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核碱基。最近发现fC在与转录和分化相关的基因中以高水平存在，并且晶体结构表明其在DNA中的存在可以强烈地扭曲双链体结构。这些发现提示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的功能至关重要，但以前的研究只关注静态方面。测量fC动力学需要简单而灵敏的测定方法，用于在相关基因组位点对fC进行时间分辨定量。然而，目前用于fC的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TALE蛋白的DNA识别的扩展可编程性的概念，所述TALE蛋白由多个串联重复序列组成，所述多个串联重复序列具有用于识别DNA中的典型核碱基和表观遗传核碱基的个体选择性。通过这个项目，为了应对fC检测的特定挑战，我们将把我们的概念从单纯的基于药物的方法推进到基于化学选择性fC交联的方法。我们将在1.）通过体内遗传编码将对乙酰基-L-苯丙氨酸（pAcF）共翻译掺入TALE的适当位点，2.）修饰的TALE与含fc的DNA靶位点的结合，和3.）在与选择性TALE-DNA复合物形成相容的条件下，通过用双官能氨氧基-接头催化肟形成，在pAcF和fC之间进行化学选择性交联。这将显著提高我们方法的选择性和灵敏度，并首次实现在用户定义的基因组位点直接和高分辨率的fC定量。我们将建立一种基于微珠的基因组亲和富集与qPCR定量相结合的方法，并将其用于研究小鼠基因组中复制后fC形成的动力学。这将首次深入了解fC的动力学，作为其生物学功能的基础，这是一个起点，旨在更好地理解fC形成在进一步氧化/修复过程中的动态后果以及关键修复和表观遗传阅读器蛋白的募集/释放的研究。

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.