Credit: Zhengyi Zhao, University of Kentucky

One of nature’s many types of molecular motors combines protein and RNA subunits to force viral (in this case, bacteriophage) DNA into a protein capsid. The understanding of the molecular mechanism by which this motor works has been advanced by the discovery that it revolves the DNA around a central channel, as the Earth revolves around the sun, rather than by rotating, as the Earth does about it axis. A hat tip to ScienceDaily for pointing to this research. From a University of Kentucky news release “Guo lab discovers new class of revolution biomotor and solves mystery in viral DNA packaging:”

Scientists at the University of Kentucky have cracked a 35-year-old mystery about the workings of natural “biomotors.” These molecular machines are serving as models for development of synthetic nanomotors that will someday pump therapeutic DNA, RNA or drugs into individual diseased cells.

The report, revealing the innermost mechanisms of these motors in a bacteria-killing virus and a “new way to move DNA through cells,” is being published online today in the journal ACS Nano.

The article, “Mechanism of One-Way Traffic of Hexameric Phi29 DNA Packaging Motor with Four Electropositive Relaying Layers Facilitating Anti-Parallel Revolution,” can be downloaded with free, open access from http://pubs.acs.org/doi/abs/10.1021/nn4002775.

Peixuan Guo, director of the UK Nanobiotechnology Center, and his colleagues explain that two motors have been found in nature: A linear motor and a rotating motor. Now they report discovery of a third type, a revolving molecular motor.

Guo points out that nanomotors will open the door to practical machines and other nanotechnology devices so small that 100,000 would fit across the width of a human hair. One major natural prototype for those development efforts has been the motor that packages DNA into the shell of bacteriophage phi29, a virus that infects and kills bacteria.

Guo’s own research team wants to embed a synthetic version of that motor into nanomedical devices that are injected into the body, travel to diseased cells and pump in medication. A major barrier in doing so has been uncertainty and controversy about exactly how the phi29 motor moves. Scientists thought that it worked by rotating or spinning in the same motion as the Earth turning once every 24 hours upon its own axis.

In their ACS Nano paper, Guo — with his team, Zhengyi Zhao, Emil Khisamutdinov, and Chad Schwartz — challenge that idea. Indeed, they discovered that the phi29 motor moves DNA without any rotational motion. The motor moves DNA with a revolving in the same motion as the Earth revolving around the sun in one orbit ever 365 days. The “revolution without rotation” model could resolve a big conundrum troubling the past 35 years of painstaking investigation of the mechanism of these viral DNA packaging motors, the report states.

Near-term application of artificial molecular motors based on this work are not difficult to imagine, such as in drug delivery or gene delivery for nanomedicine. Could motors like these be useful for more complicated molecular machine systems, such as running pulleys using DNA cables to transport components in primitive molecular manufacturing systems?
—James Lewis, PhD

(Credit: Miriam Wilson)

Nature has evolved a partial system of molecular manufacturing. Although not capable of making arbitrarily complex, atomically precise structures that faithfully represent the immense diversity of chemically feasible structures, biology very early on evolved a method of assembling a wide range of molecular machines by linking particular types of subunit molecules together in a defined sequence. The structure that makes the most complex biological molecular machines is the ribosome, a complex of two subunits 20 to 30 nm in diameter and comprising several dozen RNA and protein molecules, which works with hundreds of other RNA and protein molecules to link amino acids together as specified by genetic information to make specific protein molecules. Scientists in the UK have succeeded in mimicking this basic process using a simple artificial molecular machine bearing no resemblance to the ribosome and an order of magnitude smaller in linear dimension than the ribosome. A hat tip to Phys.org for reprinting this University of Manchester news release “Molecular machine could hold key to more efficient manufacturing“:

An industrial revolution on a minute scale is taking place in laboratories at The University of Manchester with the development of a highly complex machine that mimics how molecules are made in nature.

The artificial molecular machine developed by Professor David Leigh FRS and his team in the School of Chemistry is the most advanced molecular machine of its type in the world. Its development has been published in the journal Science [abstract].

Professor Leigh explains: “The development of this machine which uses molecules to make molecules in a synthetic process is similar to the robotic assembly line in car plants. Such machines could ultimately lead to the process of making molecules becoming much more efficient and cost effective. This will benefit all sorts of manufacturing areas as many manmade products begin at a molecular level. For example, we’re currently modifying our machine to make drugs such as penicillin.” …

Professor Leigh’s machine is based on the ribosome. It features a functionalized nanometre-sized ring that moves along a molecular track, picking up building blocks located on the path and connecting them together in a specific order to synthesize the desired new molecule.

First the ring is threaded onto a molecular strand using copper ions to direct the assembly process. Then a “reactive arm” is attached to the rest of the machine and it starts to operate. The ring moves up and down the strand until its path is blocked by a bulky group. The reactive arm then detaches the obstruction from the track and passes it to another site on the machine, regenerating the active site on the arm. The ring is then free to move further along the strand until its path is obstructed by the next building block. This, in turn, is removed and passed to the elongation site on the ring, thus building up a new molecular structure on the ring. Once all the building blocks are removed from the track, the ring de-threads and the synthesis is over.

