Current Topics in Medicinal Chemistry, 2006, 6, 913-925 913 1568-0266/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd. Chemical and Structural Diversity of siRNA Molecules Barbara Nawrot* and Katarzyna Sipa Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland Abstract: Short interfering RNAs (siRNAs) are 21-23 nt long double-stranded oligoribonucleotides which in mammalian cells exhibit a potency for sequence-specific gene silencing via an RNA interference (RNAi) pathway. It has been already proven that exogenous, chemically synthesized siRNA molecules are effective inhibitors of gene expression and are widely applied for analysis of protein function and proteomics-based target identification. Moreover, since their discovery siRNA molecules have been implemented as potential candidates for therapeutic applications. Variously modified siRNA molecules containing sugar modifications (2’-OMe, -F, -O-allyl, -amino, orthoesters and LNA analogues), internucleotide phospodiester bond modifications (phosphorothioates, boranophosphates), base modifications (s 2 U) as well as 3’-terminal cholesterol-conjugated constructs were investigated as potential candidates for effective inhibition of gene expression. This chapter reviews an impact of chemical and structural modifications of siRNA molecules on their serum and thermal stability, cellular and in vivo activity, cellular uptake, biodistribution and cytotoxicity. Functional analysis of chemically modified siRNA molecules allows for better understanding of the mechanism of the RNA interference process as well as demonstrates immense efforts in optimizing in vivo potency of siRNA molecules for RNAi-based drug design. Keywords: RNA interference, small interfering RNA, gene silencing, siRNA, modified siRNA, chemical modification, RNA structure. INTRODUCTION An initial enthusiasm paid to antisense strategy deve- loped in 80-ties of past century using DNA oligonucleotides and their analogues has not fulfilled early expectations to date [1, 2]. Only one antisense oligo(nucleoside phos- phorothioate) has been approved till now as a drug against cytomegaloviral (CMV) infection in HIV-1 infected patients (Fomivirsen, Vitravene ® , Isis Pharmaceuticals, 1998) [3]. Another oligonucleotide, active as an aptamer of vascular endothelial growth factor (VEGF) was approved by FDA in 2005 (Macugen ® , Pfizer) for age-related macular degene- ration (AMD) therapy [4]. New hopes for sequence-specific control of gene expression emerged with the discovery of RNA interference (RNAi) mechanism. Shortly after Fire’s first report on RNAi in Caernohabditis elegans [5], some of its features were found common to other mechanisms of post-transcriptional gene silencing in many different species like plants [6], fungi [7] and insects [8]. In these mechanisms common is the function of short ribonucleic acids, so far much underestimated as less stable members of nucleic acids family. The post-transcriptional gene silencing is based on degradation of mRNA complementary to exogenous double stranded RNA. Three years after discovery of the phenome- non of RNA interference, the process was intentionally induced in mammalian cells [9]. That was possible due to the use of synthetic short interfering RNA (siRNA) duplexes which do not induce antiviral defense system in eukaryotic *Address correspondence to this author at the Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland; Tel: 0048- 42-681 69 70; Fax: 0048-42-681 54 83; E-mail: bnawrot@bio.cbmm.lodz.pl cells. In spite of the fact that antiviral defense system recognizes long double stranded RNA (> 30 bp) as a signal for apoptosis and induces interferon response [10], scientists soon started to consider siRNA molecules as the most promising tool for gene silencing in mammalian cells as well as in higher organisms. Although siRNA molecules suffer from their low stabi- lity in vivo, insufficient cellular uptake and induced off- target effects, they seem to be more useful than antisense oligonucleotides for analysis of function of newly discovered genes, and in silencing of genes to combat certain diseases. Nonetheless, significant progress was made to overcome these difficulties, which capitalized on knowledge gained during attempts at chemical modification of antisense oligonucleotides. These achievements made the siRNA molecules more potent and more useful, albeit yet much unpredictable. Thus, application of the siRNA molecules requires further studies, also in respect to new chemical modifications of these tools improving their in-serum and thermal stability, intracellular and in vivo activity, cellular uptake, biodistribution and low cytotoxicity. RNAi IN MAMMALIAN CELLS In general, an RNAi phenomenon involves three major steps [see Fig. (1)]. Within the first step, the Dicer nuclease specifically hydrolyzes long double stranded RNA into siRNAs. The one strand of the resulting siRNA duplex is incorporated into a protein complex, called RISC (RNA Induced Silencing Complex). Activated in this way RISC hydrolyzes phosphodiester bond in the target mRNA in a region complementary to the guide strand of siRNA [11, 12]. The steps of Dicer-promoted formation of siRNA and unwinding of siRNA duplex just before incorporation of one strand into the RISC [13], both depend on ATP. However,