Synthetic Biology Molecules for April

This month I had pencilled in the products of the Major Histocompatibility (MHC) genes, as versatile defenders against invading pathogens. However, when I opened my electronic copy of Science last week, I changed my mind, as usual, and decided to focus on a fascinating paper which was introduced by its authors (Florian Praetorius and Hendrik Dietz, from the Physics Department and Institute for Advanced Study at the Technische Universität München) as follows. Controlling the spatial arrangement of functional components in biological systems on the scale of higher-order macromolecular assemblies is an important goal in synthetic biology. How could I resist? I have also added a section at the very end of the post which is aimed at summarising the main point for school students. First you should try reading the post which is aimed at my tutees (all undergraduate Biochemists at Sheffield) and then see if you agree with my simplified summary. Oh and let me know what you think!


As "the world" becomes increasingly excited by the transformational promises of "Synthetic Biologists", I decided to focus on the harnessing of the DNA recognition domains, abbreviated to TALs (for transcription activator like), which have been "engineered" to combine with specific DNA duplexes, to generate discrete nucleoprotein structures in a controlled manner in vitro. These structures are referred to in the title of the paper as DNA:protein hybrid nanoscale shapes. It made me think of the formidable, pre-cloning achievement of Masayasu Nomura (pictured right) when in 1966 his lab reported the reconstitution of bacterial ribosomes. Twenty years later, Tim Richmond's nucleosome group used cloning technology to generate defined nucleosome particles for crystallographic studies. The work described here, by Praetorius and Dietz differs in that the particles produced are not found in Nature, but the "ingredients" are (well, mostly!).  Coming from a Physics group, it also reveals how the groundbreaking work of the Molecular Biologists is now being exploited by Physicists (and engineers) in Synthetic Biology: a fundamentally multidisciplinary field.

The TAL proteins are referred to in their Science paper, as molecular staples, a function that they combine with single nucleotide specificity: 34 amino acids are needed to recognise a base-pair. These proteins (shown left) are engineered versions of TAL endonucleases (TALEs) secreted by Xanthomonas species when infecting plants. In Nature these genome "organisers" interfere with promoter function: you can read more about them at the above links. Interestingly, the authors utilise in vitro methods of protein synthesis (pioneered by Geoffrey Zubay at Columbia University in the 1970s), to generate their nanostructures. By combining specific DNA duplexes with recombinant TAL modules, structures of the kind shown below, can be assembled in a controlled manner and in sufficient yield for downstream analysis (I expect high resolution cryo EM structures will emerge first, since the yields may not be sufficient yet for crystallography).

With the components in hand, let's consider the spontaneous formation of such nanostructures. As with any physiological assembly of nucleoprotein complexes, ranging from viral capsids to spliceosomes; there are common tehermodynamic principles that apply. Typically, such processes occur rapidly: in seconds, if not less. It might serve you well to contemplate for a moment, the physiologically challenging biosynthesis of several thousand bacteriophage particles during the half-hour life of a regular bacterium! Such rapid assembly phenomena are thought to be "entropy driven" (take a look here) and usually involve sponatneous electrostatic neutralisation (basic proteins meet acidic polynucleotides) in tandem with a potent "hydrophobic effect". Specificity will "click in" in a later phase: in the case of TAL domains, the 34 amino acid sequences that specifiy individual nucleotides occurs in the context of a "wrap-around" arrangement (as shown above). One of the results of these TAL molecules is shown (top RHS) and demontsrates that we are now pretty competent at engineering both sequence- AND topologically- specific nucleoprotein particles. 

The authors, and its enthusiatic supporters refer to the concept of molecular "origami" throughout the narratives. In origami, however, there is a considerable engagement with the person folding the paper. Here spontaneity is programmed into the sequences (or code) of the gene encoding the TAL domains and the corresponding target DNA sequences. The use of "near physiological" in vitro conditions and components (sometimes referred to as "molecular chassis" in the world of Synthetic Biology) is aimed at demonstrating how close we have come to developing controlled production of novel, complex nanostructures in living organisms. In many ways this is a logical extension of "hijacking" host cells (bacteria, yeast, cultured cells etc) to generate recombinant proteins for Biotechnological and Medical applications, it also goes some way to satisfying the "Feynman Principle" and it is nice to see Physicists operating in "Biological Space", since they (as a community) played such a key role in developing the concepts and technologies that underpinned the development of contemporary Molecular Biology. As good a reason as any to join your national Biophysical Society?

Summary of Key Points

First of all, I have picked a protein (TAL) that has been derived from a naturally occurring gene regulatory molecule, produced by a group of microbes that infect plants. These proteins have captured the interest of Biotechnologists because it is possible to change their amino acid sequence and switch the nucleotide (ie G,A,T or C) specificity in a predictable way. In the paper discussed, these proteins are then added to specific DNA duplexes (containing the recognition sites programmed into the TALs), and they spontaneously induce the DNA double helices to form predictable (and quite remarkable) 3D structures as shown above. This is an example of the emerging field of "Synthetic Biology" in which the molecular properties found in Nature are exploited to generate novel molecules (or even cells) that could have therapeutic or other practical applications.