Professor Leigh says the current prototype is still far from being as efficient as the ribosome: “The ribosome can put together 20 building blocks a second until up to 150 are linked. So far we have only used our machine to link together 4 blocks and it takes 12 hours to connect each block. But you can massively parallel the assembly process: We are already using a million million million (10¹⁸) of these machines working in parallel in the laboratory to build molecules.”

Professor Leigh continues: “The next step is to start using the machine to make sophisticated molecules with more building blocks. The potential is for it to be able to make molecules that have never been seen before. They’re not made in nature and can’t be made synthetically because of the processes currently used. This is a very exciting possibility for the future.”

An animation illustrating how the molecular machine works is available on YouTube. Professor Leigh was the winner of the 2007 Foresight Institute Feynman Prize in Theory for the design and synthesis of artificial molecular machines.

The small molecular machine in this work is based upon a rotaxane molecule, in which a dumbbell-shaped molecule is threaded through a macrocycle. The dumbbell is constructed from two components: a “stopper”, and a “strand”, along which the amino acids to be added are linked. A chemical moiety that will link with the stopper defines the front of the strand. The order of the amino acid subunits in the peptide molecule to be manufactured is determined by the order in which the reactive building blocks are positioned along the molecular strand, with the first one nearest the front, and the last at the opposite end of the strand. Following the practice for solid state chemical synthesis of peptides pioneered by Bruce Merrifield half a century ago, the amino groups of the amino acids are protected with a chemical group that keeps them from reacting prematurely. The third component of the rotaxane is the macrocycle, which has a site at which the reactive arm of the molecular machine will be attached.

Three amino acids are linked to the strand, separated from each other by rigid spacers: phenylalanine at the front, then leucine, then alanine. As with conventional solid state peptide synthesis, the peptide will be synthesized from the C-terminal to the N-terminal end, the opposite of direction of synthesis on the ribosome. The rotaxane is assembled in a chemical reaction catalyzed by copper ions in which the macrocycle is guided over the front of the strand as the strand is linked to the stopper. The macrocycle is held in place, at the point where the stopper and strand join, by a bulky substituent on the outside of the stopper, which forms one end of the dumbbell, and by the first amino acid (phenylalanine) on the strand, which forms the other end.

A second chemical reaction added to the macrocycle the reactive arm responsible for manufacturing the peptide. The reactive arm is a tripeptide (GlyGlyCys) attached to the macrocycle by its C-terminal cysteine residue. The thiolate group of the cysteine and the N-terminus of the tripeptide are both blocked by chemical protective groups to render them non-reactive. The machine is activated by acid-catalyzed removal of the protective groups from the thiolate, from the amino terminus of the reactive arm, and from the amino groups of the three amino acids on the strand.

The liberated thiolate is now able to attack the bond linking the C-terminus of phenylalanine to the strand (the other two amino acids on the strand are too distant to react), thus freeing phenylalanine from the strand, linking it to the N-terminus of reactive arm tripeptide, and regenerating the thiolate. The reactive arm is now longer by one amino acid at its N-terminus, and the macrocycle is now free to slide along the strand until it encounters the second amino acid. The authors explain that the glycylglycine was added to the N-terminus of the cysteine residue because the thiolate and the N-terminus of the cysteine are too close to each other for the phenylalanine released from the strand to be added to the N-terminus of the cysteine. The peptide formed is thus, from the N- toward the C-terminus, PheGlyGlyCys.

The process repeats twice more until all three amino acids have been removed from the strand and attached to the N-terminus of the peptide growing on the reactive arm. With no amino acids left on the strand, the macrocycle is free to slide off the end, bearing the newly formed hexapeptide: AlaLeuPheGlyGlyCys.

Clearly this novel artificial molecular machine was able to mimic essential features of ribosomal protein synthesis, as intended. Once the reaction was initiated, the synthesis was completed without further manipulation. As noted by the authors, major limitations include the very slow rate of synthesis, the destruction of the template after the synthesis is completed, and that the size of the peptide produced my be limited by the nature of the transition state of the machine during each cycle. Nevertheless, this work has demonstrated:

… that relatively small, highly modular, artificial molecular machines can be designed to autonomously perform iterative tasks in synthesis. The principles employed in the design and operation of 1 should be broadly applicable to other types of monomer and chemical reactions …

From the standpoint of the eventual development of molecular manufacturing, the important question will be whether or not this type of molecular machine can be extended to do more general positional control of chemical synthesis, or will it be limited to linking monomers together to form various types of folding polymers?
—James Lewis, PhD

This illustration shows the structure of the molecular motors. (Credit: Saw-Wai Hla)

An international team of scientists has taken the next step in creating nanoscale machines by designing a multi-component molecular motor that can be moved clockwise and counterclockwise.

