NextGen DNA Binding Proteins Based on TAL Domains

描述（由申请人提供）：转录激活因子样（TAL）效应域是最近发现的DNA结合域。根据已报道的这些结构域的DNA识别特征，每个重复序列只使用两个可变氨基酸识别一个碱基对，这种DNA结合结构域的结构不同于先前描述的任何结构域。因此，负责特定DNA识别的蛋白质折叠和结构特征是非常新颖的，值得阐明其自身的优点。然而，看似简单的识别代码和广泛的序列明显识别具有重要意义的支架工程新的DNA结合蛋白。TAL结构域在识别特性上似乎比锌指（ZFs）更灵活，锌指的工程在基因调控、基因组工程、转基因生物和基因治疗等领域产生了变革性影响。尽管取得了成功，但高质量zf的工程设计困难是其广泛应用的重大瓶颈。我们假设TAL结构域与黄金标准的ZF结构域相比，在工程新型DNA结合蛋白方面具有优越的性能特征。我们将使用计算和生化相结合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结构域在创建基因调控和基因组工程的序列特异性工具方面的潜力（目标3）。如果成功，这项研究将提供对新的dna结合域的结构和功能的见解，并了解如何将这些见解应用于创建遗传修饰工具，这些工具将比目前的方法更广泛地可获得和更普遍的效用。关键词：蛋白-核酸相互作用，工程锌指，从头计算建模，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

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.