Adaptation of a Fluorogenic RNA to N1‐Methylpseudouridine Nucleoside Chemistry

ABSTRACT N1‐methylpseudouridine ( m Ψ) is a modified nucleoside that entirely replaces uridine in the design of many therapeutic messenger RNAs (mRNA) to alleviate their intrinsic immunogenicity while preserving their capacity to be translated into protein. However, the impact of such replacement in functional non‐coding RNAs has only been scarcely studied, and most studies have shown impairment in the function of the tested RNAs thereby limiting their potential applications. In this work, we further show that exchanging uridines by m Ψ in green‐emitting Fluorogenic Light‐up Aptamers (FLAP) is detrimental to their capacity of binding and enhancing the fluorescence of their cognate fluorogen. Moreover, combining directed evolution and rational design allowed not only to pinpoint the structural elements impacted by the replacement of uridine by m Ψ, but also to re‐establish the function of a m Ψ‐modified non‐coding RNA for the first time. This yielded Ψ‐Spinach, a FLAP suited for the precise labelling of m Ψ‐modified mRNA and usable for human cells transfection. This work thus paves the way for the adaptation of other functional non‐coding RNAs with m Ψ in view of engineering and optimizing therapeutic modified mRNAs.