As regular readers know, I see foldamer engineering as a key to next-generation atomically precise nanosystems. Valuable in themselves, foldamers can also serve as components of composite systems that exploit diverse materials and nanotechnologies of qualitatively different kinds.

“Foldamers: Accomplishments and Goals”, by Samuel Gellman, heads a collection of 59 abstracts from a recent international conference on foldamers held in Bordeaux-Pessac, France. (Note that Gellman also authored the classic 1998 paper, “Foldamers: A Manifesto”.)

This set of abstracts offers a good window into the state of a broad, dynamic, and important field.

When I picked up my copy of this week’s Chemical & Engineering News this evening, I found that the lead article begins with this:

Futuristic visions of nanobots that travel the body to treat disease and construct compounds one atom at a time got a little closer to reality this week, thanks to two advances in nanoscale robotics reported in Nature (2010, 465, 202 and 206). Using DNA as the key construction material, one group of researchers created a nanoscale robot that can autonomously walk across a track, and a different group prepared a nanofactory in which DNA robots can carry and deposit nanoparticle cargo.

The article closes with this:

“A goal of our field is to refashion and reimagine all the complex biochemical machinery of cells to suit our own purposes—to have synthetic molecules that can move around and carry cargo as protein motors do in cells, to have molecules that act as chemical factories, which make a particular product based on a particular chemical input, and above all to make these processes modular, to make them engineerable,” notes Paul W. K. Rothemund, the Caltech scientist who invented DNA origami. “These two papers mark a significant advance along this research direction.”

This is great — I can quote C&EN instead of writing my own report of the news, and by the same stroke, this makes the C&EN report part of that news.

As a bonus, the wording of the C&EN report gives me an opportunity to remind readers that the idea of constructing things “one atom at a time” is based on a misconception, serving as a common but dangerous shorthand for “atomically precise fabrication”. The mistaken idea that these are equivalent has caused endless difficulties, because chemists recognize that juggling individual atoms makes no chemical sense.

To hammer the point again: Organic synthesis is already atomically precise, and works quite well without juggling individual atoms. The same holds for prospective methods of advanced mechanosynthesis.

And yes, what we see in Nature is a step in this direction.

Self-assembled peptoid membranes, 2.7 nm thick

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“Free-floating ultrathin two-dimensional crystals from sequence-specific peptoid polymers”, K.T. Nam et al., Nature Materials (on web: April, 2010)

Fresh from Ron Zuckerman’s lab at the Molecular Foundry: a new kind of molecular membrane — thin and crystalline — made by self-assembly of peptoid oligomers. As I discussed in an earlier post, peptoids have remarkable potential as building blocks for self-assembled nanosystems. Peptoids are peptide-like structures, but with monomers that can be chosen from among thousands of readily-available building blocks (a broad class of primary amines). Some of my enthusiasm for peptoids came from an early peek at the results now reported in Nature Materials.

Reports of new nanoscale constructs turn up almost every day, and they often include claims of wondrous potential applications that are literally incredible. It’s refreshing to read a paper making bold claims like these —

The ability to efficiently create functionalized 2D crystals by spontaneous assembly will lead to many applications in device fabrication, nanoscale synthesis and imaging, membrane mimetics, sensors and separations. More generally, the ability to mimic protein architecture with synthetic polymers should eventually enable new families of robust artificial proteins with highly specific functionality.

— and to find them thoroughly credible.

News of the paper has been reported from the Berkeley Labs News Center, in Chemical & Engineering News, and at Wired.com.

Here’s view of an idealized sheet, looking across rows of chains with alternating positive and negative charge on the outer surfaces and hydrophobic phenethyl sidechains sandwiched between:

Note the modified chain at the lower right.

• Peptoids at the Molecular Foundry
• Modular Molecular Composite Nanosystems
• A Third Revolution in DNA Nanotechnology

Zinc fingers & DNA

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From “Toward Modular Molecular
Composite Nanosystems”
[talk slides, pdf]

Zinc finger technology has great promise in genetic engineering and therapeutics, with potential applications in structural DNA nanotechnology, too.

