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High affinity adapter body capable of specifically binding with saxitoxin acetate and application thereof

A saxitoxin and aptamer technology, applied in the biological field, can solve the problems of affinity constant detection, optimization and application that have not yet been reported, 4-aminopyridine has large toxic and side effects, and the safe dose range is small.

Active Publication Date: 2015-09-09
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

There is currently no specific drug for STX poisoning. Clinically, emesis, gastric lavage, and activated carbon are used to absorb toxins in the stomach.
4-aminopyridine can antagonize the toxic effect of STX and play a therapeutic effect, but 4-aminopyridine has large toxic and side effects and a small safe dose range
However, there is no relevant report on the detection, optimization and application of the affinity constant of this aptamer.

Method used

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  • High affinity adapter body capable of specifically binding with saxitoxin acetate and application thereof
  • High affinity adapter body capable of specifically binding with saxitoxin acetate and application thereof
  • High affinity adapter body capable of specifically binding with saxitoxin acetate and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Aptamer APT STX1 mutation

[0029] First, we send the APT STX1 A single point mutation is introduced into the sequence. We mutated the nucleotides at either end of the guanine nucleotides in the sequence to guanine nucleotides. By this method, we obtained 22 single-point mutation oligonucleotide sequences, the mutation positions are 3, 6, 8, 12, 13, 15, 20, 22, 25, 30, 32, 37, 41, 43, 48, 50, 55, 57, 58, 60, 61 and 64. According to the prediction results of QGRS Mapper software, there are 9 sequences (M-6, M-8, M-12, M-13, M-15, M-20, M-22, M-30 and M-32) may form a G-quadruplex structure. We synthesized the above nine sequences and evaluated their STX binding ability by biomembrane interferometry (see Table 1). Similarly, we also introduced multiple point mutations, replacing two or three nucleotides near guanine nucleotides with guanine nucleotides, and synthesized 6 sequences that may form a G-quadruplex structure, And evaluate its STX binding abili...

Embodiment 2

[0040] Example 2. Truncation optimization of aptamers

[0041] Using mfold software, we predicted the secondary structure of M-30, such as figure 1 shown. By removing the single-stranded sequences at both ends of M-30 or any of the four stem-loop structures, we obtained five sequences (M-30a, M-30b, M-30c, M-30d, M- 30e; if figure 2 ), were synthesized and their Kd values ​​were determined by biofilm interferometry (Table 3).

[0042] Among the five sequences, M-30a, M-30d and M-30e respectively remove the free nucleotides at both ends, the third stem-loop and the fourth stem-loop structure. The Kd values ​​for sequence M-30 are very close. However, after removing the first or second stem-loop structure (M-30b, M-30c), the affinity of the aptamer decreased significantly. This indicates that the first and second stem-loop structures are critical for target molecule recognition. Based on these results and our reasoning, we synthesized M-30f by removing the third and fourt...

Embodiment 3

[0045] Example 3. The affinity and specificity of M-30f binding to STX were analyzed by biofilm interference technology

[0046] After coupling 1 μM M-30f to the streptavidin-coated chip surface by using the function of biotin-streptavidin, it interacted with different concentrations of STX (5 μM, 10 μM, 20 μM) and obtained corresponding responses Curve, use the data analysis software to adopt the 1:1 binding mode to fit the response curves of different concentrations of STX, and obtain the binding and dissociation curves of the interaction between the two, such as image 3 , Kd value is 133nM. In addition, no response was observed when GTX2,3 (20 μM) was used as the target molecule for analysis by biofilm interferometry. This indicates that M-30f does not cross-react with GTX2,3 and retains its specificity for STX.

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Abstract

The invention relates to biological technical field, and provides a high affinity adapter body capable of specifically binding with saxitoxin acetate, and the sequence of single-stranded DNA adapter body as SEQ ID NO: 2. By mutation and truncation of an in vitro screened adapter body, a high affinity single-stranded oligonucleotide adapter body capable of specifically identifying the saxitoxin acetate can be obtained. The adapter body has a broad application prospect, and can be used for separation and enrichment of trace concentrations of STX (saxitoxin) in a sample, rapid detection of saxitoxin, and preparation of a drug for neutralization of saxitoxin sodium ion channel inhibition effect, and removal of toxins in water.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a high-affinity aptamer specifically bound to saxitoxin obtained through mutation and truncation optimization, which is used for the separation and enrichment of trace amounts of STX in samples, and the rapid extraction of saxitoxin The detection, the preparation of drugs that neutralize saxitoxin sodium ion channel inhibition, and the removal of toxins in water have laid the foundation. Background technique [0002] Saxitoxin (STX) belongs to the paralytic shellfish toxin family, is a guanamine neurotoxin, and is one of the most powerful small molecule neurotoxins known so far. It comes from a variety of freshwater cyanobacteria and marine dinoflagellates, and accumulates in shellfish, crabs and other aquatic products through the food chain. Neuromuscular transmission, causing paralytic shellfish poisoning in humans and marine animals. The median lethal dose LD of intraperitoneal ...

Claims

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Application Information

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IPC IPC(8): C12N15/115G01N33/53A61K31/7088A61P39/02C02F1/58A23L1/015
Inventor 郑欣胡波高顺祥刘德婧孙铭娟王梁华焦炳华
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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