Sergey N. Mikhailov

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.

Distinct mechanisms of bisphosphonate action between osteoblasts and breast cancer cells: identity of a potent new bisphosphonate analogue

While the effects of bisphosphonates on bone-resorbing osteoclasts have been well documented, the effects of bisphosphonates on other cell types are not as well studied. Recently, we reported that bisphosphonates have direct effects on bone-forming human fetal osteoblast cells (hFOB) [1]. In this report, the role of the mevalonate pathway in the actions of bisphosphonates on hFOB, and MDA-MB-231 human breast cancer cells was examined. These studies included a novel bisphosphonate analog, the anhydride formed between arabinocytidine 5′ phosphate and etidronate (Ara-CBP). Ara-CBP was the most potent inhibitor of hFOB and MDA-MB-231 cell proliferation, and stimulator of hFOB cell mineralization compared to etidronate, the anhydride formed between AMP and etidronate (ABP), pamidronate, and zoledronate. Inhibition of hFOB cell proliferation by Ara-CBP and zoledronate was partially reversed by mevalonate pathway intermediates, and stimulation of hFOB cell mineralization was completely reversed by mevalonate pathway intermediates. These results suggest that zoledronate and Ara-CBP act, at least in part, via inhibition of the mevalonate pathway in hFOB cells. In contrast, none of the mevalonate pathway intermediates reversed the inhibition of MDA-MB-231 cell proliferation by the bisphosphonates, or the effects of pamidronate on hFOB cells. As a positive control, the effects of mevastatin on hFOB and MDA-MB-231 cells were completely reversed by mevalonate. In summary, these data suggest that zoledronate and Ara-CBP induce human osteoblast differentiation via inhibition of the mevalonate pathway. In contrast, the inhibition of MDA-MB-231 cell proliferation by the bisphosphonates appears to be through mechanisms other than inhibition of the mevalonate pathway.

An efficient synthesis and physico-chemical properties of 2'-O-D-ribofuranosylnucleosides, minor tRNA components

ABSTRACT A high yield preparation of 9-(2-O-β-D-ribofuranosyl-β-D-ribofuranosyl)adenine, guanine- and the pyrimidine analogs (cytosine, thymine and uracil base moiety) has been achieved, and the conformational properties of the ring systems were investigated using NMR spectroscopy and X-ray.

New syntheses of 2′-C-methylnucleosides starting from d-glucose and d-ribose

Abstract Effective general methods have been developed for the synthesis of 2′-C-methylnucleosides starting from d -glucose and d -ribose. 3-O-benzyl-1,2-O-isopropylidene-3-C-methyl-α- d -allofuranose was prepared in 5 steps from d -glucose and converted into 1,2,3-tri-O-acetyl-2-C-methyl-5-O-p-methylbenzoyl- d -ribofuranose (5), the starting compound for nucleoside synthesis. Compound 5 was also synthesised from 2-C-hydroxymethyl-2,3-O-isopropylidene-5-O-trityl- d -ribofuranose, prepared in 3 steps from d -ribose. Condensation of 5 with the bis-trimethylsilyl derivatives of uracil, N4-benzoylcytosine, and N6-benzoyladenine in the presence of F3CSO3OSiMe3 followed by removal of the protecting acyl groups yielded the corresponding 2′-C-methylnucleosides.

Synthesis and properties of 3′-C-methylnucleosides and their phosphoric esters

Abstract 3′- C -Methyluridine and 3′- C -methylcytidine were synthesized in 11 steps starting from d -glucose. The conformation of 3′- C -methylnucleosides was studied in solution and in the crystal by using the techniques of c.d., 1 H-n.m.r. spectroscopy, and X-ray diffraction analysis. 3′- C -Methyluridine 2′,3′-cyclophosphate was prepared, and its hydrolysis with nucleases was studied. 3′- C -Methyluridine 5′- mono- and 5′-tri-phosphate were also synthesized.

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.

Ribosylation of Pyrimidine 2′-Deoxynucleosides

Abstract The previously developed method for the preparation of 2′-O-D-ribofuranosyl-nucleosides is extended to ribosylation of 2′-deoxynucleosides. The scope and limitations of this reaction are discussed.

Mapping of T7 RNA polymerase active site with novel reagents – oligonucleotides with reactive dialdehyde groups

Oligonucleotides of a novel type containing 2′‐O‐β‐ribofuranosyl‐cytidine were synthesized and further oxidized to yield T7 consensus promoters with dialdehyde groups. Both types of oligonucleotides were tested as templates, inhibitors, and affinity reagents for T7 RNA polymerase and its mutants. All oligonucleotides tested retained high affinity towards the enzyme. Wild‐type T7 RNA polymerase and most of the mutants did not react irreversibly with oxidized oligonucleotides. Affinity labeling was observed only with the promoter‐containing dialdehyde group in position (+2) of the coding chain and one of the mutants tested, namely Y639K. These results allowed us to propose the close proximity of residue 639 and the initiation region of the promoter within initiation complex. We suggest the oligonucleotides so modified may be of general value for the study of protein‐nucleic acid interactions.

Epimerization during the acetolysis of 3-O-acetyl-5-O-benzoyl-1,2-O-isopropylidene-3-C-methyl-α-d-ribofuranose. Synthesis of 3′-C-methylnucleosides with the β-d-ribo- and α-d-arabino configurations

Abstract Acetolysis of 3-O-acetyl-5-O-benzoyl-1,2-O-isopropylidene-3-C-methyl-α- d -ribofuranose with a high concentration of acetic acid yielded 1,2,3-tri-O-acetyl-5-O-benzoyl-3-C-methyl- d -arabinofuranose, which was used for the preparation of 3-C-methyl-α- d -arabinofuranosyl nucleosides. 3′-C-Methylribonucleosides were also synthesized starting from 1,2,3-tri-O-acetyl-5-O-benzoyl-3-C-methyl- d -ribofuranose.