Chemical modification of siRNAs to improve serum stability without loss of efficacy

doi:10.1016/j.bbrc.2006.02.049

Biochemical and Biophysical Research Communications

Volume 342, Issue 3, 14 April 2006, Pages 919-927

https://doi.org/10.1016/j.bbrc.2006.02.049 Get rights and content

Abstract

Development of RNA interference as a novel class of therapeutics requires improved pharmacokinetic properties of short interfering RNA (siRNA). To confer enhanced serum stability to Sur10058, a hyperfunctional siRNA which targets survivin mRNA, a systematic modification at the 2′-sugar position and phosphodiester linkage was introduced into Sur10058. End modification of three terminal nucleotides by 2′-OMe and phosphorothioate substitutions resulted in a modest increase in serum stability, with 3′ end modification being more effective. Alternating modification by 2′-OMe substitution significantly stabilized Sur10058, whereas phosphorothioate modification was only marginally effective. Through various combinations of 2′-OMe, 2′-F and phosphorothioate modifications that were directed mainly at pyrimidine nucleotides, we have identified several remarkably stable as well as efficient forms of Sur10058. Thus, our results provide an effective means to stabilize siRNA in human serum without compromising the knockdown efficiency. This advancement will prove useful for augmenting the in vivo potency of RNA interference.

Section snippets

Materials and methods

RNA and DNA oligonucleotides. 2′-OMe-, 2′-F- and phosphorothioate-modified siRNA duplexes were synthesized by Proligo (Boulder, CO) and IDT (Coralville, IA). All siRNA duplexes have the sense strand sequence of 5′-AAG GAG AUC AAC AUU UUC A dTdT-3′. The 19 nucleotides represent the target sequence in survivin (NM_001168) and dTdT is a 2-nt 3′ overhang. The antisense strand was composed of nucleotides that are complementary to the target sequence and the dTdT 3′ overhang sequence. All siRNAs were

Effect on stability and RNAi activity of 5′ and 3′ end modification

We were interested to identify specific chemical modifications which affect the structure of Sur10058 and function to greatly enhance its serum stability. In our first line of investigation, we examined the effect of nucleases on the stability of siRNA in human serum by introducing 2′-OMe or PS modification into three terminal nucleotides at 5′ and 3′ ends of Sur10058 (Fig. 1).

The unmodified Sur10058 was very unstable in 10% human serum. As a result, unmodified Sur10058 was mostly degraded

Acknowledgments

We thank Ki-Sun Kwon and Kwang-Lae Hoe for many helpful discussions. This work was supported by the International Joint R&D Program (10014823) from the Ministry of Commerce, Industry, and Energy in Korea to Y.-C. Choi.

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Chemical modification of siRNAs to improve serum stability without loss of efficacy

Abstract

Section snippets

Materials and methods

Effect on stability and RNAi activity of 5′ and 3′ end modification

Acknowledgments

Cell

Mol. Cell

J. Biol. Chem.

Curr. Biol.

Mol. Ther.

Cell

siRNAs: applications in functional genomics and potential as therapeutics

Nat. Rev. Drug Discov.

Unlocking the potential of the human genome with RNA interference

Nature

Role for a bidentate ribonuclease in the initiation step of RNA interference

Nature

Ribonuclease activity and RNA binding of recombinant human Dicer

EMBO J.

Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP

EMBO J.

Argonaute2, a link between genetic and biochemical analyses of RNAi

Science

Argonaute2 is the catalytic engine of mammalian RNAi

Science

Crystal structure of Argonaute and its implications for RISC slicer activity

Science

Validating survivin as a cancer therapeutic target

Nat. Rev. Cancer