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.
Kumar Sinniah and his team of undergraduate students at Calvin College have investigated the biomolecular interactions between insulin and a non-canonical DNA structure known as guanine-quadruplex or G-quadruplex. The DNA sequences used in their study are associated with type 1 diabetes and located in the insulin-linked polymorphic region (ILPR). Individuals predisposed to developing type 1 diabetes display 40-60 repeats of the consensus DNA sequence while healthy patients have 120-170 repeats. As well as being linked to type 1 diabetes, the ILPR is able to form the G-quadruplex structure (Figure 1). Using microcalorimetry methods and single molecule atomic force microscopy, their group has measured kinetic and thermodynamic parameters for the binding interaction (Figures 2 and 3). Most notably the binding was determined over a temperature range from 10 – 25 degrees Celsius. The results provide deep insights into the physical mechanism that control the association between insulin and G-quadruplex DNA formed by the ILPR DNA. More importantly the specific recognition of proteins such as insulin by G-quadruplex DNA additionally provides evidence of its functional role in the human genome. This research has provided training of eight female undergraduate students at Calvin College, of which three students have applied to PhD programs in chemistry, chemical engineering, and pharmacology, while the other students have applied to medical and dental schools.
