RNA nano hydrogel for lung cancer targeted therapy as well as preparation method and application thereof
A nano-hydrogel and targeted therapy technology, which is applied in the field of biochemical nanomaterials, can solve problems such as no target recognition function, and achieve the effects of weakening migration and invasion, good targeting effect, and reduced damage
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[0042] The present invention also provides the preparation method of the RNA nano hydrogel described in the above technical scheme, comprising the following steps:
[0043] 1) Mix DNA self-assembled single strands and circular DNA templates in Tris-HCl buffer for annealing treatment. The conditions of the annealing treatment are: 95°C, 5min, and then reduce to 16-16°C at a rate of 0.5°C / min. At 28°C, a three-fork self-assembled structure carrier was obtained;
[0044] 2) Mixing the three-fork self-assembly structure carrier obtained in step 1) with T4 ligase and T4 ligase buffer, and performing a ring-forming reaction at 16° C. to obtain a three-fork self-assembly structure carrier containing a circular template;
[0045] 3) Mix the three-fork self-assembly structure carrier containing the circular template obtained in step 2) with the nucleotide triphosphate mixture, RNase inhibitor, T7 polymerase and T7 polymerase buffer, and perform rolling circle transcription at 37°C Rea...
Embodiment 1
[0057] Preparation of RNA nanohydrogels:
[0058] The three DNA single strands (ASM-DNA-1, ASM-DNA-2, ASM-DNA-3) used to synthesize the three-fork self-assembly structure carrier and the Three-let7a, Three-miR 34a, The three circular DNA templates of Three-miR 145 were mixed together, and the Tris-HCl buffer was added to make the volume to 20 μL, so that the concentration of the above substances was 1×10 -6 mol / L. After annealing the above mixed solution (95°C, 5min, then drop 0.5°C per minute to 25°C), the three-pronged self-assembled structure carrier;
[0059] Add T4 ligase and its buffer solution and place at 16°C to form a circle to obtain a three-fork self-assembly structure carrier containing a circular template.
[0060] Then add nucleotide triphosphate mixture (rNTP), RNase inhibitor, T7 polymerase and its buffer to carry out RCT reaction at 37°C for 1.5h to obtain a carrier bound to circular DNA;
[0061] Mix the circular DNA-binding carrier and the targeting apta...
Embodiment 2
[0063] Using agarose gel electrophoresis, the successful synthesis of the RNA nano-hydrogel (RNANHs) prepared in Example 1 was characterized and verified, and the specific process was as follows:
[0064] First, dissolve agarose (mass fraction 2%) with TAE buffer solution and heat it to boiling in a microwave oven, then add ethidium bromide staining agent for gel staining, until the gel is completely solidified, the solidified gel Transfer to an electrophoresis machine filled with TAE buffer. Subsequently, the products of each step in the experimental steps (respectively three-fork self-assembly structure carrier, three-fork self-assembly structure carrier containing circular template, and RNA nanohydrogel for lung cancer targeted therapy) were mixed with The gel-loaded dye of bromophenol blue was mixed and dropped into the gel well, and the experiment was carried out at 110V for 40min.
[0065] The invention observes and records gel images under an ultraviolet gel imager. T...
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