Long-lived optical probes to image G-quadruplex DNA in live cells

DNA是一种多功能的生物分子，除了众所周知的双螺旋结构外，它还可以组装成各种不同的非规范结构。基因组学的最新进展已经揭示，具有形成这种非典型结构（例如发夹、三链体、四链体、连接）的潜力的DNA序列在基因组中广泛存在。有趣的是，它们的分布不是随机的：它们选择性地富集在特定的基因中，特别是在转录区域，暗示了非经典DNA结构在基本生物学功能中的作用。现在认识到，“一级结构”（即DNA序列）和观察到的表型之间的联系需要通过理解DNA的“二级”和“三级”结构在体内发挥的作用来补充。在这些非规范的DNA结构中，G-四链体-当富含鸟嘌呤的DNA序列折叠成四链螺旋时形成-在过去10年中受到越来越多的关注。这主要是由于越来越多的实验证据表明G-四链体DNA结构在端粒功能和维持、复制和基因表达调控等方面发挥着重要的生物学作用。然而，迄今为止，仍然很少有直接证据表明G-四链体在活细胞中形成并具有功能。基于我们在这个项目中的初步研究，我们建议开发新的小分子光学探针，通过磷光寿命成像显微镜（PLIM）来观察活细胞中的G-四链体。特别是，我们的目标是研究G-四链体的端粒中的形成和溶解。这种探针将提供关于这些非典型DNA结构的生物学功能的宝贵信息，并且从长远来看，是用于靶向四链体的潜在药物的细胞筛选的有用分子工具。

DNA is a versatile biomolecule which can assemble into a wide range of different non-canonical structures beyond the well-known double helix. The recent advances in genomics have revealed that DNA sequences with the potential to form such non-canonical structures (e.g. hairpins, triplexes, quadruplexes, junctions) are widespread in genomes. Interestingly, their distribution is not random: they are selectively enriched in specific genes and particularly in transcriptional regions, hinting at a role for non-canonical DNA structures in essential biological functions. It is now recognised that the link between 'primary structure' (i.e. DNA sequence) and the observed phenotypes needs to be complemented by an understanding of the roles that 'secondary' and 'tertiary' structures of DNA play in vivo. Amongst these non-canonical DNA structures, G-quadruplexes - which form when guanine-rich DNA sequences fold into quadruply-stranded helices - have received increasing attention over the past 10 years. This is mainly due to the increasing experimental evidence suggesting that G-quadruplex DNA structures play essential biological roles such in telomere function and maintenance, replication and the regulation of gene expression. However, to date there is still little direct evidence that G-quadruplexes form in live cells and are functional. Based on our preliminary studies in this project we propose to develop new small-molecule optical probes to visualize G-quadruplexes in live cells by Phosphorescence Lifetime Imaging Microscopy (PLIM). In particular, we aim to study the formation and dissolution of G-quadruplexes in the telomeres. Such probes will provide invaluable information about the biological function of these non-canonical DNA structures as well as, in the long term, be useful molecular tools for cellular screening of potential drugs that target quadruplexes.