Dinophysistoxins-1 specific binding aptamer and application thereof
A fin algae toxin and aptamer technology, which is applied in the direction of medical preparations containing active ingredients, anti-toxins, drug combinations, etc., to achieve the effects of easy operation, prevention of degradation, and high repeatability
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Embodiment 1
[0036] Embodiment 1. The design of random ssDNA library and its primers
[0037] 1. Design of ssDNA library
[0038] The DTX-1 aptamer library consists of 69 bases, with fixed regions containing 20 and 22 bases at both ends, and a random region containing 27 bases in the middle: 5'-GAGGCAGCACTTCACACGAT-N27-CATCACTACAGTCATTACGCAG- 3' (N is any one of four deoxyribonucleotide bases, A, T, G, and C, and 27 represents the number of random bases).
[0039] 2. Primer Design
[0040] Upstream primer: 5'-GAGGCAGCACTTCACACGAT-3' (SEQ ID NO.11)
[0041] Downstream primer A: 5'-CATCACTACAGTCATTACGCAG-3' (SEQ ID NO.12)
[0042] Downstream primer B: 5'-poly(dA20)-Spacer18-CATCACTACAGTCATTACGCAG-3' (SEQ ID NO.13).
[0043] 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
[0044] Example 2. Screening of DTX-1 aptamers
[0045] According to the characteristics of DTX-1 molecules, the classic method of aptamer screening—magnetic bead method is used for screening.
[0046] 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:
[0047] (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 put it in a water bath at 95°C for 10 minutes ; Then, quench in an ice bath for 5 minutes; finally, place it at room temperature for 5 minutes; at the same time, wash the DTX-1 magnetic beads several times with screening buffer, and add 20 μL to the ssDNA library that has been treated above; add a certain amount Make up the system with screening buffer to a...
Embodiment 3
[0060] Example 3. Optimization of aptamer N59
[0061] Among the selected 10 aptamers, the affinity constant (K D ) has the smallest value, which is 0.17μM. In order to obtain the core structure of the aptamer N59 and optimize the aptamer, the aptamer was truncated.
[0062] image 3 It shows the predicted secondary structure of the aptamer N59 truncated product by the online tool the mfold web server.
[0063] When the fixed regions at both ends of the aptamer N59 were removed (N59a), the aptamer could still bind to DTX-1, and K D value increased significantly. Therefore, it can be inferred that the primer-binding regions at both ends of the aptamer N59 not only do not participate in the combination with DTX-1, but may also hinder the combination of the core structure and DTX-1.
[0064] According to the prediction results of the secondary structure of aptamer N59 by the online tool the mfoldweb server, it was truncated, and aptamer N59b, aptamer N59c and aptamer N59d we...
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