SBIR-STTR Award

To many (notably Craig Venter), "synthetic biology" means simply "synthesizing large amounts of DNA". This type of synthetic biology actually began in the 1980's, when Caruthers introduced a phosphoramidite-based solid phase DNA synthesis architecture. This allowed ICI and the Benner group to synthesize complete genes encoding biomedically useful proteins and enzymes for the first time. The second example showed how biotechnological goals could better be met if the synthetic sequences were different from the sequences presented by Nature, through codon optimization and watermarking, inter alia. Subsequent developments now allow semi-routine synthesis of genes; these are used in biotechnology, gene therapy, RNA therapy, and elsewhere. Extrapolation suggests that routine synthesis of whole genomes will soon be possible, hopefully for less than the ca. $30 million spent to synthesize one in the Venter laboratory. Unfortunately, DNA has a rich biophysical chemistry that defeats any architecture that relies on autonomous assembly to make large DNA (L-DNA) constructs by simply mixing synthetic fragments, even within recombinogenic organisms. However, two innovations from the FfAME provide a new approach to creating L-DNA constructs. The first is an "artificially expanded genetic information system" (AEGIS). AEGIS is a DNA-like molecule that adds eight additional nucleobases that form four additional pairs (the Z:P, S:B, K:X, V:J pairs) to the four natural nucleotides (which form G:C and A:T pairs). By increasing information density of DNA and avoiding non-canonical interactions, AEGIS allows autonomous self-assembly of dozens of fragments to generate L-DNA. The second innovation is "transliteration" technology. Transliteration allows rule-based replacement of AEGIS nucleotides by standard nucleotides after a L-DNA assembly is complete. By converting S, B, Z, and P to T, A, C, and G (respectively), the AEGIS components can be replaced after they have served their role to guide autonomous self-assembly, converting GACTSBZP L-DNA to entirely standard GACTTACG DNA. To persuade commercial partners to engage with this technology, FfAME scientists demonstrated this strategy using the simpler GACTSB six-nucleotide AEGIS DNA to give, in one assembly step, an active, full-length, and sequence-correct gene encoding kanamycin resistance. Parallel attempts with standard 4-base DNA failed. Phase 1 project will transfer these innovations to Firebird, which will deliver L-DNA and whole plasmids by custom synthesis using an eight-letter GACTSBZP alphabet. This will require (a) adapting OligArch software to support design with this strategy, (b) creating a pipeline to synthesize DNA 60-80 nucleotide fragments using this alphabet, and (c) providing demonstration products, plasmids coding multiple enzymes for complete metabolic pathways to natural products, assembled in a single step. In Phase 2, this technology will be merged with Firebird's E. coli SEGUE strain (Second Example of Genetics Undergoing Evolution) that manages expanded DNA alphabets in cloning vehicles, with one week "order-to-clone" times for plasmids and viruses.

Public Health Relevance Statement:


Public Health Relevance:
Whole gene synthesis today has a major commercial market in biomedical research and clinical practice, with (for example) genes that include an RNA polymerase promoter being used to generate whole messenger RNA transcripts to be used in RNA-based gene therapy. The expensive part of whole gene construction is not the synthesis of the primary DNA oligonucleotide gene fragments, which have now become quite inexpensive. Rather, the cost is the assembly of those fragments to give the full gene, a process that requires considerable human involvement and risk of failure. Recently, scientists at the Foundation for Applied Molecular Evolution reported a technology that allows autonomous self-assembly of dozens of the gene fragments, largely without human attention [Merritt et al. 2014]. Once transferred to Firebird, this technology will support large DNA construction with applications ranging from biomanufacturing to diagnostics to therapy.

