Georgii V. Bobkov

A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity

The use of chemically synthesized short interfering RNAs (siRNAs) is currently the method of choice to manipulate gene expression in mammalian cell culture, yet improvements of siRNA design is expectably required for successful application in vivo. Several studies have aimed at improving siRNA performance through the introduction of chemical modifications but a direct comparison of these results is difficult. We have directly compared the effect of 21 types of chemical modifications on siRNA activity and toxicity in a total of 2160 siRNA duplexes. We demonstrate that siRNA activity is primarily enhanced by favouring the incorporation of the intended antisense strand during RNA-induced silencing complex (RISC) loading by modulation of siRNA thermodynamic asymmetry and engineering of siRNA 3′-overhangs. Collectively, our results provide unique insights into the tolerance for chemical modifications and provide a simple guide to successful chemical modification of siRNAs with improved activity, stability and low toxicity.

Detection of RNA Hybridization by Pyrene-Labeled Probes

Powerful pyrene probes: Two kinds of pyrene‐labeled oligonucleotides (HNA‐ and RNA‐skeleton probes) were explored. The enhanced fluorescence intensity in the monomer region and the disappearance of aggregate/excimer emission in duplexes has been successfully used to detect the hybridization of oligonucleotides.

Oligonucleotides Containing Disaccharide Nucleosides

Disaccharide nucleosides with 2′-O-(D-arabinofuranosyl), 2′-O-(L-arabinofuranosyl), 2′-O-(D-ribopyranosyl), 2′-O-(D-erythrofuranosyl), and 2′-O-(5-azido-5-deoxy-D-ribofuranosyl) substituents were synthesized. These modified nucleosides were incorporated into oligonucleotides (see Table). Single substitution resulted in a ΔTm of +0.5 to −1.4° for DNA/RNA and a ΔTm of −0.8 to −4.7° for DNA/DNA duplexes. These disaccharide nucleosides can be well accommodated in RNA/DNA duplexes, and the presence of a NH2−C(5″) group has a beneficial effect on duplex stability.

Effective Anomerisation of 2′‐Deoxyadenosine Derivatives During Disaccharide Nucleoside Synthesis

The formation of a disaccharide nucleoside (11) by O3′‐glycosylation of 5′‐O‐protected 2′‐deoxyadenosine or its N 6‐benzoylated derivative has been observed to be accompanied by anomerisation to the corresponding α‐anomeric product (12). The latter reaction can be explained by instability of the N‐glycosidic bond of purine 2′‐deoxynucleosides in the presence of Lewis acids. An independent study on the anomerisation of partly blocked 2′‐deoxyadenosine has been carried out. Additionally, transglycosylation has been utilized in the synthesis of 3′‐O‐β‐d‐ribofuranosyl‐2′‐deoxyadenosines and its α‐anomer.

2'-O-hydroxyalkoxymethylribonucleosides and their incorporation into oligoribonucleotides.

A simple and efficient method for the preparation of pyrimidine 2 ′-O-hydroxyethoxymethylribonucleosides and 2 ′-O-hydroxypropoxymethylribonucleosides has been developed. These modified nucleosides were incorporated into oligoribonucleotides, which were shown to form stable RNA/RNA duplexes. The effect of 2 ′ -O-modification in the antisense and sense strands of small interference RNA was evaluated in multi-drug resistant NIH 3T3 cells.

Synthesis of 2′‐O‐β‐d‐Ribofuranosylnucleosides

A simple and efficient method for the preparation of 2‐O‐β‐D‐ribofuranosylnucleosides, minor tRNA components, is described in this unit. The method consists of condensation of a small excess of 1‐O‐acetyl‐2,3,5‐tri‐O‐benzoyl‐β‐D‐ribofuranose activated with tin tetrachloride with N‐protected 3,5‐O‐tetra‐isopropyldisiloxane‐1,3‐diyl‐ribonucleosides in 1,2‐dichloroethane. Subsequent deprotection produces 2‐O‐β‐D‐ribofuranosylnucleosides in an overall yield of 46% to 72%.

UNIT 1.14 Synthesis of 2′-O-β-d-Ribofuranosylnucleosides

A simple and efficient method for the preparation of 2‐O‐β‐D‐ribofuranosylnucleosides, minor tRNA components, is described in this unit. The method consists of condensation of a small excess of 1‐O‐acetyl‐2,3,5‐tri‐O‐benzoyl‐β‐D‐ribofuranose activated with tin tetrachloride with N‐protected 3,5‐O‐tetra‐isopropyldisiloxane‐1,3‐diyl‐ribonucleosides in 1,2‐dichloroethane. Subsequent deprotection produces 2‐O‐β‐D‐ribofuranosylnucleosides in an overall yield of 46% to 72%.

Synthesis of 2'-O-beta-d-Ribofuranosylnucleosides.

A simple and efficient method for the preparation of 2‐O‐β‐D‐ribofuranosylnucleosides, minor tRNA components, is described in this unit. The method consists of condensation of a small excess of 1‐O‐acetyl‐2,3,5‐tri‐O‐benzoyl‐β‐D‐ribofuranose activated with tin tetrachloride with N‐protected 3,5‐O‐tetra‐isopropyldisiloxane‐1,3‐diyl‐ribonucleosides in 1,2‐dichloroethane. Subsequent deprotection produces 2‐O‐β‐D‐ribofuranosylnucleosides in an overall yield of 46% to 72%.

Oligonucleotides Containing Disaccharide Nucleosides: Synthesis, Physicochemical, and Substrate Properties

Abstract The efficient synthesis of oligonucleotides containing 2′-O-β-D-ribofuranosyl (and β-D-ribopyranosyl)nucleosides, 2′-O-α-D-arabinofuranosyl (and α-L-arabinofuranosyl)nucleosides, 2′-O-β-D-erythrofuranosylnucleosides, and 2′-O-(5′-amino-5-deoxy-β-D-ribofuranosyl)nucleosides have been developed.