Programmable and Chemoselective Protein-DNA Crosslinking for Sensitive Detection of 5-Formylcytosine

5-甲酰胞嘧啶(Fc)是哺乳动物基因组的第七个碱基。最近发现Fc在与转录和分化相关的基因中存在高水平，晶体结构表明它在DNA中的存在可以强烈扭曲双链结构。这些发现暗示了在基因表达调控中的作用。然而，这种潜在功能的潜在控制机制是DNA中Fc的引入和去除，即其动力学。这方面的信息对于充分理解Fc‘S的功能是至关重要的，但以往只关注静态方面的研究没有提供这方面的信息。测量Fc的动力学需要简单和灵敏的分析方法来对相关基因组座位上的Fc进行时间分辨的定量。然而，目前的FC检测方法不提供直接的序列选择性，导致在简单性和/或分辨率方面的缺点。我们最近引入了基于TALL蛋白的DNA识别的扩展可编程性的概念，TALL蛋白由具有个体选择性的多个串联重复组成，用于识别DNA中的正则和表观遗传碱基。在这个项目中，为了应对FC检测的特殊挑战，我们将把我们的概念从单纯基于识别的方法发展到基于化学选择性的FC交联方法。我们将在1年前实现这一目标。)通过体内遗传编码将对乙酰基-L-苯丙氨酸(PAcF)共翻译入故事的适当位置，2.将修饰后的TALL与含有Fc的DNA靶点结合，以及3.在与选择性TALL-DNA络合物形成相容的条件下，通过催化与双官能团氨氧基连接剂形成的肟基在pAcF和FC之间进行化学选择性的交联化。这将显著提高我们方法的选择性和灵敏度，并首次在用户定义的基因组座位上实现Fc的直接和高分辨率定量。我们将建立一种结合qPCR定量的基于珠粒的基因组亲和力测定方法，并将其用于研究小鼠基因组中复制后Fc形成的动力学。这将导致对Fc作为其生物学功能基础的动力学的第一次洞察，并为后续研究提供一个起点，旨在更好地理解Fc形成对进一步氧化/修复过程的动态影响，以及关键修复和表观遗传阅读器蛋白的招募/释放。

5-Formylcytosine (fC) is the seventh nucleobase of the mammalian genome. fC has recently been found to be present at high levels in genes associated with transcription and differentiation, and crystal structures have shown that its presence in DNA can strongly distort the duplex structure. These findings are suggestive of roles in gene expression regulation. However, the underlying control mechanism of such potential functions is the introduction and removal of fC in DNA, i.e. its dynamics. Information on this aspect is crucial for a full understanding of fC´s function, but has not been provided by previous studies that focused only on static aspects. Measuring fC dynamics requires simple and sensitive assays for the time-resolved quantification of fC at relevant genomic loci. However, current detection methods for fC do not provide a direct sequence-selectivity, resulting in disadvantages in respect to simplicity and/or resolution. We have recently introduced the concept of an expanded programmability of DNA recognition based on TALE proteins that consist of multiple concatenated repeats with individual selectivities for the recognition of both canonical and epigenetic nucleobases in DNA. With this project, to meet the specific challenges of fC detection, we will advance our concept from a solely recognition-based approach to a chemoselective fC crosslinking-based approach. We will achieve this by 1.) cotranslational incorporation of para-acetyl-L-phenylalanine (pAcF) into appropriate sites of TALEs by genetic encoding in vivo, 2.) binding of the modified TALE to the fC-containing DNA target site, and 3.) chemoselective crosslinking between pAcF and fC by catalytic oxime formation with a bifunctional aminooxy-linker under conditions that are compatible with selective TALE-DNA complex formation. This will significantly increase the selectivity and sensitivity of our approach, and for the first time enable the direct and highly resolved quantification of fC at user-defined genomic loci. We will establish a bead-based assay for genomic affinity enrichment coupled to qPCR quantification and employ it to study the kinetics of post-replicative fC formation in the mouse genome.This will lead to the first insights into the dynamics of fC as a basis of its biological function, and represents a starting point for follow-up studies aiming at a better understanding of the dynamic consequences of fC formation in respect to further oxidation/repair processes, as well as the recruitment/release of key repair and epigenetic reader proteins.