Autonomously folded α-helical lockers promote RNAi.

Christian P E Guyader,Maxim G Ryadnov,Emiliana De Santis,James E Noble,Nigel K Slater,Baptiste Lamarre

doi:10.1038/srep35012

Christian P E Guyader, Maxim G Ryadnov + Show 4 more

Open Access

https://doi.org/10.1038/srep35012

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Abstract

RNAi is an indispensable research tool with a substantial therapeutic potential. However, the complete transition of the approach to an applied capability remains hampered due to poorly understood relationships between siRNA delivery and gene suppression. Here we propose that interfacial tertiary contacts between α-helices can regulate siRNA cytoplasmic delivery and RNAi. We introduce a rationale of helical amphipathic lockers that differentiates autonomously folded helices, which promote gene silencing, from helices folded with siRNA, which do not. Each of the helical designs can deliver siRNA into cells via energy-dependent endocytosis, while only autonomously folded helices with pre-locked hydrophobic interfaces were able to promote statistically appreciable gene silencing. We propose that it is the amphipathic locking of interfacing helices prior to binding to siRNA that enables RNAi. The rationale offers structurally balanced amphipathic scaffolds to advance the exploitation of functional RNAi.

Highlights

Interfacial tertiary contacts between α-helices underpin intermolecular interactions that regulate or inhibit sub-cellular processes[1]
RNA interference (RNAi) is deemed specific as a performance test for the design series, comprising two subgroups – helices that co-fold with small interfering RNA (siRNA), i.e. responsively folded, and helices that fold without siRNA, i.e. autonomously folded
Our design rationale derives from a recognition that it is the extent to which peptides pre-fold in solution before interacting with siRNA that enables RNAi

Summary

Introduction

Interfacial tertiary contacts between α-helices underpin intermolecular interactions that regulate or inhibit sub-cellular processes[1]. The latter appears important as viral proteins do not co-fold with their cargo, but use terminal poly-cation domains to bind to it[9] Such domains lack hydrophobic interactions and do not form amphipathic structures whose hydrophobic faces may otherwise lock the bound nucleic acid (NA) inhibiting its intracellular release. Small interfering RNA (siRNA), which mediate RNAi by engaging with mRNA13, are prone to degradation[14], cannot cross cell membranes[15] and require constant protection and a reliable means for intracellular delivery[16] For these reasons, RNAi is deemed specific as a performance test for the design series, comprising two subgroups – helices that co-fold with siRNA, i.e. responsively folded, and helices that fold without siRNA, i.e. autonomously folded

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