromolar, primers were purchased from Sangon Bioengineering Company), 1 microliter (concentration: 10 millimolar) of dATP (article number: 4026Q, Baoriji Biotechnology Co., Ltd.), 1 microliter (concentration: 10 millimolar) of dATP dGTP (Cat. No.: 4027Q, Biotech Biotechnology Co., Ltd.), 1 microliter of dCTP (concentration: 10 millimolar, Cat. No.: 4028Q, Biotech Biotech Co., Ltd.), 0...

Embodiment 2

[0056] Example 2 Establishment of a single-molecule reactor system based on a DNA hairpin structure in a microfluidic reaction pool

[0057] The preparation process of the microfluidic reaction cell is as follows. Take two slides, the size is 60 mm × 24 mm. On a multi-functional grinding and engraving machine (model: TC-169), punch holes at both ends of a glass slide for liquid sampling and sampling.

[0058] Then, perform a washing step. Slides were immersed in water with dish soap and ultrasonically cleaned for 30 minutes. Replace with pure water and wash 3 times, 5 minutes each time. Replace the isopropanol and sonicate for 30 min. Replace pure water, wash 3 times, 5 minutes each time. Replace with 75% ethanol and soak for several hours. Finally blow dry with nitrogen.

[0059] Spread the nitrocellulose and fix the reference pellet. Take 1% nitrocellulose stock solution, add 250mg / 10ml monodisperse polypropylene beads (article number: PSC-03000, Big Goose (Tianjin) ...

Embodiment 3

[0064] Example 3 Normalization processing of unimolecular reaction data lines of MLL1 CXXC protein binding to CpG motif

[0065] The protein used in this example is the MLL1 CXXC domain, which can specifically recognize the CpG motif contained in the stem of the DNA hairpin structure. MLL1 CXXC protein was synthesized by Sangon Bioengineering Company. In this embodiment, when detecting the binding position of the protein on the DNA, the length of the DNA is first normalized according to the base unit. Under the test force conditions, the normalization process is based on the length of the DNA hairpin structure after it is completely unzipped. Each single-molecule data line is self-calibrated according to the length of the DNA after melting, and then a histogram is built. The middle peak in the histogram distribution indicates where the protein binds to the DNA. Convert DNA length from nanometers to bases with reference to the middle peak of the histogram distribution. This...

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Abstract

The invention relates to a single-molecule mechanical method for measuring inhibition of a small-molecule drug on protein and nucleic acid interaction. The method includes: first, designing and constructing a monomolecular reactor based on a DNA hairpin structure, the stem portion of which comprises a motif interacting with protein, and the handle portion of which is labeled with digoxin and biotin by polymerase chain reaction; secondly, preparing protein interacting with the DNA; thirdly, fixing the DNA hairpin structure between a magnetic bead and a glass sheet, and carrying out a DNA melting experiment in a microfluidic reaction tank through a monomolecular mechanics method; and finally, systematically testing the influence of the concentration of the small-molecule drug on the meltingreaction of the DNA combined with the protein, drawing a drug dose-response curve, calculating a half inhibition concentration, and evaluating the drug effect of the small-molecule drug on the proteinand nucleic acid interaction. The invention belongs to the field of biomacromolecule interaction and monomolecular pharmacology, and develops a new method for directly measuring small-molecule drug inhibition on protein nucleic acid interaction for molecular biology research, drug development and pharmacological evaluation.

Description

technical field [0001] The invention belongs to the fields of biomacromolecule interaction and single-molecule pharmacology, and in particular relates to a single-molecule mechanics method for measuring small-molecule drug inhibitory protein-nucleic acid interaction. Background technique [0002] Amino acid motifs play important roles in protein structure and function and are commonly drug targets. For example, the myeloablative drug Busulfan converts cysteines in the CXXC motif of oxidoreductase proteins to dehydroalanine and lanthionine. For another example, the protein kinase PKCθ interacts with CD28 through the CXXC motif and becomes the target of the small molecule drug dimethyl fumarate (DMF). As more and more DMF targets are identified, such as NF-kappaB and GAPDH, the diseases that DMF can treat are also expected to expand from psoriasis and multiple sclerosis to tumors. In fact, selective modification of motifs in protein targets is a popular method and strategy i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N27/74
CPCG01N33/54326G01N27/745
Inventor 于仲波马康康王泽瑜梁琳
Owner NANKAI UNIV
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