RUI: Single Molecule and Bulk Calorimetric Measurements of Insulin Binding Interactions with G-Quadruplex DNA

智力优势：G-四链体是由富含鸟嘌呤的DNA序列在一价阳离子(如钾或钠离子)存在下形成的非规范DNA结构。由于它们可能在生物过程中发挥作用，这些结构引起了人们的极大兴趣。由于人类基因组包含大量有可能形成四链的序列，与G-四链DNA结合的蛋白质可能为G-四链DNA在生物学中的作用提供线索。该项目详细研究了蛋白质、胰岛素和G-四链DNA之间的生物物理相互作用。最近有报道称，胰岛素与含有人胰岛素启动子区胰岛素连接多态区域(ILPR)序列的G-四链DNA具有高选择性结合。考虑到ILPR DNA序列在基因调控中的可能作用，胰岛素与四链DNA的结合将通过单分子力谱和整体集成技术(如微量热法)进行研究。这项研究的一个关键方面是量化从单个分子到整体的结合作用的强度和特异性。G-四链和胰岛素等四链结合蛋白的生物物理性质对于理解这些重要的生物分子系统的生物学是必不可少的。更广泛的影响：这项研究需要一种跨学科的方法，利用生物学、化学、物理学和纳米技术的想法和技能。从卡尔文学院(Calvin College)不同部门挑选的相当数量的本科生将合作工作，从参与一项跨越几个学科的研究中获得显著的教育好处。卡尔文学院主要是一所本科院校。该项目还将在研究和教育方面提供出色的培训，以解决21世纪科学所需的跨学科问题。这项研究还为学生提供了获得专门仪器和研究经验的途径，这将增强他们的分析和创造性思维能力，同时建立对他们智力能力的信心。除了培训本科生，从这项研究中获得的知识也可以用于表征其他识别G-四链的蛋白质。例如，胰岛素样生长因子等蛋白质在结构上与胰岛素相似，它们可能是在分子水平上确定其结合作用是否类似于胰岛素的良好候选者。

Intellectual Merit: G-quadruplexes are noncanonical DNA structures formed from guanine-rich DNA sequences in the presence of monovalent cations such as potassium or sodium ions. These structures are of significant interest due to their possible role in biological processes. Since the human genome contains a large number of sequences that have the potential to form quadruplexes, proteins that bind to G-quadruplex DNA may provide a clue to the role of G-quadruplex DNA in biology. This project examines in detail the biophysical interactions between the protein insulin and G-quadruplex DNA. It has recently been reported that insulin binds with high selectivity to G-quadruplex DNA that contains the sequence of the insulin-linked polymorphic region (ILPR) of the human insulin promoter region. Given the probable role of the ILPR DNA sequence in gene regulation, the insulin binding to quadruplex DNA will be investigated by single molecule force spectroscopy and bulk ensemble techniques such as microcalorimetry. A key aspect of this research is to quantify the strength and the specificity of the binding interaction from single molecule to the bulk. The biophysical properties of G-quadruplex and the quadruplex binding proteins such as insulin are essential for understanding the biology of these important biomolecular systems. Broader Impacts: This research requires an interdisciplinary approach employing ideas and skills from biology, chemistry, physics and nanotechnology. A significant number of undergraduate students chosen from different departments at Calvin College, a primarily undergraduate institution, will work collaboratively gaining significant educational benefits from participating in a study that straddles several disciplines. This project will also provide excellent training in research and education in cross-disciplinary problem solving required by twenty-first century science. The research also provides students access to specialized instrumentation and research experiences that will enhance their analytical and creative thinking skills, while building confidence in their intellectual abilities. In addition to training undergraduate students, the knowledge gained from this research can also be applied to characterize other proteins that recognize G-quadruplexes. For example, proteins such as insulin-like growth factors are structurally similar to insulin, and they could be good candidates for determining whether their binding interaction is similar to insulin at the molecular level.