Studies of Biological Activity in Response to Substrate Mechanical Strain: A Research Project at the Interface of Materials Science and Molecular Biology
Timothy D.
Champion1,
Bryce N. Chaney2, Pamela Hedrick2, Steve
McCarthy2, and Jordan C.
Poler2
1Department
of Natural Sciences, Johnson C. Smith University, Charlotte, NC
28216
2Department
of Chemistry, University of North Carolina at Charlotte, Charlotte, NC
28223
Poster Submitted to CONFCHEM, Spring
2000.
Figure 1: A l-DNA molecule is shown bound via
streptavadin (green) to a gold line on a microscope coverslip. A magnetic bead attached to the other
end provides a source of inducing strain, while restriction endonucleases (red)
are binding to the l-DNA; possibly at sites other than their
normal binding sites.
Introduction
Mechanical
changes in cellular systems resulting from differentiation are obvious. However, the contrary of this process is
not established. We intend to study
the effect of mechanical strain on a substrate DNA during controlled
endonuclease digestion. We think
this simple system (Figure 1) will provide proof of principle for our approach
so that we may examine biological activity in general as a function of
mechanical response.
Previous
studies have demonstrated a method to attach l-DNA
to Au and have studied the deformation of l-DNA
in response to stress (1-3). The
current study reproduces these results and extends these techniques to studies
of biological activity.
Methods and Preliminary Results
The
substrate is made up of l-DNA-X-Sulfur-Au/Cr/Glass
linkage (X has been a biotin-streptavadin-biotin complex; other possibilities
are being investigated). A flow
cell has been constructed using microscope slides and cover slips with the
l-DNA
substrate on the cover slip. Using
a automatic syringe, we have pumped the buffer through the flow cell and
observed the stretching of the bound l-DNA
via epi-fluorescence microscopy.
Additional
work has been done and/or is ongoing in the area of:
·
control
studies of the restriction endonuclease buffer system
·
binding
of the magnetic dynabeads to l-DNA
and their effects on restricion endonuclease digestion of bound l-DNA
·
improved
binding of the l-DNA
to the gold lines and selective binding of a magnetic dynabead to the other end
of the l-DNA.
Undergraduate Education Aspects of the Project
This project is an especially interesting and educational one for
students since it involves concepts and techniques from a broad range of
disciplines (materials science to molecular biology). Some of the techniques used to date are:
vacuum deposition of Cr and Au, photoresist masking, chemical etching, reactive
ion etching, atomic force microscopy, visible microscopy, epi-fluoresence
microscopy, restricion endonuclease digestion, DNA polymerase reaction, gel
electrophoresis and visualization.
Researchers working on the project spend some time in a clean room and
some time in the laboratory.
Regardless of their background, students working on this project are
likely to be engaged in many tasks that are new to them (and some with which
they are more familiar).
References
1. Zimmermann,
R. M. and Cox, E. C. (1994). DNA
stretching on functionalized gold surfaces. Nucleic Acids Research,
22, pp.492-497.
2. Cluzel,
P., et al. (1996). DNA: An
extensible molecule.
Science, 271, pp.792-794.
3. Bensimon,
D., et al. (1995). Stretching DNA with a receeding meniscus: Experiments and
models. Physical Review
Letters, 74, pp. 4754-4757.