DNA dynamics in biology and technology

长期以来，DNA被视为遗传信息的被动载体，其生物学相关形式是规范的b型螺旋双工。然而，越来越清楚的是，DNA动态地采用各种各样的非规范结构，这些结构涉及染色体稳定性、细胞衰老和基因表达等基本过程，并在治疗和技术方面显示出前景。一些研究最多的非典型DNA形式是鸟嘌呤四链结构（G4s）和i-基序（iMs），它们分别由富含dG和dc的DNA形成的四链结构。G4s和iMs通常是构象不均匀的，导致高度复杂的动力学行为。在大多数情况下，我们无法精确预测自然发生的富含G或c的DNA的结构或动力学，也无法预测氧化损伤和表观遗传修饰或与酶（如解旋酶和聚合酶）相互作用的影响。此外，许多目前的功能分析很大程度上是定性的。因此，尽管在人类基因组中已经确定了超过35万个假定的G4形成序列，但它们的生物学功能绝大多数是未知的。为了解决这一认识上的差距，我们需要新的定量分析来更好地表征DNA的结构、动力学和功能，并阐明一般的序列-结构-动力学-功能关系。这些信息对于理解非规范DNA在健康和疾病中的作用以及在生物技术应用中充分利用DNA至关重要。为了应对这一挑战，我们的短期目标是开发一套新的生物物理工具，以比目前可能的更高水平的细节定量表征DNA结构，动力学和功能。这些包括(i)用于测量G4s和iMs对聚合酶阻断的高灵敏度分析；（ii）精确测定DNA链中折叠和未折叠残基数目的方法；以及（iii）在单分子水平上收集高度异质结构集成的结构信息的方法。我们将使用这些新工具来阐明一些重要的非典型DNA类别的定量序列-结构-动态-功能关系：端粒和启动子G4s和iMs，适体，以及富含dG和dC的区域，我们预测这些区域具有数千或数万种不同的结构异构体，具有巨大的构象异质性。我们研究的长期愿景包括(i)理解和准确预测基于初级核苷酸序列的自然发生的非规范DNA的结构、动力学和功能相互作用，从而能够合理设计靶向DNA的治疗药物，以及（ii）基于结合协同性产生改进的基于DNA的生物传感器、治疗药物和具有新型生物物理特性的药物递送载体。

DNA was long viewed as a passive carrier of genetic information whose biologically relevant form was the canonical, B-form helical duplex. However, it is becoming increasingly clear that DNA dynamically adopts a wide variety of non-canonical structures that are implicated in fundamental processes such as chromosome stability, cell senescence, and gene expression, and have shown promise in therapeutics and technology. Some of the most-studied non-canonical DNA forms are guanine quadruplexes (G4s) and i-motifs (iMs), four-stranded structures formed by dG- and dC-rich DNA, respectively. G4s and iMs are often conformationally heterogeneous, leading to highly complex dynamical behaviour. In most cases, we are not able to predict the structures or dynamics experienced by naturally occurring G- or C-rich DNA with any precision, nor can we anticipate the effects of oxidative damage and epigenetic modifications or interactions with enzymes, such as helicases and polymerases. Furthermore, many of the current functional assays are largely qualitative. Thus, even though over 350,000 putative G4 forming sequences have been identified in the human genome, their biological functions are overwhelmingly unknown. To address this gap in our understanding, we need new quantitative assays to better characterize DNA structure, dynamics, and function, and to elucidate general sequence-structure-dynamics-function relationships. This information is critical for understanding the role of non-canonical DNA in health and disease, and for fully exploiting DNA in biotechnological applications. To address this challenge, our short-term objectives are to develop a suite of new biophysical tools to quantitatively characterize DNA structure, dynamics and function with a higher level of detail than is currently possible. These include (i) a highly sensitive assay for measuring the blockage of polymerases by G4s and iMs; (ii) a method to precisely determine the numbers folded and unfolded residues in a DNA chain; and (iii) an approach for gathering structural information at the single-molecule level for highly heterogeneous structural ensembles. We will use these new tools to elucidate quantitative sequence-structure-dynamics-function relationships for some important classes of non-canonical DNA: telomeric and promoter G4s and iMs, aptamers, as well as dG- and dC- rich regions that we predict to be hugely conformationally heterogeneous with upwards of thousands or tens of thousands of different structural isomers. The long-term vision of our research involves (i) understanding and accurately predicting the structures, dynamics, and functional interactions of naturally occurring non-canonical DNA based on the primary nucleotide sequences, enabling the rational design of therapeutics targeting DNA, and (ii) generating improved DNA-based biosensors, therapeutics, and drug delivery vehicles with novel biophysical properties based on binding cooperativity.