Friday, June 14, 2013

Building a Better Future, One Base at a Time

Synthetic Biology holds promise to revolutionize biotechnology and many other industries. Broadly defined, it is an engineering approach that seeks to design artificial genes, gene regulatory circuits, genomes, and useful and novel cellular behaviors. This includes efforts in recent years to program bacteria to specifically invade tumor cells as well as the production of microbial derived artemisinin. In addition to these impressive feats of bio-engineering, synthetic biology also gives researchers a way to better understand the design principles by which normal cells function. After all, it is much easier to understand how a system works once you have succeed in constructing one of similar complexity.

The scale of gene construction involved in synthetic biology can be quite large. In a multi-million dollar effort by the J. Craig Venter Institute, researchers successfully synthesized an entire bacterial genome and transplanted it into a cell. While the synthetic genome contained mostly natural sequence (the genome was physically synthetic, but the information contained within was a copy of what nature and evolution has already produced), future efforts may involved rewriting of vast sections of the genome. These grand examples of synthetic biology highlight a major obstacle facing this emerging discipline: the costs involved in the actual de novo synthesis and assembly of DNA.

Over the past decade and a half, tremendous advances in DNA sequencing technology have been made. The original human genome project took years of efforts by many researchers, with a final price tag estimated at approximately 4.4 billion dollars (current dollars, adjusted for inflation) or slightly more than $1 per base pair. Due to the improved scale of sequencing coupled with several technological revolutions, scientists are rapidly approaching the coveted $1000 genome, which would be approximately a million fold improvement in terms of cost (or $0.000001 per base)

DNA synthesis technology and capabilities currently sit where DNA sequencing was a decade ago, when the completion of the final draft of the human genome was announced. As a matter of fact, synthesis technology is arguably behind even this mark. The Venter Institute's creation of Mycoplasma laboratorium (also known as Mycoplasma genitalia JCVI-1.0) is estimated to have taken a small team of researchers nearly a decade and $40 million dollars to complete. Since this genome is only half a million dollars, this represents a cost of $80 a base pair. Most smaller projects demand less intense and less iterative assembly efforts, but synthesis of even short oligos is still between $0.25 and $1.0 per base, depending on the size, scope, and quality of the synthesis efforts.

Clearly, there is room for improvement in DNA synthesis technology. This improvement will be critical to the advance of the field of synthetic biology, which hold tremendous promise for a variety of industries. Where will the improvement come from? For DNA sequencing, improved scale and efficiency of existing technology was important, but not enough for the incredible leap in capability and cost-effectiveness. Both evolution and revolution (innovative second and third generation technologies) was necessary to achieve the current level of capability. 

So far, the horizon for new, ground-breaking technology for DNA synthesis is not clear. The chemistry used in the synthesis of oligomers has remained largely unchanged for years. There are a few new approaches which may hint at how DNA synthesis can be made more reliable and affordable. Here, I'll survey a few of the newest developments. This includes the MOSIC method, the research by Dr. George Church's group at Harvard and its implementation at Gen9, as well as Cambrian Genomics and some other creative ideas. 

Select 'Read More' to learn more about emerging DNA synthesis technologies.

Microchips, Gen9, and DNA laser printing

Some researchers have looked towards microchips and microarrays for cheaper ways of producing oligos for gene synthesis. Instead of column-synthesis, which is limited by the larger amounts of the precursor nucleotides and handling, microarray based synthesis allows the synthesis of many oligos in parallel for a fraction of the cost (about a 1 to 2 order of magnitude reduction, or 10 to 100 times cheaper). This approach suffers from problems such as higher error rates, as well as the creation of a complex mixture of oligos on a single chip. Separating these oligos from another, if necessary, is a challenge.

This approach has become more attractive in recent years, due in part to the research from Dr. George Church's group at Harvard. They have published several articles (some of which are freely available), which detail how the overcome the problems associated with microchip gene synthesis. This includes a particular sequence design strategy for the oligos, which enables them to anneal to partners in a complex mixture and start to stitch together (through high-fidelity PCR) into genes. For a taste of the progress they have made, check out the following papers:

Carr PA, et al (2012) Enhanced multiplex engineering through co-operative oligonucleotide co-selection. Nucleic Acid Res. Vol. 40 (17) PMID: 22638574 PMCID: PMC3458525

Matzas M, et al (2010) High-fidelity gene synthesis by retrieval of sequence-verified DNA identified using high-throughput pyrosequencing. Nature Biotechnology Vol. 28 (12) PMID: 21113166 PMCID: PMC3579223

Kosuri S, et al (2010) Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips. Nature Biotechnology Vol. 28 (12) PMID: 21113165 PMCID: PMC3139991

I especially recommend reading the Kosuri article, as it arguably gives a better overview of the current challenges of DNA synthesis than I have provided here. This approach has been explored by other researchers, and it aptly refered to by some as 'Shotgun DNA Synthesis', because it is in some ways analogous to shotgun sequencing (only in reverse). For example, see Kim H et al, (2012) 'Shotgun DNA synthesis' for the high-throughput construction of large DNA molecules. Nucleic Acids Research Vol. 40 (18) PMID: 22705793 PMCID: PMC3467036

This new approach to DNA synthesis also seems to be moving from the academic laboratory to commercial applications. Gen9 is a fledging synthesis company, founded by Dr. Church and others, that claims to have the capabilities to produce a tremendous amount of synthetic DNA at a significantly lower price (although their corporate website offers scant evidence to support these vague, qualitative claims.) Gen9, as well as others that use similar DNA synthesis technology, are beginning to utilize high-throughput sequencing to verify the sequence of the oligos or intermediate constructs made (See Matzas et al, 2010 above). The high-throughput parallel pyrosequencing complements the synthesis approach nicely and bypasses problems associated with high error rates.

