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。 这些结构由于其在生物过程中的可能作用而引起重大兴趣。 由于人类基因组包含大量具有形成四链体潜力的序列，因此与G-四链体DNA结合的蛋白质可能为G-四链体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-四链体DNA之间的生物物理相互作用。 最近有报道称，胰岛素以高选择性结合到含有人胰岛素启动子区的胰岛素连接多态性区（ILPR）序列的G-四链体DNA。 考虑到ILPR DNA序列在基因调控中的可能作用，将通过单分子力谱和批量集成技术（如微量热法）研究胰岛素与四链体DNA的结合。 本研究的一个关键方面是量化从单个分子到主体的结合相互作用的强度和特异性。 G-四链体和四链体结合蛋白（如胰岛素）的生物物理性质对于理解这些重要生物分子系统的生物学是必不可少的。 更广泛的影响：这项研究需要采用跨学科的方法，采用生物学，化学，物理学和纳米技术的思想和技能。 从卡尔文学院（一个主要的本科院校）不同部门选择的大量本科生将通过参与一项跨越多个学科的研究来协同工作，获得显著的教育效益。 该项目还将提供研究和教育方面的优秀培训，以解决二十一世纪科学所需的跨学科问题。该研究还为学生提供了专业的仪器和研究经验，这将提高他们的分析和创造性思维能力，同时建立他们的智力能力的信心。 除了培训本科生，从这项研究中获得的知识也可以应用于表征其他识别G-四链体的蛋白质。 例如，蛋白质如胰岛素样生长因子在结构上与胰岛素相似，并且它们可以是用于确定它们的结合相互作用是否在分子水平上与胰岛素相似的良好候选物。

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.