Although researchers can rotate or switch individual molecules on and off, the new study is the first to create a stand-alone molecular motor that has multiple parts, said Saw-Wai Hla, an Ohio University professor of physics and astronomy who led the study with Christian Joachim of A*Star in Singapore and CEMES/CNRS in France and Gwenael Rapenne of CEMES/CNRS.

It’s an essential step in creating nanoscale devices—quantum machines that operate on different laws of physics than classical machines—that scientists envision could be used for everything from powering quantum computers to sweeping away blood clots in arteries.

In the study, published in Nature Nanotechnology [abstract], the scientists demonstrated that they could control the motion of the motor with energy generated by electrons from a scanning tunneling microscope tip. The motor is about 2 nanometers in length and 1 nanometer high and was constructed on a gold crystal surface. [Watch an animation of the motor at the journal's website here. Credit: F. Ample and C. Joachim. (Scroll down to "Supplementary information" for links to the two animations.)]

At a temperature of minus 315 degrees Fahrenheit, the motor could move independently through thermal excitation. When scientists cooled the sample to minus 450 degrees, the motor stopped rotating. The researchers selectively applied electron energy to different parts of the motor to prompt it to move clockwise and counterclockwise.

“If we want to build an actual device based on this motor, we would install electrodes on the surface to create an energy source,” Hla said.

To construct the molecular motor, the scientific team designed a stationary base of atoms that is connected to an upper moving part by one atom of ruthenium, which serves as the “ball bearing.” The upper piece of the motor features five arms made of iron atoms. The researchers made one arm shorter than the others to be able to track the motion of the machine. The entire device is held upright by using sulfur as an “atomic glue” to secure the motor to the gold surface, Hla explained.

The scientists now plan to use this model to build more complex machines with components that could be automated, Hla said.

One of the three corresponding authors is Christian Joachim, winner of the 2005 Foresight Nanotech Institute Feynman Prize for Theoretical work and one of three winners of the 1997 Foresight Nanotech Institute Feynman Prize for Experimental work.

The molecular motor consists of a single ruthenium atom bearing connecting a tripodal stator to a five-arm rotor. to help detect the motion of the rotor, the five arms are not all equivalent. The centner of the rotor is a cyclopentadiene, one short arms is merely a toluene, while the other four long arms each comprise a phenyl group connected to a ferrocene (two cyclopentadienyl rings sandwiching an iron atom). The ferrocene groups act as reversible electron relays. The four ferrocene end groups enable control of rotation by four nanoelectrodes. The tripodal stator comprises a boron atom connected to three indazolyl groups, each with a thioether foot that binds via a sulfur atom to the gold surface upon which the motor sits.

The motor was imaged using a scanning tunneling microscope (STM) under ultra high vacuum conditions (UHV). For some samples a large bias voltage pulse was used to remove the top (rotor) part of the motor, revealing the tripod stator positioned with each leg along a [211] surface direction of the Au(111) surface. At 80 K the image of the intact motor shows a uniform circle due to the free rotation of the rotor. At 4.6 K rotation has been stopped so that the asymmetric five-lobe structure of the rotor is visible, with four long arms and one short arm.

After positioning the tungsten STM tip above the center of the rotor and then gradually increasing the voltage, rotation was observed at 0.6 V due to inelastic electron tunneling energy transfer. Stepwise rotation was achieved at 4.6 K by placing the STM tip over the molecule at a fixed height for an excitation period of less than one second. The direction of rotation was determined by where the STM tip was placed: predominantly clockwise if electrons were passing through the truncated arm, and mostly counterclockwise if electron excitation was via a ferrocene arm. The authors point out that the tripodal stator lifts the rotor high enough about the gold surface so that the surface does not influence the rotation. They present semi-empirical and density functional theory calculations to explain the detailed rotational mechanisms of the motor. The results show that asymmetries in rotational energy potential lead to the observed rotations by selective excitation of different subunits of the motor. They believe this understanding “will further accelerate the development of complex and automated nanomachinery that can be operated on a material surface.”

The combination of a multicomponent molecular motor and detailed theoretical analysis to elucidate the mechanisms responsible for the action of the motor raises the hope that this work will indeed further accelerate development of complex automated molecular machinery.
—James Lewis, PhD

The principle of contraction and extension of a telescopic polymer chain based on the supramolecular association of thousands of nano-machines. (Credit: Wiley-VCH Verlag GmbH & Co.KGaA. and CNRS)

Biology uses various types of molecular machines to produce movement, all of which are candidates to be mimicked for use in nanotechnology. Muscles produce movement through the contraction of systems of polymers, powered by the release of chemical energy. Now scientists from France’s CNRS have developed an artificial muscle that produces micrometer-scale movement through the coordinated action of thousands of individual molecular machines each producing nanometer-scale movement. A hat tip to Gene Ostrovsky at MedGadget for a story on this CNRS press release “Assembly of nano-machines mimics human muscle“:

