Visualization of de novo synthesized nascent purine and pyrimidine using carbon isotope imaging and spectral tracing...

Nitrogenous bases, namely purine and pyrimidine, and their derivatives are key metabolites for the growth and division of cells as they are an integral part of biomolecules that store genetic information, involved in protein synthesis, as energy carrier molecules, and many other metabolic processes. Purine and pyrimidine can either be synthesized (de novo pathway), recycled, and/or acquired exogenously (salvage pathway). These nitrogenous bases can be analysed with high sensitivity using mass spectrometry-based methods, which are inherently destructive. Therefore, there remains a challenge for imaging of nascent purine and pyrimidine nitrogenous bases distribution at the single-cell level.

Here, we report a novel single-cell Raman imaging framework, "carbon isotope imaging and spectral tracing (CIIST)" for mapping nascent nitrogenous bases in prokaryotic and eukaryotic microbial systems. This method helps in visualising the turnover of nascent purine and pyrimidine nitrogenous bases at the subcellular level over time. The enrichment of carbon isotope (carbon-12 or carbon-13) in the nitrogenous base pool generates Raman peaks at distinct positions, and targeting these CIIST can be used for generating spatial maps for quasi-quantitative imaging of nitrogenous base turnover in cells.

Overall findings provide the prospective utility of the CIIST technique as a highly effective non-destructive tool for multiplex and spatial biomolecular analysis of nitrogenous base metabolism at the single-cell level.