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A chemically modified nucleic acid aptamer as1411 and its use

A technology of AS1411 and nucleic acid aptamer, which is applied in the direction of medical preparations containing active ingredients, scientific instruments, pharmaceutical formulas, etc., can solve the problems of shortened action distance, increased action distance, and increased activity, so as to improve binding force and increase Inhibition, enhance the effect of inhibition

Active Publication Date: 2018-06-15
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When the incorporation site of the isonucleoside is the interaction site between the nucleic acid aptamer and the target, the change in the spatial conformation of the base will lead to a change in the spatial distance from the target. When incorporated into the binding site between the nucleic acid and the target, it will lead to an increase in the action distance with the target and a decrease in activity, while the action distance between the nucleic acid aptamer incorporated in another configuration isonucleoside and the target will be shortened, and the activity will be reduced. Increase

Method used

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  • A chemically modified nucleic acid aptamer as1411 and its use
  • A chemically modified nucleic acid aptamer as1411 and its use
  • A chemically modified nucleic acid aptamer as1411 and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 The solid-phase synthesis of AS1411 modified by isonucleoside or isonucleoside combined with 2'-deoxyinosine

[0040] DNA was synthesized using an Appllied Biosystems model 394 DNA solid-phase synthesizer.

[0041] Normal deoxynucleoside phosphorylation monomers (dT, dGAc, dABz, dCAc) were purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd.; 2'-deoxyinosine phosphoramidite monomers (compound VI) were purchased from Shanghai Zhiyan Technology Co., Ltd. Co., Ltd. (Shanghai) purchased. CPG (CPG-dG), CAP-A and CAP-B, oxidized I2 solution, Cl 3 CCOOH was purchased from Beijing Aoke Biotechnology Company; 0.25M 5-Ethylmercapto 1H-tetrazolium solution was purchased from Shanghai Zhiyan Technology Co., Ltd. (Shanghai).

[0042] According to the method of literature (HW Yu, LR Zhang, JC Zhuo, LT Ma, LH Zhang, Bioorg.Med.Chem., 1996,40,609-614), the isonucleoside compound shown in chemical formula I and chemical formula II is prepared respectively The is...

Embodiment 2

[0058] Research on the basic properties of the modified AS1411 sequence in Example 2

[0059] 1. Sample name: The 3rd, 6th, 9th, 12th, 13th, 15th or 24th position of the AS1411 sequence is mixed with the isonucleoside shown in chemical formula I or chemical formula II (wherein Base is selected from thymine T) , the modified AS1411 sequence obtained by coupling the corresponding positions instead of natural nucleosides, prepared by the method in Example 1.

[0060] 2. CD spectral analysis

[0061] The AS1411 sequence samples were dissolved in PBS, and all samples were denatured at 95°C for 5 minutes, then slowly lowered to room temperature, and then tested. The CD scanning range is from 200-400nm, the temperature is constant at 25°C, the scanning interval is 0.2nm, and the scanning speed is 100nm / min. Each sample is scanned for 3 times, and the instrument's own software is used for smoothing. See the experimental results figure 1 .

Embodiment 3

[0062] Example 3 SPR determination of isonucleoside-modified and isonucleoside combined deoxyinosine modified AS1411 sequence and nucleolin affinity

[0063] 1. Sample name: AS1411-6L, AS1411-12D, AS1411-13L, AS1411-6L / 12D, AS1411-6L / 12D / 24dI, prepared by the method in Example 1.

[0064] 2. Method

[0065] (1) The nucleolin protein was immobilized on the CM5 chip.

[0066] A certain amount of nucleolin protein was prepared into a series of solutions with appropriate pH value of sodium acetate solution, and the optimal coupling conditions of the protein on the surface of the chip were optimized. The specific operation is as follows: PBS buffer is the mobile working solution, the temperature is 25°C, the flow rate is 10 μL / min, and the baseline is stabilized. The mixture of EDC and NHS was flowed over the surface of the chip at a flow rate of 5 μL / min for 7 minutes to activate the carboxyl groups on the surface. Nucleolin protein solutions of different concentrations were pr...

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Abstract

The invention discloses a chemically modified nucleic acid aptamer AS1411 and its application. The present invention adopts the modification strategy of isonucleoside or isonucleoside combined with 2'-deoxyinosine to replace the nucleotides at different positions of the nucleic acid aptamer AS1411, thereby changing the local spatial conformation of the nucleic acid aptamer AS1411, Optimize its spatial structure to achieve the purpose of increasing molecular stability, enhancing the recognition ability with target molecules, enhancing the interaction force with targets, and improving the biological activity of nucleic acid aptamers. Studies have shown that the AS1411 modified by the method of the present invention can better bind to the target protein, the anti-breast cancer cell proliferation and DNA replication ability are significantly enhanced, and the growth inhibition effect on breast cancer xenograft tumors is more obvious, and the difference is statistically significant. significance. The computer simulation of the modified AS1411 showed that the ability to bind to the target protein nucleolin was significantly improved; at the same time, the modified AS1411 could specifically regulate some functional miRNAs, and it is expected to be developed as a biological network regulation tool.

Description

technical field [0001] The present invention relates to a modified nucleic acid aptamer (aptamer) AS1411 and its use, in particular to a chemical modification of the nucleic acid aptamer AS1411 sequence by using isonucleoside or isonucleoside in combination with 2'-deoxyinosine And the obtained product and application thereof, the present invention belongs to the field of biomedicine. Background technique [0002] Aptamer, nucleic acid aptamer, derived from the Latin word "aptus", is a single-stranded oligonucleotide (RNA) or single-stranded oligodeoxynucleotide (DNA) composed of 20-60 bases. It can specifically bind various target molecules such as proteins, small molecules, ions and cells. Nucleic acid aptamers were invented by Nobel Prize winners Szostak and Gold in 1990, and they have many advantages that antibodies cannot match. The nucleic acid aptamer is screened by SELEX technology, which can be used to screen out the nucleic acid aptamer (Aptamer) that specificall...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115A61K31/7088A61P35/00A61P35/02G01N33/574
Inventor 杨振军范鑫萌李昆峰蔡报彬张礼和
Owner PEKING UNIV
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