Engineering & Evolution of Proteins that Target Specific DNA Sites

我的项目主要研究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蛋白质支架如何以高亲和力和特异性靶向特定的DNA位点。蛋白质工程是一个不断发展的领域，吸引了越来越多的研究人员，新的多学科工具和方法正在开发，使我们的设计方法。这些工具可以来自遥远的研究领域，例如，计算机科学家和数学家开发的计算方法。**我们开发了极简杂交蛋白（minimal hybrid proteins， MHP）作为平台，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MHPs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目标。**我们的实验室还开发了新的工具，以促进制造具有所需特性的非自然蛋白质。我们以E-box DNA位点（5'-CACGTG）为靶点，该位点是Myc和Max蛋白的靶点，与50%的癌症有关。E-box网络是我们研究的理想对象，因为它是一个被充分研究的系统，有着丰富的文献。我们设计特定的突变来产生结合所需DNA目标的新蛋白质。突变可以合理设计，我们也在酵母单杂交（Y1H）和细菌单杂交（B1H）系统以及我们刚刚开始在实验室实施的新的噬菌体辅助连续进化（PACE）系统中使用了包括定向进化在内的非理性方法。**对于蛋白质：DNA络合的体内检测，我们使用了经典的Y1H，这是最早开发的用于检测活细胞中蛋白质：DNA相互作用的系统之一。由于酵母极易发出错误信号，我们试图开发一种更简单的体内检测蛋白质的工具：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）试验。初步的FRep数据（从一个更大的文库中筛选出的15个突变体）使我们能够分离出一种与E-box具有强特异性结合的蛋白质。FRep， B1H和PACE是细菌，比酵母更直接使用。**因此，本提案侧重于我的项目的两个方法学方面：1)生物物理理解FRep，我们检测蛋白质的方法：DNA相互作用，2)开发B1H，特别是PACE进化系统作为方法，使我们能够更有效和成功地生成具有所需性质的MHPs。**我们的MHP方法可能有一天会影响药物设计领域。例如，有机合成化学家已经通过生成Myc/Max蛋白/蛋白相互作用的小分子抑制剂来靶向Myc/Max转录因子。然而，经过二十多年的努力，还没有开发出有用的小分子。我们的研究可以通过为药物开发提供新的策略来补充这些努力。**我们还与玛嘉烈医院的癌症研究员Linda Penn和UofT工程学院的纳米工程师Warren Chan合作。我所有的学员都参与了这种多样化的合作，他们来自不同的化学和生物学背景。他们接触到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，并在各种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一大优势。

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.