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 (psi)-conferred SINEUP activity to naked RNA. The best combination tested in this study was fully modified with m6A and psi. 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.
