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Molecular ruler hairpin structure and method for measuring spatial scale accuracy of single-molecule magnetic tweezers by using same

A technology of spatial scale and hairpin structure, which is applied in the fields of biochemical equipment and methods, determination/inspection of microorganisms, instruments, etc., can solve the problems of the accuracy of the spatial scale of single-molecule magnetic tweezers, etc.

Active Publication Date: 2020-06-09
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no method has been reported to measure the spatial scale accuracy of single-molecule magnetic tweezers based on protein-nucleic acid complex binding events and theoretical models

Method used

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  • Molecular ruler hairpin structure and method for measuring spatial scale accuracy of single-molecule magnetic tweezers by using same
  • Molecular ruler hairpin structure and method for measuring spatial scale accuracy of single-molecule magnetic tweezers by using same
  • Molecular ruler hairpin structure and method for measuring spatial scale accuracy of single-molecule magnetic tweezers by using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1 Construction of molecular ruler hairpin structure

[0083] 1 Preparation of biotin handle and digoxin handle

[0084] 1.1 Polymerase chain reaction of biotin modification / digoxigenin modification

[0085] Perform biotin-modified / digoxigenin-modified PCR on the pbluescrIISK+ plasmid by adding:

[0086] 1 μl of primers upstream of the biotin / digoxigenin handle at a concentration of 10 μM, respectively shown in SEQ ID NO.1 and SEQ ID NO.3, (primers purchased from Jinweizhi);

[0087] 1 μl of primers downstream of the biotin / digoxigenin handle at a concentration of 10 μM, as shown in SEQ ID NO.2 or SEQ ID NO.4, respectively (primers were purchased from Jinweizhi Company);

[0088] 1 μl 10mM dATP (Catalog No.: 4026Q, Baori Medical Biotechnology Company);

[0089] 1 μl 10mM dGTP (Catalog No.: 4027Q, Baori Medical Biotechnology Company);

[0090] 1 μl 10mM dCTP (Catalog No.: 4028Q, Baori Medical Biotechnology Company);

[0091] 0.9 μl 10mM dTTP (Catalog No.: ...

Embodiment 2

[0169] Example 2 Measure and calculate the measured conversion coefficient of nano / base by applying external force with single-molecule magnetic tweezers

[0170] In this embodiment, the magnetic tweezers experiment uses a magnetic tweezers device produced by Tianjin Guangying Micro-Nano Technology Co., Ltd. The molecular ruler hairpin structure can be fixed on the glass surface of the working cell through the interaction between digoxin-digoxigenin antibody, and the biotin-streptavidin affinity binding with superparamagnetic beads (M270, item number: 65305, Invitrogen Corporation) connected.

[0171] The preparation process of the working pool is as follows: take two clean coverslips of 60mm×24mm, and use a TC-169 multi-function grinding and engraving machine to punch two holes on one piece as the water inlet and water outlet, and evenly smear 3μm polystyrene beads on one piece As a magnetic bead reference, a double-layer sealing film with a reaction channel cut in advance i...

Embodiment 3

[0174] Example 3 Calculate the theoretical conversion coefficient of nanometers / bases according to the theoretical worm model

[0175] The DNA length can be calculated by the theoretical worm model, as shown in Equation 1, where F is the force, x is the length, Lp is the persistent length, Lc is the profile length, and K B is the Boltzmann constant, and T is the temperature.

[0176]

[0177]According to Bosco, A. Under the working conditions described in the article Elastic properties and secondary structure formation of single-stranded DNA at monovalent and divalent salt conditions (PMID: 24225314), Lp is selected as 0.87 nanometers, and Lc is 0.69 nanometers / base, according to This calculated theoretical conversion factor is 0.48 nm / base.

[0178] The deviation between the measured conversion coefficient and the theoretical conversion coefficient in this embodiment is 4%. Therefore, the spatial scale accuracy of the single-molecule magnetic tweezers used in the instrume...

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Abstract

The invention relates to a molecular ruler hairpin structure and a method for measuring the spatial scale accuracy of single-molecule magnetic tweezers by using the molecular ruler hairpin structure.The method comprises the following steps: firstly, constructing a hairpin structure, namely a molecular ruler hairpin structure of which the stem part contains a plurality of CCGG sites; fixing the hairpin structure on the surface of a working pool; monitoring the position of magnetic beads in real time by using single-molecule magnetic tweezers; and performing calculation to obtain an accurate length, namely the length between adjacent CCGG sites, of the molecular ruler hairpin structure between two adjacent pause states. Through conversion of length and base logarithm, a length conversion coefficient can be accurately calculated and is compared with a theoretical value of a worm model, so that the spatial scale accuracy of the single-molecule magnetic tweezers is measured. The method canbe used for calibrating the accuracy of precision instruments, and broadens the way for measuring the accuracy of the precision instruments.

Description

technical field [0001] The invention belongs to the field of measurement and analysis of precision instruments, and in particular relates to a method for calibrating the spatial scale accuracy of a single-molecule magnetic tweezers instrument by using DNA length changes. Background technique [0002] Single-molecule force spectroscopy is an emerging precision measurement and analysis technology in the field of biophysics in recent years, which can detect the dynamic reaction process of a single molecule or molecular complex in vitro or in cells. Single-molecule magnetic tweezers is one of the popular single-molecule force spectroscopy techniques, which can apply external forces and torques to hundreds or thousands of individual molecules and track them in parallel. The length and length change of biomolecules are important indicators for the measurement of single-molecule magnetic tweezers, which can be used to calculate a series of specific parameters of protein-nucleic aci...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12Q1/686G16B25/20G16B5/00
CPCC12N15/113C12Q1/686G16B5/00G16B25/20C12N2310/531C12Q2525/301
Inventor 于仲波王泽瑜梁琳李旭王军力
Owner NANKAI UNIV
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