Engineering & Evolution of Proteins that Target Specific DNA Sites

工程

• 批准号: RGPIN-2014-05632
• 负责人: Shin, Jumi
• 金额: $ 2.55万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=665071
• 关键词: Engineering; Evolution; Proteins; Target; Specific

My program focuses on how nature uses proteins to recognize specific DNA sequences. We adopt a protein design and engineering approach to understand how the protein scaffold can be used to target a specific DNA site with high affinity and specificity. Protein engineering is a growing field that has increasingly attracted researchers, and new multidisciplinary tools and methods are being developed that enable our design approach. Such tools can come from research far afield, for example, computational methods developed by computer scientists and mathematicians.**We have developed minimalist hybrid proteins (MHP) as a platform to study how larger native proteins recognize specific DNA sequences, especially sites of 6-12 base pairs in length. Our MHPs are small (25-75 amino acids), and they mimic very well the DNA-binding function of the native transcription factors that they are based on, for they bind their targets with high DNA sequence specificity and binding affinity.**Our lab also develops new tools that facilitate making unnatural proteins with desired properties. We target the E-box DNA site (5'-CACGTG), which is targeted by proteins Myc and Max and is involved in >50% of all cancers. The E-box network is ideal for us to tackle, as it is a well-studied system with abundant literature. We design specific mutations to generate new proteins that bind a desired DNA target. Mutations can be rationally designed, and we also use nonrational methods including directed evolution in the yeast one-hybrid (Y1H) and bacterial one-hybrid (B1H) systems, and the new phage-assisted continuous evolution (PACE) system we just started to implement in the lab. **For in vivo detection of protein:DNA complexation, we have used the classic Y1H, which is one of the earliest systems developed for detecting a protein:DNA interaction in live cells. Because yeast are highly prone to giving false signals, we sought to develop a simpler in vivo tool for assaying protein:DNA interactions. We therefore developed the FRep (FRET Reporter) assay that is performed in E. coli. Preliminary FRep data (a screen of just 15 mutants from a much larger library) has allowed us to isolate a protein showing strong and specific binding to E-box. FRep, B1H and PACE are bacterial and are more straightforward to use than yeast.**This proposal is therefore focused on two methodological aspects of my program: 1) biophysically understanding FRep, our method for detection of protein:DNA interactions, and 2) developing the B1H and particularly PACE evolution systems as methods that will enable us to more efficiently and successfuly generate MHPs with desired properties.**Our MHP approach may someday impact the field of drug design. For example, organic synthetic chemists have been targeting the Myc/Max transcription factor by generating small-molecule inhibitors of the Myc/Max protein/protein interaction. However, no useful small molecule has been developed despite over two decades of work. Our research may complement these efforts by providing new strategies for drug development.**We also collaborate with Linda Penn, a cancer researcher at Princess Margaret Hospital, and Warren Chan, a nano-engineer in UofT Engineering. All of my trainees are involved in this diverse collaboration, and they come from diverse background in chemistry and biology. They are exposed to a broad range of research skills and languages including molecular biology, health-related research, chemistry, bioengineering, and nanotechnology. My trainees learn to think in an interdisciplinary manner and to communicate in various scientific and engineering disciplines. The diversity and synergistic value of this collaboration is a great strength of the training my program provides.

我的项目重点关注大自然如何利用蛋白质来识别特定的 DNA 序列。我们采用蛋白质设计和工程方法来了解如何使用蛋白质支架以高亲和力和特异性靶向特定 DNA 位点。蛋白质工程是一个不断发展的领域，越来越吸引研究人员，并且正在开发新的多学科工具和方法，以实现我们的设计方法。这些工具可以来自遥远的研究，例如计算机科学家和数学家开发的计算方法。**我们开发了极简混合蛋白（MHP）作为平台，用于研究较大的天然蛋白如何识别特定的 DNA 序列，特别是长度为 6-12 个碱基对的位点。我们的 MHP 很小（25-75 个氨基酸），它们很好地模仿了它们所基于的天然转录因子的 DNA 结合功能，因为它们以高 DNA 序列特异性和结合亲和力结合靶标。**我们的实验室还开发了新工具，有助于制造具有所需特性的非天然蛋白质。我们以 E-box DNA 位点 (5'-CACGTG) 为目标，该位点是 Myc 和 Max 蛋白的目标，并且与 > 50% 的所有癌症有关。 E-box 网络是我们解决问题的理想选择，因为它是一个经过充分研究的系统，拥有丰富的文献。我们设计特定的突变来产生结合所需 DNA 靶标的新蛋白质。突变可以合理设计，我们还使用非理性方法，包括酵母一杂种（Y1H）和细菌一杂种（B1H）系统中的定向进化，以及我们刚刚开始在实验室实施的新噬菌体辅助连续进化（PACE）系统。 **对于蛋白质：DNA 络合的体内检测，我们使用了经典的 Y1H，它是为检测活细胞中蛋白质：DNA 相互作用而开发的最早的系统之一。由于酵母很容易发出错误信号，我们试图开发一种更简单的体内工具来分析蛋白质：DNA 相互作用。因此，我们开发了在大肠杆菌中进行的 FRep（FRET 报告基因）测定。初步的 FRep 数据（从一个更大的文库中筛选出的仅 15 个突变体）使我们能够分离出一种与 E-box 表现出强烈且特异性结合的蛋白质。 FRep、B1H 和 PACE 是细菌，比酵母更容易使用。**因此，该提案重点关注我的计划的两个方法学方面：1) 从生物物理角度理解 FRep，我们检测蛋白质：DNA 相互作用的方法，以及 2) 开发 B1H，特别是 PACE 进化系统，作为使我们能够更有效、更成功地生成具有所需特性的 MHP 的方法。**我们的 MHP 方法可能 有一天会影响药物设计领域。例如，有机合成化学家一直通过生成 Myc/Max 蛋白质/蛋白质相互作用的小分子抑制剂来靶向 Myc/Max 转录因子。然而，尽管经过二十多年的努力，仍未开发出有用的小分子。我们的研究可以通过提供新的药物开发策略来补充这些努力。**我们还与玛格丽特公主医院的癌症研究员 Linda Penn 和多伦多大学工程学院的纳米工程师 Warren Chan 合作。我的所有学员都参与了这种多元化的合作，他们来自化学和生物学的不同背景。他们接触广泛的研究技能和语言，包括分子生物学、健康相关研究、化学、生物工程和纳米技术。我的学员学会以跨学科的方式思考，并在不同的科学和工程学科中进行交流。这种合作的多样性和协同价值是我的项目提供的培训的巨大优势。