Zinc finger proteins (ZFPs) are often called “game changing” because of the unprecedented way they precisely modify genes. Excitement about them is mirrored in the number of related scientific publications, which have climbed from hardly any 20 years ago to more than 360 in 2009.

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Chemical & Engineering News

A ZFP is a sequence of small protein structures (the fingers) that, taken together, bind to a specific DNA sequence. Unlike oligonucleotides, which bind to the Watson-Crick interface of single-stranded DNA, ZFPs bind in the major groove of double-stranded DNA (dsDNA), recognizing DNA base pairs from the side. They’re modular and can be engineered to target sequences chosen by a designer.

This mode of binding enables sequence-specific interactions without prying DNA strands apart, a fundamental advantage in a biological context. Binding dsDNA also opens a range of potential applications in structural DNA nanotechnology because it provides a way to bind protein structures to a dsDNA scaffold at specific sites, and with relatively high rigidity. This is an enabler for the DNA/protein/special-structure approach to modular molecular composite nanosystems.

Each finger binds with a degree of specificity and affinity, but multiple fingers must be stitched together to achieve tight, high-affinity binding to unique sequences. This engineering problem is tractable. There’s an open-source Zinc Finger Consortium (and a zinc-finger design server), as well as a thriving biotechnology company with a fat patent portfolio, Sangamo BioSciences. Sangamo aims to to produce products for medicine.

ZFPs enable a clever trick for editing DNA: A nuclease that cuts dsDNA can be inactivated by splitting into domains that regain activity when brought together. The complementary domains are linked to ZFPs that bind DNA sequences flanking a target site. Where these sequences are found together, both ZFPs bind, bringing the nuclease domains together; these bind, regain function, and cut, leaving the rest of the genome untouched.

This synergistic, cleanly targeted operation can be leveraged to inactivate or edit a selected genetic component with unmatched reliability and specificity. Powerful applications follow.

A few days ago, I wrote a brief sketch of the status and paths forward in the molecular approach to atomically precise fabrication. It offers a sampling, not a full picture:

The status of the key technologies

The technologies of biomolecular and chemical synthesis are now capable of fabricating a substantial range of complex, atomically precise structures. The most important of these are compact, polymeric structures (foldamers) and larger molecular scaffolds.

• Synthetic foldamers are now approaching the complexity of protein molecules, and can contain monomeric components with a wider range of functional properties.
• Protein engineering has recently reached the milestone of engineering new catalytic structures modeled on natural enzymes.
• Engineering molecular components that self-assemble to form new, complex crystalline materials has become routine.
• Structural DNA nanotechnology now enables the design and assembly of molecular scaffolds on a scale of millions of atoms and hundreds of nanometers.
• A rapidly developing design toolkit for self-assembly of diverse molecules and materials enables the construction of increasingly complex molecular systems.

Current research opportunities

These and related developments now make a range of experimental advances accessible. Some short-term goals and potential applications of the resulting technologies include the following:

• Demonstrate robust artificial foldamers that bind and stabilize complementary proteins.
  – Enables development of enzymatic catalysts for use in relatively harsh industrial process conditions.
• Demonstrate enzyme-like foldamers that bind and determine the activity of synthetic transition metal catalysts.
  – Enables development of highly stable and selective catalysts for the fine chemicals industry.
• Demonstrate self-assembled scaffolding structures that bind diverse components.
  – Enables the organization of nanoscale electronic. optoelectronic, and plasmonic components to form nanoscale sensors and electronic circuits.
• Demonstrate self-assembled scaffolds that promote and direct the growth of inorganic nanocrystals.
  – Enables production of atomically precise nanostructures with diverse materials and shapes for diverse applications in nanomaterials and nanosystems.

Middle-range objectives

Research opportunities today can open the door to the development of a next-generation technology platform that will, in turn, bring a new range of objectives into reach.

