Structure Tuning of Cationic Oligospermine–siRNA Conjugates for Carrier-Free Gene Silencing
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- 作者：Marc Nothisen ; Jérémy Bagilet ; Jean-Paul Behr ; Jean-Serge Remy ; Mitsuharu Kotera
- 刊名：Molecular Pharmaceutics
- 出版年：2016
- 出版时间：August 1, 2016
- 年：2016
- 卷：13
- 期：8
- 页码：2718-2728
- 全文大小：641K
- 年卷期：0
- ISSN：1543-8392
文摘
Oligospermine–siRNA conjugates are able to induce efficient luciferase gene silencing upon carrier-free transfection. These conjugates are readily accessible by a versatile automated chemistry that we developed using a DMT-spermine phosphoramidite reagent. In this article, we used this chemistry to study a wide range of structural modifications of the oligospermine–siRNA conjugates, i.e., variation of conjugate positions and introduction of chemical modifications to increase nuclease resistance. At first we examined gene silencing activity of a series of siRNA–tris(spermine) conjugates with and without chemical modifications in standard carrier assisted conditions. The three spermine units attached at one of the two ends of the sense strand or at the 3′-end of the antisense strand are compatible with gene silencing activity whereas attachment of spermine units at the 5′-end of the antisense strand abolished the activity. 2′-O-Methylated nucleotides introduced in the sense strand are compatible while not in the antisense strand. Thiophosphate links could be used without activity loss at the 3′-end of both strands and at the 5′-end of the sense strand to conjugate oligospermine. Consequently a series of oligospermine–siRNA conjugates containing 15 to 45 spermines units in various configurations were chosen, prepared, and examined in carrier-free conditions. Attachment of 30 spermine units singly at the 5′-end of the sense strand provides the most potent carrier-free siRNA. Longevity of luciferase gene silencing was studied using oligospermine–siRNA conjugates. Five day long efficiency with more than 80% gene expression knockdown was observed upon transfection without vector. Oligospermine–siRNA conjugates targeting cell-constitutive natural lamin A/C gene were prepared. Efficient gene silencing was observed upon carrier-free transfection of siRNA conjugates containing 20 or 30 spermine residues grafted at the 5′-end of the sense strand.