Kinetics of 585 Quantum Dot-HIV1 RT Complexes
Binding to DNA-DNA Duplexes
Jonathan Kawulok, D.J. Wright, Angela Rudolph, James Brozik
Washington State University, Department of Chemistry, Pullman, WA 99163
Introduction
Human Immunodeficiency Virus Reverse Transcriptase (HIV
RT) is an enzyme essential to infecting humans with HIV, the
virus responsible for AIDS. HIV RT binding to dsDNA primers
is one of the first steps in the DNA polymerization process
associated with this enzyme. In order to understand more
deeply this initial step, fluorescent probes have been
attached to the DNA and kinetically resolved Single Molecule
imaging has been used to monitor that binding process.
One Color Single Molecular Fluorescence
Microscopy Results
Association Constant of QD-bHIVRT Complex, 0 µM dGDP
In this study, bBSA was first assembled on an optical
substrate and the surface was well characterized.
Photophysical studies were then carried out on single
Quantum Dots that were modified with streptavidin and
attached to the biotinylated surface. These studies were
conducted beforehand to establish the optical properties
related to the molecular probe itself.
All studies involved step-wise assembly of surface attached
HIV RT. This was accomplished by first passivating a
borosilicate coverslip with BSA and bBSA, followed by
streptavidin, and finally biotinylated HIV RT. Quantum Dots
attached to dsDNA were then applied and concentrations
dGDP was varied. An intensified charged coupled device
(ICCD) was used to image the binding in these experiments
and the dissociation constants were determined through
direct observation.
One Color Single Molecular Fluorescence
Microscopy Experimental Setup
One Color Single Molecule Fluorescence Microscopy
Instrumental Arrangement
One Color Binding Experiment
Dissociation Constant of QD-bHIVRT Complex, 0 µM dGDP
Surface Characterization Experiments
Biotinylated BSA Surface Characterization
Association Constant of QD-bHIVRT Complex, 300 µM dGDP
Binding Kinetics
Biotinylated BSA Surface Characterization Results
Dissociation Constant of QD-bHIVRT Complex, 300 µM dGDP
Acknowledgements
Future Work
Future work includes compiling association and dissociation
constants at dGDP concentrations between 0 and 300 µM,
varying the concentration of quantum dots, and varying the
temperature at which binding is recorded. Further work may
be conducted on altering the retroviral primer and analyzing
the polymerization of HIV1 RT.
I want to thank DJ Wright for being a solid lab partner,
Angela Rudolph for being a generous mentor, and James
Brozik for being a vital manager of our experiments. This
work was supported by the National Science Foundation’s
REU program under grant number 0851502.
References
Wong L.P., A.R. Rudolph, C.M. Hartshorn, D.J. Keller and J.A. Brozik, DNA
Binding and Polymerization with Tethered HIV RT. Unpublished manuscript,
Washington State University, Pullman, WA.