Modulation of the RNA Interference Activity Using Central Mismatched siRNAs and Acyclic Threoninol Nucleic Acids (aTNA) Units.

Adele Alagia,Montserrat Terrazas,Ramon Eritja

doi:10.3390/molecules20057602

Abstract

The understanding of the mechanisms behind nucleotide recognition by Argonaute 2, core protein of the RNA-induced silencing complex, is a key aspect in the optimization of small interfering RNAs (siRNAs) activity. To date, great efforts have been focused on the modification of certain regions of siRNA, such as the 3'/5'-termini and the seed region. Only a few reports have described the roles of central positions flanking the cleavage site during the silence process. In this study, we investigate the potential correlations between the thermodynamic and silencing properties of siRNA molecules carrying, at internal positions, an acyclic L-threoninol nucleic acid (aTNA) modification. Depending on position, the silencing is weakened or impaired. Furthermore, we evaluate the contribution of mismatches facing either a natural nucleotide or an aTNA modification to the siRNA potency. The position 11 of the antisense strand is more permissive to mismatches and aTNA modification, in respect to the position 10. Additionally, comparing the ON-/OFF-target silencing of central mismatched siRNAs with 5'-terminal modified siRNA, we concluded: (i) central perturbation of duplex pairing features weights more on potency rather than silencing asymmetry; (ii) complete bias for the ON-target silencing can be achieved with single L-threoninol modification near the 5'-end of the sense strand.

Highlights

RNA interference (RNAi) is a powerful gene regulatory process that allows the inhibition of genes in a very specific way [1]
We studied the contribution of acyclic L-threoninol nucleic acid (aTNA) in slicing ability of the RNA-Induced Silencing Complex (RISC) and the possible correlation between the thermal stability of mismatches on the small interfering RNAs (siRNAs) potency and the ON/OFF-target activity
In order to evaluate the effect of the introduction of L-threoninol modification on duplex stability, we compared the melting temperature of siRNAs bearing either a natural uridine or L-threoninol-thymine (TL) (Table 1)

Summary

Introduction

RNA interference (RNAi) is a powerful gene regulatory process that allows the inhibition of genes in a very specific way [1]. Double stranded RNAs known as small interfering RNAs (siRNAs), recognized by the RNA-Induced Silencing Complex (RISC) [2], efficiently trigger the RNAi pathway. After siRNA loading into the RISC, the sense strand is cleaved and released [3]. At this stage, the active RISC, bearing only the antisense strand, can match with the corresponding complementary messenger RNA (mRNA) sequence. The cleavage of the target mRNA mediates the inhibition of the mRNA translation into the corresponding protein. Oligonucleotides have some disadvantages, such as poor nuclease stability, poor cellular uptake, off-target effects, and non-specific immune responses [7,8]

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