Fundamental and applied studies of nucleic acids

核酸基础与应用研究 30年来，马赫实验室一直在研究核酸生物学中尚未解决的问题， 生物物理学我们将继续就该领域的两大挑战开展合作。这些 挑战是故意多样化和多学科的，跨越基本和应用方面的核 酸的结构和功能。这项工作带来了独特的协同机会，并创造了一个特殊的 训练环境。 挑战1：从细菌DNA循环研究中学到的原理能否应用于阿尔蒂官方DNA 基因调控的循环 假设1.1：我们假设小的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，和c） 结构DNA结合蛋白质使DNA扭结。分子生物学实验将在体外进行， 利用活E. coli细胞。 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 细菌和真核生物系统中的阿尔蒂基因调控环。我们的研究将实施新的 通过化学诱导的异源二聚化控制的转录激活因子样效应物（TALE）蛋白， 通过创建紧密的DNA环来调节模型和内源基因，该DNA环将RNA聚合酶从基因中排除 发起人。这项工作将影响合成生物学的努力。 挑战2：我们能否识别出位于亚细胞区室的裸DNA适体？ 假设2.1：我们假设，核归巢DNA适体的机制，我们以前， 使用连接酶邻近选择识别艾德可以通过蛋白质组学来理解，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：我们假设我们新的过氧化物酶邻近选择将识别归巢DNA 特定于不同感兴趣的亚细胞区室的适体。我们将研究过氧化物酶生物素化 奖励化学，在活的培养细胞中进行选择，并探索发现归巢的机制 适体作为蛋白质和药物的可能递送剂。 支持这项建议将维持马赫实验室的生产性研究计划，解决这两个问题， 重大和未解决的挑战。该实验室的记录显示，这项投资将引发 进一步的创新，其影响超出了这两个问题。

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.