By Ben Butkus
A group led by Steven Benner of Florida's Foundation for Applied Molecular Evolution has been awarded $370,000 over the coming year from the National Institutes of Health to apply its artificial base pair technology and related methods to improve PCR-based detection of HIV, according to recently published grant information.
The eventual goal of the project, which is set to run through 2014, is to improve the sensitivity and enhance low-cost multiplexing of HIV in complex biological mixtures; and to detect emerging variants of the virus, according to the grant's abstract.
In the meantime, FAME and Benner's startup company Firebird Biomolecular Sciences continue to sell research-use-only products based on their innovations. They are also using the artificial genetic system technologies to develop commercial nucleic acid amplification-based tests in the areas of cancer, infectious disease, and forensics, Benner told PCR Insider this week.
Under the latest grant, administered by the National Institute of Allergy and Infectious Diseases, Benner and colleagues will combine four innovations made by the laboratory in recent years in order to improve molecular detection of HIV: an artificially expanded genetic information system, or AEGIS; a self-avoiding molecular recognition system, or SAMRS; methods to conduct so-called orthogonal PCR within the framework of AEGIS and SAMRS; and reversible terminators for single nucleotide variation detection.
The first innovation, AEGIS, describes the development of artificial bases to expand on the four natural DNA bases and support so-called "six-nucleotide PCR," which allows independent amplification of small amounts of HIV RNA without interference from other DNA in the environment.
The roots of the AEGIS technology extend to Benner's days as a scientist with the Swiss Federal Institute of Technology in Zurich, and later as co-founder of EraGen Biosciences. Benner, widely credited as being one of the first innovators of the field of synthetic biology, in 2005 founded the Gainesville-based FAME, where his laboratory has since continually worked to improve the AEGIS technology.
According to Benner, one of the biggest challenges facing nucleic acid amplification-based molecular testing is that it is highly non-linear, "so if you try to multiplex it … you have different amplifications going at slightly different rates. If it's one percent faster and you do the cycle 50 times, you have a lot more of one product than the other." In addition, "AT-rich primers bind at different temperatures than GC-rich primers, so you have a hard time getting multiplexing going at the same time."
Further, most PCR-based testing requires an excess of primers that, if not sufficiently specific, bind either non-target nucleic acids or bind each other, creating primer-dimers.
"If you want to do PCR right, make the primers so they don’t bind to anything in the natural world," Benner said. His laboratory has developed eight additional bases, or four additional base pairs — dubbed Z-P, V-J, K-X, and S-B — that pair with each other but not with natural DNA. FAME will only use one pair in its HIV study.
Since Benner first conceived of AEGIS, many other groups have developed artificial base pair technologies. Most recently, scientists from Japan's Riken and spinout company TagCyx, led by Ichiro Hirao, described a new unnatural base pair system and demonstrated that it could be consistently used for PCR amplification – with the caveat that it requires a specially engineered polymerase (PCR Insider, 12/1/2011).
Hirao's based pairs "are much more different from natural DNA, and the polymerases that have evolved for billions of years to take AGCT don't like it," Benner said. "Ours, they just love. Now, we cheated too, we had to engineer the polymerases … so they are not exactly the ones you have from billions of years of biological evolution. But the fact is you can do PCR with our six-letter genetic alphabet, where the primers are found nowhere in the natural genome, and therefore don't bind off target, no matter how many of them you add."
Meantime, Benner and colleagues have developed the SAMRS technology to "support essentially unlimited multiplexing in DNA probing, priming, and multiplexed PCR amplification;" as well as procedures to convert standard DNA into AEGIS-containing DNA and support "downstream orthogonal capture that allows DNA-targeted assays to be flexible and adaptive, possibly allowing new targets to be added to a multiplexed assay kit without demanding a reworking of the parts of that kit already targeted," according to the grant's abstract.
Lastly, Benner's group has developed reversible terminators that are hypothesized to allow detection and relative quantitation of variant HIV sequences.
"One of the problems with HIV infection is to try and understand whether you have an infection that's chronic … or a newly incident disease," Benner said. "You can tell the difference if you have multiplexed detection, especially if it allows you to detect single nucleotide variations."
The reversible terminators are 3' modifications of a nucleoside triphosphate, "where a polymerase will add the base to the growing primer, and stop. But unlike dideoxynucleotides … here the stop is reversible. So we can actually do PCR after we stop, but only if we have stopped at a base that is indicating a mutation that you're interested in," Benner explained. The reversible terminator technology, Benner noted, has also been licensed to Intelligent Biosystems for next-generation sequencing purposes.
According to the grant's abstract, the Benner group will perform a "staged series of assay development, adding each of these innovations in series to increasingly challenging problems in the detection of HIV target sequences, starting with … detection of single HIV targets in relatively simple environments, adding innovations as we lower the amount of target molecules, [increasing] the level of multiplexing, and [making] the environment more complex."
At each stage, the group notes, it will "drive the system to fail, and note the parameters … at which the system fails," thus providing a metric for progress. Lastly, the researchers plan to deliver kits of primers, probes, and detection capture beads to be provided to HIV researchers.
Benner and FAME's delivery vehicle is Firebird Biomolecular Sciences, which currently sells various reagents for DNA sequencing, amplification, and analysis for research use only; as well as bioinformatics software for genomic database management.
Even though the recent NIAID funding will support HIV assay development, Benner and colleagues are already eyeing a number of molecular testing applications that could be improved by the same technological innovations.
Benner said the applications include detecting single nucleotide variations in colon cancer and lung cancer; and detecting targets related to insect-borne infectious diseases such as dengue fever, yellow fever, encephalitis, and West Nile virus. "We have also just prepared some [technologies] for bacterial diseases [such as] Rickettsia disease, typhus, and Q fever," he said.
Firebird and FAME have also used some basic research funding from the US Defense Threat Reduction Agency to develop a forensic analysis kit based on the expanded genetic alphabet, and are working to get that into the hands of early adopters.
As far as clinical testing goes, Benner said Firebird will likely seek external partners, or at least external advice, to help shepherd it through the US Food and Drug Administration process. Benner noted that one of his contacts at the Defense Advanced Research Projects Agency, Daniel Wattendorf, head of DARPA's Defense Sciences office, has experience with diagnostic development and the approval process. "We are certainly going to take advantage of whatever experience he has to try and move some of these products through FDA approval," Benner said.
"It's an easy thing to do when you're talking about insect-borne infectious disease. You always have a public health use," he added. "But the funding agencies … don't just want toy technologies, either. They would like at some point to have this stuff end up in the clinic, and that's something we're certainly going to try and do now."
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