Nanopore CapZyme-Seq, a method for detecting and quantifying metabolite caps on RNA
Venkata Sai Vaishnavi Prathivadi Bhayankaram
MS Student, Bioinformatics and Computational Biology, CBCB
University of Delaware
Abstract: Our laboratory is interested in a new class of RNA 5’-cap composed of cellular metabolites such as nicotinamide adenine dinucleotide (NAD) and Coenzyme A (CoA). These RNA caps are found in all branches of the tree of life including in gram negative and positive bacteria, humans, yeast, plants and even archaea. Unlike the canonical eukaryotic methyl-guanosine (m7G) cap, which is added to the growing RNA co-transcriptionally, metabolite caps are primarily added by RNA polymerases, which utilize these metabolites as non-canonical initiating nucleotides (NCINs) during transcription initiation. As transcription is the first step in gene expression, this ab initio capping represents a new mechanism for the regulation of gene expression based on cellular metabolic state. It has been previously shown that both E. coli cellular transcripts and S. cerevisiae mitochondrially encoded transcripts exhibit remarkably high levels of NAD capping. To better understand the functions of these NAD caps, it is necessary to know what transcripts carry the caps. The project is focused on developing a new and easy to perform, fast sequencing method for the detection and quantification of NAD-capped RNAs called CapZyme-Seq. This method will utilize the NCIN de-capping enzyme NudC, and other Oxford Nanopore direct RNA sequencing to identify which RNAs carry the NCIN caps, and to also quantify the amount of RNA that is capped. As a part of the method development, we are trying to sequence and analyze RNAs from the model bacteria E. coli, using the Nanopore cDNA-PCR library kit.
Bio: Vaishnavi is a master’s student in the Bioinformatics and Computational Biology program at UD. She received her bachelor’s degree in biotechnology from GITAM University, Vizag, India. Thereafter at Azico Biophore, a pharmaceutical company, she worked as a Trainee Biochemist focusing on regulation of water quality systems and microbiome analysis. Currently, she is working as a research assistant in Dr. Jeremy Bird’s laboratory, department of biological sciences. Her current research is focused on developing an easy to perform, fast sequencing method for the detection and purification of NAD-capped RNA’s.