Single-molecule visualisation of PARP trapping by PARP inhibitors

PARP抑制剂是用于治疗癌症的药物。正如其名称所示，它们靶向PARP蛋白，PARP蛋白在修复受损DNA中起着重要作用。PARP抑制剂的功效与在DNA断裂位点捕获PARP 1相关。我们最近发现PARP 1可以使受损的和未受损的DNA浓缩;该酶的辅因子NAD+可以使受损DNA的这种浓缩恢复;并且PARP抑制剂奥拉帕尼甚至在NAD+存在下也能使DNA保持缩合构象。总的来说，这些发现表明PARP-DNA结合、DNA延伸和PARP被抑制剂捕获之间存在复杂的相互作用，以及它们的治疗功能。为了解开这些不同的影响，我们需要解决PARP结合到不同构象的较长的DNA片段，在不同阶段的PARP激活和抑制。在这个项目中，我们将开发和应用新的原子力显微镜（AFM）方法，可逆地捕获，可视化和研究DNA在不同构象的PARP抑制剂，在纳米分辨率和水溶液中，总的目的是可视化和理解PARP结合诱导的构象变化（亚）分子细节与抑制剂和无抑制剂。具体目标如下：优化表面功能化方法，以促进AFM成像，同时仍然允许大量的分子灵活性。PARP与溶液中DNA结合和成形的纳米分辨率成像。提供PARP诱导的DNA缩合的机理见解。提供PARP抑制剂对PARP的影响的机理见解。AFM是一种物理技术，已被广泛用于以纳米分辨率对DNA进行成像。然而，由于技术原因，它的使用主要限于牢固附着在固体载体表面的DNA。本项目将开发能够改变DNA形状和力学的原位解析方法，并将其应用于药物对DNA-蛋白质结合的影响的研究。通过开发以纳米分辨率研究DNA-蛋白质相互作用的新方法，本项目符合BBSRC生物科学技术开发优先事项。

PARP inhibitors are pharmaceutical drugs used to treat cancers. As indicated by the name, they target PARP proteins, which play an important role in repairing damaged DNA. The efficacy of PARP inhibitors correlates with trapping of PARP1 at the site of a DNA break. We have recently discovered that PARP1 condenses both damaged and undamaged DNA; that the enzyme's co-factor NAD+ can revert this condensation for damaged DNA; and that the PARP inhibitor olaparib acts to retain the DNA in a condensed conformation even in the presence of NAD+.Overall, these findings indicate a complex interplay between PARP-DNA binding, DNA extension and PARP trapping by inhibitors, and presumably their therapeutic function. To disentangle these various effects, we need to resolve PARP bound to longer DNA segments in different conformations, in different stages of PARP activation and inhibition.In this project, we will develop and apply novel atomic force microscopy (AFM) methods to reversibly trap, visualise and study DNA in different conformations with and without bound PARP inhibitors, at nanometre resolution and in aqueous solution, with the overall aim to visualise and understand conformational changes induced by PARP binding with and without inhibitors at (sub)molecular detail.The specific objectives are as follows:Optimisation of surface functionalisation methods to facilitate AFM imaging while still allowing substantial molecular flexibility.Nanometre-resolution imaging of PARP binding to and shaping of DNA in solution.Providing mechanistic insight into PARP-induced DNA condensation.Providing mechanistic insight into the effect of PARP inhibitors on PARP-DNA interactions.AFM is a physical technique that has been widely used to image DNA at nanometre resolution. However, for technical reasons, its use is largely limited to DNA that is firmly attached to a solid support surface. In this project, we will develop methods to achieve such resolution on DNA that can change its shape and mechanics in situ, and apply it to study the effect of pharmaceutical drugs on DNA-protein binding.By the development of new methodology to study DNA-protein interactions at nanometre resolution, this project is aligned to the BBSRC priority of Technology development for the biosciences.