Patrick P.A. Bertrand

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.

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.

The detection of small molecules (e.g., pesticides, pollutants, antibiotics, drug residues) is critical for human health and environmental surveys as well as for many industrial applications. Fluorogenic RNA-based biosensors (FRBs) are aptamer-based tools specifically reporting on the presence of such targets by converting a specific binding event into fluorescence emission, thus making them of particular interest for on-site sensing applications. However, new sensor aptamers suited for FRB development are tedious to develop. In this study, we show that Capture-SELEX used in tandem with our microfluidic-assisted screening technology (μIVC) accelerates de novo FRB development by reprogramming the specificity of an existing sensor aptamer, followed by fluorescence-based functional selection. Furthermore, we show that these new sensor aptamers can be repurposed into functional biotechnological tools, such as gene-regulating aptazymes, thus demonstrating the versatility of our pipeline for the development of sensor aptamers adapted for various applications.