Method for assembling and morphology regulating of double-chain functional nucleic acid nanoflower and application thereof
A technology of functional nucleic acid and assembly method, applied in biochemical equipment and methods, nanotechnology, nanotechnology, etc., can solve the problems of destroying the integrity of nanostructures and loose structures
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Embodiment 1
[0053] Example 1 Controllable assembly of double-stranded functional nucleic acid nanoflowers
[0054] dNTPs, rTaq DNA polymerase, and 10×PCR buffer were purchased from TaKaRa Company, DNA purification kit DP204-02 was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., MgCl 2 ·6H 2 O, K 4 P 2 o 7 The solution grade is analytically pure.
[0055] After cultivating Salmonella, use the kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to carry out genomic extraction of bacterial genomic DNA, using the Salmonella genome as an amplification template, the upstream primer SEQ ID NO: 1 and the downstream primer SEQ ID NO: 2 pair the genome Amplify.
[0056] Wherein, the sequences of the upstream primer 1 SEQ ID NO: 1 and the downstream primer 1 SEQ ID NO: 2 are respectively:
[0057] Upstream primer 1 SEQ ID NO: 1: 5'-GGGGGGAAGAGGGAAGGTT-oxyethyleneglycolbridge-TTGTGAAATTATCGCCACGTTCGGGCAA-3'
[0058] Downstream primer 1 SEQ ID NO:2: 5'-GGGGGGAAGAGGGAAGGT...
Embodiment 2
[0067] Example 2 Nucleic acid quantitative analysis of double-stranded functional nucleic acid nanoflowers
[0068] Since the double-stranded functional nucleic acid nanoflowers are not tolerant to Tris-HCl buffer (pH 5.0), the experiment performed a morphology verification on the dissolution process of the double-stranded functional nucleic acid nanoflowers. After incubating the prepared nanoflower samples with 0.05 mM Tris-HCl buffer (pH 5.0) in a cell culture incubator at 37 °C for 24 h, the morphology of the nanoflowers was observed by scanning electron microscopy, and the nanoflowers almost disappeared. SEM picture see figure 2 . The double-stranded nucleic acid of the functional nucleic acid nanoflower can be obtained.
[0069] Next, create a standard curve. The purified PCR amplicon (concentration: 95.218 ng / μL) diluted by 10 times was used as the amplification template for quantitative analysis of real time PCR (Tiangen Biochemical Technology (Beijing) Co., Ltd., S...
Embodiment 3
[0075] Example 3 Morphology regulation of double-stranded functional nucleic acid nanoflowers
[0076] First, the concentration of double-stranded PCR purifiers chemically modified with oxyethyleneglycol bridge was used to verify the regulation of nucleic acid nanoflower morphology, that is, the final concentrations of nucleic acid purifiers were 95.218 μg / L, 95.218 ng / L, 95.218pg / L, Then with 10 mM Mg 2+ , at a final concentration of 1.0 mM K 4 P 2 o 7 After incubating in a metal bath at 30°C for 18 hours, they were centrifuged and washed with water, dried overnight, and photographed with a Gemini SEM 300 electron microscope to observe the morphology of nucleic acid nanoflowers.
[0077] The specific incubation system is as follows:
[0078]
[0079] The experimental results show that when the concentration of pyrophosphate, the type and concentration of metal ions are constant, as the concentration of nucleic acid decreases, the particle size of nucleic acid nanoflowe...
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