For the first time, an assembly of thousands of nano-machines capable of producing a coordinated contraction movement extending up to around ten micrometers, like the movements of muscular fibers, has been synthesized by a CNRS team from the Institut Charles Sadron. This innovative work, headed by Nicolas Giuseppone, professor at the Université de Strasbourg, and involving researchers from the Laboratoire de Matière et Systèmes Complexes (CNRS/Université Paris Diderot), provides an experimental validation of a biomimetic approach that has been conceptualized for some years in the field of nanosciences. This discovery opens up perspectives for a multitude of applications in robotics, in nanotechnology for the storage of information, in the medical field for the synthesis of artificial muscles or in the design of other materials incorporating nano-machines (endowed with novel mechanical properties). This work has been published in the on-line version of the journal Angewandte Chemie International Edition [abstract].

… Even though synthetic chemists have made dazzling progress over the last few years in the manufacture of artificial nano-machines (the mechanical properties of which are of increasing interest for research and industry), the coordination of several of these machines in space and in time hitherto remained an unresolved problem.

Not anymore: for the first time, Giuseppone’s team has succeeded in synthesizing long polymer chains incorporating, via supramolecular bonds (1), thousands of nano-machines each capable of producing linear telescopic motion of around one nanometer. Under the influence of pH, their simultaneous movements allow the whole polymer chain to contract or extend over about 10 micrometers, thereby amplifying the movement by a factor of 10,000, along the same principles as those used by muscular tissues.

… These results, obtained using a biomimetic approach, could lead to numerous applications for the design of artificial muscles, micro-robots or the development of new materials incorporating nano-machines endowed with novel multi-scale mechanical properties.

UT Dallas researchers have made artificial muscles from carbon nanotube yarns that have been infiltrated with paraffin wax and twisted until coils form along their length. (Credit: UT Dallas)

A different flavor of nanotechnology involving nanostructures that are not defined to atomic precision has produced a different type of artificial muscle that looks very robust in that it does not require changing solutions of electrolytes, responds very quickly, and is far stronger than human muscles of the same size. These muscles are composed of a yarn of carbon nanotubes filled with wax. The research was published in Science [abstract]. A hat tip to KurzweilAI for reprinting this news release from UT Dallas “Wax-Filled Nanotech Yarn Behaves Like Super-Strong Muscle“:

New artificial muscles made from nanotech yarns and infused with paraffin wax can lift more than 100,000 times their own weight and generate 85 times more mechanical power than the same size natural muscle, according to scientists at The University of Texas at Dallas and their international team from Australia, China, South Korea, Canada and Brazil.

The artificial muscles are yarns constructed from carbon nanotubes, which are seamless, hollow cylinders made from the same type of graphite layers found in the core of ordinary pencils. Individual nanotubes can be 10,000 times smaller than the diameter of a human hair, yet pound-for-pound, can be 100 times stronger than steel.

“The artificial muscles that we’ve developed can provide large, ultrafast contractions to lift weights that are 200 times heavier than possible for a natural muscle of the same size,” said Dr. Ray Baughman, team leader, Robert A. Welch Professor of Chemistry and director of the Alan G. MacDiarmid NanoTech Institute at UT Dallas. “While we are excited about near-term applications possibilities, these artificial muscles are presently unsuitable for directly replacing muscles in the human body.”

Described in a study published in the Nov. 16 issue of the journal Science, the new artificial muscles are made by infiltrating a volume-changing “guest,” such as the paraffin wax used for candles, into twisted yarn made of carbon nanotubes. Heating the wax-filled yarn, either electrically or using a flash of light, causes the wax to expand, the yarn volume to increase, and the yarn length to contract.

The combination of yarn volume increase with yarn length decrease results from the helical structure produced by twisting the yarn. A child’s finger cuff toy, which is designed to trap a person’s fingers in both ends of a helically woven cylinder, has an analogous action. To escape, one must push the fingers together, which contracts the tube’s length and expands its volume and diameter.

“Because of their simplicity and high performance, these yarn muscles could be used for such diverse applications as robots, catheters for minimally invasive surgery, micromotors, mixers for microfluidic circuits, tunable optical systems, microvalves, positioners and even toys,” Baughman said.

Muscle contraction – also called actuation – can be ultrafast, occurring in 25-thousandths of a second. Including times for both actuation and reversal of actuation, the researchers demonstrated a contractile power density of 4.2 kW/kg, which is four times the power-to-weight ratio of common internal combustion engines. …

“The remarkable performance of our yarn muscle and our present ability to fabricate kilometer-length yarns suggest the feasibility of early commercialization as small actuators comprising centimeter-scale yarn length,” Baughman said. “The more difficult challenge is in upscaling our single-yarn actuators to large actuators in which hundreds or thousands of individual yarn muscles operate in parallel.”

