Bio-nanotechnology – Page 7 – Soft Machines, by Richard Jones

Nanotechnology gets complex

The theme of my book Soft Machines is that the nanomachines of biology operate under quite different design principles from those we are familiar with at the macroscale. These design principles exploit the different physics of the nanoworld, rather than trying to engineer around them. The combination of Brownian motion – the relentless shaking and jostling that’s ubiquitous in the nanoworld, at least at normal temperatures – and strong surface forces is exploited in the principle of self-assembly. Brownian motion and the floppiness of small scale structures are exploited in the principle of molecular shape change, which provides the way our muscles work. We are well on our way to exploiting both these principles in synthetic nanotechnology. But there’s another design principle that’s extensively used in Nature, that nanotechnologists have not yet exploited at all. This is the idea of chemical computing – processing information by using individual molecules as logic gates, and transmitting messages through space using the random motion of messenger molecules, driven to diffuse by Brownian motion. These are the mechanisms that allow bacteria to swim towards food and away from toxins, but they also underly the intricate way in which cells in higher organisms like mammals interact and differentiate.

One argument that holders of a mechanical conception of radical nanotechnology sometimes use against trying to copy these control mechanisms is that they are simply too complicated to deal with. But there’s an important distinction to make here. These control systems and signalling networks aren’t just complicated – they’re complex. Recent theory of the statistical mechanics of this sort of multiply connected, evolving networks is beginning to yield fascinating insights (see, for example, Albert-László Barabási’s website). It seems likely that these biological signalling and control networks have some generic features in common with other complex networks, such as the internet, and even, perhaps, free market economies. Rather than being the hopelessly complicated result of billions of years of aimless evolutionary accretion, we should perhaps think of these networks as being optimally designed for robustness in the noisy and unpredictable nanoscale environment.

It seems to me that if we are going to have nanoscale systems of any kind of complexity, we are going to have to embrace these principles. Maintaining rigid, central control of large scale systems always seems to be a superficially good idea, but such control systems are often brittle and fail to adapt to unpredictability, change and noise. The ubiquity of noise in the nanoscale world offers a strong argument for using complex, evolved control systems. But we still lack some essential tools for doing this. In particular, biological signalling relies on allostery. This principle underlies the operation of the basic logic gates in chemical computing; the idea is that when a messenger molecule binds to a protein, it subtly changes the shape of the protein and affects its ability to carry out a chemical operation. Currently synthetic analogues for this crucial function are very thin on the ground (see this abstract for something that seems to be going the right way). It would be good to see more effort put in this difficult, but exciting, direction.

Nanomedicine gets clinical

Everyone agrees that some of the key applications of nanotechnology will be in medicine. Within medicine, drug delivery is an obvious target. So when can we expect to see nano-enabled medicines on the pharmacy shelves? The answer, as usual, depends on what you mean by nanotechnology. Many people have welcomed Abraxane™, which received FDA approval for use for breast cancer earlier this year, as the first nano-drug. But a number of other drugs already in clinical use have just as much right to the nano- label.

Ruth Duncan gives a useful list of nano-medicines in current clinical use in an article in Nano Today – Nanomedicine gets clinical (I’ve already referred to this article here). We can summarise the key functions that nano-engineering confers on these products as packaging and targeting – the active drug molecules need to be protected from the body’s systems for repelling foreign materials, and if possible they need to be actively targetted to the parts of the body at which the therapy is directed. For the anti-cancer therapeutics that dominate this list, this target is the tumour.

One approach to targetting is to wrap the molecule up in a liposome – a nanoscale container that is formed, by self-assembly, when soap-like lipid molecules form a bilayer sheet which folds over on itself to make a bag. These are the same structures that are already incorporated in some cosmetics. DaunoXome® consists of the anti-cancer drug daunorubicin encapsulated in liposomes, and is used for the treatment of HIV–related Kaposi’s sarcoma. Doxil® and Caelyx® are liposomal preparations of the related drug doxorubicin, and are used for advanced ovarian cancers. Simple liposomes have quite a short lifetime in the body; in Doxil the surfaces of the liposome are modified by being coated by the water soluble polymer polyethylene glycol.