• The use of molecular scaffolds to bind and organize diverse components could be developed and elaborated to provide nanoelectronic fabrication methods for the post-Moore’s-law era.
• Devices that link multiple catalytic centers (analogous to polyketide and polypeptide synthases) could be developed and elaborated to provide “molecular assembly lines” that convert small feedstock molecules into high-value macromolecular products in a single, integrated process.
• A capacity for directing the growth of nanocrystals and other non-polymeric structures could be developed and elaborated to provide a capacity for building entirely new classes of complex, high-performance, atomically precise nanoscale components and systems.

Accelerators

Progress toward these objectives can be accelerated by increasing the capacity for innovative design, and for reducing innovations to routine practice. The greatest needs today comprise:

• Design-oriented software that integrates levels of description and physical analysis that range from quantum chemistry through molecular mechanics to the continuum mechanics of materials.
• Design-oriented data repositories that describe available nanoscale and molecular components and fabrication methods.

See also:

• Toward Integrated Nanosystems:
  Fundamental Issues in Design and Modeling [pdf]
• The Molecular Machine Path to Molecular Manufacturing (1):
  Foldamers and Brownian Assembly
• Modular Molecular Composite Nanosystems
• The Physical Basis of High-Throughput Atomically Precise Manufacturing

Unnatural amino acids compatible with ribosomes

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(circled: azide, alkyne,
and biotin derivative)

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From Neumann et al., 2010 and Dougherty, 2000.

All ribosomes read genetic data as three-letter words that encode 20 standard amino acids (give or take a few anomalies). This is equally true of the ribosomes in deep-sea bacteria living at 120°C, and the ones in your thumb. This universal code has been a wall that bounds the scope of biosynthetic polypeptide engineering — until now.

Recent developments have cracked the wall by tweaking the code, but Jason Chin’s group in the UK has blasted a wide hole by expanding the address space.

From the abstract of a paper soon to be published in Nature:

[E]very triplet codon in the universal genetic code is used in encoding the synthesis of the proteome….Here we synthetically evolve an orthogonal ribosome (ribo-Q1) that efficiently decodes a series of quadruplet codons…. By creating mutually orthogonal aminoacyl-tRNA synthetase–tRNA pairs and combining them with ribo-Q1 we direct the incorporation of distinct unnatural amino acids…. it will be possible to encode more than 200 unnatural amino acid combinations using this approach.

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H Neumann et al., “Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome”, Nature (early online publication).

I’ve selected some examples (image above) to illustrate the scope of these methods. Each of these amino acids (some highly unnatural) has already been used as a building block in ribosomal polypeptide synthesis. Together, they provide a glimpse of the vast new world now opening to molecular engineers. Polypeptides (of the sort usually called “proteins”) are already a family of versatile, high-performance engineering polymers, and an expanded set of building blocks can be exploited to increase thermodynamic stability, extend useful functionality, facilitate self assembly, and enable more systematic design.

Realizing this potential for expanding the scope of protein engineering will require extensive development of new tools, including new aminoacyl-tRNA synthetase–tRNA pairs. Because these are themselves proteins, there will be increasing opportunities for bootstrapping, using the new tools to facilitate development of those that follow. For example, could task-specific side chains (perhaps resembling PNA oligomers) facilitate the development of new aminoacyl-tRNA synthetases? There are complex constraints, but wide room for maneuver.

• Cell-free synthetic biology
• A High-Performance Polymer for Nanosytems Engineering
• Modular Molecular Composite Nanosystems
• Macromolecular Modeling for Molecular Systems Engineering

“Cells”
(Courtesy, Robert Hooke)

Synthetic biology doesn’t require cells, and in several ways, cells are liabilities.

Cells can make engineering difficult. Cell membranes and bacterial walls stand between new genes and the machinery needed to transcribe and translate them. They are barriers to liberating gene products. They contain systems that are complex products of eons of evolutionary history, not systems streamlined to simplify engineering. They are easily poisoned by what would be, to us, useful raw materials and products.

