The past few years have brought important advances in our ability to
study the structure and dynamics of molecules--the scanning tunneling microscope
and its relatives, new kinds of nuclear magnetic resonance analysis, and
femtosecond-resolution laser chemistry, to name three. More recently, the
pace has quickened: hardly a month now passes without some remarkable new
technique being reported. Let us look at a few of these.
J.M.R. Weaver and colleagues at IBM have developed a method that, in effect,
images materials at optical wavelengths but at 1 nanometer resolution. Impossible,
you say? Not if you cast light on the sample, and detect the light's effects
on individual molecules with a scanning tunneling microscope (STM). In practice,
the sample is illuminated with monochromatic light of a desired wavelength;
some of the light is absorbed by particular atoms or groups of atoms, where
it changes the shapes and positions of the electron clouds surrounding them;
these changes are detected by the STM. The arrival of this technique is
like the arrival of a lamp in a darkened room where formerly objects had
to be studied by touch. Among its many potential applications is the sequencing
of DNA: a judicious choice of wavelength should permit the different nucleotides
to be distinguished by color.
[Nature 342:783-785,14Dec89]
Another new approach to subwavelength optical imaging is called molecular
exciton imaging (MEM). Developed by K. Lieberman at the Hebrew University
in Jerusalem and others, the technique makes use of crystals that store
light energy as excitons--bound pairs of electrons and "holes."
A crystal of anthracene is grown in the tip of a micropipette less than
100 nanometers in diameter. When illuminated from inside the pipette, the
crystal concentrates the light energy and then emits it from the tip of
the pipette as a very compact beam of photons. If a sample is brought to
within a few nanometers of the pipette tip, a photon of suitable wavelength
will be absorbed with almost 100% probability--an increase of about 109
as compared with ordinary light sources. When combined with high resolution
scanning, MEM may lead to optical microscopes capable of resolving molecules.
Furthermore, the technology promises to be very inexpensive.
[Science 247:59-61,5Jan90]
A very different imaging technology has been developed by Z. Vager at the
Weizmann Institute of Science, and others. Called "coulomb explosion
imaging" (CEI), it has already elucidated the structures of molecules
resistant to other analytical methods. The sample to be analyzed is accelerated
to about 2% of the speed of light and made to pass through a plastic film
3 nanometers thick. The film strips all of the bonding electrons from the
molecule, leaving the individual atoms positively charged. These atoms now
repel each other as they continue to travel toward a detector where their
positions and arrival times are recorded. Roughly speaking, the effect is
to magnify the configuration of the molecule at t0, the moment
of passage through the film. Working backward from the recorded data, one
can determine the precise arrangement of the atoms in the molecule at t0;
even the vibrational and rotational displacements are accurately represented.
Since the sample consists of many molecules caught in different phases of
motion at t0, the recorded data contains not just the geometry
of one particular molecule but a full representation of the possible configurations
for the molecular species being studied.
[Science 244:426-431,28Apr89]
It remains to be seen whether CEI can be used to study the structure
of large molecules like enzymes, but even if it can't it promises to revolutionize
our understanding of smaller ones, particularly molecular ions and molecules
in excited states.
Who would have thought that detailed structural information could be gleaned
from the pieces that fly out of a surface after an ion crashes into it?
Nicholas Winograd of Penn State thought so, and he was right. The technique
is called "secondary ion mass spectroscopy" (SIMS), and consists
of directing a beam of ions at the surface of interest, then measuring the
angles and energies of the particles that emerge. Although it is not possible
to use these measurements to compute backward to determine the original
state of the surface, one can compare the measurements to the results calculated
from various theoretical models and then reject the models that give the
wrong answers. SIMS is expected to reveal details of chemical reactions
taking place on surfaces; catalysis is of particular interest.
[Science 246:995-997,24Nov89]
Just think of it: four new techniques... how might they affect progress
toward nanotechnology? The first--STM imaging of photon-stimulated materials--should
greatly simplify the task of identifying molecules and parts of molecules.
In combination with MEM, it may become an efficient optical probe for doing
spectroscopic studies on local regions within molecules. Such a probe might
even enable researchers to make and break specific bonds: STM information
would be used to steer the MEM probe to the desired target, where the probe
would release one or more photons having energies appropriate for controlling
the desired reaction.
CEI promises to bring rapid understanding of the structure and dynamics
of small molecules and molecular fragments--just the sort of understanding
that is needed if the design and construction of nanomachines is to become
more than a hit-or-miss affair.
SIMS should help to elucidate the structure of surfaces, making possible
the rational design of moving parts for nanomachinery.
