Recently we reported that intracellular trafficking of oligonucleotides could be controlled by conjugation with nuclear export signal (NES) and nuclear localization signal (NLS) peptides and that siRNA-NES conjugates showed drastically enhanced silencing of BCR/ABL chimeric gene in human chronic myelogenous leukemia cell line K562 [1, 2, 3].

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.

Syntheses of siRNA-NES conjugates were achieved by SPFC as previously described .[1]

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 for C1 and HIV-1 rev for C2, 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. [2] 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]

The author is grateful for financial support by JSPS KAKEN Grant Number 22550159, MAFF/AFFRCS International Joint Project 2017-03, JMRF-RFBR Japan-Russia Joint Research Project 2018-P2 and Kindai Research Grant KD201704.