Create Ultralong DNA Constructs in One Assembly Step

Create Ultralong DNA Constructs in One Assembly Step Firebird Biomolecular Sciences LLCSteven ABenner Foundation for Applied Molecular EvolutionShuichi Hoshika AbstractFrost andampSullivan found aglobal market for DNA oligos at $million$million for genesPrivate investment in DNA synthesis companies like TwistGinkgoand DNA Script give collective valuations of several billion dollarsFederal public investment by the NIHDARPAand others in andquot synthetic biologyandquotthat depends on DNA synthesis exceeds $million annuallyThese numbers stand behind this project to develop two innovations toadeliverunder a custom synthesis modellong DNAL DNAassembliesbsecure a licensing platformandccreate collaboration and buyout opportunitiesThese technologies areArtificially expanded genetic information systemsAEGISwhich addnucleotides formingadditional orthogonally binding nucleobase pairsZ P and S Bto the pairsC G and T Afound in natural DNAEightletter DNA increases the number of sequence accurate fragments that can be autonomously assembledTransliterationwhich converts ZPS and B to CGT and A respectivelygiving an entirely natural LDNA construct by removing the AEGIS components after they have done their job assembling fragmentsHighlights of Phase I results includeaOLIGARCHTM software predicting stability ofletter GACTZPSB DNA duplexesbFidelity of DNA products made by AEGIStransliteration as good as in commercial G blockscConstructed genes for kanamycin resistance and green fluorescence protein were active in Ecoli cellsThese successes shift the cost quality burden for L DNA synthesis towards residual error managementAimManage residual error usingas experiments suggestC glycosides to eliminate depurination and depyrimidinylationshould these cause residual errorEnzymatically removable protecting groups to eliminate chemical damage during deprotectionCapturable capping groups to achieve simple andgtremoval of truncated speciesEnzymatic DNA synthesis to eliminate all andquot chemicalandquotdegradation in andquot harshandquotphosphoramidite synthesisResidual error will be further managed using MutS and Surveyor nuclease error correctionAimCreate synthetic pipelines to prepare the building blocks and reagents used to manage residual errorAimDevelop array based phosphoramidite synthesis of fragments with continuous error evaluationReproducibility will be ensured by making the reagents themselves available for saleThis is a source of immediate revenue as well as a major part of our marketing strategyAlreadyreagent sales to satisfied customers have yielded licensing deals worth over $MMFor commercializationFirebird just executed an agreement with DNA Scripta pioneer for non templated enzymatic DNA synthesis and its automationto go forward after Phaseshould enzyme based DNA synthesis be preferred to manage residual errorThis includes licensing Firebirdandapos s patents for enzymatic cyclic reversibly terminated untemplated DNA synthesis Create Ultralong DNA Constructs in One Assembly StepFirebird Biomolecular Sciences LLCSteven ABennerFoundation for Applied Molecular EvolutionShuichi Hoshika Narrative Whole gene synthesis today has markets in excess of $millionand annual public investment of perhaps $million in the synthetic biology dependent on itand corporate valuationse gTwistGinkgoDNA Scriptapproaching $billionThe medical relevance of DNA synthesis extends from the synthesis of therapeutic DNA molecules to the production of natural productsas in the NIHGenomes to Natural ProductsandquotUensembleto the creation of biotechnology platformsas in the $million DARPA andquot Living Foundriesandquotprogramto drug discoverytarget evaluationand basic researchThe expensive part of whole gene construction is not the synthesis of the primary DNA oligonucleotide gene fragmentswhich have now become quite inexpensiveRatherthe cost is the assembly of those fragments to give the full genea process that requires considerable human involvement and risk of failurePhaseof this STTR collaboration has shown how two technologies can significantly lower the cost for long DNAL DNAsynthesisdefined as plasmid to virus length assembliesThis will lower public and private investment costs substantiallyand allow Firebird to develop a custom synthesis business to deliver L DNA assemblieslicensing its platformand create collaboration and buyout opportunities