A series of nucleobase-modified siRNA duplexes containing “rare” nucleosides, 2-thiouridine (s²U), pseudouridine (Ψ), and dihydrouridine (D), were evaluated for their thermodynamic stability and gene silencing activity. The duplexes with modified units at terminal positions exhibited similar stability as the nonmodified reference. Introduction of the s²U or Ψ units into the central part of the antisense strand resulted in duplexes with higher melting temperatures (Tm). In contrary, D unit similarly like wobble base pair led to the less stable duplexes (ΔTm 3.9 and 6.6°C, respectively). Gene-silencing activity of siRNA duplexes directed toward enhanced green fluorescent protein or beta-site APP cleaving enzyme was tested in a dual fluorescence assay. The duplexes with s²U and Ψ units at their 3′-ends and with a D unit at their 5′-ends (with respect to the guide strands) were the most potent gene expression inhibitors. Duplexes with s²U and Ψ units at their 5′-ends were by 50% less active than the nonmodified counterpart. Those containing a D unit or wobble base pair in the central domain had the lowest Tm, disturbed the A-type helical structure, and had more than three times lower activity than their nonmodified congener. Activity of siRNA containing the wobble base pair could be rescued by placing the thio-nucleoside at the position 3′-adjacent to the mutation site. Thermally stable siRNA molecules containing several s²U units in the antisense strand were biologically as potent as their native counterparts. The present results provide a new chemical tool for modulation of siRNA gene-silencing activity.

The RNA phosphodiester bond cleavage activity of a series of 16 thio‐deoxyribozymes 10–23, containing a P‐stereorandom single phosphorothioate linkage in predetermined positions of the catalytic core from P1 to P16, was evaluated under single‐turnover conditions in the presence of either 3 mmMg²⁺or 3 mmMn²⁺. A metal‐specificity switch approach permitted the identification of nonbridging phosphate oxygens (proR_PorproS_P) located at seven positions of the core (P2, P4 and P9–13) involved in direct coordination with a divalent metal ion(s). By contrast, phosphorothioates at positions P3, P6, P7 and P14–16 displayed no functional relevance in the deoxyribozyme‐mediated catalysis. Interestingly, phosphorothioate modifications at positions P1 or P8 enhanced the catalytic efficiency of the enzyme. Among the tested deoxyribozymes, thio‐substitution at position P5 had the largest deleterious effect on the catalytic rate in the presence of Mg²⁺, and this was reversed in the presence of Mn²⁺. Further experiments with thio‐deoxyribozymes of stereodefined P‐chirality suggested direct involvement of both oxygens of the P5 phosphate and theproR_Poxygen at P9 in the metal ion coordination. In addition, it was found that the oxygen atom at C6 of G₆contributes to metal ion binding and that this interaction is essential for 10–23 deoxyribozyme catalytic activity.

Four series of phosphorus dendrimers (generations 1 and 4) having various types of amine terminal groups (pyrrolidine, morpholine, methyl piperazine, or phenyl piperazine) are synthesized. After protonation, the fourth generations of three of them are found water-soluble, and used for several biological experiments. First, the cytotoxicity of these polycationic dendrimers towards three cell strains (one healthy: HUVEC, and two cancerous: HEK 293 and HeLa) is assayed and found low. Second, their ability to interact with DNA is tested by electrophoresis: the dendrimer terminated by pyrrolidinium groups is found efficient to form dendriplexes. Finally, the polycationic dendrimers are used as transfection agents to deliver single- and double-stranded DNA into the three above-mentioned cell strains. Here also the dendrimer having pyrrolidinium groups is found the most efficient.

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 (s2U) 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.