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Single-stranded DNA aptamer capable of specifically recognizing O-GlcNAc modification and screening method and application of single-stranded DNA aptamer

A screening method and aptamer technology, applied in the field of biochemical detection, can solve problems such as easy shedding, low specificity of lectin method, small molecular weight, etc., to achieve the effect of ensuring specificity and broadening the scope of application

Active Publication Date: 2020-08-18
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The primary method to study O-GlcNAc modification is to identify and enrich proteins with this modification. However, there are many difficulties in the identification of O-GlcNAc modification, because the modification is not only small in molecular weight and low in abundance, but also in the In highly dynamic changes, this modification is easy to fall off when preparing mass spectrometry samples, etc.
Although there are a variety of methods for detecting and identifying O-GlcNAc modifications, none of them can satisfy the large-scale identification research on O-GlcNAc modifications.
For example, the specificity of the lectin method is low, and the existing O-GlcNAc modified antibodies are not only expensive but also have amino acid sequence dependence, etc.

Method used

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  • Single-stranded DNA aptamer capable of specifically recognizing O-GlcNAc modification and screening method and application of single-stranded DNA aptamer
  • Single-stranded DNA aptamer capable of specifically recognizing O-GlcNAc modification and screening method and application of single-stranded DNA aptamer
  • Single-stranded DNA aptamer capable of specifically recognizing O-GlcNAc modification and screening method and application of single-stranded DNA aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 SELEX technology screens out nucleic acid aptamers that specifically bind to O-GlcNAc modified polypeptides

[0041]1. First, prepare the screening target. According to the characteristic that the sulfhydryl group (-SH) on cysteine ​​can react with gold nanoparticles (GNPs) under mild conditions to form a stable gold sulfhydryl bond, and its UV absorption peak will shift after the reaction. It is loaded on the surface of nano gold for solid-liquid separation. After incubating the polypeptide and gold nanoparticles at room temperature overnight under neutral conditions, the O-GlcNAc-modified polypeptide can be attached to the surface of gold nanoparticles (with a particle size of 13nm), and the unlinked polypeptide is removed by centrifugation to complete the preparation of the screening target.

[0042] 2. A relatively stable single-stranded DNA aptamer library is used for screening. The library information is 5'-GCGGAATTCAACAGTCCGAGCC-N30-GGGTCAATGCGTCATA-3...

Embodiment 2

[0047] Example 2 Identification of aptamers that specifically bind O-GlcNAc polypeptides

[0048] The secondary library with the highest affinity in the screening was subjected to high-throughput (HGS) sequencing, and the sequencing results were used as the basis for analysis to determine candidate aptamers. Then find the aptamers that can specifically bind to the O-GlcNAc polypeptide from the candidate aptamers for identification.

[0049] 1. Identification of aptamers that can specifically bind O-GlcNAc polypeptides among candidate aptamers

[0050] 1. According to the sequence information and secondary structure in the sequencing results, analyze the aptamers with more repetitions in the results, and find out ten representative aptamers as candidate aptamers for subsequent experiments;

[0051] 2. After the single strand of the candidate aptamer is sent to the biological company for synthesis, a symmetric PCR is performed first to obtain a full-length double-stranded aptam...

Embodiment 3

[0061] Example 3 ELONA method to detect the affinity of single aptamer and O-GlcNAc polypeptide

[0062] By analyzing the results of MST detection, it was found that the aptamers aptO-19, aptO-31 and aptO-14 could specifically bind to the O-GlcNAc modified polypeptide, but not to the unmodified polypeptide. The affinity of the three aptamers to the O-GlcNAc modified polypeptide was further determined by K d Value representation.

[0063] (1) Use the correctly sequenced aptamer plasmid as a template, and use the biotin-labeled downstream primer P2 as a primer to perform asymmetric PCR to obtain biotin-labeled single aptamers, and recover them by ethanol precipitation;

[0064] (2) The recovered aptamers were diluted in multiples with screening buffer, and the concentrations were 1uM, 0.5uM, 0.25uM, 0.125uM, 0.0625uM, 0.031uM, and 0uM. After dilution, keep at 95°C for 10 minutes, and immediately put it on ice for later use.

[0065] (3) Coat a detachable 96-well plate with 10...

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Abstract

The invention discloses a single-stranded DNA aptamer capable of specifically recognizing O-GlcNAc modification and a screening method and application of the single-stranded DNA aptamer, and belongs to the technical field of biochemical detection. Specifically, an exponentially enriched ligand phylogenetic technology (SELEX) is used as a basis, O-GlcNAc modified polypeptide is used as a screeningtarget, and unmodified polypeptide is used as reverse screening. After multiple rounds of screening, the DNA aptamer capable of specifically recognizing O-GlcNAc modification is obtained. The aptameris used as a core, and the DNA aptamer labeled by biotin and the like is used for detecting the O-GlcNAc modification level in protein. The invention provides a screening method of an O-GlcNAc modified single-stranded DNA aptamer. The DNA aptamer labeled by biotin and the like is used for detecting protein O-GlcNAc modification, a new method is provided for research of O-GlcNAc modification, a newdirection is opened up for application of the aptamer in post-translational modification, and the DNA aptamer has potential application prospects.

Description

technical field [0001] The invention relates to the technical field of biochemical detection, in particular to a single-stranded DNA aptamer capable of specifically recognizing O-GlcNAc modification, a screening method and application thereof. Background technique [0002] Protein O-GlcNAc modification widely exists in a variety of proteins and is a post-translational modification in response to nutrition and stress. Since its discovery, more than 4,000 proteins have been identified with O-GlcNAc modification, and these proteins are involved in almost all physiological processes, such as the stabilization of protein structure, signal transduction, regulation of transcriptional activity, regulation of epigenetics, regulation of neuronal function, and protein Interactions, stress responses, etc. all play a very important role. Abnormal modification of protein O-GlcNAc can also lead to the occurrence of various diseases. Such as tumors, diabetes, cardiovascular diseases, neur...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12N15/10G01N33/53G01N33/68
CPCC12N15/1048C12N15/115C12N2310/16G01N33/53G01N33/68
Inventor 章晓联王咏
Owner WUHAN UNIV
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