Rather than putting the drug in a liposome, and then coating the liposome with polymer, it is possible simply to attach polyethylene glycol directly to the drug. This is the basis of “polymer therapeutics” (this is Ruth Duncan’s own field). Examples in clinical use include Oncaspar®, for acute lymphoblastic leukemia, and Neulasta®, used to decrease infection in patients receiving chemotherapy. Both these drugs consist of a protein drug molecule which is disguised from the body by being coated in a diffuse cloud of polyethylene glycol (PEG). How PEG works is still not entirely clear, but the basis of the effect is that it forms a diffuse layer which resists protein adsorption.

Mylotarg®, a drug licensed in the USA for acute myeloid leukemia, is a (currently rather rare) example of a targetted drug. The drug itself – a potent anti-tumor antibiotic – is chemically linked to an antibody – a protein molecule which specifically binds to chemical groups on the outside of the target cells. In Abraxane™, it is the drug molecule itself, paclitaxel, that is nanoengineered – it is prepared in a nanoparticulate form to improve its solubility; the nanoparticles are coated with the blood protein albumin.

So what we see now are a number of products which use individual tricks of nanoengineering to improve their effectiveness. What we will probably see in the future is the combination of more than one of these functions in a single product – moving beyond clever formulation to integrated nanodevices.

Model Railways

I’ve been in Leeds for a few days for the biennial conference of the Polymer Physics Group of the UK’s Institute of Physics. Among many interesting talks, the one that stood out for me was the first – an update from Andrew Turberfield on his efforts to make a molecular motor from DNA.

Turberfield, who is at the Oxford IRC in Bionanotechnology, is building on the original work from Ned Seeman, exploiting the remarkable self-assembling properties of DNA to make nanoscale structures and devices. A few years ago, Turberfield, working with Bernie Yurke at Lucent Bell Labs, designed and built a DNA nano-machine (see here for a PDF preprint of the original Nature paper), and in 2003 they published a paper describing a free-running motor powered by the energy released when two complementary strands of DNA meet to make a section of double helix (abstract here).

This motor doesn’t actually do anything, apart from sit around in solution cyclically changing shape. What Turberfield wants to do now is make something a bit like the linear motors common in cell biology, in which the motor molecule moves along a track, often carrying a cargo. To make this kind of molecular railway, Turberfield’s scheme is to prepare a track along a surface by grafting strands of DNA to it. The engine is another DNA molecule; what needs to be done is get some scheme whereby the engine molecule is systematically passed along from strand to strand.

His first effort, in collaboration with Duke University’s John Reif, involves using enzymes to alternately cut DNA strands and rejoin them in a sequence that has the effect of making a short strand of DNA move linearly in one direction. In this case, it’s the energy used by the enzyme that joins two bits of DNA that makes the motor run. The full paper is here (PDF). In motor mark 2, it’s a so-called nicking enzyme that makes the engine move, and the directionality is imposed by the fact that the track is destroyed in the path of the engine (abstract here, subscription probably required for full article). What Andrew really wants to do, though, is have a motor that is solely powered by the energy released when DNA strands make a helix, which doesn’t chew up the track behind it, and which doesn’t involve the use of any biological components like enzymes. He has a scheme, and he is confident that it’s not far off working.

These motors are inefficient and slow in their current form. But they are important, because they work on the same basic principles as biological motors, principles which are very different to the mechanical principles that underly the motors we are familiar with. They rely on the Brownian motion and stickiness of the nanoscale environment. But because of the simplicity of the base pair interaction, the calculations you need to do to predict whether the motor will work or not are feasibly simple. By learning to make model railways from these simple, modular components, we’ll learn the design rules that will enable us to make a wider variety of practical nanoscale motors.

Making life from the bottom up

I wrote below about Craig Venter’s vision of synthetic biology – taking an existing, very simple, organism, reducing its complexity even further by knocking out unneccessary genes, and then inserting new genetic material to accomplish the functions you want. One could think of this as a kind of top-down synthetic biology; one is still using the standard mechanisms and functions of natural biology, but one reprogrammes them as desired. Could there be a bottom-up synthetic biology, in which one designs entirely new structures and systems for metabolism and reproduction?

One approach to this goal has been pioneered by Steven Benner at the University of Florida. He’s been concentrating on creating synthetic genetic systems by analogy with DNA, but he’s not shy about where he wants his research to go: “The ultimate goal of a program in synthetic biology is to develop chemical systems capable of self-reproduction and Darwinian-like evolution.” He’s recently written a review of this kind of approach in Nature Genetics Reviews (subscription only): Synthetic biology.

