Transcript Bionano

Bionano
Part 1.
Biocatalytic Synthesis of Polymers with Precisely Defined
Structures, Deming, Conticello & Tirrell, in Nanotechnology,
G. Timp, ed., Springer-Verlag, New York, 1999.
Part 2.
Measuring molecular machines (Stephen Block website).
Part 3 (yet to be completed)
Mixing bio building blocks with synthetic ones (Seth Fraden)
Part 1
In Nanotechnology, ed. Gregory L. Timp, Springer-Verlag, New York, 1999
Uniform polymers
Dendrimer
Merrifield: good for ~60 residues, no
proofreading
Templated synthesis, as in protein biosynthesis.
Details of
protein biosynthesis
Getting E. Coli to
do our work
Why not just let living animals or
plants make the proteins and isolate?
Well, first of all there is the waste management
problem! 40 kilos per day fed to an elephant
produces a mess all its own!
Some proteins cannot be isolated. For example,
the adhesive proteins used by barnacles are
secreted and soon crosslinked, becoming
impossible to isolate. These “glue” proteins
work in seconds, even under water.
Some naturally-occurring research targets
Elastin: protein of arteries and lungs that
lasts a lifetime of stretching and
contracting without degradation.
Elastin is a molecular spring, and can
undergo stretching of 300%.
Viral spike proteins: fiber-like sequences
at surfaces copied and can make liquid
crystalline solutions and fibers with
properties good enough for commercial
use.
Commercial interest
Protein Polymer Technologies
• Silk
• Silk with fibronectin-like patches, useful to coat
glassware used in cell culture. Available in >500g
quantities.
Allied-Signal
• Collagen
DuPont
• Viral spike proteins
Military interest
U.S. Army: coiled coil (probably for fibers of
good tensile and compressive strength)
Making polymers that nature never
intended to simulate structures nature
makes all the time.
Conventional Synthesis of the rigid, helical
rod, PBLG=poly(benzylglutamate)
O
O
n
O
O
+
NH2
N
H
-n CO2
O
O
H
N
H
n
O
O
CH2
NHC6H13
Mw/Mn ~ 1.2
Bioengineering approach to PBLG
• Code the nucleic acid for exact repeats of
glutamic acid
• Include an occasional aspartic acid to prevent
enzymatic cleavage
• Get E. Coli to make poly(glutamic acid)-co(aspartic acid)
• Convert the acid groups to the benzyl ester
Unnatural amino acids,
including fluorinecontaining amino
acids—a genetic
engineering approach
to low surface tension
materials, hydrolytic
stability, solvent
resistance and low
friction.
One-piece protein-based nanodevices
Functional part. The first one to be
tried will mimic a phosphotriesterase
enzyme that can detect and detoxify
nerve toxins and some pesticides.
Self-assembling
base made of lysine
or cysteine residues
to attach to negative
surfaces or gold,
respectively.
Part 2. Another aspect of bionano is
measuring and emulating biomachines.
The following material is taken from the
website of the Stephen Block group at
Stanford.
This group is proficient in “grabbing” a large
nanoparticle (e.g., latex) with light tweezers.
That idea is associated with the name Ashkin
and helped to win a Nobel Prize for Steven
Chu (who initially did nothing related to
polymers).
Kinesin
Cartoon of a kinesin experiment. The
kinesin walks along the microtubule
towards its plus-end, while being
subjected to a retarding force by the
optical trap.
Cartoon of a kinesin experiment. The kinesin walks along the
microtubule towards its plus-end, while being subjected to a
retarding force by the optical trap.
In order to track kinesin motion, we attach the molecules to microscopic beads. Kinesin
itself is much too small to see in the optical microscope, so the beads serve as markers that
can be tracked with very high precision (to 1 nm or better). The beads also act as "handles",
through which we can apply force using an optical trap. Applying tension reduces
Brownian motion of the bead, making it clear that kinesin moves in a stepwise fashion, in
8-nm increments (see below, and Svoboda et al., Nature 1993). For each 8-nm step, kinesin
uses a single fuel molecule, hydrolyzing one ATP molecule into ADP and inorganic
phosphate (Schnitzer and Block, Nature 1997).
Kinesin
Kinesin steps under constant forward load. The position of a kinesin
motor in nanometers versus time at low ATP concentration. In this
graph, the 8-nm steps that kinesin takes along the microtubule can
readily be seen. This step size corresponds to the spacing of the
tubulin dimers that make up the microtubule (Figure from Lang et al.,
Biophys. J. 2002).
Energy Wells in which Kinesin Operates
3D cartoon of the effect of sideways loads. By using a 2D
optical force clamp, we have been able to probe the multidimensional potential energy landscape that governs the kinetics
of kinesin motion.
DNA Helicase—Unwinds ds-DNA
Static representation of a DNA helicase as it unwinds a piece
of double stranded DNA. (Picture from Taekjip Ha, UIUC).
Cartoon of a DNA unzipping experiment. The optical trap
applies a force to the hairpin causing it to unzip.
We are investigating the mechanical properties of
nucleic acids by focusing in particular on hairpins.
These structures consist of single strands of DNA
or RNA whose ends are self-complementary, such
that they loop back on themselves to form a duplex
"stem" connected to a single-stranded loop (inset
below). Hairpins not only provide a model system
for studying DNA unzipping, but are also important
in their own right (for example, they are a principal
motif of secondary structure in RNA, and they play
a role in gene transcription and regulation). The
aim of our research is to understand, at a
fundamental level, the factors that affect the
stability of hairpins. We use a DNA "handle" to
attach a hairpin to a small bead, which we can then
manipulate with optical tweezers. As illustrated in
the figure below, pulling on the bead with the
tweezers applies a force to the hairpin, causing it
eventually to unzip. By measuring the force at
which the hairpin is pulled apart as well as the
amount of time the hairpin spends in open and
closed states at a given time, we are trying to map
out the energetics of the hairpin while
systematically varying parameters such as the neck
length, neck sequence, and buffer conditions.
Simultaneous fluorescence/tweezers
The combination of single molecule fluorescence and optical
tweezers in a hairpin unzipping experiment. The addition of the
fluorescence signal allows us to better locate, both in space and
time, a known structural event relative to an event observed with
the optical tweezers (Figure from Lang et al. J. Biology 2003).
RNA polymerase reading DNA
Single molecule RNA Polymerase
experiment using optical tweezers.
RNAP is attached to a micron-sized polystyrene bead. The bead is then held in an
optical trap equipped with an interference-based detection system. Tension is
applied to the DNA, via an attachment to either the coverslip or a second bead (not
shown). Transcription of RNAP affects the position of the bead.
Jerky motion of RNA polymerase
under tension
Single E. coli RNA polymerase molecule transcribing under force. The position of
the polymerase in base pairs (bp) is shown as a function of time. The motion of the
polymerase is frequently interrupted by pauses of variable duration, between which
the transcriptional velocity is constant.
Watching a nanomotor at work
As the RNAP transcribes along the DNA, the two beads get closer together. Here
is a movie of a single RNAP transcribing along the DNA at 1 mM
ATP,CTP,GTP,UTP sped up 30 times [RNAP Dumbell 30x.avi 178 KB].