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Polypeptide and nucleic acid coupling compound used for targeted therapy

A technology for coupling compounds and nucleic acids, applied in the field of biomedicine, can solve problems such as safety issues, technical difficulties, and complicated processes

Inactive Publication Date: 2016-08-17
北京动生科技集团有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In these applications, genetic technology is used, including complex steps such as gene cloning, bacterial expression, separation and purification, the process is complicated, the technology is difficult, and it may bring some safety problems to the human body and the environment.

Method used

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  • Polypeptide and nucleic acid coupling compound used for targeted therapy
  • Polypeptide and nucleic acid coupling compound used for targeted therapy
  • Polypeptide and nucleic acid coupling compound used for targeted therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Azide-labeled GnRH polypeptide has targeting and specificity

[0031] Hela cells with high expression of GnRHR were selected as experimental cells to verify the binding of [D-Lys6]GnRH to different concentration gradients of Hela cells. Hela cells were seeded in 6-well plates, and when the confluence of the cells reached 70% to 80%, different concentrations of fluorescently labeled [D-Lys 6 (FITC)] GnRH (purchased from Beijing Zhongke Yaguang Biotechnology Co., Ltd.), after 6 hours, the cells were digested with trypsin, and the supernatant was discarded by centrifugation; resuspended in PBS, and the supernatant was discarded by centrifugation (repeat three times), Removal of unbound [D-Lys 6 (FITC)] GnRH, finally add 0.5mL PBS, resuspend, and detect by flow cytometry (BD company), flow cytometry results are analyzed using FlowJo 7.6.1 software, the results are shown in figure 1 . From figure 1 Visible, [D-Lys 6 (FITC)] GnRH binds to receptors on cells in ...

Embodiment 2

[0034] Example 2 [D-Lys 6 ] Synthesis, isolation, purification and verification of GnRH-siRNA

[0035] This embodiment synthesizes [D-Lys by click reaction 6 ] GnRH-siRNA, and then the product was isolated and purified by UPLC, and the product was verified by UPLC-MS.

[0036] Experimental materials and reagents: [D-Lys 6 (azidopentanonic acid)] GnRH (synthesized by Beijing Zhongke Yaguang Biotechnology Co., Ltd.), PLK1 siRNA modified by butynyl at the 5' end of the sense strand (sense strand: 5'-CH≡C-CH 2 -CH 2 (mU)GAAGAAGA(mU)(mC)A(mC)(mC)(mC)(mU)(mC)(mC)(mU)(mU)A dTdT-3'("m"in the sequence represents the 2' position Methoxy modification is 2'-OMe, the same below, see SEQ ID No: 2), antisense strand: 5'-UAAGGAGGGUGAUCUUCUUCA dTdT-3' (SEQ ID No: 3), purchased from Guangzhou Ruibo Biotechnology Co., Ltd. ), Dimethylsulfoxide (DMSO), CuSO 4 ·5H 2 O, tert-butanol (t-BuOH), TBTA (tris(benzyltriazolylmethyl)amine), UPLC, ESI-Orbitrap MS.

[0037] Chromatographic column: Ju...

Embodiment 3

[0040] Example 3 Validation of [D-Lys6]GnRH-siRNA activity after CLICK reaction

[0041]In order to verify the biological activity of the product [D-Lys6]GnRH-siRNA after the click reaction, we detected the expression level of plk1 by qPCR after transfecting [D-Lys6]GnRH-siRNA into Hela cells. Hela cells were seeded into 24-well plates one day before transfection, and the number of cells per well was 5×10 4 , The cell density should reach 30%-50% during transfection. During transfection, the cells were divided into four groups on average, which were blank group (Blank), negative control group (NC), PLK1 group (P+) and click product group (Click+). The blank group did not undergo any treatment, and the negative control group was transfected with lipofectamine 2000 (abbreviated as lip2000 hereinafter) with a stable irrelevant sequence siRNA (sense strand 5'-UUCUCCGAACGUGUCACGUdTdT-3' (SEQ ID No: 4); antisense strand: 5 '-ACGUGACACGUUCGGAGAAdTdT-3' (SEQ ID No: 5)), P+ group was...

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Abstract

The invention discloses a polypeptide and nucleic acid coupling compound used for targeted therapy. An alkynyl group is introduced to nucleic acid used as an effect molecule, an azido group is introduced to polypeptide [D-Lys<6>]GnRH used as a target molecule, and a click chemical reaction is carried out in order to synthesize the polypeptide and nucleic acid coupling compound with a targeting effect. The polypeptide and nucleic acid coupling compound can be applied to targeted therapy of tumors.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a coupling compound of polypeptide and nucleic acid, a preparation method thereof, and an application of the compound in targeted disease therapy. Background technique [0002] In 1998, Fire et al. discovered that RNA interference (RNA interference, RNAi) can specifically degrade the mRNA of the target gene (Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specificgenetic interference by double- Stranded RNA in Caenorhabditis elegans. Nature. 1998; 391:806-11.). This phenomenon is mediated by double-stranded siRNA. In 2001, Elbashir et al. successfully silenced the expression of related genes in mammalian cells with double-stranded siRNA synthesized in vitro, which laid the foundation for the development of siRNA gene therapy drugs (Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cult...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K1/107A61K47/48A61K31/7088A61P35/00
CPCA61K31/7088C07K7/06C07K19/00
Inventor 李军陈中华朱德生
Owner 北京动生科技集团有限公司
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