Venu Vandavasi, PhD, director of the Biophysics Core Facility in the Frick Chemistry Laboratory at Princeton University, talks to contributing editor Tanuja Koppal, PhD, about the changes he is witnessing in the technology and applications for biophysical characterization of molecules. He also discusses the current challenges and what can be done to mitigate some of them.
Q: Can you tell us about your work and the types of techniques you use in your biophysics core facility?
A: Our core facility houses a wide variety of techniques to help with the biophysical characterization of diverse molecules. We use analytical ultracentrifugation, biolayer interferometry, circular dichroism spectroscopy, differential scanning calorimetry, dynamic light scattering, isothermal titration calorimetry, fluorescence spectroscopy, microscale thermophoresis, surface plasmon resonance, and more. The biophysical interactions that we study involve molecules such as proteins, nucleic acids (DNA and RNA), small molecules, and lipids. The small molecules include synthetic chemicals and drug-like molecules or peptides that are extracted from living organisms or synthesized in a lab. We also work with proteins, DNA, and RNA that are labeled with various chemical tags.
Our users want to study how these molecules interact, and we characterize them both qualitatively and quantitatively. In qualitative analysis, we find out if these molecules are interacting or binding with each other. For quantitative analysis, we evaluate how strongly they are interacting and determine the affinities of binding. Further, we characterize the thermodynamic parameters and kinetics of these interactions, or sometimes, we study the secondary or tertiary structure of proteins and changes in the structure caused by these interactions, which is often important for their function. We also study the oligomeric states of biological molecules and how they are affected by other molecules.
Q: What are some of the main trends that you are seeing in biophysical analysis, in terms of technology and applications?
A: Biophysics core facilities have equipment that use the principles of math, physics, and chemistry to characterize biological molecules and address biological problems. These instruments tend to be quite expensive and require technical expertise. The lifespan of these instruments in a core facility is about five to six years, by which time they either start to break down or a new technology comes along to replace it. Some of these techniques have been in existence for about 50 years, but access to these instruments has been quite limited until about 10 years ago, when core facilities were set up to share resources. The biophysics core at Princeton University was established and started its operations in 2018.
Previously, the applications of these biophysical techniques were mostly related to basic science projects. However, in recent years the projects have moved toward applied research, and in the past year many of them were related to COVID-19. Advances in synthetic chemistry have made it possible to have huge libraries of small molecules. Biophysical screening of these molecules against drug targets (proteins or nucleic acids) helps determine which of these molecules have potential to be new therapeutic drugs. These potential molecules are called “hits.” Similarly, with advances in biology, new drug targets are being discovered and screened to identify which ones are “druggable.” The hits need to be analyzed and characterized in detail to understand what kind of drug-target interactions are taking place. What is the kinetics of those interactions? How will it affect the structure and stability of the bio molecule? Answering each of these questions needs a different technique and we have them available in our biophysics core. Automation of the instrumentation and advances in the software have saved a lot of time for performing experiments and made it simpler to analyze the data.
Q: What are some of the big challenges in biophysical characterization today?
A: The core facilities typically house many different types of instruments and the lab managers need to have a good understanding of their strengths and limitations. Knowing which technique is appropriate to address a certain biological problem is never easy. The rationale for picking the right technique is subjective, given many different techniques can give similar or same answers at times, and this comes with experience and thorough knowledge of what each of them has to offer. Keeping up to date with the new technologies is always challenging. To understand the instrumentation, you must have a good understanding of the math, physics, and chemistry, but the problems you are dealing with are biological and that needs a good understanding of the biological context as well. Each biomolecule is different, and each problem is different.
The other big challenge is managing the quality of the sample that comes in for testing. We always suggest some pilot experiments and quality control tests be done to ensure that samples are good. We have techniques that help users quickly determine the sample purity and concentrations before they conduct a laborious and time-consuming experiment. For core facilities that function as a contract research organization, things are streamlined, automated, and mostly done by trained personnel. However, in an academic setting, a lot of decisions must be made in real time. We train about a few hundred students every year with different levels of expertise and scientific backgrounds, and they run their own experiments while we provide consultation and help troubleshoot issues with instrumentation. This can be a challenge because each one handles the instrument differently. While we can predict the lifespan of an instrument, it is not always accurate due to user variability. Hence, there are always challenges with maintenance of the instruments and getting funding.
In terms of investing in new techniques, the objective must be very clear, and the equipment should sustain its own running cost. Be up to date with the technologies and follow the trend in high impact journals to see what techniques the scientists are using. Avoid buying expensive instruments that will appeal only to a small group of users. If you do buy a very expensive instrument, be prepared to have to do some of the work yourself. For a core lab, it is always a good idea to consider which instrument can make things simple and reduce the time for analysis.
Venu Vandavasi obtained his PhD in Biophysics and Structural Biology from Saha Institute of Nuclear Physics, India, in 2011. He performed his postdoctoral research at the Life Sciences Institute at the University of Michigan, Ann Arbor, and at the Oak Ridge National Laboratory, Oak Ridge, TN. Since 2018, he has led the Biophysics Core Facility at Princeton University and lectures advanced biophysical chemistry at Princeton. He acts as a subject matter expert, provides consultancy, and necessary training to researchers in the areas of biophysics at Princeton. His research interests include biophysics and structural biology of proteins with therapeutic and industrial importance.