title: D-Isonucleotide (isoNA) incorporation around cleavage site of passenger strand promotes the vibration of Ago2-PAZ domain and enhances in vitro potency of siRNA.
abstract: It has been demonstrated that passenger strand cleavage is important for the activation of RNA-induced silencing complex (RISC), which is a crucial step for siRNA-mediated gene silencing. Herein, we report that isonucleotide (isoNA) modification around the cleavage site of the passenger strand would affect the in vitro potency of modified siRNAs by altering the motion pattern of the Ago2-PAZ domain. According to western blotting, q-PCR and antiviral test results, we proved that D-isonucleotide (isoNA) modification at the position 8 of the passenger strand (siMek1-S08D), which is adjacent to the cleavage site, markedly improved the in vitro potency of the modified siRNA, whereas siRNAs with D-isoNA incorporation at position 9 (siMek1-S09D) or L-isoNA incorporation at positions 8 and 9 (siMek1-S08L, siMek1-S09L) displayed lower activity compared to native siRNA. Kinetics evaluation of passenger strand cleavage induced by T. thermophilus Ago (Tt-Ago) showed that D-isoNA modification at position 8 of the passenger strand had no significant influence on the cleavage rate, but L-isoNA modification at position 8 slowed the cleavage rate markedly. Moreover, the results of molecular dynamics simulations showed that D-isoNA modification at position 8 affected the open-close motion of the PAZ domain in the Ago/siRNA complex, which may promote the loading of RISC and release of a passenger strand cleavage product, and consequently accelerate the activation of RISC and enhance silencing activity. However, D-isoNA modification at position 9 or L-isoNA modification at position 8 or 9 exerted opposite influences on the motion of the Ago-PAZ domain.
authors: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China. yangzj@bjmu.edu.cn.
authors: Ye
authors: Y
authors: Huang
authors: Miao
authors: M
authors: Tian
authors: Yichao
authors: Y
authors: Zhang
authors: Gang
authors: G
authors: Sheng
authors: Zhuo
authors: Z
authors: Chen
authors: Yuan
authors: Y
authors: Ma
authors: Yue
authors: Y
authors: Chen
authors: Yihong
authors: Y
authors: Peng
authors: Yi-Lei
authors: YL
authors: Zhao
authors: Yanli
authors: Y
authors: Wang
authors: Lihe
authors: L
authors: Zhang
authors: Zhenjun
authors: Z
authors: Yang
mesh_terms: D060565:Argonaute Proteins / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016963:Thermus thermophilus / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection
chemical_list: D060565:Argonaute Proteins; D001426:Bacterial Proteins; D034741:RNA, Small Interfering