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Exploring PAZ/3′-overhang interaction to improve siRNA specificity. A combined experimental and modeling study

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Abstract

The understanding of the dynamical and mechanistic aspects that lie behind siRNA-based gene regulation is a requisite to boost the performance of siRNA therapeutics. A systematic experimental and computational study on the 3′-overhang structural requirements for the design of more specific and potent siRNA molecules was carried out using nucleotide analogues differing in structural parameters, such as sugar constraint, lack of nucleobase, distance between the phosphodiester backbone and nucleobase, enantioselectivity, and steric hindrance. The results established a set of rules governing the siRNA-mediated silencing, indicating that the thermodynamic stability of the 5′-end is a crucial determinant for antisense-mediated silencing but is not sufficient to avoid sense-mediated silencing. Both theoretical and experimental approaches consistently evidence the existence of a direct connection between the PAZ/3′-overhang binding affinity and siRNA’s potency and specificity. An overall description of the systems is thus achieved by atomistic simulations and free energy calculations that allow us to propose a robust and self-contained procedure for studying the factors implied in PAZ/3′-overhang siRNA interactions. A higher RNAi activity is associated with a moderate-to-strong PAZ/3′-overhang binding. Contrarily, lower binding energies compromise siRNA potency, increase specificity, and favor siRNA downregulation by Ago2-independent mechanisms. This work provides in-depth details for the design of powerful and safe synthetic nucleotide analogues for substitution at the 3′-overhang, enabling some of the intrinsic siRNA disadvantages to be overcome.

Graphical abstract: Exploring PAZ/3′-overhang interaction to improve siRNA specificity. A combined experimental and modeling study

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Publication details

The article was received on 02 Jan 2018, accepted on 15 Jan 2018 and first published on 15 Jan 2018


Article type: Edge Article
DOI: 10.1039/C8SC00010G
Citation: Chem. Sci., 2018, Advance Article
  • Open access: Creative Commons BY license
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    Exploring PAZ/3′-overhang interaction to improve siRNA specificity. A combined experimental and modeling study

    A. Alagia, A. F. Jorge, A. Aviñó, T. F. G. G. Cova, R. Crehuet, S. Grijalvo, A. A. C. C. Pais and R. Eritja, Chem. Sci., 2018, Advance Article , DOI: 10.1039/C8SC00010G

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