RNA Chemistry and the RNA World

生命的起源是生物学中的一个基本问题。RNA在核糖体核心的存在，核苷酸辅因子的广泛使用，以及从RNA前体合成DNA和一些氨基酸，表明RNA曾经是占主导地位的生物催化剂和遗传聚合物。这种“RNA世界”假说假定RNA能够进行维持和进化“核糖体”所需的所有反应。我的实验室的长期目标之一是分离出一种RNA复制酶核酶，它能够复制和转录自己的基因组，从而在实验室中实现自我维持的基于RNA的进化。这样的系统可以被称为“活的”，并将化学的基本简单性与生物学的复杂性结合起来。这种以实验室为基础的对进化的理解将前所未有地告知这个星球上生命的曙光。必须分阶段进行这一雄心勃勃的努力。半保守复制需要能够将单链模板前体转化为双链体产物的聚合酶。在我之前的发现基金中，我提出并开发了一种完全由RNA构建的依赖启动子的RNA聚合酶。这种核酶可以在单链RNA模板周围形成拓扑钳，然后将其填充到RNA双链体中（Cojocaru和Unrau，Science 2021）。这样的拓扑钳在蛋白质聚合酶中很常见，但是还需要什么才能使RNA通过RNA复制呢？在现存的基因组复制中，持续合成能力和链置换是紧密结合的，但是链置换的机制是如何在进化的早期进化出来的呢？本实验室将把现有的RNA聚合酶核酶进化成能够侵入RNA双链体的核酶。RNA滚环复制可能在进化的早期发挥了重要作用。事实上，如果将滚环复制中的RNA替换为复制叉中的DNA，这两个复制过程看起来是相同的：聚合酶侵入下游双链体，将前导单链复制成双链体，同时释放滞后链。三个目标将从进化的角度探讨这一复杂的机制：I。通过严格拓扑选择提高夹持聚合酶核酶的加工能力。我们的拓扑夹RNA聚合酶核酶的持续合成能力将使用一种新的体外选择方法来提高。二.能够进行链置换的核酶聚合酶的直接选择。热棘轮将演变为促进下游链侵入。三.选择易位聚（A）聚合酶核酶。将选择使用“动力冲程”来易位并且在机制上类似于现存蛋白质聚合酶的核酶聚合酶。这项工作将开发尖端的加拿大RNA生物化学方法，培养HQP，并对了解地球上生命的进化具有全球意义。

The origin of life is a fundamental problem in biology. The presence of RNA at the core of the ribosome, the extensive use of nucleotide cofactors, and the synthesis of DNA and some amino acids from RNA precursors suggests that RNA was once the dominant biological catalyst and genetic polymer. This `RNA World' hypothesis assumes that RNA is capable of all the reactions required to maintain and evolve a `ribo-organism'. One of my laboratory's long-term objectives is to isolate an RNA replicase ribozyme capable of copying and transcribing its own genome and thus enabling self-sustaining RNA based evolution in the laboratory. Such a system could be called `alive' and would marry the underlying simplicity of chemistry to the complexity of biology. This laboratory-based understanding of evolution would inform, as never before, about the dawn of life on this planet. Such an ambitious endeavour must be approached in stages. Semi-conservative replication requires a polymerase able to convert single-stranded template processively into a duplex product. In my previous Discovery grant, I proposed and then developed a promoter-dependent RNA polymerase built entirely from RNA. This ribozyme can form a topological clamp around a single-stranded RNA template and then fill it in to RNA duplex (Cojocaru and Unrau, Science 2021). Such topological clamps are commonly found in protein polymerases, but what is still needed to enable RNA replication by RNA? Processivity and strand displacement are tightly coupled in extant genomic replication, but how could the mechanism of strand displacement have evolved early in evolution? My laboratory will evolve our existing RNA polymerase ribozyme into one able to invade RNA duplex. RNA rolling circle replication could have played a fundamental role early in evolution. Indeed, if one replaces the RNA in rolling circle replication with the DNA in a replication fork, the two replicative processes look identical: A polymerase invades downstream duplex copying the leading single-strand into duplex while simultaneously liberating the lagging-strand. Three aims will explore this complex mechanism from an evolutionary perspective: I. Increasing the Processivity of the Clamping Polymerase Ribozyme by Stringent Topological Selection. The processivity of our topologically clamping RNA polymerase ribozyme will be improved using a new in vitro selection approach. II. Direct Selection for Ribozyme Polymerases able to Perform Strand Displacement. A thermal ratchet will be evolved to facilitate downstream strand invasion. III. Selection for translocating poly(A) polymerase ribozymes. A ribozyme polymerase that uses a `power stroke' to translocate and that is mechanistically similar to extant protein polymerases will be selected. This work will develop cutting edge Canadian RNA biochemical methods, train HQP, and is of global significance to understanding the evolution of life on this planet.