Additional coverage, including a video of these micro-muscles in action, is included in Science News coverage by Sarah C. P. Williams “Wax-Filled Nanotubes Flex Their Muscles“. These two very different approaches to providing nanoscale motion that can scale to microscale or even macroscale robotic motion have different features that will probably suit them to different applications. It will be interesting to see if any applications are developed that will be relevant to nanoscale atomically precise manufacturing.
—James Lewis, PhD

… the question biology has to face is: what is the optimal bond strength for a given mechanical function? This issue is tackled by Henry Hess of Columbia University, US, in a paper that is stimulating fresh thinking about molecular machines [abstract]. Consider a kinesin motor protein ‘walking’ along a tubulin track. The objective is to transfer impulse from the protein’s motor stroke – a conformational change driven by hydrolysis of adenosine triphospate – to the protein–tubule interface, propelling the molecule forward. Hess compares it to a car (kinesin) stuck in mud (tubulin). Anyone who has ever faced this situation knows how delicately the coupling must be managed, by engaging the clutch to just the right degree. Too much and the wheel just spins: the bond snaps. Too little, and the wheel’s coupling to the engine is insufficient to generate movement. The optimal point is found where the wheel–mud adhesion is just about to cease.

… Hess shows that as the load on a bond is increased, the transfer of impulse across the bond has a peak. The position of this peak depends on the distance to the transition state for bond rupture along the reaction coordinate. In other words, here is a design criterion for the ideal molecular machine that transfers energy during reversible binding: the bond should be just strong enough to be likely to survive during the power stroke. …

Proposals of how to advance from current nanotechnology to atomically precise manufacturing (see, for example, the Technology Roadmap for Productive Nanosystems) embody a range of proposals for different stages of development, from biological molecular machines based on networks of weak noncovalent bonds, to nanoscale versions of macroscopic machines constructed from hard materials like diamond comprising dense networks of strong covalent bonds. An important question to be clarified is how (or if) the design rules for molecular machine systems change at various points along this continuum.
—James Lewis, PhD

January 11-13, 2013
Crowne Plaza Cabana Hotel, Palo Alto, CA USA

Over 30 speakers will present reviews and research on a wide variety of groundbreaking atomic- and molecular-scale science and technology, interesting intrinsically and for aiding the development of atomically precise technologies, devices and materials. Events will include an opening reception with a special panel discussion on Friday night and the Feynman Prize Awards Banquet on Saturday night.

Conference Co-Chairs
J. Fraser Stoddart, Board of Trustees Professor, Northwestern University
Larry S. Millstein, President, Foresight Institute

Conference Sponsors
The Thiel Foundation
Autodesk

Sessions
Atomic Scale Devices
Chair: John Randall – President, Zyvex Labs
Molecular Machines & Non-Equilibrium Processes
Chair: J. Fraser Stoddart – Board of Trustees Professor of Chemistry, Northwestern University
Self-Organizing & Adaptive Systems
Chair: Lee Cronin – Gardiner Chair of Chemistry, Glasgow University
Commercially Implemented Single Molecule Technologies
Chair: Steve Turner – Founder/CTO, Pacific Biosciences
Computation and Molecular Nanotechnology
Chair: Alexander Wissner-Gross – Harvard and MIT Media Lab

Look for further details on the conference, the speakers and the events in the coming weeks and months. Registration will open in late July or early August.

At macro-/meso-scale, an additional perturbation with long-range time correlation is required for unidirectional transport in asymmetric systems. However, at nanoscale, because thermal noise has a significantly long autocorrelation time, unidirectional transport is feasible in asymmetric systems, even in the situation that thermal noise is the only perturbation. In such a situation, extra energy is required to sustain the asymmetry of the system against thermal noise. (Credit: © Science China Press)

A fundamental advance in the theoretical understanding of the role of thermal noise in molecular motors has been made by Chinese scientists. From EurekAlert, this news release from Science in China Press “Thermal noise molecular ratchet mechanism found by researchers in the Chinese Academy of Sciences“:

Designing machines that can be driven by thermal noise is a dream for scientists. In 1912, Smoluchowski presented a gedankenexperiment consisting of an asymmetric ratchet with a pawl that could harness work from thermal noise but the concept was disproved. In 1997, Kelly et al. experimentally designed a molecule and observed spontaneous unidirectional rotations of the molecule (Angew. Chem. Int. Ed. Engl. 36, 1866 (1997)). Later, in a paper entitled “Tilting at Windmills? The Second Law Survives” Davies argued that this observation did not challenge the second law of thermodynamics because the spontaneous unidirectional rotations happened only within a limited angle (Angew. Chem. Int. Ed. 37, 909 (1998)). In 2007, Fang et al. theoretically proposed a charge-driven molecular water pump, in which water spontaneously flows from one side to the other through a nanochannel with asymmetrically distributed charges that are adjacent to the nanochannel (Nat Nanotechnol. 2, 709 (2007)). This pump has been empirically queried, from the viewpoint of whether the second law of thermodynamics holds. Recently, Professor Fang Haiping and his group from the Shanghai Institute of Applied Physics, Chinese Academy of Sciences theoretically showed that asymmetric transport is feasible in nanoscale systems experiencing thermal noise, without the presence of external fluctuations. The key to this theoretical advance is the recognition that thermal noise, previously considered to be white noise, is not white at the nanoscale, i.e., the autocorrelation time of thermal noise becomes significantly long in nanoscale systems. Their work, entitled “Asymmetric transportation induced by thermal noise at the nanoscale”, was published in SCIENCE CHINA Physics, Mechanics & Astronomy. 2012, doi: 10.1007/s11433-012-4695-8.