One company, Cambrian Genomics, has a different twist on the microchip-pyrosequencing tandem synthesis and sequencing approach. This twist, DNA Laser Printing, captures sequence-verified oligos from pyrosequencing wells using laser capture microdissection. These oligos can then be transferred to separate wells to facilitate the ordered construction of a synthetic gene. Alternatively, the now separated oligos can be utilized for unrelated projects. This twist will enable Cambrian Genomics to synthesize oligos for multiple investigators in parallel on demand, instead of making a small number of oligo mixtures that all contribute to the same synthesis project.

The MOSIC Method

If an analogy is to be made between synthesis and sequencing technologies, then the column-synthesis method is akin to Sanger sequencing. The second generation synthesis and sequencing technologies, the microchip and pyrosequencing respectively, share some similarities in that they are both high-throughput, highly parallel approaches. As highlighted above, microchip gene synthesis relies upon pyrosequencing in a complementary fashion to verify the accuracy of synthesis. What then is the synthesis equivalent of third generation sequencing technologies, such as nano-pore sequencing

Nanopore sequencing promises to further revolutionize the field of DNA sequencing, by again drastically lowering the cost. It relies on threading DNA molecules through a protein pore in a lipid membrane, and measuring the changes in electric current as particular bases pass through. This new sequencing approach has a distinct 'biological' flavor as opposed to the more mechanical Sanger and pyrosequencing technologies. In this author's humble opinion, this sort of biological-nanotechnology (after all, isn't molecular biology just nanotechnology crafted by nature?) is the sort of approach that will dominate in the future, leading to extremely effective solutions to sequencing, synthesis, and problems in a variety of different industries.

Looking towards a more biological solution to the DNA synthesis problem, a research group Swedish Medical Nanoscience Center has just recently developed a method they term MOSIC, for 'MOnoclonal StoIChiometric' oligo synthesis. In the MOSIC method, desired oligo sequences are engineered into a plasmid, separated by cleavable DNA hairpins. This method is in some ways complementary to the microchip assembly methods, since the desired sequenced need to be engineered into the plasmid to begin with. Plasmid DNA is recovered from a sequenced verified clone, and many copies of the plasmid sequence are generated through rolling-circle amplification (RCA). Enzymatic cleavage of the DNA hairpin release the oligos from this large concatenated DNA.

The MOSIC method has definite advantages in that particular oligo sequences, once engineered into the plasmid and sequence verified, can be produced in parallel at defined stoichiometric amounts. Although the initial engineering requires some solid-state (column or microchip) synthesis, the production of larger quantities of the same oligos is simple, fast, and cheap. While this approach doesn't represent a breakthrough third generation synthesis technology that outstrips microchip synthesis, it represents an useful step in this direction. You can read more about MOSIC in the recently published paper by Ducani C, et al, (2013) Enzymatic production of 'monoclonal stoichiometric' single-stranded DNA oligonucleotides. Nature Methods PMID: 23727986. This work was done by the laboratory group of  Dr. Bjorn Hogberg, whom specializes in DNA origami (for which MOSIC has particular utility).

The future of DNA synthesis

While great strides have been made towards a future in which DNA synthesis is cheap, reliable and available to all researchers, much more work remains. In addition to refining the emerging second generation microchip gene synthesis methods, researchers will need to develop even more powerful technologies which currently appear to be beyond the foreseeable horizon. If the $1000 genome becomes a reality for not only sequencing but synthesis as well, the field of synthetic biology will be empowered to tackle biomedical, agricultural, and industrial problems on a scale that would not otherwise be possible.

What might future DNA synthesis technologies look like? If I had more insight than what I provided above, then I would probably be writing a SBIR grant or working in a laboratory somewhere trying to develop such a technology (the commercial potential would be great, especially in the coming decade). A scheme using modified telomerase might be possible, since telomerase synthesis DNA based upon an integral template (not really de-novo synthesis, but is is close). Other solutions that employ a modified or controllable polymerase probably represent a more powerful approach than the step-wise chemical synthesis currently used. If only the bases could be made to lined up along the electrodes and gates that are triggered when a research types a particular string of A, C, G, and Ts. Alas, I doubt such a nanotechnology solution is currently feasible, let alone theoretically possible.

What emerging technologies have you heard about (or thought up) that will revolutionize DNA synthesis? How cheap, fast, and accuracy do you think de-novo DNA synthesis can become? Please share your thoughts in the comments section!

No comments:

Post a Comment