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，匹兹堡大学的卡比鲁尔·伊斯拉姆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、细胞和生物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改酶的保护和忠实解码，其中许多酶仍有待发现。因此，从真核生物中确定这类酶的特征是至关重要的。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。拟议的活动将把高中生、本科生和研究生，包括来自代表性不足群体的学生纳入研究，为从事科学研究和开发和(或)教育学职业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取得重要里程碑。PI提出了一项计划，以提高化学领域的博士生留存率。支持真核生物发展的基因表达的变化主要是通过对遗传物质的化学修饰来调节的。这个项目的重点是研究一种特殊类型的碱基修饰，即胞苷甲基化。目前在DNA中建立、识别和移除胞苷修饰的酶还远远不是详尽的，部分原因是缺乏适当的药理探针。这项研究将专注于开发一流的基于寡核苷酸的具有亲电功能的活性探针，这些探针将被用于通过胚胎干细胞的互补反应来捕获DNA修饰因子。随后，将进行化学蛋白质组和生物信息学分析，以及生化验证，以确定构成细胞分化、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广泛提供给有兴趣解决DNA中的化学修饰如何存储和调节仅存在于初级序列中的关键信息的研究人员。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.