Smoluchowski’s gedankenexperiment in 1912 is widely considered as the first identifiable contribution to ratchet theory. Later, Feynman recapitulated and extended this device in his lectures (The Feynman Lectures on Physics Vol. 1, Chapter 46). Ratchet theory for meso- and macroscopic systems was established based on this idea, in which long-range time correlation of the perturbation and broken spatial inversion symmetry in the systems are two necessary conditions for biased motion (Europhys. Lett. 28 459 (1994)). In this theory, thermal noise is regarded as white noise, and white noise alone cannot result in biased motion in an asymmetric meso-/macroscopic system.

However, down at the nanoscale, is thermal noise still unable to induce biased motion in asymmetric nanoscale systems, as is the case in meso- or macroscopic systems? If we stick to the traditional ratchet theory, thermal noise alone cannot induce biased motion in asymmetric systems because thermal noise is treated as white noise (zero autocorrelation time). Actually, thermal noise cannot be regarded as white noise for nanoscale systems, because at room temperature, the time duration between two collisions of molecules is on the order of 10 to 100 picoseconds, which, although negligibly small at a meso- or macroscopic view, is significantly large at the nanoscale. Based on a very simple model at nanoscale, Prof. Fang and his group show the feasibility of unidirectional transport in nanoscale systems with thermal noise by considering the finite autocorrelation time of the thermal noise. Although the autocorrelation time of thermal noise has not been measured directly by experiment, they find that the thermal noise in bulk water at room temperature has an autocorrelation time of the order of 10 ps from molecular dynamics (MD) simulations.

Now, one may argue that such a nanoscale system with an asymmetric structure comprises a perpetual motion machine of the second kind, because the biased motion is induced from a single heat bath. Prof. Fang said: “At nanoscale, we cannot keep the asymmetry of the systems against thermal motion without any input of extra energy. This is different from the situation of meso- and macroscopic systems, in which the length scale of the asymmetry is large enough to ignore the thermal motion. Thus, the biased motion driven by the thermal noise in the asymmetric nanoscale systems accompany with the extra energy-input to maintain the asymmetry does not violate the second law of thermodynamics.”

This work provides a seminal contribution to the study of the unique behavior of nanoscale systems, which usually have spatial inversion asymmetry. It is expected to be of fundamental importance in the understanding and prediction of the behaviors of nanoscale systems, including molecular motors. The results should be of interest to a wide-ranging community of scientists in the fields of physics, chemistry, biology, and nanotechnologies. However, experiments are called for to determine the distribution of the autocorrelation time of thermal noise and more studies are required on energy transformations in such nanoscale processes.

The simple take-home lesson from this is from the last sentence of the researchers’ abstract: “Our observation does not violate the second law of thermodynamics, since at the nanoscale, extra energy is required to keep the asymmetric structure against thermal fluctuations.” [Free Fulltext Article] Advances in the theoretical understanding of molecular ratchets are very welcome because a major issue in designing artificial molecular machines is whether in a particular case it should take advantage of Brownian motion, as do biological molecular machines, or deterministic motions transmitted by stiff molecular components, as in nanofactory designs.
—James Lewis, PhD

Floyd E. Romesberg, associate professor at Scripps Research Institute (Credit: The Scripps Research Institute)

Back in 1992, in chapter 15 of Nanosystems, Eric Drexler suggested that it would be easier to design proteins that fold predictably, an important step on the road to advanced nanotechnology (or molecular manufacturing, or atomically precise manufacturing) if additional amino acids beyond the 20 that are genetically coded could be incorporated into proteins. Chemical synthesis of peptides has provided a way to accomplish this for small amounts of short proteins, but to obtain large amounts of long proteins, it would be very convenient to expand the genetic alphabet to encode additional amino acids. This long-standing effort has taken a major step forward with the discovery of how artificial DNA base pairs can be replicated. A hat tip to ScienceDaily for reprinting this Scripps Research Institute news release “Scripps Research Institute study suggests expanding the genetic alphabet may be easier than previously thought“:

A new study led by scientists at The Scripps Research Institute suggests that the replication process for DNA—the genetic instructions for living organisms that is composed of four bases (C, G, A and T)—is more open to unnatural letters than had previously been thought. An expanded “DNA alphabet” could carry more information than natural DNA, potentially coding for a much wider range of molecules and enabling a variety of powerful applications, from precise molecular probes and nanomachines to useful new life forms.

