Development of Activity-Based Probes for Genome-Modifying Enzymes

在化学系生命过程化学（CLP）项目的支持下，匹兹堡大学的Kabirul Islam博士正在研究保护和控制遗传信息的DNA修饰酶。DNA是在分子、细胞和有机体水平上定义物种的基本蓝图。遗传信息受到广泛的基因组修饰酶的保护和忠实地解码，其中许多仍有待发现。因此，从真核生物中鉴定这些酶具有根本的重要性。该项目将采取跨学科的方法，结合有机化学，机械生物化学，细胞生物学，蛋白质组学和生物信息学，目的是发现新的基因组修饰酶。拟议的活动将使高中生、本科生和研究生，包括代表性不足群体的学生，参与研究，为从事科学研究与发展和/或教育学提供适当的培训经验。该教育计划旨在为博士生建立一个指导平台，以克服最初的挑战，并在博士研究和发展道路上沿着达到重要的里程碑。 PI提出了一项计划，以提高博士生在化学领域的保留。基因表达的变化，真核生物的发展，主要是通过对遗传物质的化学修饰介导的。这个项目的重点是研究一种特殊类型的核碱基修饰，即胞苷甲基化。目前建立、识别和去除DNA中胞苷修饰的酶的库远未穷尽，部分原因是缺乏适当的药理学探针。这项研究将集中在开发一流的基于磷脂酰肌醇的活性探针携带亲电功能，这是用来捕获DNA修饰因子通过胚胎干细胞的互补反应。随后，将进行化学蛋白质组学和生物信息学分析以及生物化学验证，以确定细胞分化，谱系承诺以及最终生物体发育的新酶和信号通路。在这些研究中开发的新工具将被广泛提供给有兴趣解决DNA中的化学修饰如何储存和调节超出主要序列的关键信息的研究人员。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

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.