Molecular motors and flagella are in the news again:
J. Howard at the Univ. of Calif. at San Francisco, and others, have been
studying the kinesin motor proteins that move organelles along microtubules
inside cells (see Update No. 7, Progress). They are developing
methods for measuring the force exerted on a microtubule by a single kinesin
motor, and the amount by which the motor moves along a microtubule during
each stroke.
[Nature 342:154-158,9Nov89]
Bacteria swim by using rotary motors to turn helical filaments extending
from their surfaces. These filaments, called "flagella," are composed
of repeating subunits of the protein "flagellin." A map of a flagellar
filament has now been made at 20 Å resolution by Keiichi Namba and
others of ERATO in Japan. Flagellin molecules, it appears, form a flagellum
by stacking in a helical pattern with approximately 11 subunits per two
turns of the helix. The center of the filament is a hole 60 Å in diameter--thought
to be the channel through which pre-folded flagellin molecules travel during
flagellar assembly. The researchers plan to investigate the mechanisms by
which bacterial flagella change shape in response to chemical and physical
changes such as pH, ionic strength, or the direction of motor rotation.
[Nature 342:648-654,7Dec89]
What might we want to do with flagella? Use them to drill holes? Let
them pull loads along some microscopic byway? Attach special molecules to
their tips and use them as robot arms? Since flagella have evolved as bacterial
propellors, they will likely not have all the right characteristics for
doing any of these things. But recent work with enzymes has shown that it
can be surprisingly easy to re-engineer existing proteins, radically improving
them for given tasks. Bacterial flagella have a lot to offer as starting
points for molecular engineering: they self-assemble, they are equipped
with motors, and their helical parameters can be controlled by external
stimuli.
The road to nanometer-sized diodes appears to be open. At IBM's T.J. Watson
Research Center, In-Whan Lyo and colleagues have demonstrated negative differential
resistance (NDR) in sites this small on treated silicon surfaces. NDR is
the essential property that allows fast switching in quantum-well devices
and Esaki diodes. The investigators used a scanning tunneling microscope
to create a tunneling current between the STM tip and a silicon surface
containing isolated boron atoms as defects. NDR appeared when the tip was
located over such defects.
One of the great themes of the 21st Century, in my opinion, will be the
generalizing of traditional biological motifs. We are already seeing the
early harbingers: artificial hearts, mice with human immune systems, bacteria
that can produce plastic, cotton with bacterial genes for insect resistance.
But in the laboratory, more fundamental generalizations are already underway.
Let us look now at three exciting examples.
An enzyme is a molecule (or molecular complex) that accelerates a chemical
reaction by binding the reactant(s) into positions and circumstances that
make the reaction more probable. Biological enzymes are generally proteins,
but nonprotein enzymes can (and have) been made that are much smaller and
simpler; until now these have been designed for reactions involving only
one reactant. T. Ross Kelly and others at Boston College have now constructed
a rudimentary nonprotein enzyme that binds two reactants, fosters
the formation of an amide bond between them, then releases the product back
into solution. The binding is accomplished by patterns of hydrogen bonds
between groups on the enzyme and matching groups on the intended substrate
molecules. Having established that the enzyme works, Kelly's group now intends
to alter the reaction rate by fiddling with the geometry of the system and
to design enzymes for other kinds of reactions.
[J. Am. Chem. Soc. 111(10):3744-3745,1989]
About 20 kinds of amino acids make up the vast array of traditional proteins
that play so many roles in the biological world. Why only 20? Because every
cell must either contain the machinery for making each such amino acid or
have a 100% reliable source of it. So there is an advantage in keeping the
number low, even though a larger number might be much better from an engineering
point of view. Human technology, however, is under no such constraints.
Hence, we find that Christopher J. Noren and his colleagues at the Univ.
of Calif. at Berkeley have developed a general method for getting bacteria
to make proteins that include nonstandard amino acids. Their strategy makes
use of the codon TAG--a triplet of DNA bases that normally stops protein
synthesis when encountered by a cell during the translation of DNA, because
it corresponds to no amino acid. Noren's group prepared a special transfer-RNA
molecule by attaching an amino acid of their own choosing to a transfer-RNA
bearing a recognition site for the TAG codon. They also prepared a mutant
DNA gene for the protein they wanted to make by putting the codon TAG at
a place in the DNA corresponding to the place in the protein chain where
they wanted their special amino acid to be. When this DNA was used as the
program for protein synthesis, the desired protein was produced.
[Science 244:182-188,14Apr89]
Nonstandard proteins should be of great use in studies of protein structure
and function. The method's principal limitation stems from its dependence
on traditionally unused codons--since there are only three of these, and
one is needed as a stop signal, only two novel amino acid type can be used
in a given protein.
