NextGen DNA Binding Proteins Based on TAL Domains

描述(申请人提供)：最近发现转录激活物样(TAL)效应域是DNA结合域。根据已报道的这些结构域的DNA识别特征，这些结构域是一长串重复，每个重复只使用两个可变氨基酸识别一个碱基对，这种DNA结合域的结构不同于之前描述的任何结构。因此，负责特定DNA识别的蛋白质折叠和结构特征具有很高的新颖性，值得阐明其各自的优点。然而，看似简单的识别密码和对广泛序列的明显识别，对于设计新的DNA结合蛋白具有重要的意义。TAL结构域在识别特性上似乎比锌指(ZF)更灵活，锌指(ZF)的工程在基因调控、基因组工程、转基因生物和基因治疗领域产生了革命性的影响。尽管它们取得了成功，但制造高质量ZF的难度是其广泛应用的一个重大瓶颈。我们假设TAL结构域在设计新型DNA结合蛋白方面将比黄金标准的ZF结构域具有更好的性能特征。我们将使用一种结合计算和生物化学的方法来检验我们的假设，以检查蛋白质折叠和重复组装(目标1)，阐明DNA识别的机制和程度(目标2)，并研究TAL结构域用于创建基因调控和基因组工程的序列特异性工具的潜力(目标3)。如果成功，这项研究将提供对一种新的DNA结合域的结构和功能的见解，并理解如何将这些见解应用于创造比目前方法更广泛、更普遍的遗传修改工具。关键词蛋白质-核酸相互作用，工程锌指，从头算建模，蛋白质结构，蛋白质折叠，结构-功能关系，基因组工程，基因治疗，计算设计。 与公共卫生相关：在过去的二十年里，蛋白质被设计成调节活细胞的DNA并对其进行精确的改变，导致我们研究和治疗人类疾病的能力取得了革命性的进步。这些方法是基于重新编程锌指蛋白的DNA结合特异性的能力，这是困难的，因此限制了它们的使用。在这里，我们将探索新发现的TAL DNA结合基序，它似乎克服了这些限制，这应该能够更多地获得更强大的医学研究和治疗工具。

DESCRIPTION (provided by applicant): Transcription activator-like (TAL) effector domains were recently discovered to be DNA binding domains. Based on the reported DNA recognition features of the these domains, long arrays of repeats that each recognize one base pair using only two variable amino acids, the structure of this DNA binding domain is unlike any that have been previously described. As such, the protein fold and structural features responsible for specific DNA recognition are highly novel and worthy of elucidation for their own merits. However, the seeming simple recognition code and apparent recognition of a wide spectrum of sequences has important implications as a scaffold for engineering new DNA binding proteins. TAL domains appear to be even more flexible in their recognition properties than zinc fingers (ZFs), the engineering of which have had transformative impact in the areas of gene regulation, genome engineering, genetically modified organisms, and gene therapy. Despite their successes, the difficultly of engineering high-quality ZFs represents a significant bottleneck to their widespread application. We hypothesize that TAL domains will have superior performance characteristics compared to the gold-standard ZF domains for engineering novel DNA- binding proteins. We will test our hypothesis using a combined computational and biochemical approach to examine the protein fold and repeat assembly (Aim 1), elucidate the mechanism and extent of DNA recognition (Aim 2), and investigate the potential of TAL domains for the creation of sequence-specific tools for gene regulation and genome engineering (Aim 3). If successful, this study will provide insights into the structure and function of a novel DNA-binding domain, and an understanding of how those insights can be applied to create tools for genetic modification that would be more broadly accessible and of greater general utility than current methods. KEY WORDS Protein-nucleic acid interactions, engineered zinc fingers, ab initio modeling, protein structure, protein folding, structure-function relationship, genome engineering, gene therapy, computational design. PUBLIC HEALTH RELEVANCE: Over the past two decades, proteins have been engineered to regulate and make precise changes to the DNA of living cells, leading to transformative advances in our ability to study and treat human diseases. These methods are based on the ability to reprogram the DNA binding specificity of zinc finger proteins, which is difficult and consequently has restricted their use. Here we will explore the newly discovered TAL DNA binding motif that seems to overcome these limitations, which should enable greater access to more powerful tools for medical research and therapy.