A Protein Sensor For Detecting Single Stranded DNA Antony Research Group Chemistry and Biochemistry Department A Protein Sensor For Detecting Single Stranded DNA By Shania Bitsoie and Crystal Vejar Advanced Biotech 2014

Outline Objectives What is SSB (Single Strand DNA Binding Protein). Our approach Experiments and results Uses for biomedical research Conclusion

Experimental Objective Objectives Experimental Objective We want to detect single stranded DNA in solution. Being able to detect the presence of single stranded DNA and RNA viruses in body fluids. Application

What is SSB? Single Strand DNA binding protein is a protein that binds specifically to single stranded DNA It is present in all organisms from bacteria to humans Function: It protects ssDNA from being digested by nucleases Prevents premature annealing in DNA Removes secondary structures from the DNA to allow enzymes to function efficiently Its functions in many processes on the DNA in the cell: replication, recombination, and repair.

What do we want to do with this SSB protein? SSB binds to ssDNA 4 identical subunits (homotetramer) ssDNA wraps around the tetramer Wraps like seams on a tennis ball What do we want to do with this SSB protein? - In all of three days 

Our Goal Make a fluorescent version of SSB. Hypothesis: Upon binding to ssDNA, the fluorescence will change (hopefully)

Where to attach the fluorophore on SSB? What is a fluorophore? A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation. Where to attach the fluorophore on SSB? The fluorophore will bind to the cysteines.

Experiment 1: Labeling SSB We added the Fluorescein to the protein and let it sit for about an hour Then we filtered it through a column containing small beads The protein exited the column before the free Fluorescein We ran a gel and determined that we correctly labeled 18.4% of the protein

Experiment 2: Purification of Fluorescently labeled SSB Pf-SSB Free Label

SDS-PAGE gel to check labeled protein

Experiment 3: SSB protein after purification Coomassie Image: Stains all the protein Fluorescence Image: Shows labelled-SSB

Testing our hypothesis Make a fluorescent version of SSB. Hypothesis: Upon binding to ssDNA, the fluorescence will change (hopefully) Experiment: Add ssDNA to labeled-SSB and test if the fluorescence changes.

Experiment 4: Fluorescence of SSB increases with ssDNA Fluorimeter Hypothesis: Upon binding to ssDNA, the fluorescence will change (hopefully) – IT WORKED!!

Experiment 5: Can SSB selectively bind to ssDNA

In our research we: Summary of Results Correctly labeled 18.4% of our protein Measured fluorescence in SSB with and without DNA Proved that SSB specifically binds to ssDNA

Application in biomedical research Labeled SSB could be used to detect ssDNA in bodily fluids like blood. Many viruses have ssDNA or ssRNA.

Acknowledgements