The Analytical and Surface Chemistry Program, with co-funding from the Molecular and Cellular Biosciences Genes and Genome Systems Cluster, supports Prof. Patrick Limbach at the University of Cincinnati for work targeting a mass spectrometry-based approach to higher throughput sequencing of transfer ribonucleic acids (tRNAs). Many types of RNAs are involved in key cellular functions in all living cells. Among all known bio-organic molecules within living cells, RNA molecules are the only ones that store genetic information and act as catalysts. In the course of this research, Dr. Limbach and his group will establish new methods to fractionate mixtures of tRNAs; will develop a strategy for tRNA enrichment allowing rare tRNAs to be expressed and sequenced; will use a combination of normal and limited RNase digestion for increasing tRNA sequencing read length; and will enhance the RNA database of signature digestion products. The project will provide researchers with additional tools that can examine transcription and translation processes, complementing existing genomic and proteomic strategies. These developments will provide enabling advances for RNA researchers and can be translated to related areas of nucleic acid analysis (e.g., xenobiotic modifications).
In addition to the technical impacts outlined above, the work offers broader impacts by providing students with education and training in key areas of biological mass spectrometry. Dr. Limbach strives to expand the number of underrepresented minorities engaged in science in general, and mass spectrometry in particular.
The research proposed in NSF CHE 0910751 builds upon our previous developments in NSF CHE 0602413 where we demonstrated a new approach for identifying non-protein coding ribonucleic acids (ncRNAs). These ncRNAs are important participants in a variety of biochemical reactions within the cell. Our new approach now allows researchers to identify the specific ncRNAs that are present in any cell based on their analysis by a technique known as mass spectrometry (MS). In addition, this method can determine whether these ncRNAs contain modifications to the original sequences. During CHE 0910751, we sought to address a limitation that was found with the method developed in CHE 0602413. The limitation with the prior method is that it could not be broadly implemented unless additional sequence information was obtained from more types of ncRNAs. Thus, our research focused on new developments in the field of mass spectrometry that would improve the quantity and quality of sequence information obtained from ncRNAs. The outcomes of our research resulted in new strategies for preparing ncRNAs prior to analysis by mass spectrometry. We also investigated a different mass spectrometry platform and found that it yielded a richer set of data on ncRNAs than the original platform. Because low abundance ncRNAs may be biologically important, we developed a research strategy for enhancing the cellular levels of these ncRNAs so that we could characterize them by our new methods. Furthermore, throughout this funding period we collaborated with researchers throughout the US and Europe to identify new types of sequence modifications to ncRNAs, to identify the enzymes responsible for these modifications, and to expand the utility of our ncRNA sequencing methods to organisms beyond bacteria. While accomplishing these research goals, we also trained the next generation of scientists in modern biochemical research. These students ranged from middle-school to undergraduates to graduate students as well as post-doctoral researchers. Such students have become better prepared to tackle new challenges in biochemical research.
