Nucleic acid aptamer capable of being combined with prometryn and application of nucleic acid aptamer
A nucleic acid aptamer and proturon technology, which is applied in the field of environmental pollutant detection, can solve the problems of less research on nucleic acid aptamers, unfavorable proturon detection work, and no research reports.
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Embodiment 1
[0068] Example 1: Screening of nucleic acid aptamers that can bind to promethazin
[0069] In order to obtain nucleic acid aptamers that can bind proturon with high specificity, the inventors conducted a capture SELEX experiment for screening. The screening process includes:
[0070] (1) Initial library design: The initial library sequence is 5'-ATTGGCACTCCACGCATAGG-N(40)-CCTATGCGTGCTACCGTGAA-3' (SEQ ID NO.17). Among them, N(40) is a random sequence, and both ends are fixed sequences, which is convenient for amplifying the nucleic acid aptamer and hybridizing the aptamer with a biotin-labeled primer. The primer sequences are shown in Table 1.
[0071] Table 1. Primers for nucleic acid aptamer screening
[0072]
[0073] (2) Initial screening: first use 0.1 M PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM NaCl 2 HPO 4 , 1.4 mM KH 2 PO 4 ) After dissolving 1 OD initial library in a 1 mL sterile EP tube, add 14 µL of pre-dissolved 100 µM primer S1-CS. Use a PCR instrument for sl...
Embodiment 2
[0081] Example 2: Proturon affinity and specificity experiments of candidate nucleic acid aptamers
[0082] This embodiment mainly uses nano-gold colorimetric method to measure the affinity and specificity of candidate nucleic acid aptamers to proturon, and establishes a nano-gold colorimetric method for proturon. This method utilizes nano-gold under the protection of aptamers The principle of aggregation not induced by high concentration NaCl. The specific determination process includes the following steps:
[0083] (1) Preparation of gold nanoparticles
[0084] Gold nanoparticles were prepared by sodium citrate reduction method. First, 100 mL of ultrapure water was heated to boiling, then 1 mL of 1% chloroauric acid solution was added, and 10 mL of 1% sodium citrate solution was added after 1 min, the solution turned from light yellow to gray, and finally to wine red. After heating for 5 min, turn off the heat source and cool down at room temperature.
[0085] (2) Nano-g...
Embodiment 3
[0091] Example 3: Structural prediction of nucleic acid aptamers
[0092] In order to analyze the structural principle of the binding of the candidate aptamers to promethazin and to find the nucleotide sequence binding to protamizone, the structure prediction of the candidate nucleic acid aptamers was carried out. The prediction was performed using the online prediction tool UNA-fold, where the folding temperature was 25 °C, Na + The concentration is 0.1 M.
[0093] The predicted structure of the nucleic acid aptamer P60 is as follows image 3 As shown, it was predicted that the nucleic acid aptamer P60 formed a stem-loop structure, in which nucleotides 3-8 and 30-35 formed the first stem S1, and nucleotides 11-15 and 24-28 formed the second Stem S2, positions 8-11 and 28-30 form the first loop L1, positions 15-24 form the second loop L2, and positions 35-55 form the third loop L3.
[0094]Since the deletion of the third loop L3 has no effect on the affinity of promethazin ...
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