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Nucleic Acid Base modifications for gene silencing with RNAi

Properly designed antisense oligonucleotides (AONs) or siRNAs allow the cleavage of specific messenger RNA (mRNA) strands.

AONs and siRNAs take advantage of endogenous cellular pathways to silence the expression of particular genes. Oligonucleotide-directed approaches target mRNAs directly before translation, eliminating the need for protein or enzyme inhibition using small molecules. However, some obstacles can prevent siRNA-based oligonucleotide therapies.

These obstacles include: 

(1) their poor extracellular and intracellular stability, 

(2) low efficiency of intracellular delivery to target cells or tissues, and 
(3) the potential for ‘‘off-target’’ gene silencing, immune stimulation, and other side effects.

The incorporation of modified nucleic acids into AONs or siRNAs may potentially allow to circumvent these obstacles. 

In 2007, Sipa et al. tested a series of siRNA duplexes containing the “rare” nucleosides, 2-thiouridine (s2U), pseudouridine (Ψ), and dihydrouridine (D), for their thermodynamic stability and gene silencing activity.

The study found that oligonucleotide duplexes with modified nucleic acid bases at terminal positions showed similar stability as a nonmodified reference duplex. The introduction of the s2U or Ψ units into the central part of the antisense strand resulted in duplexes with higher melting temperatures (Tm). However, adding D units resulted in less stable duplexes. 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.

Table 1: Base Modifications and their Effect.



ΔTm duplex per modification

Impact on the efficiency of RNAi


2′ thiouridine


7% s2U are tolerated by RNAi.
s2U can change thermal asymmetry of the duplex and increase the efficiency of siRNA in vitro.

s2U slightly increases nuclease resistance in vitro.


−1 to +1°C

One Ψ is tolerated by RNA.

Stabilizes 3′endo ribose conformation.
Reduces the PKR-induced interferon response.




The nonaromatic nucleobase disrupts base stacking.

D unit similarly like wobble base pair lead to less stable duplexes.

R = ribose residue.


Chernikov IV, Vlassov VV, Chernolovskaya EL. Current Development of siRNA Bioconjugates: From Research to the Clinic. Front Pharmacol. 2019 Apr 26;10:444. [PMC]

Glen F. Deleavey, Masad J. Damha; Designing Chemically Modified Oligonucleotides for Targeted Gene Silencing. Chemistry & Biology, Volume 19, Issue 8, 2012, Pages 937-954. [PubMed]

Katarzyna Sipa, Elzbieta Sochacka, Julia Kazmierczak-Baranska, Maria Maszewska, Magdalena Janicka, Genowefa Nowak, and Barbara Nawrot;  Effect of base modifications on structure, thermodynamic stability, and gene silencing activity of short interfering RNA. RNA 2007. 13: 1301-1316. [PMC]


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