Probing the Identification of Single Modification Sites in Short RNA Strands

Le résumé fourni par la source

ABSTRACT RNA modifications play critical roles in epitranscriptomic processes such as RNA stability, localization, translation efficiency, and splicing by altering RNA structures and interactions. Identifying these modifications at single‐base resolution is highly desirable for understanding their functions and mechanisms. However, traditional sequencing‐based methods (such as antibody enrichment or chemical derivatization) suffer from indirect labeling and bulk analysis limitations, whereas the nanopore sequencing method lacks spatial resolution for distinguishing adjacent modified nucleotides. Tip‐enhanced Raman spectroscopy (TERS) offers a promising solution to this end by combining high spatial resolution with sensitive chemical specificity. Unlike sequencing‐based methods, TERS can directly probe the intrinsic vibrational fingerprint of RNA molecules without chemical labeling, enabling real‐space imaging of modification sites in their native configurations. Recent advancements in TERS have demonstrated its ability to spectrally resolve nucleobases in single‐stranded DNA, but direct real‐space identification of RNA modifications with single‐base resolution still remains out of reach experimentally. Here, we explore the capability of TERS to identify the modification sites of pseudouridine (Ψ) in two exemplified RNA molecules that are artificially synthesized. By measuring the TERS spectra of unmodified uracil (U) and modified Ψ, their structural differences can be distinguished through respective vibrational fingerprints, particularly the characteristic vibrational modes associated with the splitting of C=O stretching peaks. Spatially resolved TERS spectra further enable both the identification of Ψ and its localization in a long RNA sequence. Our results demonstrate the potential of TERS as a tool for identifying different types of RNA modifications.

Ce résumé expose les affirmations des auteurs. BNTIC ne l’interprète pas comme une validation indépendante des résultats.