Project Terms:
Architecture; Attention; base; Base Pairing; Biomanufacturing; Biomedical Research; biophysical chemistry; biophysical properties; Biotechnology; Businesses; clinical practice; Cloning; Code; Codon Nucleotides; Collaborations; Color; commercialization; Computer software; cost; Custom; deep sequencing; density; design; Development; Diagnostic; DNA; DNA biosynthesis; DNA-Directed RNA Polymerase; Enzymes; Escherichia coli; Evolution; Failure; Foundations; gene correction; gene synthesis; gene therapy; Genes; Genetic; genetic information; Goals; Human; improved; Information Systems; innovation; Kanamycin Resistance; Laboratories; Length; Letters; Ligase; Marketing; meetings; Messenger RNA; Metabolic Pathway; Modeling; Molecular Evolution; Motivation; Natural Products; Nature; novel strategies; nucleobase; Nucleotides; Occupations; Oligonucleotides; Organism; Phase; phosphoramidite; plasmid DNA; Plasmids; prevent; Procedures; Process; promoter; Proteins; public health relevance; Publishing; Reagent; Reporting; resistance gene; Risk; RNA; Role; Science; Scientist; seal; self assembly; Services; Single-Stranded DNA; Site; Small Business Technology Transfer Research; Software Design; Solid; Staging; synthetic biology; Technology; Time; Transcript; Virus; whole genome

Create Ultralong DNA Constructs in One Assembly Step Firebird Biomolecular Sciences LLC Steven A. Benner Foundation for Applied Molecular Evolution Shuichi Hoshika Abstract Frost & Sullivan found a 2014 global market for DNA oligos at $241 million, $137 million for genes. Private investment in DNA synthesis companies like Twist, Ginkgo, and DNA Script give collective valuations of several billion dollars. Federal public investment by the NIH, DARPA, and others in synthetic biology that depends on DNA synthesis exceeds $100 million annually. These numbers stand behind this project to develop two innovations to (a) deliver, under a custom synthesis model, long DNA (L-DNA) assemblies (b) secure a licensing platform, and (c) create collaboration and buyout opportunities. These technologies are: 1. Artificially expanded genetic information systems (AEGIS), which add 4 nucleotides forming 2 additional orthogonally binding nucleobase pairs (Z:P and S:B) to the pairs (C:G and T:A) found in natural DNA. Eight- letter DNA increases the number of sequence accurate fragments that can be autonomously assembled. 2. Transliteration, which converts Z, P, S and B to C, G, T and A respectively, giving an entirely natural L- DNA construct by removing the AEGIS components after they have done their job assembling fragments. Highlights of Phase I results include: (a) OLIGARCHTM software predicting stability of 8-letter GACTZPSB DNA duplexes. (b) Fidelity of DNA products made by AEGIS + transliteration as good as in commercial G-blocks. (c) Constructed genes for kanamycin resistance and green fluorescence protein were active in E. coli cells. These successes shift the cost/quality burden for L-DNA synthesis towards residual error management. Aim 1. Manage residual error using, as experiments suggest: 1.1 C-glycosides to eliminate depurination and depyrimidinylation, should these cause residual error. 1.2 Enzymatically removable protecting groups to eliminate chemical damage during deprotection. 1.3. Capturable capping groups to achieve simple >99.999% removal of truncated species. 1.4 Enzymatic DNA synthesis to eliminate all chemical degradation in harsh phosphoramidite synthesis. Residual error will be further managed using MutS and Surveyor nuclease error correction. Aim 2. Create synthetic pipelines to prepare the building blocks and reagents used to manage residual error. Aim 3. Develop array-based phosphoramidite synthesis of fragments with continuous error evaluation. Reproducibility will be ensured by making the reagents themselves available for sale. This is a source of immediate revenue as well as a major part of our marketing strategy. Already, reagent sales to satisfied customers have yielded licensing deals worth over $2.5 MM. For commercialization, Firebird just executed an agreement with DNA Script, a pioneer for non-templated enzymatic DNA synthesis and its automation, to go forward after Phase 2, should enzyme-based DNA synthesis be preferred to manage residual error. This includes licensing Firebird's patents for enzymatic cyclic reversibly terminated untemplated DNA synthesis.