Computational Studies of AEGIS Molecular Components

合成生物学在过去25年中最重要的成果之一是认识到，经过40亿年的生物进化，生物聚合物并不是唯一可能支持遗传、遗传、进化和催化的分子。开发新的生物聚合物并表征其在活生物体中的特性不仅对测试我们关于现有“生命分子”的功能优化的想法具有重要意义，而且还为生物技术开辟了新的机会。这项工作也使我们得以深入了解地球生物学的独特性，以及宇宙其他地方的生命是否可以使用替代化学物质来构建。仅考虑DNA和RNA（统称为xNA），现在很清楚的是，xNA的四个不同的“标准”构建块（A，G，C，T及其等价物U）并没有穷尽指导天然核酸中沃森-克里克配对的两个规则所施加的约束。例如，核碱基对的数目可以仅通过重排氢键供体和受体基团而从2增加到6。在实践中使用化学合成来实现这一观察结果的努力（到目前为止）已经导致了两“代”新的杂环，其可以被并入适合用于自动化xNA合成的前体中，从而产生人工扩增的遗传信息系统（AEGIS）。虽然利用改变的氢键模式来获得与A：T和G：C（原则上）“正交”的新型核碱基对似乎很简单，但这些想法的实际实现已被证明存在令人惊讶的问题。例如，一些潜在的杂环具有高度聚集的互变异构体形式，其具有改变的氢键模式;这些可以与双链体DNA中或聚合酶的活性位点内的标准核碱基碱基配对，从而在复制期间引起意外的突变或AEGIS核碱基的丢失。在化学合成之前能够预测溶液中或酶活性位点内的互变异构体群体将是提高发现新核碱基对的效率的重要一步。即使这些“设计”问题得到解决，很少有人知道如何将这些非天然的核碱基纳入xNA影响这些复杂分子的构象偏好和动力学性质，这是基本的“标准”xNA与蛋白质，如聚合酶，和转录因子的相互作用。我们注意到，一直缺乏研究，旨在了解如何AEGIS核碱基，这改变了静电特性（偶极矩，电荷分布），可能会扰乱xNA结构在自由溶液中，当绑定在聚合酶活性位点。最后，验证AEGIS核碱基建模所需的力场参数，例如，xNA与蛋白质相互作用自由能的计算值与实验值的比较尚未见报道。因此，本项目的工作将通过（i）开发和验证新的计算方法，用于确定AEGIS核碱基在水和蛋白质环境中的互变异构形式的群体，和（ii）使用先进的MD为基础的方法，以了解AEGIS核碱基对的掺入如何影响双链体DNA的构象和动力学性质及其与DNA结合蛋白和聚合酶的相互作用。特别是，自由能微扰方法将被用来研究如何取代“标准”沃森-克里克碱基的AEGIS核碱基对改变的人SETMAR转录因子的DNA结合结构域的亲和力。这一目标的成功实现将为获得新的能切割含AEGIS双链DNA的内切酶奠定基础。

One of the most important outcomes of the last quarter-century of synthetic biology is the recognition that the biopolymers that have been delivered to us by 4 billion years of biological evolution are not the only molecules that might support genetics, inheritance, evolution, and catalysis. Developing new biopolymers and characterizing their properties in living organisms not only has significance for testing our ideas about the functional optimization of the existing "molecules of life" but also opens new opportunities in biotechnology. Such work also permits insights into questions of the uniqueness of terrean biology and whether life in other parts of the universe could be constructed using alternate chemistries. Considering just DNA and RNA (collectively xNA), it is now clear that the four distinct "standard" building blocks (A, G, C, T and its equivalent U) for xNA do not exhaust the constraints imposed by the two rules guiding Watson-Crick pairing in natural nucleic acids. For example, the number of nucleobase pairs can be increased from two to six by merely rearranging hydrogen bond donor and acceptor groups. Efforts to implement this observation in practice using chemical synthesis, have (so far) led to two "generations" of novel heterocycles that can be incorporated into precursors suitable for use in automated xNA synthesis, thereby yielding artificially expanded genetic information systems (AEGIS). Although exploiting altered patterns of hydrogen bonding to obtain novel nucleobase pairs that are (in principle) "orthogonal" to A:T and G:C appears straightforward, the practical realization of these ideas has proven surprisingly problematic. For example, some potential heterocycles have highly populated tautomeric forms with altered hydrogen bonding patterns; these can base pair with standard nucleobases in either duplex DNA or within the active sites of polymerases, thereby giving rise to unanticipated mutations or the loss of the AEGIS nucleobases during replication. Being able to predict tautomer populations in solution or within enzyme active sites prior to chemical synthesis would be a significant step in improving the efficiency with which new nucleobase pairs can be discovered. Even were these "design" problems to be resolved, little is known about how the incorporation of these non-natural nucleobases into xNA affects the conformational preferences and dynamical properties of these complex molecules, which are fundamental to the interaction of "standard" xNA with proteins, such as polymerases, and transcription factors. We note that there has been a dearth of studies aimed at understanding how AEGIS nucleobases, which have altered electrostatic properties (dipole moments, charge distribution), might perturb xNA structure in both free solution and when bound within polymerase active sites. Finally, the validation of the force field parameters needed to model AEGIS nucleobases by, for example, comparing calculated free energies of interaction between xNA and proteins with experimental measurements has not yet been reported.Work in this project will therefore seek to address the problems outlined above by (i) developing and validating new computational methods for determining the populations of tautomeric forms of AEGIS nucleobases in water and in protein environments, and (ii) using advanced MD-based methods to understand how the incorporation of AEGIS nucleobase pairs affects the conformational and dynamical properties of duplex DNA and its interactions with DNA-binding proteins and polymerases. In particular, free energy perturbation methods will be used to study how replacing "standard" Watson-Crick bases by an AEGIS nucleobase pair changes the affinity of the DNA-binding domain of the human SETMAR transcription factor. The successful accomplishment of this aim will lay a foundation for obtaining novel endonucleases capable of cleaving AEGIS-containing duplex DNA.