Development of Activity-Based Probes for Genome-Modifying Enzymes

University Of Pittsburgh, Pittsburgh PA

Investigators

Abstract

With the support of the Chemistry of Life Processes (CLP) Program in the Division of Chemistry, Dr. Kabirul Islam from University of Pittsburgh is investigating DNA-modifying enzymes that protect and control genetic information. DNA is the essential blueprint that defines species at the molecular, cellular and organismal level. Genetic information is protected and faithfully decoded by a wide range of genome-modifying enzymes, many of which remain to be discovered. It is, thus, of fundamental importance to characterize such enzymes from eukaryotic organisms. This project will take an interdisciplinary approach, combining organic chemistry, mechanistic biochemistry, cell biology, proteomics and bioinformatics with the aim of uncovering novel genome-modifying enzymes. The proposed activity will integrate high school, undergraduate and graduate students, including those from under-represented groups, into the research to provide appropriate training experience for a career in scientific research and development and/or pedagogy. The educational plan is geared toward establishing a mentoring platform for doctoral students to overcome initial challenges and reach important milestones along the doctoral research and development pathway. The PI puts forth a plan to improve retention of PhD students in the field of chemistry. Changes in gene expression that underlie the development of eukaryotic organisms are primarily mediated by chemical modifications on the genetic material. The focus of this project is to study a particular type of nucleobase modification; namely cytidine methylation. The current repertoire of enzymes that establish, recognize and remove cytidine modifications in DNA is far from exhaustive, due in part to the lack of appropriate pharmacological probes. This research will focus on developing first-in-class oligonucleotide-based activity probes carrying electrophilic functionalities, which are to be employed to capture DNA-modifying factors via complementary reactivity from embryonic stem cells. Subsequently, chemoproteomic and bioinformatic analyses, and biochemical validation will be conducted to identify novel enzymes and signaling pathways that underlie cellular differentiation, lineage commitment, and ultimately, organismal development. The new tools developed in these studies will be made broadly available to researchers interested in addressing how chemical modifications in DNA store and regulate critical information beyond that which is present in the primary sequence alone. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.