Advanced Biotech

2014

A Protein Sensor For Detecting Single Stranded DNA

By Shania Bitsoie and Crystal Vejar

Antony Research GroupChemistry and Biochemistry Department

Outline

0Objectives0What is SSB (Single Strand DNA Binding Protein). 0Our approach 0Experiments and results0Uses for biomedical research0Conclusion

Objectives

0We want to detect single stranded DNA in solution.

0Being able to detect the presence of single stranded DNA and RNA viruses in body fluids.

Application

Experimental Objective

What is SSB?

0 Single Strand DNA binding protein is a protein that binds specifically to single stranded DNA

0 It is present in all organisms from bacteria to humans

Function:0 It protects ssDNA from being digested by

nucleases0 Prevents premature annealing in DNA0 Removes secondary structures from the DNA

to allow enzymes to function efficiently 0 Its functions in many processes on the DNA in

the cell: replication, recombination, and repair.

SSB binds to ssDNA

04 identical subunits (homotetramer)0 ssDNA wraps around the tetramer0Wraps 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)

What is a fluorophore?

Where to attach the fluorophore on SSB?

A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation.

The fluorophore will bind to the cysteines.

Experiment 1: Labeling SSB

0We added the Fluorescein to the protein and let it sit for about an hour

0Then we filtered it through a column containing small beads0The protein exited the column before the free Fluorescein0We ran a gel and determined that we correctly labeled 18.4%

of the protein

0

0.2

0.4

0.6

0.8

1

1.2

1.4

40 80 120 160 200 240 280

Abs

Time (min)

Labeled Pf-SSB

Free Label

Experiment 2: Purification of Fluorescently labeled SSB

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

Experiment: Add ssDNA to labeled-SSB and test if the fluorescence changes.

Make a fluorescent version of SSB. Hypothesis: Upon binding to ssDNA, the fluorescence will change

(hopefully)

2 105

4 105

6 105

8 105

1 106

1 106

1 106

2 106

2 106

500 510 520 530 540 550

No DNA+DNA (dT)70

Emission Wavelength (nm)

Experiment 4: Fluorescence of SSB increases with ssDNA

Hypothesis: Upon binding to ssDNA, the fluorescence will change (hopefully) – IT WORKED!!

Fluorimeter

0

5 104

1 105

1.5 105

2 105

0 0.5 1 1.5 2 2.5 3

[DNA]/[SSB]

+ssDNA

+dsDNA

Experiment 5: Can SSB selectively bind to ssDNA

Summary of Results

In our research we:0Correctly labeled 18.4% of our protein0Measured fluorescence in SSB with and without DNA0Proved that SSB specifically binds to ssDNA

Application in biomedical research

0Labeled SSB could be used to detect ssDNA in bodily fluids like blood.

0Many viruses have ssDNA or ssRNA.

Acknowledgements