Valentini Paola, Pierattini Bianca, Zacco Elsa, Mangoni Damiano, Espinoza Stefano, Webster Natalie A, Andrews Byron, Carninci Piero, Tartaglia Gian Gaetano, Pandolfini Luca, Gustincich Stefano
Central RNA Laboratory, Istituto Italiano di Tecnologia (IIT), 16152 Genova, Italy.
Area of Neuroscience, International School for Advanced Studies (SISSA), 34136 Trieste, Italy.
Mol Ther Nucleic Acids. 2022 Feb 2;27:1092-1102. doi: 10.1016/j.omtn.2022.01.021. eCollection 2022 Mar 8.
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 transcribed SINEUP for therapeutics applications, SINEUP RNAs were synthesized 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.
SINEUPs是一类新型的天然和合成非编码反义RNA分子,能够增加靶标mRNA的翻译。它们具有模块化结构,包括一个无结构的反义靶标特异性结构域,该结构域决定了每个SINEUP的特异性,以及一个结构化的效应结构域,负责增强翻译。为了设计用于治疗应用的全功能转录SINEUP,合成了具有多种化学修饰的SINEUP RNA,并在转染后筛选它们对内源靶标mRNA的活性。三种修饰核糖核苷酸的组合——2'-O-甲基-ATP(Am)、N6-甲基-ATP(m6A)和假尿苷三磷酸(ψ)——赋予了裸RNA SINEUP活性。本研究中测试的最佳组合用m6A和ψ进行了完全修饰。除了功能性外,这种组合在转染时赋予了更高的稳定性和更高的热稳定性。通过圆二色性鉴定了活性的常见结构决定因素,定义了通过不同修饰组合实现的核心功能结构。