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ssDNA aptamer capable of specifically recognizing N-acetylneuraminic acid and application thereof

A technology of acetylneuraminic acid and aptamer, which is applied in the direction of recombinant DNA technology, DNA/RNA fragments, fluorescence/phosphorescence, etc., can solve the problems of limiting the application of immunoassay methods, limited types of enzyme-labeled antibodies, production and application restrictions, etc. , to achieve the effect of high affinity, easy modification and labeling, and low cost

Active Publication Date: 2021-07-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are some ELISA kit products for the detection of Neu5Ac residues on the market, but the price is expensive, and the types of enzyme-labeled antibodies are limited, and because Neu5Ac is a small molecule compound, it is not immunogenic itself, and there are many restrictions on production and application.
These have largely limited the application of immunoassay methods in the detection of Neu5Ac

Method used

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  • ssDNA aptamer capable of specifically recognizing N-acetylneuraminic acid and application thereof
  • ssDNA aptamer capable of specifically recognizing N-acetylneuraminic acid and application thereof
  • ssDNA aptamer capable of specifically recognizing N-acetylneuraminic acid and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] (1) Construction of random ssDNA library and its primers:

[0030] (a) Construct a random ssDNA library with a length of 79 bases

[0031] 5'-TAGGGAATTC GTCGACGGAT CC-N35-CTGCAGGTCG ACGCATGCGC CG-3', wherein N represents any one of the bases A, T, C, and G.

[0032] (b) Synthetic forward primer

[0033] Forward primer 1: 5′-TAGGGAATTC GTCGACGGAT-3′;

[0034] Forward primer 2: 5′-FAM-TAGGGAATTC GTCGACGGAT-3′;

[0035] (c) Synthetic reverse primer

[0036] Reverse primer 1: 5'-CGGCGCATGC GTCGACCTG-3';

[0037] Reverse primer 2: 5'-biotin-CGGCGCATGCGTCGACCTG-3'.

[0038] (2) In vitro screening of nucleic acid aptamers:

[0039] In order to screen out ssDNA aptamers with high affinity and specificity to Neu5Ac, a total of 10 rounds of nucleic acid aptamer screening were carried out.

[0040] (a) The 25 μL PCR amplification system is shown in Table 1.

[0041] Table 1 Substances and dosages of 25 μL PCR amplification system

[0042] raw material concentr...

Embodiment 2

[0052] Determination of dissociation constant K of aptamer sequence by fluorescence method d value

[0053] The stability and secondary structure of multiple aptamer sequences obtained in Example 1 were analyzed by Mfold online software. Add different concentrations of aptamer sequences modified with the fluorescent group 6-carboxyfluorescein (FAM) to the binding buffer, and supplement the volume with binding buffer to 200 μL, denature at 90°C for 10 minutes, rapidly ice-bath for 10 minutes, and place at room temperature 10min. Then add 10 μmol Neu5Ac into the solution, shake slightly, react at room temperature for 2 hours, then add graphene oxide, incubate at room temperature for 2 hours, and mix the solution at 13000r·min -1 Centrifuge for 5 minutes and take the supernatant. Finally, all the supernatant was added to a 96-well plate to measure its fluorescence intensity with a microplate reader. The content of the aptamer sequence is proportional to the fluorescence inten...

Embodiment 3

[0057] Specific results of detection of ssDNA aptamer sequence ap1 by fluorescence method

[0058] Add 200 μL of binding buffer to the aptamer api of the same concentration modified by the fluorescent group 6-carboxyfluorescein (FAM), denature at 90°C for 10 minutes, rapidly ice-bath for 10 minutes, and place at room temperature for 10 minutes. Then add 1 μmol of Neu5Ac, N-glycolylneuraminic acid (Neu5Gc), urosonic acid (KDN), glucose, sucrose, and maltose solution into the solution, shake slightly, react at room temperature for 2 hours, and then add graphite oxide ene, incubated at room temperature for 2h, and the mixture was heated at 13000r·min -1 Centrifuge for 5 minutes and take the supernatant. Finally, all the supernatant was added to a 96-well plate to measure its fluorescence intensity with a microplate reader.

[0059] Specific test results such as image 3 As shown, in the presence of Neu5Ac, the detected fluorescence intensity is significantly higher than that o...

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Abstract

The invention relates to an ssDNA aptamer capable of specifically recognizing N-acetylneuraminic acid and application thereof. According to the invention, 4 ssDNA aptamer sequences capable of being highly specifically combined with Neu5Ac are obtained through screening of a magnetic bead-SELEX (systematic evolution of ligands by exponential enrichment) technology, and sequence ap1 has the highest affinity and specificity; and moreover, sequence ap1 is stable in structure. On the basis of sequence ap1, the sequence is truncated according to a secondary structure and a molecular simulation docking result to obtain one aptamer ap2 with the sequence length of 21nt, and the affinity is improved compared with that before truncation. The ssDNA aptamer disclosed by the invention is relatively high in binding specificity and affinity with Neu5Ac, and is high in sensitivity when being applied to Neu5Ac detection.

Description

technical field [0001] The invention relates to the fields of biochemistry and molecular biology, analytical chemistry and combinatorial chemistry, in particular to ssDNA aptamers specifically recognizing N-acetylneuraminic acid and applications thereof. Background technique [0002] N-acetylneuraminic acid (Neu5Ac) is the most studied and intensively studied sialic acid, and it is a precursor for the synthesis of other classes of sialic acid. As an important part of glycoconjugates, sialic acid can promote mutual repulsion between cells, stabilize cell membranes, increase the viscosity of coupled glycoproteins, etc. due to the negative charge it carries. Sialic acid is also an important biological information transmission molecule, such as participating in the recognition of host and pathogen and the reaction between cells, and participating in the phagocytosis of macrophages on aging red blood cells. It has been previously demonstrated that, on the one hand, the abnormal ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/53G01N21/64
CPCC12N15/115G01N33/5308G01N21/6428C12N2310/16G01N2021/6432
Inventor 周楠迪岳辉张雨婷王晓丽
Owner JIANGNAN UNIV
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