Probing Nascent RNA with Metabolic Incorporation of Modified Nucleosides

Area of Science:

• Biochemistry and Molecular Biology
• Chemical Biology
• RNA Biology

Background:

• Growing interest in RNA's functional roles necessitates advanced tools for studying RNA molecules.
• RNA metabolic labeling, incorporating unnatural nucleobases, enables tracking of RNA synthesis, localization, and enrichment.
• Existing methods face challenges including analogue toxicity, limited conjugation chemistries, and background labeling.

Purpose of the Study:

• To develop improved RNA metabolic labeling techniques addressing current limitations.
• To showcase novel vinyl nucleosides and orthogonal chemical reactions for enhanced RNA analysis.
• To enable precise and simultaneous investigation of multiple RNA-related cellular processes.

Main Methods:

• Utilized cellular pathways for RNA metabolic labeling and studied nucleoside-kinase interactions.
• Employed vinyl nucleosides, specifically 5-vinyluridine (5-VUrd), demonstrating reduced toxicity.
• Developed cell-specific labeling strategies using phosphoribosyl transferase (PRT) enzymes and optimized inverse-electron-demand Diels-Alder (IEDDA) reactions.

Main Results:

• Identified uridine monophosphate synthase (UMPS) as a source of background labeling with modified uracil nucleobases.
• Developed a mutant uracil PRT (UPRT) for highly specific RNA metabolic labeling with 5-vinyluracil (5-VU).
• Established mutually orthogonal chemical reactions for selective labeling of 5-VUrd and 2-vinyladenosine (2-VAdo).

Conclusions:

• The developed methods overcome key challenges in RNA metabolic labeling, including toxicity and background incorporation.
• Vinyl nucleosides and orthogonal reactions offer precise tools for chemical modification and analysis of newly synthesized RNA.
• The ability to simultaneously label multiple RNA species opens new avenues for studying complex cellular RNA dynamics.