David Deamer, from UC Santa Cruz, has a slightly different take on the same problem in another recent review, this time in Trends in Biotechnology (again, subscription only, I’m afraid). “A giant step towards artificial life?” concentrates on the idea of creating artificial cells by using self-assembling lipids to make liposomes (the very same creatures that L’Oreal uses in its expensive face creams). Encapsulated within these liposomes are some of the basic elements of metabolism, such as the mechanisms for protein synthesis. How close can this approach get to creating something like a living, reproducing organism? In Deamer’s words: “Everything in the system grows and reproduces except the catalytic macromolecules themselves, the polymerase enzymes or ribosomes. Every other part of the system can grow and reproduce, but the catalysts get left behind. This is the final challenge: to encapsulate a system of macromolecules that can make more of themselves, a molecular version of von Neumann’s replicating machine.” He sees a glimmer of hope in the work of David Bartel at MIT, who has made a RNA enzyme that synthesizes short RNA sequences, pointing the way to RNA-based self-replication.

But all these approaches still follow the pattern set by the life we know about on earth; they depend on the self-assembling properties of a familiar repertoire of lipids and macromolecules, like DNA, RNA and proteins, in watery environments. Could you do without water entirely? Benner is quoted in an article by Philip Ball in this week’s Nature (Water and life: Seeking the solution, subscription required) arguing that you can: “Water is a terrible solvent for life…. We are working to create alternative darwinian systems based on fundamentally different chemistries. We are using different solvent systems as a way to get a precursor for life on Earth.”

Commercialising synthetic biology

What’s going to be the quickest way of achieving some kind of radical nanotechnology, in which sophisticated nanoscale machines carry out complex chemical tasks? Since nature has evolved sophisticated and effective nanomachines that are optimised for the nanoscale environment, an obvious approach is to take components from living systems and reassemble them to do the tasks you want. This is the approach of bionanotechnology. But we could take this logic further. Rather than rebuilding systems from individual biological components, we could take a complete organism, strip out the functions we don’t want, and patch in the genetic code for the components we need. This top-down approach to bionanotechnology is exactly what is being proposed by a new company, Synthetic Genomics Inc, founded in June by Craig Venter. Venter is, of course, the scientist behind the private sector venture to sequence the human genome. The initial focus will be on the use of these partly synthetic organisms to make alternative fuels such as hydrogen and ethanol.

The vehicle for these strange hybrids is likely to be the parasitic bacteria Mycoplasma genitalium, an unwelcome inhabitant of some people’s urinary tracts, which currently has the distinction of having the smallest known genome. This is contained on a mere 580,000 base pairs of DNA, coding for about 480 proteins and 40 RNA molecules. Venter’s group systematically knocked out genes from this organism in an attempt to find a so-called minimal genome. One can think of this as the simplest possible fully functioning life-form (of course, such an organism would be very restricted in the environment it can live in). In Venter’s 1999 paper in Science, Global transposon mutagenesis and a minimal mycoplasma genome, a further 100 proteins were eliminated without fatally compromising the organisms’ existence. Having stripped the organism down to a minimal level of complexity, the idea would be to reinsert synthetic genes coding for whatever machinery you require.

There are two questions to ask about this: will it work, and should it be done? It’s certainly a very bold commitment to a very reductionist view of life: in their words “using the genome as a bio-factory, a custom designed, modular cassette system will be developed so that the organism executes specific molecular functions”. As for the ethics of the enterprise, I’m sure even the most enthusiastic technophile would at least pause to think about the implications of attempting to re-engineer life on this scale. Indeed, Venter’s group commissioned their own bioethicists to think about the issues, and this ethical commentary accompanied their original Science article. This is just the beginning of a very big story.

Cheap designer genes

The kind of DNA-based nanotechnology pioneered by New York University’s Ned Seeman is currently the closest thing we have to the radical aim of making nanoscale structures and machines with atomic precision, but the development of the technology is limited by cost. DNA is an expensive molecule – currently it costs about $5000 a gram to make short, synthetic DNA sequences.

The cost of synthetic DNA has been dropping, but a new company is promising orders of magnitude drops in cost for much longer sequences of DNA. The company, Codon Devices, is commercialising methods developed in George Church’s group at Harvard Medical School – the method is describe in this Nature paper (subscription required for full paper): Accurate multiplex gene synthesis from programmable DNA microchips.

It’s not DNA nanotechnology that the company cites as its major potential market, though. Their ambition is to make synthetic genes for synthetic organisms, in the emerging field of synthetic biology.

