Wu Li, Wang Jie, Tang Xinjing
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, China.
School of Chemistry and Chemical Engineering, University of Chinese Academy of Sciences, Beijing, China.
Curr Protoc Nucleic Acid Chem. 2015 Jun 1;61:6.12.1-6.12.15. doi: 10.1002/0471142700.nc0612s61.
Photolabile small interfering RNA (siRNA) oligonucleotide duplexes are becoming a powerful tool for photoregulation of gene expression through an RNA interference (RNAi) mechanism. Terminal or statistical labeling of siRNAs has been previously achieved. Recently, we have shown a new strategy for site-specific incorporation of a photolabile group (1-(2-nitrophenyl)ethyl [NPE]) at any phosphate position of siRNA strands for photomodulation of their gene-silencing activity. In this unit, we first describe in detail the syntheses of four new NPE protected nucleoside phosphoramidites (dA 0, dG 0, dC 0, dT 0) and 2-cyanoethyl-1-(2-nitrophenyl)ethyl-N,N'-diisopropylphosphoramidite (N 0). They are then site specifically incorporated into any position of RNA oligonucleotides according to standard phosphoramidite chemistry. Phosphate-caged siRNA duplexes are then prepared by hybridization of single-stranded RNAs containing the 1-(2-nitrophenyl)ethyl caging moiety on the phosphate group with their complementary RNA.
光不稳定的小干扰RNA（siRNA）寡核苷酸双链体正成为一种通过RNA干扰（RNAi）机制对基因表达进行光调节的强大工具。之前已经实现了siRNA的末端或随机标记。最近，我们展示了一种新策略，可在siRNA链的任何磷酸位置位点特异性引入光不稳定基团（1-（2-硝基苯基）乙基[NPE]），以光调节其基因沉默活性。在本单元中，我们首先详细描述四种新型NPE保护的核苷亚磷酰胺（dA 0、dG 0、dC 0、dT 0）和2-氰基乙基-1-（2-硝基苯基）乙基-N，N'-二异丙基亚磷酰胺（N 0）的合成。然后根据标准亚磷酰胺化学方法将它们位点特异性地掺入RNA寡核苷酸的任何位置。随后，通过将在磷酸基团上含有1-（2-硝基苯基）乙基笼蔽部分的单链RNA与其互补RNA杂交，制备磷酸笼蔽的siRNA双链体。
