Research Projects

Would in vitro biomolecular structure corroborate with in-cell studies? How does the cellular milieu influence the conformational properties? Would the drug binding features be altered within a cell? These persistent questions have baffled biophysical chemists. The past two decades have showcased enormous strides, especially with solution-state NMR spectroscopy characterizing protein molecules in cells. Our proposal aims to develop and apply available NMR methodologies to characterize the structurally polymorphic G-quadruplexes (G4s). G4s are essential gene regulators commonly formed by guanine-rich DNA and RNA sequences and are implicated in various processes, including diseases such as cancer. The project objectives are to develop NMR and optimize sample preparation methods to study in-cell conformational stability and folding of G4s, alleviating several impediments that limit interpretation in currently available methodologies. The results will enable us to understand how small molecules (potential drug leads) interact with these systems, furthering our understanding of creating drug molecules specifically targeting G4s. Biophysical studies of DNA G4s in the past decades have been predominantly carried out in vitro conditions and have highlighted the rugged folding landscape. For instance, a G-rich DNA sequence can potentially sample 26 different three-plane intramolecular G4 structures, with 14 experimentally observed thus far. The question has remained whether such a diverse range of structures is feasible in-cell, as they are involved in a critical part of the genome. This proposal shall first test the hypothesis whether all the diverse topologies observed in vitro are sampled inside the cell. To this end, the ¹³C/¹H chemical shift method developed in my lab (JPCL 2020) promises to be a reliable, rapid, and least invasive way of accessing G4s in-cell. As in-cell conditions are more challenging (molecular crowding, increased viscosity, background signals), new NMR methods must be optimized to maximize sensitivity per unit time, wherein multiple 2D experiments shall provide an overall structural characterization at near-atomic resolution. The initial characterization of the G4 topologies would pave the way to understand how promising molecular binders fare in-cell. This project pushes biophysical studies beyond the realms of the lab and test tube and attempts to take as close to reality as possible. Currently the methods that study G4 in-cell rely on (a) chemically modified nucleotides (alter folding landscape substantially, are expensive, multiple samples to study one system) (b) acquiring the ¹⁵N/¹H region of the NMR spectrum (imino chemical shifts are not effective reporters of topology), (c) use optical tags (severely alters topology and potentially changes the systems studied) and (d) lack time course of how binding is affected (as most techniques are invasive). Most importantly, understanding in-cell binding characteristics is crucial, as many promising in vitro binders perform poorly in real life. This proposal attempts to take on these shortcomings by using the ¹³C/¹⁵N isotopic enrichment of nucleotides (without any unnatural chemical modifications) that has proven not to affect biomolecular structure or conformational dynamics. NMR, being non-invasive, would allow for charting any changes that may come up in topology and/or alterations to binding as a function of time. With the advancement in NMR hardware, novel methods would allow the obtaining of multiple datasets that complement conformational characterization, with the best possible sensitivity. The results from the proposal stand to answer a long-standing question, whether G4s are stable/formed inside cells and help in evolving more molecular binders as a pursuit to various diseases.