An Optimized Enzyme-Nucleobase Pair Enables In Vivo RNA Metabolic Labeling with Improved Cell-Specificity

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Transcriptome-wide analyses reveal numerous regulatory RNAs with cell-type-specific expression patterns.
• Accurate identification of cell-specific nascent RNA is crucial for understanding cellular function and disease.
• Existing RNA labeling methods may lack sufficient cell-specificity for complex biological systems.

Purpose of the Study:

• To develop an improved RNA metabolic labeling technique with enhanced cell-specificity.
• To validate the selective incorporation of a modified nucleobase into nascent RNA.
• To demonstrate the utility of this method for profiling cell-specific nascent RNA in vivo.

Main Methods:

• Engineered cells expressing an optimized uracil phosphoribosyltransferase (UPRT) enzyme.
• Metabolic labeling using a modified nucleobase (5-vinyuracil) for incorporation into nascent RNA.
• Validation using dot blot, quantitative PCR (qPCR), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and microscopy.

Main Results:

• Demonstrated highly selective metabolic incorporation of 5-vinyuracil into nascent RNA in engineered cells.
• Validated the selective incorporation across multiple analytical techniques, confirming method accuracy.
• Successfully applied the method in a metastatic human breast cancer mouse model for cell-specific RNA profiling.

Conclusions:

• The optimized UPRT-based RNA metabolic labeling strategy offers improved cell-specificity.
• This method provides a robust tool for studying cell-specific nascent RNA dynamics.
• The approach has significant potential for applications in cancer research and other biological fields.