1 research outputs found
An Optimized Enzyme-Nucleobase Pair Enables <i>In Vivo</i> RNA Metabolic Labeling with Improved Cell-Specificity
Current transcriptome-wide analyses have identified a
growing number
of regulatory RNA with expression that is characterized in a cell-type-specific
manner. Herein, we describe RNA metabolic labeling with improved cell-specificity
utilizing the in vivo expression of an optimized
uracil phosphoribosyltransferase (UPRT) enzyme. We demonstrate improved
selectivity for metabolic incorporation of a modified nucleobase (5-vinyuracil)
into nascent RNA, using a battery of tests. The selective incorporation
of vinyl-U residues was demonstrated in 3xUPRT LM2 cells through validation
with dot blot, qPCR, LC-MS/MS and microscopy analysis. We also report
using this approach in a metastatic human breast cancer mouse model
for profiling cell-specific nascent RNA