Nucleic acid aptamer with high affinity to okadaic acid and dinophysistoxin

A technology of okadaic acid and nucleic acid aptamers, which can be applied in the fields of climate change adaptation, biochemical equipment and methods, and resistance to vector-borne diseases, etc., and can solve problems such as the lack of sequence reports of various marine toxin aptamers. achieve good affinity

Active Publication Date: 2021-06-11
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At this stage, there have been a few reports on a specific marine toxin aptamer sequence detection method, however, there are no reports on a variety of marine toxin aptamer sequences

Method used

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  • Nucleic acid aptamer with high affinity to okadaic acid and dinophysistoxin
  • Nucleic acid aptamer with high affinity to okadaic acid and dinophysistoxin
  • Nucleic acid aptamer with high affinity to okadaic acid and dinophysistoxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1O

[0026] The optimization of embodiment 1OA original aptamer OA-1234

[0027] The DNA sequence of the original OA adapter OA-1234 is (as shown in SEQ ID NO.3):

[0028] 5'-GGTCACCAACAACAGGGAGCGCTACGCGAAGGGTCAATGTGACGTCATGCGGATGTGTGG-3', a total of 60 bases.

[0029] The secondary structure of the original aptamer OA-1234 was predicted by the online analysis tool the mfold web server, and it was found that the secondary structure contained 4 stem-loop structures, which were numbered sequentially from the 5' end, as shown in figure 1 shown.

[0030] The affinity constant (Kd) between the original OA aptamer OA-1234 and OA was 1.32 μM. In order to obtain a more optimized aptamer, the sequence of the aptamer was truncated and split.

[0031] The stem-loop structure plays an important role in the combination of the aptamer and the target. According to the simulation of the secondary structure of the aptamer, one stem-loop was removed in turn, and the aptamers after removing the ste...

Embodiment 2

[0036] Example 2 Aptamer DSP-134, DSP-1,34 specificity experiment

[0037] In order to verify the specificity of the modified aptamers DSP-134 and DSP-1,34, specificity experiments were performed with other toxin analogs that are easily enriched in shellfish.

[0038] The modified aptamers DSP-134 and DSP-1,34 were tested by biomembrane interference molecular interaction instrument for STX, neo-STX, and GTX respectively, and the results showed that none of them could produce specific binding. The results are as follows: Figure 4 , Image 6 shown.

[0039] The aptamers DSP-134 and DSP-1,34 were tested by biomembrane interference molecular interaction instrument for DTX and OA respectively. Good affinity.

[0040] The specific information of DSP-134 and DSP-1,34 in this example is shown in Table 2.

[0041] Table 2 DSP-134 and DSP-1,34 specificity information

[0042] name target k DSP-134 STX - DSP-134 neo-STX - DSP-134 GTX - DSP...

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Abstract

The invention discloses a nucleic acid aptamer with high affinity to okadaic acid and dinophysistoxin, which comprises two sections of split type aptamers DSP-1 and DSP-34, and the nucleotide sequences of the two sections of split type aptamers DSP-1 and DSP-34 are as shown in SEQ ID NO.1 and SEQ ID NO.2. The invention further discloses another nucleic acid aptamer, namely, DSP-134, which has high affinity to okadaic acid and dinophysistoxin, and the nucleotide sequence of the nucleic acid aptamer is shown as SEQ ID NO.5. The Kd value of the aptamer DSP-1 and the aptamer DSP-34 combined with OA is 11.6 nM, and the Kd value of the aptamer DSP-1 and the aptamer DSP-34 combined with DTX is 43.7 nM; the Kd value of the aptamer DSP-134 combined with OA is 133nM, and the Kd value of the aptamer DSP-134 combined with DTX is 228nM; and the nucleic acid aptamer has no recognition capability on marine toxins such as saxitoxin, neosaxitoxin, gonyautoxin and the like. According to prediction of a secondary structure of an original aptamer, a transformation mode of firstly truncating and then cracking is adopted, so that the transformed aptamer has better affinity to a target and has recognition capability to both OA and DTX.

Description

technical field [0001] The invention relates to a nucleic acid aptamer with high affinity for okadaic acid and fin algae toxin, and belongs to the field of food safety biotechnology. Background technique [0002] Diarrhetic Shellfish Poisoning (DSP), first discovered and named in Japan in 1976, is a type of marine microalgae mainly composed of Prorocentrum spp. and Dinophysis spp. Produced, heat-stable secondary metabolites of polycyclic polyether lipids can be enriched in shellfish such as emerald mussels (Pernaviridis), bay scallops (Argopecten irradians), and razor clam (Sinonovacul constricta). According to research, DSP is one of the most threatening algal toxins in the world, and the poisoning incidents caused by it account for 5% of the global shellfish poisoning incidents. Among them, okadaic acid (OA) and fin algae toxin (dinophysistoxin, DTX) is the most common diarrheal shellfish toxin. Ingestion of shellfish contaminated by DSP will cause diarrhea, nausea, vomi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/53
CPCC12N15/115G01N33/53C12N2310/16G01N2333/43504Y02A50/30
Inventor 王赛毛相朝王乐乐阎晓琛赵颍琳李玲亓晓燕马蕊
Owner OCEAN UNIV OF CHINA
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