Self-assembled nucleic acid aptamer dna nano-train and its preparation method and application
A nucleic acid aptamer and self-assembly technology, which is applied in the fields of biology and medicine, can solve the problems of complex application, complicated preparation process, and small drug loading capacity, and achieve simple and easy preparation methods, simple reaction conditions, and time-consuming short effect
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Embodiment 1
[0038]Example 1 Synthesis and purification of self-assembled aptamer DNA nanotrains loaded with doxorubicin
[0039] (1) Preparation of TA6NT-AKTin-DOX
[0040] 1) Preparation and purification of DNA-polypeptide complexes
[0041] Using trichloroethyl phosphate (TCEP) as the reducing agent, 100 μL of disulfide-modified M1 and M2 at a concentration of 20 μM were mixed with 50 μL of TCEP reducing beads, respectively, and the reaction was shaken at 37 °C for 2 h. The samples were then centrifuged at 1000 xg for 5 min. Take the supernatant containing the reduced M1 and M2, add the same volume of 200 μM maleimide-modified polypeptide AKTin to the supernatant, 37 ° C, shake for 4 h to carry out the conjugation reaction, and then use the before and after conjugation. The difference in retention time (the retention times of M1, M1-AKTin, M2 and M2-AKTin are 10.6min, 12.9min, 8.4min and 12.4min, respectively, as figure 1 ) using preparative high performance liquid chromatography to ...
Embodiment 2
[0046] Example 2 Mammosphere formation test
[0047] BCSCs were given DOX and TA6NT-AKTin-DOX, respectively, incubated at 37 °C for 12 h, and then digested with BCSCs at 1 × 10 4 The density of each well was inoculated into a 6-well culture plate, and incubated at 37°C in a 5% carbon dioxide environment, and the number of mammospheres formed was observed after 14 days. like Figure 5 As shown, the number of mammospheres in the DOX group did not change significantly compared with the control group, while the number of mammospheres in the TA6NT-AKTin-DOX group was reduced by about 60%, indicating that TA6NT-AKTin-DOX could inhibit the self-renewal capacity of BCSCs.
Embodiment 3
[0048] Example 3 MTT assay for cell viability assay
[0049] Digest BCSCs in good condition and dilute to 5×10 with culture medium 4 cells / mL cell density, blow evenly, add 200 μL of cell suspension to each well of a 96-well plate, and incubate at 37°C in a 5% carbon dioxide environment for 24h. Use 200 μL DOX and TA6NT-AKTin-DOX drug-containing culture medium instead, set 5 parallel wells for each concentration, and set up a blank control group at the same time. ) 20 μL, continue to incubate for 4 h, aspirate the culture medium, add 150 μL of DMSO, shake on a microplate shaker for 30 min at room temperature, and measure the absorbance of each well at 490 nm with an enzyme-linked immunosorbent assay. Taking the control cells as 100%, the survival rate of the cells in the administration group relative to the survival of the control cells was determined as the percentage.
[0050] The cytotoxic effects of the two drugs on BCSCs were investigated by MTT method, the cell viabili...
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