Helicase activity determination method based on double-stranded DNA and application of helicase activity determination method

Abstract

The invention discloses a double-stranded DNA (deoxyribonucleic acid)-based helicase activity determination method and application thereof. The method mainly realizes detection based on a nucleic acid secondary structure obtained by single-stranded hybridization of at least two DNAs. The tail end of at least one DNA single chain in the nucleic acid secondary structure is connected with a tail sequence, and the tail ends of any two different DNA single chains in the nucleic acid secondary structure are respectively connected with two substances or groups which are not interfered with each other and are specifically combined. Compared with other helicase detection methods, the detection method provided by the invention has a more accurate helicase activity quantitative effect, does not need tedious steps such as electrophoresis and membrane transfer, shortens the detection time by 3-4 hours, allows simultaneous detection of ATP consumption in the same solution system, and makes helicase activity evaluation more comprehensive.

Description

technical field [0001] The invention belongs to the field of enzyme activity detection methods, in particular to a double-stranded DNA-based helicase activity detection method and application thereof. Background technique [0002] At the molecular level, helicases are motor proteins that move directionally along the nucleic acid phosphodiester backbone and are able to use energy from the hydrolysis of adenosine triphosphate (ATP) to unfold folded nucleic acid strands. In the related art, the activity of helicase has been shown to affect most of the metabolic processes of nucleic acids in cells, such as DNA replication and repair, gene transcription and translation, chromosome segregation and telomere maintenance, etc. Deletion or mutation of helicase can also lead to various diseases and susceptibility to cancer. [0003] There are multiple important nucleic acid secondary structures and corresponding helicases in living organisms, and exploring these different unwinding pr...

AI Technical Summary

Problems solved by technology

However, these methods also have many limitations and defects.

EMSA can only achieve semi-quantitative detection, and cannot further achieve accurate quantification, and the detection method is relatively cumbersome, with low throughput, and cannot achieve rapid detection of large batches of samples

The fluorescence method is limited by the use of fluorescent labels. Fluorophores can only be labeled at specific positions on the two DNA strands, and the distance between the two fluorophores must be accurately designed, and these necessary technical requirements greatly limit Expanding the design of the DNA substrate model makes it lack of universality. Moreover, the assay based on the fluorescence method is susceptible to the interference of proteins in the sample, which may interfere with the fluorophore or even directly cause the fluorescence quenching, resulting in inaccurate detection. exact question

However, single-molecule technology also has the disadvantages of high requirements for use, many requirements for operators, high technical difficulty, high detection cost, and low throughput. It is difficult to meet the needs of most general laboratories or simple detection. Therefore, it is urgent to develop Novel assay for convenient and accurate quantification of helicase activity

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