〈PAPER〉Synthesis, Delivery and Gene Silencing of siRNA-NES Conjugates

Herein we described the synthesis of siRNA-NES (nuclear export signal) peptide conjugates by solid phase fragment coupling (SPFC) and application of them to silencing of bcr/abl chimeric gene in human leukemia cell line K562. Synthesis of siRNA-NES conjugates was achieved by SPFC. As a result, two types of siRNA-NES conjugates C1 and C2 were prepared in which 5' -end of sense strand was covalently linked to N-terminus of the NES peptides derived from TFIIIA and HIV-1rev, respectively. Silencing effects of C1 and C2 against bcr/abl mRNA in human leukemia cell line K562 were evaluated by quantitative PCR. The expression of bcr/abl gene was suppressed to 30.2％ at 200nM and 36.3％ at 50 nM by native siRNA. Significant enhancement of silencing efficiency was observed with C1 and C2. siRNA-TFIIIA NES (C1) suppressed the expression of bcr/abl gene to 8.3％ at 200 nM and 11.6％ at 50nM and siRNA-HIV-1rev NES (C2) suppressed to 4.0％ at 200 nM and 6.3％ at 50nM. Previously, we reported that DNA-HIV-1 rev NES peptide conjugate was localized in cytoplasm of Jurkat cell. The large enhancement of the silencing efficiency of siRNA-NES conjugates could be reasonably ascribed to the localization of siRNA-NES conjugates in cytoplasm. It can be also pointed out that modification of 5' -endo of sense strand reduced off-target effect by minimizing the extent of the sense strand incorporation into RISC. (3) Unfortunately, siRNA-NES conjugates could not penetrate the cellular membrane by itself and required a transfection reagent to be taken up into cells, while oligodeoxyribonucleotide-NES conjugates were internalized into cells without any transfection reagents. It can be elucidated the double stranded structure of siRNA retarded the penetration through cellular membrane.