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Bradley K.M.,Foundation for Applied Molecular Evolution | Bradley K.M.,Westheimer Institute for Science and Technology | Benner S.A.,Foundation for Applied Molecular Evolution | Benner S.A.,Westheimer Institute for Science and Technology | Benner S.A.,Firebird Biomolecular Sciences, LLC
Beilstein Journal of Organic Chemistry | Year: 2014

Synthetic biologists wishing to self-assemble large DNA (L-DNA) constructs from small DNA fragments made by automated synthesis need fragments that hybridize predictably. Such predictability is difficult to obtain with nucleotides built from just the four standard nucleotides. Natural DNA's peculiar combination of strong and weak G:C and A:T pairs, the context-dependence of the strengths of those pairs, unimolecular strand folding that competes with desired interstrand hybridization, and non-Watson-Crick interactions available to standard DNA, all contribute to this unpredictability. In principle, adding extra nucleotides to the genetic alphabet can improve the predictability and reliability of autonomous DNA self-assembly, simply by increasing the information density of oligonucleotide sequences. These extra nucleotides are now available as parts of artificially expanded genetic information systems (AEGIS), and tools are now available to generate entirely standard DNA from AEGIS DNA during PCR amplification. Here, we describe the OligArch (for "oligonucleotide architecting") software, an application that permits synthetic biologists to engineer optimally self-assembling DNA constructs from both six- and eight-letter AEGIS alphabets. This software has been used to design oligonucleotides that self-assemble to form complete genes from 20 or more single-stranded synthetic oligonucleotides. OligArch is therefore a key element of a scalable and integrated infrastructure for the rapid and designed engineering of biology. © 2014 Bradley and Benner; licensee Beilstein-Institut. Source


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 149.69K | Year: 2015

DESCRIPTION provided by applicant The public health challenges from RNA viruses are at a tipping point with dengue moving up Florida human transmission of chickungunya now established in Florida and transmitted by different mechanism Ebola While these have long been a challenge their incidence has been predominantly overseas In addition the health hazards of other RNA viruses better known in the United States remains unabated Eastern equine encephalitis is again emerging in New England with its mortality rate West Nile virus continues as an important health hazard Further given the speed of international travel and the ease with which the sequences of RNA viruses mutate a public health official can easily have a mosquito that carries an RNA virus from one of these classes that is nevertheless not detected by PCR with standard primers This creates a need for exactly the product that Firebirdandapos s technologies enable These technologies are Self avoiding molecular recognition systems SAMRS SAMRS primers do not interact with each other This allows unlimited multiplexing of PCR Further SAMRS allows additional targets to be added to an assay without the multiplex collapsing SAMRS also suppresses primer dimers in isothermal amplification e g RPA HDA making possible in Phase point of sampling kits SAMRS therefore supports a highly adaptable molecular diagnostics able to detect dozens of RNA species for essentially the cost of detecting one Artificially expanded genetic information systems AEGIS AEGIS adds nucleotides to the four in standard DNA and RNA collectively xNA Thus AEGIS generates primers that cannot complement any natural DNA no matter how complex the sample supporting assays with very low noise and very few false positives Universal base technology Biversals RNA viruses easily mutate exemplifying the consequences to public health recent surveillance of HIV infected patients with the goal of measuring incidence found that the viruses in of the isolates were not PCR amplified by standard primers Firebirdandapos s evolutionary analyses show that mutations often occur in silent sites in coding regions Thus Firebird scientist invented andquot biversalandquot nucleobases that prime on either A or G the Y biversal or on either T or C the R biversal Phase will generate a kit to detect common mosquito borne RNA viruses allowing public health staff to screen on a Luminex platform for all of these for less than $ less than the cost of detecting any two RNA viruses separately In Phase these will be moved to simpler platforms Firebirdandapos s pipeline investments allow us to prepare AEGIS and SAMRS building blocks in multi gram amounts and AEGIS and SAMRS oligonucleotides on demand Because surveillance assays are not heavily regulated and since its customers for those kits public health staff are well trained this product fits well within Firebirdandapos s current product line However strong performance of a low cost highly multiplexed easily adaptable and low false positive surveillance product will undoubtedly help Firebird raise capital for patient targeted applications of these technologies PUBLIC HEALTH RELEVANCE The public health challenges presented by RNA viruses are this year reaching a tipping point with dengue moving its way up the Florida peninsula human to human transmission of chickungunya now established in the United States which until this year was always imported from an exotic tropical locale Eastern equine encephalitis now being detected in New England with a mortality rate and of course operating by an entirely different mechanism of transmission Ebola which is uncontrolled at this moment in Western Africa This creates a need for exactly the product that Firebirdandapos s technologies enable a highly adaptable molecular diagnostics assay that is able to detect dozens of RNA species for essentially the same cost as detecting a single RNA virus design to manage the rapid divergence of these species and capable of extraordinarily few false positives This project will develop kits that allow a single public health official in a single step to test real biological samples for example mosquitoes caught in a single trap for all RNA viruses at the same time and with the same total cost as a test for any one of them separately Since such surveillance kits have few regulatory barriers Firebird will be able to bring these kits to market on its own before the end of the Phase project period


