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

DNA是一种多用途的生物分子，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表明，具有形成这种非规范结构（如发夹、三联体、四联体、连接）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：它们在特定基因中选择性地富集，特别是在转录区域，这暗示了非规范DNA结构在基本生物功能中的作用。现在认识到，“一级结构”（即DNA序列）与观察到的表型之间的联系需要通过对DNA“二级”和“三级”结构在体内发挥作用的理解来补充。在这些非规范的DNA结构中，g -四重结构——当富含鸟嘌呤的DNA序列折叠成四股螺旋时形成的——在过去的10年里受到了越来越多的关注。这主要是由于越来越多的实验证据表明g -四重体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，到目前为止，仍然很少有直接证据表明g -四联体在活细胞中形成并具有功能。在本项目初步研究的基础上，我们提出开发新的小分子光学探针，利用磷光寿命成像显微镜（PLIM）观察活细胞中的g -四联体。特别是，我们旨在研究端粒中g -四联体的形成和溶解。这种探针将提供关于这些非规范DNA结构的生物学功能的宝贵信息，并且从长远来看，是针对四联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.