Towards SINEUP-based therapeutics: Design of an in vitro synthesized SINEUP RNA

Paola Valentini,Bianca Pierattini,Elsa Zacco,Damiano Mangoni,Stefano Espinoza,Natalie A Webster,Byron Andrews,Piero Carninci,Gian Gaetano Tartaglia,Luca Pandolfini,Stefano Gustincich

doi:10.1016/j.omtn.2022.01.021

Abstract

SINEUPs are a novel class of natural and synthetic non-coding antisense RNA molecules able to increase the translation of a target mRNA. They present a modular organization comprising an unstructured antisense target-specific domain, which sets the specificity of each individual SINEUP, and a structured effector domain, which is responsible for the translation enhancement. In order to design a fully functional in vitro transcribed SINEUP for therapeutics applications, SINEUP RNAs were synthesized in vitro with a variety of chemical modifications and screened for their activity on endogenous target mRNA upon transfection. Three combinations of modified ribonucleotides—2′O methyl-ATP (Am), N6 methyl-ATP (m6A), and pseudo-UTP (ψ)—conferred SINEUP activity to naked RNA. The best combination tested in this study was fully modified with m6A and ψ. Aside from functionality, this combination conferred improved stability upon transfection and higher thermal stability. Common structural determinants of activity were identified by circular dichroisms, defining a core functional structure that is achieved with different combinations of modifications.

Highlights

SINEUPs are a new class of antisense long non-coding RNAs able to enhance the translation of a target mRNA.[1]
Unmodified IVT SINEUP RNA is not functional In order to study the functionality of modified and unmodified in vitro transcribed SINEUPs, we used as a model molecule a validated SINEUP, named miniSINEUP-DJ-1,1,3 targeting PARK7-DJ1, a gene found mutated in familial forms of Parkinson’s disease (PD)
In this study, we have defined different combinations of natural modifications that can be added to an IVT SINEUP RNA in order to reproduce the functionality of an endogenously transcribed SINEUP

Summary

Introduction

SINEUPs are a new class of antisense long non-coding RNAs (lncRNAs) able to enhance the translation of a target mRNA.[1]. The structure-activity relationship of both domains has been extensively investigated.[1,3,5–10] This information allowed the design of synthetic SINEUPs with artificial binding domains, capable of binding virtually any target gene of interest, defining a novel class of RNA therapeutics able to increase the protein synthesis of specific targets. Increasing understanding of the structural determinants of activity has allowed the progressive miniaturization of SINEUPs, with the aim of designing a fully functional, shorter version.[3,8,9]. Such short molecules are more convenient for drug delivery and display more predictable pharmacology and pharmacokinetics,[12] accelerating translation to clinical applications Increasing understanding of the structural determinants of activity has allowed the progressive miniaturization of SINEUPs, with the aim of designing a fully functional, shorter version.[3,8,9] Such short molecules are more convenient for drug delivery and display more predictable pharmacology and pharmacokinetics,[12] accelerating translation to clinical applications

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Discussion

Conclusion