title: Structure Tuning of Cationic Oligospermine-siRNA Conjugates for Carrier-Free Gene Silencing.
abstract: 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.
authors.affiliation: Laboratoire V-SAT, CAMB, UMR 7199, Université de Strasbourg and CNRS, Faculté de Pharmacie , F-67401 Illkirch, France.
authors.forename: Marc
authors.initials: M
authors.lastname: Nothisen
authors.affiliation: Laboratoire V-SAT, CAMB, UMR 7199, Université de Strasbourg and CNRS, Faculté de Pharmacie , F-67401 Illkirch, France.
authors.forename: Jérémy
authors.initials: J
authors.lastname: Bagilet
authors.affiliation: Laboratoire V-SAT, CAMB, UMR 7199, Université de Strasbourg and CNRS, Faculté de Pharmacie , F-67401 Illkirch, France.
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Behr
authors.affiliation: Laboratoire V-SAT, CAMB, UMR 7199, Université de Strasbourg and CNRS, Faculté de Pharmacie , F-67401 Illkirch, France.
authors.forename: Jean-Serge
authors.initials: JS
authors.lastname: Remy
authors.affiliation: Laboratoire V-SAT, CAMB, UMR 7199, Université de Strasbourg and CNRS, Faculté de Pharmacie , F-67401 Illkirch, France.
authors.forename: Mitsuharu
authors.initials: M
authors.lastname: Kotera
mesh_terms: D000072283:A549 Cells; D002412:Cations / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D020868:Gene Silencing / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D034904:Lamin Type A / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009841:Oligonucleotides / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000502:physiology; D013096:Spermine / Q000737:chemistry*
chemical_list: D002412:Cations; D034904:Lamin Type A; D009841:Oligonucleotides; D034741:RNA, Small Interfering; D013096:Spermine; D008156:Luciferases
keywords: carrier-free transfection; cationic oligospermine−siRNA conjugate; chemically modified siRNA; siRNA delivery