Non-Destructive Chemoselective Labeling and Bioorthogonal Enrichment of Pseudouridine-Containing RNA via Facile N 1-Selective Functionalization

Abstract Pseudouridine (Ψ), often referred to as the “fifth nucleotide”, constitutes the most abundant post-transcriptional RNA modification and plays pivotal roles in regulating RNA structure and function. However, the precise mapping and functional investigation of Ψ remain challenging due to the lack of mild, nondestructive detection methods. Herein, we report a robust chemical proof-of-concept strategy utilizing α-methylene-γ-butyrolactone (MBL) derivatives to regioselectively functionalize Ψ at its N1 position under aqueous conditions through a phosphate-promoted reaction. Crucially, this N1-selective modification preserves the Watson–Crick base-pairing interface of Ψ, retaining native base-complementarity information while remaining compatible with reverse transcription in the tested model RNA system. To evaluate the applicability of this chemistry in a macromolecular context, we coupled chemoselective MBL labeling with a bioorthogonal azide–alkyne click ligation and a cleavable biotin pull-down workflow. Using a 66-mer RNA model system, we achieved a 20-fold target enrichment, while downstream Sanger sequencing supported the preservation of native base complementarity under the tested conditions. Ultimately, this nondestructive and modular chemical toolkit lays a solid structural and mechanistic foundation for future advancements toward the high-confidence profiling of Ψ within complex biological transcriptomes.