to investigate biological systems at the
single-molecule level.
INTENDED OUTCOME AND BACKGROUND
The Quantum Physics Division investigates
important biological systems at the
single-molecule level, thus leveraging
our measurement expertise and experience
with atomic and quantum systems.
Accordingly, we are evolving a part of
our research program in this direction to
help NIST contribute to the scientific
revolution taking place in biophysics.
The Division's strengths are the foundation
of our efforts to contribute to
groundbreaking research on single biomolecules.
These strengths include our
ability to build institutional bridges to
renowned university departments, a
superlative infrastructure, experience
in manipulating and measuring atomic
and quantum mechanical systems, and
a reputation that attracts the best and
brightest of today's young scientists.
Our new biophysics program is being
implemented in close collaboration with
the Department of Molecular, Cellular,
and Developmental Biology, and the
Biochemistry Division of the Department
of Chemistry, at the University
of Colorado. By integrating additional
departments into JILA, we will enhance
the very productive, interdisciplinary
character of the institute. Most importantly,
because of our existing expertise,
our biophysics program should rapidly
build on the successes achieved by our
other programs.
Accomplishments
Single-Molecule Fluorescence
Microscopy: Biomolecular
Conformational Dynamics
This biometrology project probes conformational
dynamics (e.g., folding and
unfolding) of biomolecules in chemically
active states. We focus on simplifying
complex RNA structures to understand
the mechanisms that stabilize specific
structural folds. This information is
crucial to understanding RNA-based
enzymes, or ribozymes.
We use ultrasensitive time-, color-, and
polarization-resolved fluorescence detection
of single RNA molecules in a confocal
microscope. A mode-locked laser is
focused into an aqueous, dilute sample
of RNA molecules, with less than a single
molecule in the detection region.
The resulting weak fluorescence is sorted
by both polarization and color, and is
imaged onto single-photon-counting
avalanche photodiodes. The individual
photon events are recorded as a function
of time after the incident laser pulse,
achieving an 11 order-of-magnitude
range of kinetic time scales.
The fast-fluorescence behavior monitors
the local environment of the RNA and
can be extracted via time-correlated, single-
photon counting. Using fluorescence
resonant energy transfer (FRET), we
can measure distances (2 nm to 8 nm)
between specifically labeled sites on the
RNA. This allows us to investigate the
folding kinetics for RNA in real time at
the single-molecule level.
Thus far, studies have required tethering
the RNA to a glass coverslip. To eliminate
possible surface effects, we are
developing methods for studying "free"
RNA by exploiting "burst-mode" single-molecule
microscopy. This technique
detects species diffusing into and out
of the confocal region, but it limits the
time that we can probe single biomolecules.
Consequently, we are also working
on methods to slow down diffusion.
We plan to isolate single RNA molecules
in liposomes, which could be
manipulated by optical tweezers and
viewed microscopically for arbitrary
lengths of time.
Single-Biomolecule
Electrophoresis
We have begun exploring single-molecule
electrophoresis. The apparatus for
these studies is based on wide-field
microscopy through a thin gel-electrophoresis
cell. In this technique,
weak electric fields are used to coax
single DNA molecules through a
micrometer-scale field of view.
The DNA motion can be studied by
labeling it with highly fluorescent dyes
that are illuminated by a laser and
detected by imaging on an intensified
CCD-array camera. This method is sensitive
enough to image at a 10 Hz frame
repetition rate, and allows us to monitor
single-DNA electrophoresis dynamics in
real time.
We can automatically determine the
location of the DNA as a function of
time, and thereby track its progress and
mobility during electrophoresis. These
studies permit direct visualization and
detailed tests of biopolymer percolation
dynamics. In the future, they will help
us to learn more about improving separation
efficiency and the kinetics/dynamics of protein-DNA binding.
The latter are relevant to cell regulatory
processes and would be amenable to
study at the single-molecule level.
Single-Molecule
Measurement with
Nanometer Resolution
Figure 4. Individual 0.4 nm steps measured in
a new differential back-focal-plane detection,
optical-trapping microscope. The fact that the
data points lie on lines demonstrates that
0.1 nm stability is achieved over several seconds.
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We are studying molecular motors at
the level of single molecules. Our efforts
are prompted by the insight that motor
proteins generate measurable force and displacement.
Determining the elementary step size of
motor proteins is crucial to understanding
their mechanisms. The enzymes
involved in DNA replication, DNA
transcription, and RNA translation
work, in a literal sense, as molecular
motors; once bound to their nucleic
acid substrates, they translocate in a
more-or-less unidirectional fashion,
carrying out hundreds to millions of enzymatic cycles.
We have recently developed an instrument
with subnanometer spatial resolution
that has significantly improved the
displacements that optical trapping
experiments can detect. (See Fig. 4.)
Besides studying DNA-based molecular
motors, such as helicases, we are using
our new subnanometer technology to
study transcription factors, which bend
DNA rather than moving along it.
Transcription factors turn on and off
the production of messenger RNA
and thereby protein expression.
We are also starting a new program to
study the unfolding of single molecules
of RNA. This work will bring together
JILA's expertise in laser and precision
measurement with CU's expertise in RNA research.
First strategic focus |
Second strategic focus |
Third strategic focus |
Fourth strategic focus
"Technical Activities 2004" - Table of Contents |
