Structure-Based Design of Novel Chemical Modification of the 3′-Overhang for Optimization of Short Interfering RNA Performance

Abstract

Short interfering RNAs (siRNAs) are broadly used to manipulate gene expression in mammalian cells. Although chemical modification is useful for increasing the potency of siRNAs in vivo, rational optimization of siRNA performance through chemical modification is still a challenge. In this work, we designed and synthesized a set of siRNAs containing modified two-nucleotide 3'-overhangs with the aim of strengthening the interaction between the 3'-end of the siRNA strand and the PAZ domain of Ago2. Their efficiency of binding to the PAZ domain was calculated using a computer modeling program, followed by measurement of RNA-Ago2 interaction in a surface plasmon resonance biochemical assay. The results suggest that increasing the level of binding of the 3'-end of the guiding strand with the PAZ domain, and/or reducing the level of binding of the sense strand through modifying the two-nucleotide 3'-overhangs, affects preferential strand selection and improves siRNA activity, while we cannot exclude the possibility that the modifications at the 3'-end of the sense strand may also affect the recognition of the 5'-end of the guiding strand by the MID domain. Taken together, our work presents a strategy for optimizing siRNA performance through asymmetric chemical modification of 3'-overhangs and also helps to develop the computer modeling method for rational siRNA design.

Therapeutic potential of chemically modified siRNA: Recent trends

“…Analysis of the crystal structure of the PAZ domain from human Argonaute elF2c1 (Ago2) bound to both ends of 9‐mer siRNA‐like duplex (pdb entry 1SI2) was performed, and the results are shown in Figures and . The PAZ domain consists of a β barrel and αβ components that together form a conserved pocket structure (Figure a).…”

Guide Strand 3′‐End Modifications Regulate siRNA Specificity

“…The 1-ER analogue, bearing an ethynes ubstituent in place of the nucleobase, is likely to have aw eaker stacking interaction with Phe294.P ast reports showed that the 3'-end binding pocket of the PAZd omain can accommodate av ariety of large hydrophobic modifications such as pyrene, anthracene, and naphthalene, [15] as wella sn ucleoside analogues containing phenyl groups. [16] However, although dockings tudies predicted that pyrene would have the highest PAZd omain binding affinity,i t resultedi nl ow knockdown efficiency.T he authors attributed this to the Ago cleavagem echanism, which requires the 3'-end of the guide strand to be released from the PAZd uring the reaction, thus suggesting that too much stabilization could result in ad ecreasei nR NAi activity. [15] It is possible that the 1-ER modification tested here facilitates the PAZd omain binding, release, and re-binding cycle, but it is less effective at anchoring the 3'-end in the PAZd omain.…”

“…The 1-ER analog, bearing an ethyne substituent replacing the nucleobase, is likely to have a weaker stacking interaction with Phe 294. Past reports showed that the 3′ end binding pocket of the PAZ domain can accommodate a variety of large hydrophobic modifications such as pyrene, anthracene and naphthalene 20 , as well as nucleoside analogs containing phenyl groups 21 . However, while docking studies predicted that pyrene would have the highest PAZ domain binding affinity, it resulted in low knockdown efficiency.…”

“…Other groups have incorporated aromatic derivatives at the 3'-ends of siRNA guide strands and observede nhanced binding affinity to the Ago2 PAZw ithout compromising RNAi activity. [15,16] In contrast, ac omputational analysis of av ariety of 3'-end modifications suggested that weaker PAZd omain binding correlates with high RNAi activity. [17] However,there have been no reports on how guide strand 3'-end modification can affect silencing of miRNA-like targets.…”

“…However, since Ago2 catalytic turnover is not required for miRNA-like gene silencing and release of the 3′ end from the PAZ domain is apparently not necessary for seed-only binding, guide strands with altered PAZ domain binding properties may have reduced miRNA-like off target effects. Other groups have incorporated aromatic derivatives at the 3′ ends of siRNA guide strands and observed enhanced binding affinity to the Ago2 PAZ without compromising RNAi activity 2021 . In contrast, a computational analysis of a variety of 3′ end modifications suggested that weaker PAZ domain binding correlates with high RNAi activity 22 .…”