Grant
Agency: Department of Defense | Branch: Army | Program: STTR | Phase: Phase I | Award Amount: 100.00K | Year: 2011

Because of chemical behaviors of real DNA molecules, the nanotechnology envisioned by the Defense Department in this Solicitation will be possible only if design automation software creates nanostructures that exploit artificially expanded genetic information systems (AEGIS). AEGIS DNA molecules have more than the four nucleotides (GACT) found in natural DNA. Our software will accommodate nanoarchitectures built from these four, plus eight additional orthogonally pairing AEGIS nucleotides. By evading the computational challenges associated with natural DNA nanostructures, AEGIS will allow delivery at the end of Phase I, ahead of schedule, software that helps nanotechnologists design nanostructures that incorporate binding and catalytic DNA molecules, sensing functionality, and optical signaling elements. Additional benefits of AEGIS DNA nanostructures are their enhanced stability and potential to support continuous environmental monitoring. Also ahead of schedule, our Phase I work will deliver a physical example of a nanostructure containing these functional elements. This will allow Phase II to focus on higher level nanostructure performance, including dynamic architectures, interfaces to electrical output, and amorphous computing. This project overlaps Firebird's business, which supports human diagnostics based on reagent innovations like AEGIS. Adding nanotechnology to its existing business makes commercial sense, and will help"dual uses"emerge from this project.


Grant
Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 102.75K | Year: 2007

DESCRIPTION (provided by applicant): The proposed research will develop dynamic combinatorial chemistry as a new and innovative strategy to allow a DNA or RNA target in a biological sample to assemble its own template under conditions of dynamic equilibrium. This chemistry incorporates element that allows the template-created primer to primes the synthesis of a strand of DNA complementary to the target. The selectivity of the process is such that a single DNA sequence in a genome as complex as the human genome can be targeted. The architecture of the process, however, allows it to discriminate against single nucleotide changes with the effectiveness of small duplexes. Consistent with the basic research/business mission/plan of the Foundation for Applied Molecular Evolution and Firebird Biomolecular ("We do not make DNA assays. We develop chemistry that makes DNA assays better"), this will create a platform technology that meets specifications that are unavailable at the present time: selectivity of a probe as if it were a long oligonucleotide, but discrimination by the probe as if it were a short oligonucleotide. This platform technology should have application in many assays, including in human diagnostics and research markets. In preliminary studies, a proof of concept showed that this combinatorial chemistry architecture met the novel specifications when the target was DNA. The Phase 1 milestone will be passed if similar success can be obtained with RNA as a target. Potential commercial applications in human medicine are substantial, starting with applications in biomedical research, where these tools will allow us to detect messenger RNA, RNAi, and other RNA molecules important in biology. Several important diagnostics and drug development tools been driven in the past decade through the creation of platform chemical technologies by scientists at the Foundation for Applied Molecular Evolution. Such technologies have commercial impact far beyond their development costs because they deliver a set of chemical performance specifications that is unavailable by existing chemistry. The proposed work will develop another platform technology, this one allowing the detection of nucleic acid targets through a novel combination of dynamic combinatorial chemistry, enzymology, and nucleic acid science.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 150.00K | Year: 2014

Firebird will deliver multiplexed assays that inexpensively detect and distinguish coccidioidomycosis, blastomycosis, and histoplasmosis by targeting the genomic DNA of their causative agents. This assay uses six Firebird technologies: 1. Artificially expanded genetic information systems (AEGIS), which support ultra-clean DNA amplification with ultra-low noise despite the complexity of realbiological samples. 2. Self-avoiding molecular recognition systems (SAMRS), which permit unlimited multiplexing of xNA­ targeted assays, and (consequently) low cost and extreme adaptability in such assays. 3. Conversion technologies that create AEGIS-containing amplicons easily detected by Luminex without interference from any naturally found xNA. 4. Evolution-based software to select genomic targets for genus and species discrimination. 5. Novel reagent-enzyme combinations that make fungal xNA accessible to amplification. 6. Novel isothermal amplification methods to allow the assays to work near points-of-care. The initial product will address the interest of the NCEZID in analyzing environmental samples, to avoid costly FDA regulation. Firebird's investment in pipelines allows us to prepare AEGIS and SAMRS building blocks in multi-gram amounts, and their oligonucleotides on demand. Thus, this product fits well within Firebird's current product line. Specific diagnosis of fungal infections will enhance public health by providing a mechanism to save lives and prevent death serious disease.

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