The traditional "genetic alphabet" of DNA has only 4 "letters"--A,
T, C, and G--representing the four nucleotides from which DNA molecules
are composed. Joseph A. Piccirilli and others at Zurich's Laboratory for
Organic Chemistry have now added at least two new letters: kappa and pi.
Starting with a larger collection of candidate base-pairs, the researchers
subjected each to tests of stability and acceptability to DNA and RNA polymerases
(the biological proteins responsible for replication). Kappa and pi emerged
as winners--they pair with each other and not with A, T, C, or G; and they
are recognized and dealt with by DNA polymerases almost as well as are A,
T, C, and G.
[Nature 343:33-37,4Jan90]
A genetic code like Earth's leads to a Rube-Goldberg biosphere--most
of the active machinery (proteins) has to be built from only a few types
of components (amino acids). An amino acid is specified by a triplet of
letters taken from a 4-letter alphabet; thus, Earth's genetic code is limited
to specifying at most 64 kinds of amino acids (actually 63, since one triplet
is needed as a stop signal). In practice, the need for redundancy has reduced
this number to 20.
We would have at least 48 new amino acids to work with in
a given organism
A 6-letter genetic code would increase the theoretical number
of amino acids to 216 (i.e., 63); the useable number would be
about 68 if present levels of redundancy are retained. Assuming that the
existing 4-letter code is kept as a subset for "upward compatibility,"
we would have at least 48 new amino acids to work with in each organism.
Effective use of an extended genetic code requires the development of
a set of transfer-RNAs to specify the translation of the new triplets, and
a set of synthetases to load these transfer-RNAs with the new amino acids.
This is a major undertaking and will not be accomplished overnight.
The most obvious application of an extended genetic code would be to
simplify existing proteins by replacing sections of their protein chains
by shorter chains containing nonstandard amino acids. Similarly, one might
improve the stability, specificity, or activity of enzymes. Carrying this
strategy a little further might lead to endowing proteins with novel properties
not achievable with standard amino acids. Such improved proteins would be
developed as industrial catalysts, new materials, research tools, and the
like.
Another interesting application would be in ensuring the safety of engineered,
self-replicating organisms. An organism that meets the following three criteria
could not survive without being fed by its employer: (1) some of the organism's
essential proteins require nonstandard amino acids; (2) the organism lacks
the apparatus needed to synthesize these amino acids; (3) these amino acids
are not found in the environment. See Engines of Creation
for discussion of an analogous concept for nanoreplicators.
If an era of multiple, mutually incompatible genetic codes lies ahead
then there are profound philosophical and historical implications to be
discussed.... but not in this column.
Dr. Mills's background is in biophysics; he is currently a businessman
and a volunteer at the Foresight Institute.
John Chiplin of Biosym: "The Conference brought together a fascinating
collection of people. The presentations relevant to the molecular CAD field
actively represented the current state-of-play and also the future challenges
that lie ahead for us--particularly in the protein/structure field. I look
forward to future meetings."
Michael Ward of Du Pont: "In addition to being the most well organized
meeting I have attended, I found it to be one of the most stimulating as
well."
Prof. Josef Michl of University of Texas at Austin, Dept. of Chemistry:
"It was marvelous to have an opportunity to meet people in related
fields and to listen to what they have to say."
A sample of the comments from the conference evaluation forms:
Best aspect of the meeting: "Broad, high-quality technical
presentations, superb organization." "The quality of the attendees."
"Outstanding speakers and coherence among subjects." "Broad
range of areas described by leaders in the field." "Cast of stars--so
many top people." "Interdisciplinary contact." "Informal
discussions." "Heterogeneity of participants." "Open
discussion--informality." "Diversity." "Breadth of coverage."
"Good mix of scientific/technical disciplines." "Caliber
of speakers and guests." "The speakers acknowledged the diversity
of backgrounds and started from basics." "Extensive opportunities
to interact informally." "Very thought provoking" "Success
in bringing together people of different disciplines for serious discussion
of nanotechnology." "Clearly a meeting of quality people who wouldn't
otherwise meet each other easily." "Small enough to mix and mingle."
"Good overview. Emphasis of interdisciplinary aspects." "Legitimized,
for me, the field of nanotechnology."
Worst aspect of the meeting: "Need better meeting
rooms." "Visibility of screen from side seating." "Inadequate
time for informal discussion toward the end of the meeting." "Program
too long." "Too short!" "Expensive!" "I ate
too much. The food was too good."
