Development of a Biomolecular
NanoToolbox: Current research in micropatterning
in the Chilkoti Group has largely focused on the development
and optimization of an ensemble of fabrication techniques
that are compatible with biomolecules. Our research plans
in this area for the near future will focus on the development
of an ensemble of fabrication techniques that are designed
for, and compatible with biomolecules and will enable both
bio and abio components to be precisely positioned in 3-D
with nanometer resolution using both top-down and bottom-up
approaches. This is a wide-open area of research, and I
will simply summarize some of the directions moving in with
illustrative examples of “excavating” “building”
and “capturing” molecules with nanoscale precision
on a surface.
Excavating Biomolecules at the
Nanoscale. The ability to carry out biochemical
reactions catalyzed by enzymes with nanoscale precision
at a surface is an important goal in the development of
bottom-up nanofabrication. We chose enzymes to catalyze
biochemical reactions at a surface with nanoscale spatial
resolution, because: (1) enzymes are the nanoscale factories
of biology, in their ability to catalyze the conversion
of myriad substrates into products; (2) a large number of
enzymes are readily available as off-the-shelf reagents,
so that diverse biochemical manipulations should be possible
by enzyme-driven nanolithography; and (3) enzymes are among
the most widely studied class of biomolecules, so that their
use to catalyze reactions at the nanoscale can benefit from
the detailed structure-function studies of these enzymes
that are available
My group recently took a small step
forward in the realization of this goal by describing proof-of-principle
of an enzyme–catalyzed reaction on an immobilized
substrate –a self-assembled monolayer (SAM) presenting
a terminal oligonucleotide – without the constraint
of using an enzyme that is tethered to an atomic force microscope
(AFM) tip [J.
Am. Chem. Soc. 126: 4770-4771, 2004].
Physisorbed nanopatterns of DNase I deposited by dip-pen
nanolithography (DPN) were shown to locally digest the immobilized
oligonucleotide substrate with nanoscale spatial resolution
at the surface. Circumventing the constraint of having to
couple the enzyme to the AFM tip demonstrated by this study
is important because it will enable nanoscale surface chemistry
to be performed in situ with considerably greater flexibility
and throughput than would be possible by rastering an AFM
tip with a single tethered enzyme across a surface.
“Building-Up”
with Enzymes with Nanoscale Spatial Resolution.
Nucleic acid nanostructures are useful as templates for
generating composite molecular ensembles in materials science,
molecular electronics, and biosensing. Although nucleic
acid modifying enzymes are extensively studied, commercially
available, and widely used in solution reactions, the application
of these biomolecular catalysts in surface-initiated polymerization
of nucleic acids has not been previously exploited. We recently
demonstrated that terminal deoxynucleotidyl transferase,
which repetitively adds mononucleotides to the 3' end of
a short DNA initiator, could be used to rapidly fabricate
DNA nanostructures up to 120 nm high with lateral dimensions
from 0.1 to 4 mm [J.
Am. Chem. Soc. 2005 (127) 14122-14123].
To our knowledge, this is the first example of enzyme-directed
surface initiated polymerization on a surface. In future
wok, we will explore how these DNA nanostructures could
be used to direct the step-wise formation of composite molecular
ensembles consisting of natural or unnatural nucleotides
and serve as a structural component for more complicated
two- or three-dimensional nanostructures. In addition, the
capability to enzymatically extend the programmable DNA
scaffolds makes it possible to selectively dock other molecules
along the z-direction with nanometer-level precision.
Capturing Proteins with
Nanoscale Precision on a Surface. In work that
combines our interest in the design of Bioinspired Materials
and Biointerface Science performed as a collaboration with
Stefan
Zauscher
(Mech. Engr. and Materials.
Sci.) we recently demonstrated that stimulus responsive
fusion proteins could be reversibly bound to a nanoscale
spatial address by exploiting the phase transition behavior
of these proteins. We grafted stimulus responsive elastin-like
polypeptide (ELP) nanostructures onto substituted thiolates
that were patterned onto gold by dip-pen nanolithography
(DPN). The ELP phase transition was exploited to reversibly
immobilize a thioredoxin-ELP (Trx-ELP) fusion protein onto
the ELP nanopattern above the lower critical solution temperature
(LCST). Subsequent binding of an anti-thioredoxin monoclonal
antibody (anti-Trx) showed the biological activity of the
protein nanoarray. The resulting Trx-ELP/anti-Trx complex
was released below the LCST, demonstrating the potential
of using ELP nanostructures for the on-chip capture and
release of as few as several hundred protein molecules in
integrated nanoscale bioanalytical devices. The molecular
mechanism underlying the interactions that enable the nanoscale
capture and release of an ELP fusion protein from a surface
was also investigated by measurement of the height changes
that accompany the binding and desorption steps as well
as by adhesion force measurements using atomic force microscopy