The state of the art in cell-free synthetic biology is already advanced, and moving forward rapidly:

Time and again, decreasing the dependence on cells has increased engineering flexibility with biopolymers and self-copying systems….

Current in vitro methods for synthesizing proteins and evolving protein, nucleic acid, and small-molecule ligands will be improved to accelerate production of new reagents, diagnostics, and drugs. New methods will be developed for synthesizing circular DNAs, modified RNAs, proteins containing unnatural amino acids, and liposomes.

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Forster and Church, “Synthetic biology projects in vitro”.

A glimpse of some recent developments:

Cell-free systems offer a unique platform for expanding the capabilities of natural biological systems for useful purposes, i.e. synthetic biology. They reduce complexity, remove structural barriers, and do not require the maintenance of cell viability. Cell-free systems, however, have been limited by their inability to co-activate multiple biochemical networks in a single integrated platform. Here, we report the assessment of biochemical reactions in an Escherichia coli cell-free platform designed to activate natural metabolism, the Cytomim system….

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Jewett et al., “An integrated cell-free metabolic platform
for protein production and synthetic biology”.

“Porous, Crystalline, Covalent
Organic Frameworks”
Côté et al.

Atomically precise self-assembly of complex structures can be engineered by providing for multiple binding interactions that

1. Cooperate to stabilize the correct configuration, in a thermodynamic sense, and
2. Do not stabilize any other configuration, in a kinetic sense

Roughly speaking, in the correct configuration, the parts fit together to allow all the binding interactions to operate simultaneously, and the system doesn’t get stuck in other configurations. It’s easy to see how weak interactions and cooperative binding can implement these conditions, but there are alternatives.

As I’ve discussed elsewhere, recent advances in biomimetic self assembly based on peptide and nucleic acid polymers provide a platform for developing complex, functional self-assembled systems, and in the right environments, some of these structures can be surprisingly robust. However, most of their characteristic binding interactions (hydrogen bonds, hydrophobic interactions, van der Waals interactions in well-packed structures, etc.) are weak in terms of both binding energy and mechanical strength.

Proteins structures, however, often include disulfide bonds (R₁–S–S–R₂), and these are covalent and strong. Their role in protein folding illustrates a key point:

Disulfide bonds can shuffle among different pairings through thiol/disulfide exchange,

a process that can be fast in the presence of R–S^– ions. A well-folded structure will strongly favor correct pairings by holding a momentarily displaced R–S^– in a position to reform the bond. In thermodynamic terms, this decreases the entropy cost of the bond-forming reaction, and in kinetic terms, it increases the effective concentration that drives the forward reaction, typically accelerating it by an large factor (> 10³). Exchange can be shut off by decreasing pH or removing free thiols from the folding environment.

The formation and hydrolysis of boronate esters can play a similar role in artificial self-assembling systems. A sample chapter from Boronic Acids (2005, posted by Wiley-VCH Verlag) provides an extensive discussion of the chemistry of boronic acid derivatives; it notes that boronic acids (at high pH, as hydroxyboronate anions) react with diols to form boronate esters with forward rate constants in the 10³ – 10⁴ M ^–1s^–1 range. Hydrolysis is likewise fast. Boronate esters can be stabilized by reducing pH or removing water. They, and boronic acids, are generally biocompatible, and have even been developed as drugs, where they serve to bind carbohydrate moieties.

Here are some recent papers on self assembled systems that discuss boronic acid chemistry, along with other covalent chemistries of similar utility:

• “An Iminoboronate Construction Set for Subcomponent Self-Assembly”
• “Complex Systems from Simple Building Blocks via Subcomponent Self-Assembly”

And a dissertation:

• “Self-Assembly of Boron-Based Supramolecular Structures” [pdf]

Self assembly need not be biomimetic.

• Effective Concentration in Self Assembly,
  Catalysis, and Mechanosynthesis (1)
• Effective Concentration in Self Assembly,
  Catalysis, and Mechanosynthesis (2)
• Self-assembling nanostructures: Building the building blocks