Non-terminal conjugation of small interfering RNAs with spermine improves duplex binding and serum stability with position-specific incorporation

The conjugation of small interfering RNAs (siRNAs) has been studied using lipid and ligand conjugates for efficient delivery. However, most conjugates have been inserted at the terminal position; very few have been inserted at non-terminal positions. Herein, we synthesized a 4 & PRIME;-C-propyllevulinate-2 & PRIME;-O-methyluridine analog for non-terminal conjugation of spermine into the passenger strand of siRNA. Solid-phase oligonucleotide synthesis using this analog was successful, with the conjugation of one or two spermine molecules. The siRNAs conjugated with spermine displayed improved thermodynamic stability and resistance against nucleases, which depended on the site of conjugation in each case. Circular dichroism spectroscopy revealed that the A-type helical structure of the RNA duplex was not altered by these modifications. However, the gene-silencing activity of conjugated siRNAs was reduced and further decreased when the number of spermine molecules was increased. Hence, this work supplies valuable information and provides scope for the further development of drug-delivery systems through non-terminal conjugation.

Automated summary

A novel method of non-terminal conjugation of spermine into the passenger strand of siRNA was synthesized and evaluated. The conjugation at non-terminal positions resulted in increased stability and resistance against nucleases, but showed reduced gene-silencing activity.