The new study, which appears in the June 3, 2012 issue of Nature Chemical Biology [abstract], solves the mystery of how a previously identified pair of artificial DNA bases can go through the DNA replication process almost as efficiently as the four natural bases.

“We now know that the efficient replication of our unnatural base pair isn’t a fluke, and also that the replication process is more flexible than had been assumed,” said Floyd E. Romesberg, associate professor at Scripps Research, principal developer of the new DNA bases, and a senior author of the new study. The Romesberg laboratory collaborated on the new study with the laboratory of co-senior author Andreas Marx at the University of Konstanz in Germany, and the laboratory of Tammy J. Dwyer at the University of San Diego.

Adding to the DNA Alphabet

Romesberg and his lab have been trying to find a way to extend the DNA alphabet since the late 1990s. In 2008, they developed the efficiently replicating bases NaM and 5SICS, which come together as a complementary base pair within the DNA helix, much as, in normal DNA, the base adenine (A) pairs with thymine (T), and cytosine (C) pairs with guanine (G).

The following year, Romesberg and colleagues showed that NaM and 5SICS could be efficiently transcribed into RNA in the lab dish. But these bases’ success in mimicking the functionality of natural bases was a bit mysterious. They had been found simply by screening thousands of synthetic nucleotide-like molecules for the ones that were replicated most efficiently. And it had been clear immediately that their chemical structures lack the ability to form the hydrogen bonds that join natural base pairs in DNA. Such bonds had been thought to be an absolute requirement for successful DNA replication‑—a process in which a large enzyme, DNA polymerase, moves along a single, unwrapped DNA strand and stitches together the opposing strand, one complementary base at a time.

An early structural study of a very similar base pair in double-helix DNA added to Romesberg’s concerns. The data strongly suggested that NaM and 5SICS do not even approximate the edge-to-edge geometry of natural base pairs—termed the Watson-Crick geometry, after the co-discoverers of the DNA double-helix. Instead, they join in a looser, overlapping, “intercalated” fashion. “Their pairing resembles a ‘mispair,’ such as two identical bases together, which normally wouldn’t be recognized as a valid base pair by the DNA polymerase,” said Denis Malyshev, a graduate student in Romesberg’s lab who was lead author along with Karin Betz of Marx’s lab.

Yet in test after test, the NaM-5SICS pair was efficiently replicable. “We wondered whether we were somehow tricking the DNA polymerase into recognizing it,” said Romesberg. “I didn’t want to pursue the development of applications until we had a clearer picture of what was going on during replication.”

Edge to Edge

To get that clearer picture, Romesberg and his lab turned to Dwyer’s and Marx’s laboratories, which have expertise in finding the atomic structures of DNA in complex with DNA polymerase. Their structural data showed plainly that the NaM-5SICS pair maintain an abnormal, intercalated structure within double-helix DNA—but remarkably adopt the normal, edge-to-edge, “Watson-Crick” positioning when gripped by the polymerase during the crucial moments of DNA replication.

“The DNA polymerase apparently induces this unnatural base pair to form a structure that’s virtually indistinguishable from that of a natural base pair,” said Malyshev.

NaM and 5SICS, lacking hydrogen bonds, are held together in the DNA double-helix by “hydrophobic” forces, which cause certain molecular structures (like those found in oil) to be repelled by water molecules, and thus to cling together in a watery medium. “It’s very possible that these hydrophobic forces have characteristics that enable the flexibility and thus the replicability of the NaM-5SICS base pair,” said Romesberg. “Certainly if their aberrant structure in the double helix were held together by more rigid covalent bonds, they wouldn’t have been able to pop into the correct structure during DNA replication.”

An Arbitrary Choice?

The finding suggests that NaM-5SICS and potentially other, hydrophobically bound base pairs could some day be used to extend the DNA alphabet. It also hints that Evolution’s choice of the existing four-letter DNA alphabet—on this planet—may have been somewhat arbitrary. “It seems that life could have been based on many other genetic systems,” said Romesberg.

He and his laboratory colleagues are now trying to optimize the basic functionality of NaM and 5SICS, and to show that these new bases can work alongside natural bases in the DNA of a living cell.

“If we can get this new base pair to replicate with high efficiency and fidelity in vivo, we’ll have a semi-synthetic organism,” Romesberg said. “The things that one could do with that are pretty mind blowing.”

Now that these scientists have demonstrated that DNA replication is far more flexible than had been thought, it will be fascinating to see what researchers do to expand the genetic alphabet, what additional amino acids they choose to incorporate into what proteins, and what this expansion of the set of amino acids composing proteins means for predicable protein folding and for artificial protein molecular machines.
—James Lewis, PhD

Darpa's Living Foundries program is looking to transform biology into an engineering practice. Photo: VA

Synthetic biology promises near-term breakthroughs in medicine, materials, and energy, and is also one promising development pathway leading to advanced nanotechnology and a general capability for programmable, atomically-precise manufacturing. Darpa (US Defense Advanced Research Projects Agency) has launched a new program that could greatly accelerate progress in synthetic biology by creating a library of standardized, modular biological units that could be used to build new devices and circuits. A hat tip to KurzweilAI.net for pointing to a recent article in Wired Danger Room “Darpa, Venter launch assembly line for genetic engineering“:

… The program, called “Living Foundries,” was first announced by the agency last year. Now, Darpa’s handed out seven research awards worth $15.5 million to six different companies and institutions. Among them are several Darpa favorites, including the University of Texas at Austin and the California Institute of Technology. Two contracts were also issued to the J. Craig Venter Institute. Dr. Venter is something of a biology superstar: He was among the first scientists to sequence a human genome, and his institute was, in 2010, the first to create a cell with entirely synthetic genome.

