Fundamental and applied studies of nucleic acids

核酸的基础与应用研究

基本信息

批准号：
10323099
负责人：
LOUIS JAMES MAHER
金额：
$ 29.94万
依托单位：
MAYO CLINIC ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-01 至 2027-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10323099
关键词：
Address Architecture Bacterial DNA Biology Biophysics Biotinylation Cell Nucleus Cells Chemicals Chemistry Chromatin Loop Collection Cultured Cells DNA DNA-Binding Proteins DNA-Directed RNA Polymerase Elements Environment Escherichia coli Exhibits Gene Expression Regulation Genes Heterodimerization Home Homing In Vitro Investments Laboratories Lac Operon Learning Ligase Modeling Molecular Biology Mus Nuclear Nucleic Acids Peroxidases Pharmaceutical Preparations Polymerase Gene Proteins Proteomics RNA Regulator Genes Research Rewards Sequence-Specific DNA Binding Protein Superhelical DNA Synthetic Genes System Tissues Training Transcription Coactivator Work aptamer design experimental study in vivo innovation interest kinked DNA multidisciplinary novel novel therapeutics nucleic acid structure programs promoter student training synergism synthetic biology tool

项目摘要

Fundamental and applied studies of nucleic acids For 30 years the Maher laboratory has been investigating unresolved problems in nucleic acids biology and biophysics. We will continue our collaborative efforts related to two major challenges in the ﬁeld. These challenges are deliberately diverse and multidisciplinary, spanning fundamental and applied aspects of nucleic acid structure and function. This work brings unique opportunities for synergy and creates an exceptional training environment. Challenge 1: Can principles learned from studies of bacterial DNA looping be applied to artiﬁcial DNA looping for gene regulation? Hypothesis 1.1: We hypothesize that small DNA loops form more easily in vivo than in vitro because a) proteins bridge DNA loops to reduce required DNA bending, b) DNA supercoiling pre-bends DNA, and c) architectural DNA binding proteins kink DNA. Molecular biology experiments will be performed in vitro and using elements of the lac operon in living E. coli cells. Hypothesis 1.2: We hypothesize that designed sequence-speciﬁc DNA binding proteins can be used to create artiﬁcial gene regulatory loops in bacterial and eukaryotic systems. Our studies will implement novel Transcription Activator-like Effector (TALE) proteins controlled by chemically-induced heterodimerization to regulate model and endogenous genes by creating tight DNA loops that exclude RNA polymerase from gene promoters. This work will impact synthetic biology efforts. Challenge 2: Can we identify naked DNA aptamers that home to sub-cellular compartments? Hypothesis 2.1: We hypothesize that the mechanism of nucleus-homing DNA aptamers we previously identiﬁed using Ligase Proximity Selection can be understood by proteomics and this selection concept extended to discover naked DNA aptamers that exhibit tissue-speciﬁc nuclear homing in mice. Hypothesis 2.2: We hypothesize that our new Peroxidase Proximity Selection will identify homing DNA aptamers speciﬁc for different sub-cellular compartments of interest. We will study peroxidase biotinylation reward chemistry, undertake selections in live cultured cells, and explore the mechanism of discovered homing aptamers as possible delivery agents for proteins and drugs. Support for this proposal will sustain the Maher laboratory's productive research program addressing these two important and unresolved challenges. The laboratory's track record shows that this investment will trigger further innovations with impact beyond these two problems.

核酸的基本和应用研究 30年来，Maher实验室一直在研究核酸生物学和生物物理学。我们将继续与该领域的两个主要挑战有关的协作工作。挑战是故意的潜水和多学科，涵盖了核的基本和应用方面酸的结构和功能。这项工作为协同作用带来了独特的机会，并创造了一个杰出的机会培训环境。挑战1：从细菌DNA循环研究中学到的原则可以应用于艺术性DNA 基因调控循环？假设1.1：我们假设小型DNA环在体内比体外更容易形成，因为a）蛋白质桥接DNA环减少所需的DNA弯曲，b）DNA超螺旋前弯曲的DNA，C）建筑DNA结合蛋白扭结DNA。分子生物学实验将在体外进行，并且使用LAC歌剧的元素在活的大肠杆菌细胞中。假设1.2：我们假设设计的序列特异性DNA结合蛋白可用于创建细菌和真核系统中的艺术家基因调节环。我们的研究将实施小说转录激活剂样效应子（Tale）蛋白，该蛋白通过化学诱导的异二聚化控制通过创建将RNA聚合酶排除在基因的紧密DNA环中调节模型和内源基因发起人。这项工作将影响合成生物学的工作。挑战2：我们可以识别出裸露的DNA适体，这些适体源于亚细胞隔室的家园？假设2.1：我们假设我们先前的核与DNA适体的机制使用连接酶接近度选择识别可以通过蛋白质组学和此选择概念来理解扩展到发现裸露的DNA适体，可暴露小鼠组织特异性核归巢。假设2.2：我们假设我们的新的过氧化物酶接近度选择将鉴定归巢DNA 适体特异性针对不同感兴趣的亚细胞隔室。我们将研究过氧化物酶生物素化奖励化学，在活培养的细胞中进行选择，并探索发现的归巢的机制适体的蛋白质和药物输送剂。对该建议的支持将维持Maher实验室的产品研究计划，以解决这两个方面重要且尚未解决的挑战。实验室的记录显示，这项投资将触发进一步的创新与这两个问题超出了影响。