title: Synthesis of site-specifically phosphate-caged siRNAs and evaluation of their RNAi activity and stability.
abstract: A complete set of new photolabile nucleoside phosphoramidites were synthesized, then site-specifically incorporated into sense or antisense strands of siRNA for phosphate caging. Single caging modification was made along siRNA strands and their photomodulation of gene silencing were examined by using the firefly luciferase reporter gene. Several key phosphate positions were then identified. Furthermore, multiple caging modifications at these key positions led to significantly enhanced photomodulation of gene silencing activity, suggesting a synergistic effect. The caging group on both the terminally phosphate-caged siRNA and the single-stranded caged RNA has comparatively high stability, whereas hydrolysis of the caged group from the internally caged siRNA was observed, irrespective of the presence of Mg(2+). Molecular dynamic simulations demonstrated that enhanced hydrolysis of the caging group on internally phosphate-caged siRNAs was due to easy fragmentation of the caging group upon formation of the pentavalent intermediate of the phosphotriester with attack by water. The caging group in the terminally phosphate-caged siRNA or single-stranded caged RNA prefers to form π-π stacks with nearby nucleobases. In addition to providing explanations for previous observations, this study sheds further light on the design of caged oligonucleotides and indicates the direction of future development of nucleic acid drugs with phosphate modifications.
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191 (China), Fax: (+86) 10-8280-5635; College of Chemistry and Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049 (China).
authors.forename: Li
authors.initials: L
authors.lastname: Wu
authors.forename: Fen
authors.initials: F
authors.lastname: Pei
authors.forename: Jinhao
authors.initials: J
authors.lastname: Zhang
authors.forename: Junzhou
authors.initials: J
authors.lastname: Wu
authors.forename: Mengke
authors.initials: M
authors.lastname: Feng
authors.forename: Yuan
authors.initials: Y
authors.lastname: Wang
authors.forename: Hongwei
authors.initials: H
authors.lastname: Jin
authors.forename: Liangren
authors.initials: L
authors.lastname: Zhang
authors.forename: Xinjing
authors.initials: X
authors.lastname: Tang
mesh_terms: D006801:Humans; D015394:Molecular Structure; D010755:Organophosphates / Q000138:chemical synthesis*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000138:chemical synthesis*
chemical_list: D010755:Organophosphates; D034741:RNA, Small Interfering
keywords: RNA hydrolysis; caged siRNA; nucleosides; photoactivation; regioselectivity