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.

核酸的基础与应用研究 30 年来，Maher 实验室一直在研究核酸生物学中未解决的问题，我们将继续针对该领域的两个主要挑战进行合作。挑战非常多样化和跨学科，涵盖核酸的基础和应用方面这项工作带来了独特的协同作用并创造了特殊的机会。训练环境。挑战 1：从细菌 DNA 成环研究中学到的原理能否应用于人工 DNA 循环进行基因调控？假设 1.1：我们发现小 DNA 环在体内比在体外更容易形成，因为 a) 蛋白质桥接 DNA 环以减少所需的 DNA 弯曲，b) DNA 超螺旋预弯曲 DNA，以及 c) 结构性 DNA 结合蛋白扭结 DNA 的分子生物学实验将在体外进行。在活的大肠杆菌细胞中使用紫胶操纵子的元素。假设 1.2：我们勇敢地认为设计的序列特异性 DNA 结合蛋白可用于创建我们的研究将在细菌和真核系统中实现新的人工基因调节环。由化学诱导的异二聚化控制的转录激活样效应器 (TALE) 蛋白通过创建紧密的 DNA 环（将 RNA 聚合酶排除在基因之外）来调节模型和内源基因这项工作将影响合成生物学的工作。挑战 2：我们能否识别出位于亚细胞区室的裸露 DNA 适体？假设2.1：我们捕捉到了之前我们研究过的核归巢DNA适体的机制使用连接酶邻近选择识别可以通过蛋白质组学来理解，并且这种选择概念扩展到发现在小鼠中表现出组织特异性核归巢的裸DNA适体。假设 2.2：我们勇敢地说，我们的新过氧化物酶邻近选择将识别归巢 DNA 我们将研究过氧化物酶生物素化作用。奖励化学，在活培养细胞中进行选择，并探索发现的归巢机制适体作为蛋白质和药物的可能递送剂。对该提案的支持将维持马赫实验室针对这两个问题的富有成效的研究计划该实验室的记录表明，这项投资将引发重要且尚未解决的挑战。进一步创新的影响超越了这两个问题。