Molecular probe for nucleic acid detection and preparation method and application thereof
A molecular probe and nucleic acid technology, applied in the field of oligonucleotide molecular probes, can solve problems such as probe detection accuracy and safety limitations, and achieve the effects of high raw material utilization, less production and waste, and accurate nucleic acid detection.
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Embodiment 1
[0119] Example 1: Preparation of Cell Penetrating Peptide-VSP Molecular Probe Carrier
[0120] The inventors have found through research that, compared with linear cell-penetrating peptides, the effect of cell transfection obtained by using cyclic cell-penetrating peptides is better. In an exemplary embodiment of the present invention, a cyclic peptide (hereinafter referred to as "CP") having an amino acid sequence as shown in SEQ ID NO: 1 is used as a cell-penetrating peptide for the following experiments.
[0121] In this embodiment, the contrast agent VSP-320 (GE Healthcare, product number 1181607CHN) was used as the detectable marker.
[0122] A). VSP structure and modification
[0123] Adopt VSP-320, VSP-320 has the structure shown in following formula (I):
[0124]
[0125] Modification is carried out on the basis of VSP-320 of formula (I) to add a carbon chain whose end is modified as a carboxyl group to form a new Visee Parker (cVSP), which has the structure shown...
Embodiment 2
[0136] Example 2: Preparation of cell-penetrating peptide-VSP-TTR targeting siRNA molecular probe
[0137] The siRNA (custom-synthesized by Gemma Gene) targeting liver fibrosis-specific marker gene TTR is used, and its sequence is as follows:
[0138] Sense strand: 5'-mCAGmUGmUmUmCmUmUGmCmUmCmUAmUAAdTdT-3'
[0139] Antisense strand: 5'-UmUAmUAGAGmCAAGAAmCACUGdTdT-3'
[0140] Wherein, m represents the 2' hydroxymethylation modification of the corresponding base.
[0141]Preparation process of molecular probe:
[0142] A). Add siRNA to DEPC water to make the final concentration 5 μM / mL, and set aside;
[0143] B). Mix the molecular probe carrier VSP-CP and siRNA according to the molar ratio of 20:1, and incubate at room temperature for 15-20 minutes;
[0144] C). Molecular probe carrier VSP-CP and siRNA are non-covalently combined to prepare a complete molecular probe.
[0145] The obtained molecular probe can be used immediately for in vitro cell detection or aerosol inhal...
Embodiment 3
[0146] Example 3: Target nucleic acid detection using cell-penetrating peptide-VSP-TTR targeting siRNA molecular probe
[0147] A). Rat BRL3A cells (purchased from ATCC) were seeded in a six-well plate at a cell density of 1.2×10 5 / well, add 1.5mL MEM containing serum medium, 37°C, 5% CO 2 Incubate for 24 hours under conditions to make the cells reach 40-70% confluency;
[0148] B). Prepare the molecular probes required for transfection:
[0149] a. Dilute siRNA in solution A with serum-free medium, with a final volume of 600 μL and a concentration of 0.4 nm / μL;
[0150] b. Dilute VSP-CP with serum-free medium in solution B, with a final volume of 600 μL and a concentration of 8 nm / μL;
[0151] c. Mix solutions A and B, mix gently, and place at room temperature for 15-20 minutes to form a VSP-CP-siRNA molecular probe;
[0152] C). Transfection preparation: Wash the cells twice with serum-free medium, add 1.3 mL / well of serum-free medium to the cells;
[0153] D). Add the...
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