Nucleic acid aptamer combined with DKK1 protein and application thereof
A nucleic acid aptamer and protein technology, applied in the biological field, can solve the problems affecting the convenience of preservation and application of nucleic acid aptamers, cannot be truncated, and cannot be defined, and achieves easy preservation and labeling, easy preservation, and inhibition of antagonism. effect of action
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Embodiment 1
[0066] Example 1: Screening of ssDNA nucleic acid aptamers that bind to DKK1 protein
[0067] The method for screening ssDNA nucleic acid aptamers that bind to DKK1 protein in this embodiment includes the following steps:
[0068] 1. Synthesize the random single-stranded DNA library and primers shown in the following sequence:
[0069] Random single-stranded DNA library:
[0070] 5'-CTACGGTGCCTTGAAGTGAC-36N-CATAGCAGGTCACTTCCAGG-3' (SEQ ID NO. 5);
[0071] Here, "36N" represents a sequence in which 36 arbitrary nucleotide bases are linked. The library was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.
[0072] The primer information is shown in Table 1, which was synthesized by Nanjing GenScript Biotechnology Co., Ltd.
[0073] Table 1. Primers and their sequences
[0074]
[0075] Among them, the S in the primer name represents the forward primer, the A in the primer name represents the reverse primer, the 19 A in the sequence represents the polyA tail compose...
Embodiment 2
[0098] Example 2: Comparison between ZZK1-1 after truncation and before truncation
[0099] After truncating ZZK1-1, the nucleic acid aptamer shown in SEQ ID NO. 3 was obtained, and after verification, it had an ideal binding affinity to DKK1, and was named ZZK1-17. SPR was used to detect the change of ZZK1-17's ability to recognize DKK1 protein, and the comparison of the aptamer with only 17 nucleotides after truncation and before truncation for binding to DKK1 or DKK1-CRD2 was verified.
[0100] The experimental results show that the aptamer (SEQ ID NO.3) containing only 17 nucleotides after truncation has affinities of 6.7nM and 8.3nM with DKK1 and DKK1-CRD2 respectively, compared with the nucleic acid before truncation. Although the affinity of the aptamer has not changed significantly, it significantly improves the specificity of the aptamer and greatly reduces the difficulty of synthesis, indicating that the core region of the key domain of the aptamer ZZK1 is located in...
Embodiment 3
[0101] Example 3: Effects of different screening methods on screening nucleic acid aptamers
[0102] In this example, the method provided in Example 1 is used to screen nucleic acid aptamers. In the process of magnetic bead screening, the following four methods are used for screening: 1. No serum is added; 2. Each addition is started in the third round. 50% serum; 3. Add 50% serum in the sixth round; 4. Add 50% serum in the eighth round; 5. Add 10% serum in the third round, add 25% serum in the fourth round, and add 25% serum in the fifth round 50% serum was added; 6. 10% serum was added in the sixth round, 25% serum was added in the seventh round, and 50% serum was added in the eighth round; the serum was purchased from Beijing Soleibo Technology Co., Ltd., item number: SL010; The screening results are shown in Table 4.
[0103] Table 4. Effects of different screening methods on screening nucleic acid aptamers
[0104] group Filter results 1 Screening mult...
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