There are currently several active projects in our laboratory. These projects are essentially focused on either on DNA/RNA structure or interaction of DNA with relevant proteins. We use single molecule fluorescence microscopy techniques as the main tool. In particular, single molecule Förster Resonance Energy Transfer (FRET) and FRET-PAINT are the methods commonly used in our laboratory. Our methods take advantage of the high signal to noise ratio provided by total internal reflection to achieve single molecule resolution.
Telomeres and G-quadruplexes
Most of our work is focused on interactions of various proteins with G-quadruplex (GQ) structures. GQ structures (GQs) are non-canonical DNA second structures that could form at chromosome ends (telomeres), within the genomic DNA, or in RNA. Depending on where they form, they perform different functions including protection and capping of telomeres, transcription or translation level gene expression regulation.
As GQs are thermodynamically very stable, they typically require protein action to be unfolded during replication, repair, or transcription. Single stranded DNA (ssDNA) binding proteins, such as Replication Protein A (RPA) and Protection of Telomere 1 (POT1), and helicases, such as Bloom and Werner helicases, are capable of unfolding GQs with varying efficiency. The understanding the interactions of such physiologically significant proteins with GQs and deciphering the underlying principles of protein mediated GQ unfolding forms a significant aspect of our research efforts.
Targeting G-quadruplexes with CRISPR
Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins, particularly Cas9, have provided unprecedented programmable control on targeting specific sequences. An active CRISPR complex requires near-perfect complementarity between its guide RNA and the target DNA.
We investigate the capabilities and limitations of Cas9 for targeting sequences that are prone to folding into secondary structures, such as G-quadruplexes. In recent publications, we demonstrated examples of how GQs could inhibit target recognition, distort the structure, and inhibit Cas9-mediated DNA cleavage. We perform systematic single molecule and bulk biophysical studies to determine how GQs located in the vicinity of Cas9 target site impact critical aspects of its function.
DNA-based Liquid Crystals
The capability of chemically synthesizing DNA provides extreme control on designing and generating molecular structures with different sizes, shapes and geometries. Such DNA molecules form different phases and attain different characteristics and long range structures when they are condensed to high concentrations. Liquid crystal phases, where materials demonstrate both crystal-like and liquid-like characteristics, are some of the fundamentally important phases formed by DNA molecules.
In our approach, DNA molecules that have both rigid and flexible regions, such as two double stranded DNA molecules that are connected by a single strand, are particularly potent at demonstrating different liquid crystal phases at different concentrations. These phases are typically studied by small angle or wide angle X-ray scattering and polarized optical microscopy.