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A method for determining the conformation of sulfhydryl-modified linear DNA or nucleic acid aptamers on the surface of gold nanoparticles

A nucleic acid aptamer and sulfhydryl modification technology, applied in the biological field, can solve problems affecting conformational research, large sample volume, large experimental error, etc., and achieve the effect of fewer chemical groups, reduced sample volume, and simple structure

Active Publication Date: 2017-06-06
CAPITAL NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although agarose gel electrophoresis technology can be used to study the conformational changes of linear DNA, there are many defects in this method: 1. The sample size required by agarose gel electrophoresis technology is relatively large, and the samples cannot be reused, which is time-consuming and laborious. Suitable for kinetic studies; 2. Gold nanoparticles modified with short-chain DNA (less than 40 nucleotides) are common in practical applications, but agarose gel electrophoresis cannot obtain the resolution of a single short-chain DNA; 3. Gel method The experimental error of the calculated DNA / AuNP particle size is relatively large, which is affected by the gel ratio and requires multiple measurements, which is time-consuming and laborious; 4. Nano-gold needs to be modified with small molecules to improve the stability of nano-gold before agarose gel electrophoresis to prevent Aggregation occurs during electrophoresis, but in practical applications, the existence of these small molecules will interfere with the actual conformation of DNA on the surface of gold nanoparticles, so that the correct conformational information cannot be obtained; 5. Aptamer DNA with a secondary conformation will fold into Tight secondary structure, which affects electrophoretic mobility and thus the study of conformation

Method used

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  • A method for determining the conformation of sulfhydryl-modified linear DNA or nucleic acid aptamers on the surface of gold nanoparticles
  • A method for determining the conformation of sulfhydryl-modified linear DNA or nucleic acid aptamers on the surface of gold nanoparticles
  • A method for determining the conformation of sulfhydryl-modified linear DNA or nucleic acid aptamers on the surface of gold nanoparticles

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1. DLS real-time monitoring of the hydration particle size of linear DNA without sulfhydryl modification and thiol-modified linear DNA and nucleic acid aptamer and 13nm nano-gold complex in the aging process.

[0040] With 5' terminal thiol modification, linear DNA of different lengths: polyAl5, polyA30, polyA44, polyA51, and aptamer DNAs of different lengths: TBA30, TBA44 (adapter to bovine thrombin protein, Thrombinbinding aptamer), CBA45, CBA51 (Cocaine binding aptamer) 40μM, were reduced with 40mM dithiothreitol (DTT) in 2% (V / V) triethylamine (TEA) solution for 30 minutes and then used NAP- 5 columns for two purifications. The ratio of the purified DNA to the input material of nano gold is 500:1. At the same time, the amount ratio of linear DNA single-stranded polyA15, polyA30, polyA44, polyA51 without sulfhydryl modification to gold nanometer (AuNP) was 500:1. The 12 strands were coated with phosphate buffer (10mM PB, pH 7.4) at room temperature (25°C) a...

Embodiment 2

[0042] Example 2. Determination of the relationship between the coating amount of linear DNA on gold nanoparticles and the hydrated particle size of linear DNA / AuNP during the aging process.

[0043] The f-polyA30 modified with the sulfhydryl group at the 5' end and the fluorescent FAM group at the 3' end was reduced and coated with gold nanoparticles. During the aging process, select points of 0.2, 0.5, 1, 2, 6, 10, and 17 hours to centrifuge the sample at 15,000 rpm, and redissolve the precipitate in 1 () () uL, 1 M dithiothreitol (DTT) phosphate buffer The solution was left overnight and centrifuged to get the supernatant, and the probe concentration in the supernatant was determined according to the standard curve and the coating amount was calculated. And observe the relationship between the coating amount and particle size change.

Embodiment 3

[0045] Example 3. Fitting the adsorption kinetic model of linear DNA on the surface of 13nm gold nanometers.

[0046] According to the linear relationship q between particle size and coating amount during aging e = kd e q t = kd t Equation 2), through the pseudo-second-order kinetic equation (Formula 1) to get (Formula 3) (where Ks' is kKs). Select different lengths of thiol-modified polyA15, polyA30, polyA44, and polyA51 at the time points of 0.2, 0.5, 1, 2, 6, 10, and 17 hours (h) and the particle size growth bands at the corresponding time when the nano-gold surface is aged and coated into formula 3.

[0047] The results show that t / dt has a linear relationship with t ( Figure 4 A ), and the calculated qe is highly consistent with the experimentally obtained qe (Table 2), that is, the adsorption process of thiol-modified DNA on the surface of gold nanoparticles conforms to the pseudo-second-order adsorption kinetic model; The rate-limiting step of the kinetic co...

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Abstract

The invention relates to a method for measuring confirmations of a sulphydryl-modified linear DNA (deoxyribonucleic acid) or nucleic acid aptamer on a nano-gold surface. The method comprises the following steps of measuring the hydrate particle size of a linear DNA or nucleic acid aptamer and a nano-gold composite in the assembling process in a dynamic light scattering way, and measuring the confirmations of the linear DNA or nucleic acid aptamer on the nano-gold surface under different surface coating amounts by combining adsorption kinetics respectively. According to the method, under the condition of medium surface probe capacity, one end, connected with a sulphydryl, of the confirmation of the linear DNA on the nano-gold surface winds the nano-gold surface, and the other end of the confirmation of the linear DNA outwards extends perpendicularly to the surface; along with the increase of the surface probe capacity, the length of each DNA probe winding the nano-gold surface is continuously decreased, the outwards-extending tail of each DNA probe becomes longer and longer, and however, the nano-gold surface is kept in a DNA basic group adsorbed-wrapped low-surface free energy state; the confirmations can be described as DILOT (dynamic inner-layer and outer-tail) models significant on guiding the applications of nucleic acid sensor probe designs, DNA-hybridization-based nano-assembling and the like.

Description

technical field [0001] The invention relates to a method for measuring the conformation of sulfhydryl-modified linear DNA or nucleic acid aptamer on the surface of nano-gold, belonging to the field of biotechnology. Background technique [0002] Gold nanocomposites (DNA / AuNP) modified by sulfhydryl linear DNA or nucleic acid aptamers have very important applications in the fields of self-assembly material preparation, biosensing technology, and drug delivery. However, recent studies have shown that DNA bases will interact non-specifically with the surface of gold nanoparticles, and some of them will be adsorbed on the surface of gold nanoparticles, which will reduce the efficiency of linear DNA hybridization and reduce the recognition ability of nucleic acid aptamers and their targets. Therefore, an in-depth understanding of the conformation of linear DNA or aptamer probes on the surface of gold nanoparticles is of great value for optimizing probe design. At this stage, the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N15/02
Inventor 娄新徽王文杰丁小凡
Owner CAPITAL NORMAL UNIVERSITY