Expanding the chemical range of RNA-mediated catalysis : structure and mechanism

扩大RNA介导的催化的化学范围：结构和机制

• 批准号: EP/X01567X/1
• 负责人: David Lilley
• 金额: $ 70.32万
• 依托单位: University of Dundee
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FX01567X%2F1
• 关键词: Expanding; chemical; range; RNA; mediated

All chemical reactions that take place in living cells are catalysed by enzymes, the great majority of which are made of protein. However, a small subset of enzymes are made of RNA, called ribozymes. These are very important for a number of reasons. First, they catalyse some very important biological reactions, such as protein synthesis. Second, they probably played a key role in the early development of life on the planet, when RNA likely served as both the carrier of genetic information and the catalyst for metabolic reactions. Third, understanding how the chemically-simple (compared to combinatorial complexity of protein) RNA can act as a catalyst is a challenge to the biological chemist, and offers general insight into the mechanisms of biocatalysis. Lastly, RNA catalysts could potentially provide a source of useful and novel reagents and tools for organic chemistry, biotechnology and medicine. The great majority of known, natural ribozymes catalyse reactions making or breaking phosphorus-oxygen bonds. How the limited chemical resources of RNA are exploited to catalyse these phosphoryl transfer reactions is understood to some degree, but important questions remain. New ribozymes continue to be found in nature, such as the LINE-1 ribozyme that occurs in a human transposable element. These offer a new perspective on the existing mechanistic classification of ribozymes.A primitive RNA-catalysed metabolism would have required a much greater range of reactions to be accelerated, including 'difficult' reactions such as the formation of carbon-carbon and carbon-nitrogen bonds. In vitro selection provides a source of ribozymes that catalyse different chemical reactions. We have recently solved the crystal structure of a selected RNA that catalyses the transfer of a methyl or alkyl group from O6-methyl guanine or O6-alkyl guanine to a specific nitrogen atom on an adenine nucleotide of the RNA. Methyl transferase ribozymes are currently creating a lot of interest in biological chemistry as the full extent of methylation of RNA and its functions are still being elucidated. We are now using a combination of structural and mechanistic approaches to elucidate fully the mechanism of this ribozyme, and our present information already indicates that the ribozyme uses a sophisticated chemical mechanism to accelerate the reaction. We propose to develop this alkyl transferase ribozyme into a tool to engineer site-specific modification of RNA, including fluorescent probes and sites for crosslinking, first in vitro and subsequently in vivo. Although RNA can carry out remarkable feats of catalysis, with rate acceleration of a million-fold or more, the range of chemistry that is possible will be limited by the relative chemical simplicity of RNA. However the catalytic repertoire of RNA might be greatly expanded by the recruitment of co-enzymes. These are small molecules that bind to enzymes and participate in the reaction that is catalysed. RNA is an excellent receptor for binding small molecules, and this is exploited in biology by riboswitches in mRNA that bind metabolites to control adjacent genes. Many riboswitches bind powerful co-enzymes like S-adenosyl methionine, thiamine pyrophosphate and nicotinamide adenine dinucleotide that could greatly expand the catalytic repertoire of ribozymes, and we have hypothesised that some coenzyme-binding riboswitches have evolved from RNA world ribozymes. We propose to test this idea by reverse-engineering such riboswitches into ribozymes using in vitro selection. Such ribozymes could potentially catalyse a wide variety of chemical conversions including the formation of C-C bonds and oxidation-reduction reactions. Such novel catalysts could have applications in chemical synthesis and diagnostics.

在活细胞中发生的所有化学反应都是由酶催化的，其中绝大多数由蛋白质组成。然而，一小部分酶是由RNA组成的，称为核酶。这些都是非常重要的，原因有很多。首先，它们催化一些非常重要的生物反应，如蛋白质合成。其次，它们可能在地球生命的早期发展中发挥了关键作用，当时RNA可能既是遗传信息的载体，又是代谢反应的催化剂。第三，理解化学上简单的（与蛋白质的组合复杂性相比）RNA如何充当催化剂对生物化学家来说是一个挑战，并提供了对生物催化机制的一般见解。最后，RNA催化剂可能为有机化学、生物技术和医学提供有用的新型试剂和工具。绝大多数已知的天然核酶催化生成或断裂磷-氧键的反应。如何利用有限的RNA化学资源来催化这些磷酰基转移反应在一定程度上得到了理解，但重要的问题仍然存在。新的核酶在自然界中不断被发现，例如在人类转座因子中出现的LINE-1核酶。这为现有的核酶机制分类提供了一个新的视角。原始的RNA催化代谢需要更大范围的反应来加速，包括“困难”的反应，如碳-碳和碳-氮键的形成。体外选择提供了催化不同化学反应的核酶的来源。我们最近已经解决了所选RNA的晶体结构，该RNA催化甲基或烷基从O 6-甲基鸟嘌呤或O 6-烷基鸟嘌呤转移到RNA的腺嘌呤核苷酸上的特定氮原子。甲基转移酶核酶目前在生物化学中引起了很大的兴趣，因为RNA的甲基化及其功能的完整程度仍在阐明中。我们现在正在使用结构和机制的方法来充分阐明这种核酶的机制，我们目前的信息已经表明，核酶使用一种复杂的化学机制来加速反应。我们建议将这种烷基转移酶核酶开发成一种工具，以工程师的RNA的位点特异性修饰，包括荧光探针和交联位点，首先在体外，随后在体内。尽管RNA可以进行非凡的催化，其速率可以加速一百万倍或更多，但可能的化学范围将受到RNA相对化学简单性的限制。然而，RNA的催化库可能会通过辅酶的募集而大大扩展。这些小分子与酶结合并参与催化反应。RNA是一种结合小分子的优秀受体，在生物学中，这一点被mRNA中的核糖开关所利用，核糖开关可以结合代谢物来控制相邻的基因。许多核糖开关结合强大的辅酶，如S-腺苷甲硫氨酸，焦磷酸硫胺素和烟酰胺腺嘌呤二核苷酸，可以大大扩展核酶的催化库，我们假设一些辅酶结合核糖开关已经从RNA世界核酶进化而来。我们建议测试这一想法的反向工程核糖开关到核酶使用体外选择。这种核酶可以潜在地催化多种化学转化，包括C-C键的形成和氧化还原反应。这种新型催化剂可以在化学合成和诊断中应用。