Delivering interfering RNA

RNA interference is one of the most fascinating biological discoveries of the last few years, and there’s excitement that it could lead to a new class of powerful drugs which would be an absolutely specific treatment both for viral diseases and cancers. But these drugs, based on short lengths of RNA, need to be introduced into the target cell. A recent paper in Nature Biotechnology – Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs by Morissey et al (subscription required) – suggests that encapsulating the RNA in a liposome can do the job.

In the normal process of gene expression, the genetic code for is transferred from the cell’s DNA, where the information is stored, to the ribosome where the corresponding protein is made in the form of a molecule of RNA – messenger RNA. It turns out that there’s a naturally occurring cellular process that destroys messenger RNA when it’s been marked with a short piece of RNA which binds to it. This RNA interference process was named Science Magazine’s breakthrough of the year in 2002 (needs free registration). These short interfering RNA molecules can thus be used to inactivate one individual gene. To quote from a January 2004 article by Richard Robinson in Public Library of Science: Biology – RNAi Therapeutics: How Likely, How Soon? – “The clinical applications appear endless: any gene whose expression contributes to disease is a potential target, from viral genes to oncogenes to genes responsible for heart disease, Alzheimer’s disease, diabetes, and more.”

But bits of free RNA floating around the body are soon identified and destroyed – after all, they are most likely to originate in viruses. And the highly charged RNA molecule can’t penetrate the lipid bilayer that separates a cell from its surroundings. To quote from the Robinson article again: “stability and delivery are also the major obstacles to successful RNAi therapy, obstacles that are intrinsic to the biochemical nature of RNA itself, as well as the body’s defenses against infection with foreign nucleotides.” The Nature Biotechnology article describes the work of scientists from a pharmaceutical company trying to bring this technology to the clinic – Sirna therapeutics. They have shown that by using a lipid-based nanoparticle delivery system they can get good results treating hepatitis B virus in an animals. The delivery system is essentially a liposome, a self-assembled hollow shell formed by a phospholipid sheet which has folded round on itself to form an enclosed surface, but I suspect there’s quite a lot of art to selecting the mixture of lipids to use. This includes charged lipids which probably bind to the RNA, lipids to promote uptake of the delivery device by the cell, and lipids bound to protective polyethylene glycol hairs to disguise the liposomes from the body’s defenses.

Soft Machines at the Foresight Conference

The newly relaunched Foresight Institute – now officially the Foresight Nanotech Institute, with a mission of “Advancing Beneficial Nanotechnology” – holds its annual conference from October 22 to 27th in San Francisco. I was very pleased to get an invitation to talk in the first part of the meeting – the Vision Weekend. I’ll be taking the opportunity to set out some of my more speculative thoughts about how we might learn lessons from nature to make a radical nanotechnology based on some of the design principles used by cell biology.

Bacterial nanowires

Electrical phenomena are important in biology, as Galvani discovered long ago when he learnt to make dead frogs twitch. But in biology electrical currents are generally carried by currents of ions rather than electrons. The transport of electrons is important in processes like photosynthesis, but the distances over which the electrons are transported are very small – the nanometer or two that defines the thickness of a lipid membrane. So the discovery of what look like electrically conducting nanowires in a soil bacterium is rather surprising. The discovery, from a group at UMASS Amherst (press release here), was reported in Nature (subscription required for full article) a few weeks ago.

The bacteria in question are soil bacteria that make their living by metabolising iron; to do this they seem to have evolved electrically conducting filaments called pili that allow them to do electrochemistry at a distance on a particle of iron oxide. Pili are common in many types of bacteria; they’re used by pathogenic bacteria to inject toxins into host cells, and for transfer of DNA between bacteria. They’re composed of protein molecules which self-assemble into long filaments, which are anchored into the bacterial cell wall by a large protein complex.

This report still leaves some unanswered questions in my mind. The conductivity of the pili was measured using atomic force microscope based conductance mapping of a graphite surface decorated with pili that had been broken off bacterial surfaces; it would be more convincing (though much more difficult) to quantify the conductivity along the length of the filament, rather than across the thickness. More importantly, perhaps, it doesn’t yet seem to be clear what is the structural feature of the pilus-making protein in this particular bacteria that leads to its electrical conductivity (as opposed to pili from other types of bacteria, which are shown in the paper to be non-conductive). It’s still a remarkable and suggestive result, though.

Thanks to Jim Moore for a comment drawing my attention to this press release.