Research

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 high resolution particle tracking 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.

1) A guanine-rich sequence in the promoter region of Thyrosine Hydroxylase regulates its activity
Structure of a 3-layer G-Quadruplex
The goal of this project is to deconvolute the role of sequences that could form a dynamic structure of one or more G-Quadruplexes (GQs) in the regulation of Tyrosine Hydroxylase (TH) at the transcription level.
Aberrant TH expression is linked to many psychiatric problems including bipolar disorder, manic depression, Parkinson’s disease, and schizophrenia. To address this problem, we are studying a 45 nt segment of human TH promoter containing seven stretches of Gs’ located within a conserved region and is just 24 nucleotide upstream of the transcription start site.  It is hypothesized that this G-rich segment can adopt multiple GQ structures that vary in stability and conformation and is a key regulatory element in TH promoter.  Preliminary studies on this 45 nt DNA and a subset of its fragments suggest the presence of multiple GQs that vary in stability and conformation.  This project aims to characterize the dynamicity and variability in conformations associated with the ensemble of GQs formed in the 45 nt segment by utilizing the unique capabilities of single molecule FRET in combination with other biophysical and biochemical techniques. 

2) G-Quadruplex unfolding activity of Replication Protein A

RPA in complex with ssDNA
Replication Protein A (RPA) is the most abundant single strand DNA binding protein in eukaryotes and has very high affinity to ssDNA. RPA has been shown to unfold double stranded DNA and GQ structures at rates that depend on the stability of these structures. In this project, we study the unfolding of various GQ structures by RPA using single molecule methods. The RPA unfolding rate of GQ structures of different number of layers, loop lengths, and overhanging sequences is studied in order to identify properties that would make a GQ structure physiologically viable in the presence of destabilizing proteins. Traditionally the stability of GQ structures has been characterized via their thermal melting temperatures however, our preliminary work demonstrates that thermodynamic stability may not always be a proper gauge in the context of protein-DNA interactions. In the Figure below TR-3 is significantly more stable against RPA unfolding despite its lower thermal melting temperature than TH-2 (melting temperatures are 69 C and 78 C for TR-3 and TH-2, respectively).

RPA unfolding of three GQ structures of different thermodynamic stability

        

A schematic of the single molecule FRET setup.







Prism type total internal reflection.



A photo of our single molecule fluorescence microscope.




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