RNA interference with 2 0 ,4 0 -bridged nucleic acid analogues S. M. Abdur Rahman a,b , Hiroyuki Sato a , Naoto Tsuda a , Sunao Haitani a , Keisuke Narukawa a , Takeshi Imanishi a , Satoshi Obika a, * a Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan b Department of Clinical Pharmacy and Pharmacology, Faculty of Pharmacy, University of Dhaka, Dhaka 1000, Bangladesh article info Article history: Received 29 January 2010 Revised 26 March 2010 Accepted 27 March 2010 Available online 3 April 2010 Keywords: 2 0 ,4 0 -Bridged nucleic acid siRNA Serum stability Argonauate cleavage site abstract In this study, a number of 2 0 ,4 0 -BNA- and 2 0 ,4 0 -BNA NC -modified siRNAs were designed and synthesized. Their thermal stability, nuclease resistance and gene silencing properties against cultured mammalian cells were evaluated and compared with those of natural siRNAs. The 2 0 ,4 0 -BNA- and 2 0 ,4 0 -BNA NC -modi- fied siRNAs (named siBNA and siBNA NC , respectively) showed very high T m values, were remarkably sta- ble in serum sample and showed promising RNAi properties equal to those exhibited by natural siRNAs. Thermally stable siBNAs composed of slightly modified sense and antisense strands were capable of sup- pressing gene expression equal to that of natural siRNA. A number of modifications on the sense strand by 2 0 ,4 0 -BNA or 2 0 ,4 0 -BNA NC , either consecutively or separated by natural RNA nucleotides, is tolerable in RNAi machinery. Modifications at the Argonauate (Ago2) cleavage site of the sense strand (9–11th posi- tions from the 5 0 -end of the sense strand) produced variable results depending on siRNA composition. Mostly, modification at the 10th position diminished siRNA activity. In moderately modified siRNAs, modification at the 11th position displayed usual RNAi activity, while modification at the 9th position showed variable results depending on siRNA composition. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Since its discovery in 1998, 1 RNA interference (RNAi) has emerged as a very important and powerful molecular biological tool for regulation of gene expression. 2–6 RNAi technology utilizes short 21 bp double-stranded RNA (dsRNA) with a two nucleotide (nt) 3 0 -overhang generally termed as small interfering RNA (siR- NA), which upon incorporation to RNA-induced silencing complex (RISC) interacts with specific mRNA and ultimately suppresses it. 7,8 In this procedure, the sense strand (passenger strand) of siRNA is typically cleaved at 9 nt from the 5 0 -end of the sense strand by Arg- onauate 2 (Ago2) endonuclease, 9,10 and the activated RISC contain- ing the antisense strand (guide strand) binds with target mRNA through Watson–Crick base pairing to cause degradation or trans- lational block of target RNA. Although natural siRNAs elicit promising RNAi activity in cell culture, 11–15 their in vivo use as a drug remains questionable because of obstacles such as low biostability and undesirable toxicity (off-target effects). To overcome these problems and to improve the pharmacokinetics and delivery of siRNA, efforts to use chemically modified siRNAs are increasing day by day. Various types of chemical modifications applied to siRNA have been nicely presented in some recent reports and reviews. 16–21 Chemical modification in siRNAs in many cases has improved the serum sta- bility of siRNAs, albeit often at the expense of RNAi activity. 22–26 However, careful placement of some specific modified residues has also enhanced siRNA biostability without loss of siRNA potency, even enhancing siRNA potency in some cases. 27–32 Some specific modifications have reduced siRNA side effects, such as induction of recipient immune responses and inherent off-target- ing effects. 33–35 Among the various chemically modified siRNAs investigated, bridged nucleic acid 36 such as 2 0 ,4 0 -methylene bridged nucleic acid (2 0 ,4 0 -BNA, 37 also known as locked nucleic acid or LNA, Fig. 1) 38 modified siRNAs (named siLNAs) are some of the most promising Figure 1. Chemical structures of natural RNA, 2 0 ,4 0 -BNA, and 2 0 ,4 0 -BNA NC . 0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2010.03.076 * Corresponding author. Tel.: +81 6 6879 8200; fax: +81 6 6879 8204. E-mail address: obika@phs.osaka-u.ac.jp (S. Obika). Bioorganic & Medicinal Chemistry 18 (2010) 3474–3480 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc