New DNA polymerases for the direct detection of epigenetic marks in RNA

在过去的几十年里，核酸作为序列信息唯一载体的观点是不准确的。特别是，已经发现RNA具有相当广泛的细胞功能，包括催化生化反应、控制转录后修饰和调节基因表达。事实上，已经在细胞RNA中鉴定了超过150种酶促引入和化学上不同的核苷酸修饰。修饰的核苷酸增加了RNA分子的结构多样性和化学多样性，以实现其多种多样的细胞功能。通过这种方式，他们可以微调RNA折叠以及RNA-RNA和RNA-蛋白质相互作用。此外，蛋白质编码RNA中高度动态的核苷酸修饰被认为可以控制mRNA代谢，从而调控基因表达，但沿着的一个瓶颈是，现有的高精度检测这些修饰的方法非常费力，从而阻碍了进展。该项目的目标是开发广泛适用的方法，允许可靠地检测RNA内的RNA修饰。在第一个资助阶段，我们成功地“进化”了一种逆转录酶，这种酶在遇到m6 A-RNA修饰时表现出特定的逆转录特征。通过与SPP内的Mark Helm（美因茨大学）和Yuri Motorin（L 'Université de Lorraine，Nancy）的密切合作，我们可以证明工程酶在不同序列背景下在m6 A位点提供突出的RT-签名，同时不会对未修饰的核苷酸和大多数其他修饰的核苷酸产生较高的错误率。在下一个资金阶段，我们的目标是a）优化进化的第一代酶，使其能够广泛应用于m6 A位点的检测，以及B）产生对假尿苷有反应的逆转录酶变体。mRNA中的假尿苷化已被认为在mRNA代谢中发挥重要的调节作用，因此，允许检测假尿苷的可靠技术将刺激该领域。因此，我们将遵循在第一个资助阶段开发的实验路线，包括组合蛋白质设计，筛选和生化表征。在项目结束时，我们将能够为社区提供针对m6 A和假尿苷位点选择性检测的酶，这将有利于未来的应用。此外，我们将获得更多的了解，在许多生物技术应用中使用的DNA聚合酶的功能。

During the last decades the view of nucleic acids as sole carrier of sequence information was inaccurate. In particular, RNA has been found to bear a considerably broader range of cellular functions, including the catalysis of biochemical reactions, the control of post-transcriptional modifications, and the regulation of gene expression. In fact, more than 150 enzymatically introduced and chemically distinct nucleotide modifications have been identified in cellular RNA. Modified nucleotides increase the structural versatility and chemical diversity of RNA molecules to allow for their multifarious cellular functions. In that way, they can fine-tune RNA folding as well as RNA-RNA and RNA-protein interactions. Moreover, highly dynamic nucleotide modifications in protein-coding RNAs are proposed to control mRNA metabolism and thereby regulate gene expression.A bottleneck along these lines is that available methods for the high-precision detection of these modifications are very laborious thereby hampering progress. The objective of this project is to develop widely applicable means that allow for the reliable detection of RNA modifications within RNA. In the first funding phase we were successful in “evolving” a reverse transcriptase enzyme that exhibits specific reverse transcription-signatures as a response to encountering an m6A-RNA modification. In close collaboration with the group of Mark Helm (University Mainz) and Yuri Motorin (L’Université de Lorraine, Nancy) within the SPP we could show that the engineered enzyme delivers prominent RT-signatures at m6A sites in different sequence contexts, while not exerting elevated error rates opposite unmodified nucleotides and the majority of the other modified nucleotides. This allows mapping of m6A sites by next generation sequencing.In the next funding phase we aim at a) the optimisation of the evolved 1st generation enzyme for enabling broad applications in the detection of m6A sites and b) Generation of a reverse transcriptase variant that is responsive to pseudouridine. Pseudouridinylation in mRNA has been suggested to perform important regulatory roles in mRNA metabolism, and thus, reliable techniques that allow detection of pseudouridine will spur the field. Thereby we will follow the experimental routes that were developed in the first funding phase including combinatorial protein design, screening and biochemical characterization. At the end of the project, we will be able to provide the community with enzymes that are tailored for the site selective detection of m6A and pseudouridine which will be beneficial for future applications. In addition, we will have gained more insight into the function of a DNA polymerase that is used in many biotechnological applications.