At the Pollack lab we study interesting biophysical problems involving RNA, DNA. We take a bottoms-up all inclusive approach- from protein expression and nucleic acid synthesis, biochemical characterization and modification, instrumentation and beamline development, all the way to computational techniques development.
We utilize a combination of techniques to study biological systems, such as Förster resonance energy transfer (FRET), Fluorescence Correlation Spectroscopy (FCS), Circular Dichroism (CD), analytical gel filtration chromatography, among others. Our technique of choice, however, is Small Angle X-Ray Scattering (SAXS).
SAXS enables us to obtain low resolution structural information of nucleic acids and their complexes while in solution. In a SAXS experiment, X-rays from a synchrotron scatter from biomolecule in solution onto an area detector. These images are captured in a detector which is then averaged by rings of constant scattering vector (q) to obtain a scattering insensitive curve. This curve contains structural information that yield a lot of insight into the biophysics of the molecule, and since the molecules are in solution we can study the solvent conditions around it or couple it to a mixer to see the dynamics of nucleic acids, proteins and their interactions.
At Pollack lab we are committed to pushing the boundaries SAXS through a combination of biochemistry, engineering, and computational techniques, and we have applied it to study some fascinating properties of several biological systems:
Nucleosome-DNA unwrapping
A system of particular interest to us is the interaction between DNA and histone proteins. Four protein varieties combine to form an octamer core, which wraps DNA around itself in two turns to form a nucleosomal complex. These nucleosomes are the fundamental organization and storage system of DNA in cells and interact to form more complex storage structure. In order to understand this interaction, we use SAXS to observe the unwrapping of the DNA for various modifications of the DNA and protein core.
RNA and DNA properties
RNAs are flexible molecules and their flexibility can be tuned by the internal structures, sequences, and solvent conditions [1]. Knowledge of how conformations of simple RNA constructs respond to different ionic strengths can be generalized to complex functional RNA molecules and their folding energy landscape [2].
Ribosomal Proteins and RNAs
RNA and protein molecules form the ribosome, a large macromolecular machine that reads the mRNA sequence and stitches together polypeptide chains to make proteins. RNA molecules constitute the functional units of the ribosome, so that it is essentially a large ribozyme or RNA enzyme. In the Pollack Lab, we are studying the folding dynamics of the ribosomal RNA fragment, the 58 nucleotide GAC RNA [3] and using contrast variation x-ray scattering techniques to understand how the protein that bind the GAC RNA affects its folding and function.
Riboswitches
We want to see how molecules react to changes in solution conditions: adding magnesium ions causes RNA to collapse and fold into its most compact three dimensional structures, adding a small-molecule ligand causes structural change within an RNA riboswitch.
Viruses and their Assembly
At Pollack lab we have utilized Small Angle X-Ray Scattering, to study some of the dynamics of viral assembly and disassembly. We are particularly interested in the properties +ssRNA viruses.
References
- Sutton at al. Tuning RNA Flexibility with Helix Length and Junction Sequence. Biophysical Journal, 2015. doi: 10.1016/j.bpj.2015.10.039
- Chen et al. How the Conformations of an Internal Junction Contribute to Fold an RNA Domain. The Journal of Physical Chemistry B, 2018. doi: 10.1021/acs.jpcb.8b07262
- Welty et al. Divalent ions tune the kinetics of a bacterial GTPase center rRNA folding transition from secondary to tertiary structure. RNA, 2018. doi: 10.1261/rna.068361.118