Aptamer capable of being specifically combined with nodularin-R and application of the aptamer

An aptamer and specific technology, which is applied in the field of aptamers that specifically bind to nodularin-R, achieves the effects of high repeatability, simplified construction of technical routes, and low cost

Active Publication Date: 2018-12-07
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no related reports on NOD-R aptamers

Method used

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  • Aptamer capable of being specifically combined with nodularin-R and application of the aptamer
  • Aptamer capable of being specifically combined with nodularin-R and application of the aptamer
  • Aptamer capable of being specifically combined with nodularin-R and application of the aptamer

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1. The design of random ssDNA library and its primers

[0038] 1. Design of ssDNA library

[0039] The NOD-R aptamer library consists of 80 bases, both ends are fixed regions containing 20 bases, and the middle is a random region containing 40 bases: 5'-AAGGAGCAGCGTGGAGGATA-N40-TTAGGGTGTGTCGTCGTGGT-3' (N is any one of the four deoxyribonucleotide bases of A, T, G, and C, and 40 represents the number of random bases).

[0040] 2. Primer Design

[0041] Upstream primer: 5'-AAGGAGCAGCGTGGAGGATA-3' (SEQ ID NO.13)

[0042] Downstream primer A: 5'-ACCACGACGACACACCCTAA-3' (SEQ ID NO.14)

[0043] Downstream primer B: 5'-poly(dA20)-Spacer18-ACCACGACGACACACCCTAA-3' (SEQ ID NO.15).

[0044] Among them, the downstream primer A is mainly used for the amplification of ssDNA in the screening, and the downstream primer B is mainly used for cloning and sequencing.

Embodiment 2

[0045] Example 2. Screening of NOD-R aptamers

[0046] According to the characteristics of NOD-R molecules, the classical aptamer screening method—magnetic bead method was used for screening.

[0047] Such as figure 1 As shown, the screening process mainly includes four steps, namely incubation, separation, elution and amplification. Table 1 shows the specific screening scheme, and the specific process is as follows:

[0048] (1) Incubation: Take 3 nmol ss DNA library (the second to fifth round library volume is 200 pmol, and the sixth to twelfth round library volume is 120 pmol) for denaturation treatment, that is, first heat in a 95°C water bath. Bath for 10 minutes; then, quench in an ice bath for 5 minutes; finally, place at room temperature for 5 minutes; at the same time, wash the NOD-R magnetic beads several times with screening buffer, and add 20 μL to the ssDNA library that has been treated above; add a certain amount of screening buffer to a total volume of 600 μL ...

Embodiment 3

[0061] Example 3. Optimization of aptamer N13

[0062] Among the selected 10 aptamers, the affinity constant (K D ) has the smallest value, which is 0.138μM. In order to obtain the core structure of aptamer N13, the aptamer was truncated and optimized.

[0063] image 3 It shows the predicted secondary structure of the truncated aptamer N13 by the online tool the mfold web server.

[0064] When the fixed regions at both ends of the aptamer N13 are removed (N13-T), the aptamer can still bind to NOD-R, and K D The value is the same as before truncation. Therefore, it can be inferred that the primer-binding regions at both ends of the aptamer N13 are not involved in the combination with NOD-R.

[0065] According to the prediction results of the secondary structure of aptamer N13-T by the online tool the mfoldweb server, it was further truncated, and aptamer N13-T-O1, aptamer N13-T-O2, aptamer N13-T-O2, Aptamer N13-T-O3 and Aptamer N13-T-O4. Among the four aptamers, except ...

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Abstract

The invention relates to the technical field of bio-medicine engineering, and provides an aptamer capable of being specifically combined with nodularin-R and an application of the aptamer. A sequencegeneral formula of the aptamer is 5'-AAGGAGCAGCGTGGAGGATA-N40-TTAGGGTGTGTCGTCGTGGT-3', wherein N is any one of the four deoxyribonucleotide basic groups A, T, G and C, the number 40 refers to the number of random basic groups, preferably a sequence represented as anyone from the SEQ ID No.1 to the SEQ ID No.7, most preferably, anyone from the SEQ ID No.9 to SEQ ID No.11. The aptamer can be used for preparing an aptamer sensor or a detection reagent applied to detection of the NOD-R for a drinking water sample. The products also can be a basis for preparing medicines for preventing or treatingNOD-R poisoning and removing the NOD-R from water body or aquatic products.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, in particular to an aptamer specifically binding to nodularin-R and its application. Background technique [0002] Nodularin-R (Nodularin-R, NOD-R) is a strong cyanobacterial hepatotoxin, mainly produced by the filamentous planktonic cyanobacteria Nodularia spumigena. Studies have shown that NOD-R not only has a cancer-promoting effect, but also is a carcinogen, and its mechanism of action is to inhibit the activity of serine / threonine protein phosphatase 1 (PP1) and 2A (PP2A), thereby exerting toxic effects. Since 1988, researchers have discovered ten kinds of NOD, among which NOD-R is the most abundant and the only commercialized toxin. NOD-R is easily soluble in water, and has strong heat resistance, and the toxin cannot be destroyed by heating and boiling. People are mainly poisoned by drinking contaminated water and eating contaminated aquatic products. Symptoms of poisoning...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/68A61K31/7088A61P39/02C02F1/58C02F101/30
CPCA61P39/02G01N33/68C12N15/115C02F1/58C02F2101/30G01N2410/00C12N2310/16
Inventor 欧阳生群胡波刘德婧周蓉李振钢李真焦炳华王梁华
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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