Fundamental and applied studies of nucleic acids

核酸的基础和应用研究 30年来，马赫实验室一直在研究核酸生物学和 生物物理学。我们将继续共同努力，应对fi领域的两大挑战。这些 挑战是刻意多样化和多学科的，跨越核学的基础和应用方面。 酸的结构和功能。这项工作带来了独特的协同机会，创造了一个不同寻常的 训练环境。 挑战1：从细菌dna环路研究中学到的原理是否可以应用于人工dna？ 为了基因调控而循环？ 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超卷曲预弯DNA，以及c) 建筑DNA结合蛋白扭结DNA。分子生物学实验将在体外和 在活的大肠杆菌细胞中使用乳胶操纵子的成分。 假设2：我们假设设计的特定序列的fic dna结合蛋白可以用来产生 细菌和真核系统中的ArtifiSocial基因调控环。我们的研究将实施新颖的 转录激活因子样效应子(TALE)蛋白受化学诱导的异源二聚体调控 通过创建将RNA聚合酶排除在基因之外的紧密DNA环来调节模型和内源性基因 推动者。这项工作将影响合成生物学的努力。 挑战2：我们能识别出存在亚细胞室的裸露DNA适配子吗？ 假设2.1：我们假设核归巢DNA适体的机制与我们之前的 使用连接酶邻近选择的Identified可以被蛋白质组学理解，并且这个选择概念 扩展到发现裸露的dna适配子，在小鼠身上表现出组织特异性的fic核定位。 假设2.2：我们假设我们的新的过氧化物酶邻近选择将识别归宿DNA 不同亚细胞室的适体专化fic。我们将研究过氧化物酶的生物素化 奖励化学，在活的培养细胞中进行选择，并探索已发现的归巢机制 适体作为蛋白质和药物的可能递送剂。 对这项提议的支持将维持马赫实验室解决这两个问题的生产性研究计划 重要且悬而未决的挑战。实验室的记录表明，这笔投资将引发 具有超越这两个问题的影响的进一步创新。

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.