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 phosphor-
amidite 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.
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