Small interfering RNAs (siRNAs) which mediate sequence-specific gene silencing through RNA interference have been harnessed for therapeutic applications. Here, we found combinations of modest 2′-ribose siRNA modifications that permit effective gene silencing and improve siRNA stability in human serum. Introduction of as few as two different or identical 2′-ribose modifications at a specific position of either strand, which has been previously suggested to suppress unintended off-target effects, enhances the thermal and thermodynamic stability of siRNA duplexes, and does not alter the overall A-form helical geometry that is required for silencing activity. Our results provide a useful template for the design for therapeutic siRNAs.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Price excludes VAT (USA)
Tax calculation will be finalised during checkout.
Breslauer KJ (1995) Extracting thermodynamic data from equilibrium melting curves for oligonucleotide order-disorder transitions. Methods Enzymol 259:221–242
Chiu YL, Rana TM (2002) RNAi in human cells: basic structural and functional features of small interfering RNA. Mol Cell 10:549–561
Chiu YL, Rana TM (2003) siRNA function in RNAi: a chemical modification analysis. RNA 9:1034–1048
De Paula D, Bentley MV, Mahato RI (2007) Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting. RNA 13:431–456
Elmen J, Thonberg H, Ljungberg K, Frieden M, Westergaard M, Xu Y, Wahren B, Liang Z, Orum H, Koch T, Wahlestedt C (2005) Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality. Nucleic Acids Res 33:439–447
Haupenthal J, Baehr C, Kiermayer S, Zeuzem S, Piiper A (2006) Inhibition of RNAse A family enzymes prevents degradation and loss of silencing activity of siRNAs in serum. Biochem Pharmacol 71:702–710
Hoerter JA, Walter NG (2007) Chemical modification resolves the asymmetry of siRNA strand degradation in human blood serum. RNA 13:1887–1893
Jackson AL, Burchard J, Leake D, Reynolds A, Schelter J, Guo J, Johnson JM, Lim L, Karpilow J, Nichols K, Marshall W, Khvorova A, Linsley PS (2006) Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing. RNA 12:1197–1205
Libonati M, Gotte G (2004) Oligomerization of bovine ribonuclease A: structural and functional features of its multimers. Biochem J 380:311–327
Ma JB, Ye K, Patel DJ (2004) Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain. Nature 429:318–322
Ma JB, Yuan YR, Meister G, Pei Y, Tuschl T, Patel DJ (2005) Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein. Nature 434:666–670
Matranga C, Tomari Y, Shin C, Bartel DP, Zamore PD (2005) Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 123:607–620
Pham JW, Pellino JL, Lee YS, Carthew RW, Sontheimer EJ (2004) A Dicer-2-dependent 80 s complex cleaves targeted mRNAs during RNAi in Drosophila. Cell 117:83–94
SantaLucia J Jr, Turner DH (1997) Measuring the thermodynamics of RNA secondary structure formation. Biopolymers 44:309–319
Turner JJ, Jones SW, Moschos SA, Lindsay MA, Gait MJ (2007) MALDI-TOF mass spectral analysis of siRNA degradation in serum confirms an RNAse A-like activity. Mol Biosyst 3:43–50
We are grateful to Prof. S. S. Park for critical reading of this manuscript. This research was supported by a grant (07132KFDA689) from the Korea Food and Drug Administration, in 2007.
Ik Sang Cho and Jung Kim contributed equally to this work.
Rights and permissions
About this article
Cite this article
Cho, I.S., Kim, J., Lim, D.H. et al. Improved serum stability and biophysical properties of siRNAs following chemical modifications. Biotechnol Lett 30, 1901–1908 (2008). https://doi.org/10.1007/s10529-008-9776-4
- A-form helix
- Melting temperature
- 2′-Ribose modification
- RNA interference