Computational Studies of AEGIS Molecular Components

在合成生物学的最后四分之一个世纪中，最重要的成果之一是认识到，通过40亿年的生物进化提供给我们的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在活体中表征它们的性质，不仅对于检验我们关于现有生命分子功能优化的想法具有重要意义，而且还为生物技术打开了新的机遇。这样的工作还可以洞察地球生物的独特性以及宇宙其他部分的生命是否可以使用替代化学物质来构建的问题。仅考虑DNA和RNA(统称为XNA)，现在很明显，XNA的四个不同的“标准”构件(A、G、C、T及其等价物U)并没有用尽指导天然核酸中Watson-Crick配对的两个规则施加的限制。例如，只需重新排列氢键供体和受体基团，就可以将碱基对的数量从2个增加到6个。到目前为止，利用化学合成将这一观察结果落实到实践中的努力已经导致了两代新的杂环，它们可以并入适合用于XNA自动合成的前体中，从而产生了人工扩展的遗传信息系统(宙斯盾)。尽管利用氢键的改变模式来获得(原则上)与A：T和G：C“正交”的新型碱基对似乎很简单，但这些想法的实际实现已被证明是令人惊讶的问题。例如，一些潜在的杂环具有高度填充的互变异构体，其氢键模式发生变化；这些互变异构体可以与双链DNA中的标准碱基或聚合酶活性位点中的标准碱基配对，从而导致意外突变或复制过程中宙斯盾碱基的丢失。能够在化学合成之前预测溶液中或酶活性部位内的互变构体数量，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，关于将这些非天然碱基结合到XNA中如何影响这些复杂分子的构象偏好和动力学性质，人们也知之甚少，而这些复杂分子是“标准”XNA与蛋白质(如聚合酶和转录因子)相互作用的基础。我们注意到，一直缺乏旨在了解宙斯盾碱基如何改变静电性质(偶极矩、电荷分布)的研究，它可能会在自由溶液中和结合在聚合酶活性部位时扰乱XNA结构。最后，通过比较XNA和蛋白质之间相互作用的自由能和实验测量来验证建模宙斯盾核苷酸碱基所需的力场参数还没有报道。因此，这个项目的工作将寻求通过(I)开发和验证新的计算方法来确定水和蛋白质环境中的宙斯盾核苷酸碱基互变异构体的数量，以及(Ii)使用先进的基于分子动力学的方法来了解宙斯盾核苷酸碱基对的结合如何影响双链DNA的构象和动力学性质以及它与DNA结合蛋白质和聚合酶的相互作用。特别是，自由能微扰方法将被用于研究用宙斯盾碱基对取代“标准”Watson-Crick碱基如何改变人类SETMAR转录因子的DNA结合域的亲和力。这一目标的成功实现将为获得能够切割含有宙斯盾的双链DNA的新型内切酶奠定基础。

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.