Alkyne-Functionalized Coumarin Compound for Analytic and Preparative 4-Thiouridine Labeling

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• RNA bioconjugation is crucial for studying post-transcriptional modifications.
• 4-thiouridine (s⁴U) is a key modified nucleoside in bacterial RNA, used for metabolic labeling and cross-linking.
• Existing methods for s⁴U labeling and analysis have limitations.

Purpose of the Study:

• To develop a novel chemical probe for selective bioconjugation of 4-thiouridine in RNA.
• To enable quantitative monitoring of s⁴U metabolic incorporation.
• To facilitate site-specific functionalization of RNA for analytical purposes.

Main Methods:

• Design and synthesis of 4-bromomethyl-7-propargyloxycoumarin (PBC), a coumarin derivative with a terminal alkyne.
• Application of PBC for selective reaction with 4-thiouridine in RNA.
• Utilizing click chemistry for secondary bioconjugation of the alkyne handle.
• Quantitative analysis of s⁴U incorporation and site-specific fluorophore labeling of bacterial tRNA.

Main Results:

• PBC demonstrated high selectivity for 4-thiouridine.
• The method allowed for quantitative monitoring of s⁴U metabolic labeling in RNA.
• Site-specific introduction of a fluorophore into bacterial tRNA at position 8 was achieved.
• The labeled tRNA was used to determine the binding constant to an RNA-modification enzyme.

Conclusions:

• The novel coumarin probe PBC provides an efficient and selective tool for 4-thiouridine bioconjugation.
• This approach expands the capabilities for studying RNA modifications and functions.
• PBC facilitates both quantitative metabolic labeling and site-specific functionalization of RNA.