“Living Foundries” aspires to turn the slow, messy process of genetic engineering into a streamlined and standardized one. Of course, the field is already a burgeoning one: Scientists have tweaked cells in order to develop renewable petroleum and spider silk that’s tough as steel. And a host of companies are investigating the pharmaceutical and agricultural promise lurking — with some tinkering, of course — inside living cells.

But those breakthroughs, while exciting, have also been time-consuming and expensive. As Darpa notes, even the most cutting-edge synthetic biology projects “often take 7+ years and tens to hundreds of millions of dollars” to complete. Venter’s synthetic cell project, for example, cost an estimated $40 million.

Synthetic biology, as Darpa notes, has the potential to yield “new materials, novel capabilities, fuel and medicines” — everything from fuels to solar cells to vaccines could be produced by engineering different living cells. But the agency isn’t content to wait seven years for each new innovation. In fact, they want the capability for “on-demand production” of whatever bio-product suits the military’s immediate needs.

To do it, Darpa will need to revamp the process of bio-engineering — from the initial design of a new material, to its construction, to its subsequent efficacy evaluation. The starting point, and one that agency-funded researchers will have to create, is a library of “modular genetic parts”: Standardized biological units that can be assembled in different ways — like LEGO — to create different materials.

Once that library is created, the agency wants researchers to come up with a set of “parts, regulators, devices and circuits” that can reliably yield various genetic systems. After that, they’ll also need “test platforms” to quickly evaluate new bio-materials. Think of it as a biological assembly line: Products are designed, pieced together using standardized tools and techniques, and then tested for efficacy. …

The Darpa Living Foundries solicitation will remind long-term Nanodot readers of discussions of the need for an engineering perspective in the development of advanced nanotechnology centered on molecular manufacturing:

The Microsystems Technology Office (MTO) of the Defense Advanced Research Projects Agency (DARPA) is sponsoring an Industry Day for “Living Foundries,” a new DARPA program. The goal of the Living Foundries program is to apply an engineering framework to biology to harness its use as a technology and drive its advance as a manufacturing platform. In turning biological production into an engineering space where the only limit is the creativity of the designer, Living Foundries aims to enable on-demand production of new and high-value materials, devices and capabilities for the Department of Defense and establish a new manufacturing capability for the United States.

Because of the multidisciplinary nature of Living Foundries, DARPA is looking to engage the wider research community from fields both outside and inside the biological sciences to develop new ideas, approaches and tools to overcome current limitations and to create revolutionary capabilities.

Current, primitive examples of engineering biology rely on an ad hoc, laborious, trial-and-error process, wherein one successful project does not inform subsequent, new designs. This approach combined with the complexity of biological systems restricts current, one-off efforts to modifying only a small set of genes and constructing simple, isolated genetic circuits and metabolic pathways. Consequently, we are limited to producing only a small fraction of the vast number of possible chemicals, materials, and living systems that would be enabled by the ability to truly engineer biology. Through an engineering-driven approach to biology, Living Foundries aims to create a rapid, reliable manufacturing capability where multiple cellular functions can be fabricated, mixed and matched on demand and the whole system controlled by integrated circuitry, opening up the full space of biologically produced materials and systems. Key to success will be the democratization of the biological design and manufacturing process, breaking open the field to those outside the biological sciences.

In order to achieve the vision of Living Foundries, new tools, technologies and methodologies must be developed to transform biology into an engineering practice, decoupling design from fabrication and speeding the biological design, build, test cycle. These include: design tools that span from high-level description to fabrication in cells; modular genetic parts that allow a combination of systems to be designed and reproducibly assembled; methods for developing and fine-tuning new genetic parts and systems; well-understood test platforms, “cell-like” systems and chassis that readily integrate new genetic designs in a predictable fashion; next generation DNA synthesis and assembly techniques; and tools that allow for routine system characterization and debugging, among others. Further, these technological advances and innovations must be integrated to prove-out and push the boundaries of biological design towards the ultimate vision of point-of-use, on-demand, mass-customization biological manufacturing. …

If Darpa’s Living Foundries program achieves its ambitious goals, it should create a methodology, toolbox, and a large group of practitioners ready to pursue a synthetic biology pathway to building complex molecular machine systems, and eventually, atomically precise manufacturing systems.
—James Lewis, PhD