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Lucigen Team Publishes Details of Thermostable Viral DNA Polymerase for One-Enzyme RT-PCR

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Scientists from genomics tools provider Lucigen have published a paper describing the discovery, characterization, and performance of a thermostable viral DNA polymerase that enables true one-step, single-enzyme reverse-transcriptase PCR.

According to the researchers, the paper is the first peer-reviewed report of a reagent enzyme produced from a viral metagenomic library; of a viral polymerase shown to be fully thermostable in vitro; and of a single-enzyme RT-PCR method with high sensitivity and specificity comparable to commercial two-enzyme systems.

Lucigen is now hoping that the publication, along with recently issued patents surrounding the technology, will spur interest in the enzyme from potential customers and licensees, and drive its use in a wide variety of applications — including Lucigen's own fledgling molecular diagnostics program, Thomas Schoenfeld, vice president of enzyme discovery at Madison, Wis.-based Lucigen, told PCR Insider this week.

Schoenfeld was involved with the initial discovery and subsequent development of the polymerase, called 3173 Pol, which, he said, began with a "leap of faith … 12 years ago that there was a significant population of viruses in hot springs and that their genomes would encode useful thermostable DNA polymerases."

Schoenfeld and colleagues worked with "leading viral ecologists" along the way to develop methods and tools for viral metagenomics that would enable the discovery of the new polymerase.

Specifically, as described in a paper published last week in PLoS One, the researchers constructed a viral metagenomic library from Octopus hot spring in Yellowstone National Park, and analyzed more than 20,000 Sanger sequence reads from the library to identify hundreds of potential pol genes.

Then, they tested these candidates using a primer extension assay to identify the most thermostable clones, eventually identifying 3173 Pol as the most thermostable of the lot.

The enzyme belongs to a family of thermostable viral polymerases that have the strongest sequence similarity to Pol I-type enzymes from the Aquificales family, and shares 32 percent amino acid identity with Thermocrinis albus Pol I, according to the researchers.

In addition, the enzyme shares no significant sequence similarity to any previously described viral protein; and indeed, Lucigen's scientists have yet to discern the natural physiological role of the polymerase or determine the virus it belongs to or the host that virus infects.

However, Lucigen's scientists have determined that the polymerase exhibits physiological properties that make it unlike any polymerase described to date and conducive to use in a variety of research and molecular diagnostic applications.

"We went to the hot springs hoping to discover ten enzymes with good uses; and basically discovered one enzyme with 10 good uses," Schoenfeld said.

The most important characteristic of 3173 Pol is the fact that it can enable single-enzyme RT-PCR, something that scientists have been trying to achieve by modifying well-characterized enzymes such as Moloney Murine Leukemia Virus reverse transcriptase and Thermus thermophilus Pol I — with mixed success, since the modified versions of these enzymes often do not have sufficient thermal stability, accuracy, or sensitivity.

"I think of this as direct detection," Schoenfeld said. "Normally when one detects RNA you're actually quantifying the cDNA that has been reverse-transcribed by MMLV reverse transcriptase." With 3173 Pol, "you're directly interrogating," he added. "The same enzyme is reverse-transcribing and detecting at the same time. Because the enzyme has both activities, it is directly detecting the product."

"There are some other advantages, as well," Schoenfeld added. "Because you're not doing that extra step, it's faster. And also, something we're working on is a hot-start that affects not just the amplification step but also the reverse transcription step, so there are some other benefits, as well.

Another major advantage of the enzyme is its thermal stability, which allows it to be easily incorporated into master mixes prior to experiments.

"There aren't too many master mixes out there … and the reason for that is some people have had trouble … with stability [of other enzymes] with the pre-mix and master mix," Schoenfeld said. "Some have become available … but MMLV is not very stable in solution, so there have been problems, and that's something we tried to improve upon."

This is particularly important when researchers are conducting high-volume or high-throughput RT-PCR experiments in which it would save both time and money to create a pre-mix with the enzyme instead of adding it just prior to the RT-PCR step.

Lucigen has been aware of these properties, among others, for several years, and already markets 3173 Pol as a standalone enzyme called PyroPhage 3173 DNA polymerase; and as an RT-PCR master mix called PyroScript.

However, even though Lucigen has discussed the enzyme with potential customers at various scientific meetings, and has inked a few scientific collaborations to investigate various applications for 3173 Pol, interest from the scientific community has been less enthusiastic than the company had hoped, primarily because researchers had not, until last week, published a peer-reviewed paper fully describing the technology and its application to RT-PCR.

In their PLoS One paper, Schoenfeld and colleagues compared 3173 Pol to one of the most commonly used one-enzyme RT-PCR systems (T. thermophilus); and to three commercial two-enzyme RT-PCR systems employing MMLV reverse transcriptase with Taq polymerase: Life Technologies' SuperScript III One-Step RT-PCR system with Platinum Taq; Quanta Biosciences' qScript One-Step SYBR Green qRT-PCR kit; and Roche's Transcriptor One-Step RT-PCR kit.

Specifically, they compared the various enzymes for their ability to quantitatively detect MS2 RNA, influenza RNA, and mRNA targets, and found that the 3173 Pol had higher specificity and sensitivity than T. thermophilus Pol, and comparable sensitivity and specificity to the three common two-enzyme systems.

"We tried to be fair …. And to use the respective manufacturers' conditions in every case," Schoenfeld said. The upshot of the comparison studies, he added, is that Lucigen's enzyme is at least as effective as three leading commercial brands but with the benefit of being easier and potentially cheaper to use.

"I think it's going to be quite cost-effective," he said, declining to provide a specific pricing comparison. "Because it's one enzyme, we can provide it at a better price. There are some targets that we've heard anecdotally … that people haven't been able to get with RT-PCR with the other two-enzyme systems, but we don't have that data in the paper. So it's the ease of use, and it's probably going to be price, because right now the sensitivity seems comparable to the other systems."

Lucigen's commercial enzyme and master mix actually use a modified version of the wild-type 3173 Pol that eliminates its innate proofreading 3'-5' exonuclease activity in order to make it easier to use for routine applications. However, Lucigen also plans to release the wild-type as a standalone enzyme that offers extremely high fidelity due to this proofreading activity.

"For preparative applications where you're going to be cloning or sequencing the product, there is a major advantage to having the higher fidelity," Schoenfeld said. "For analytic amplification, you don't really care if some percentage of amplicons you're detecting actually have a mutation. It doesn't affect the result."

Furthermore, in May, Lucigen said it had been awarded a $2.8 million Small Business Innovation Research Grant from the National Institutes of Health to develop a point-of-care diagnostic test for influenza.

The test will be able to test multiple RNA pathogens — such as influenza A and B and respiratory syncytial virus — in a fast isothermal reaction without the use of microfluidics or valves, and will be powered by 3173 Pol.

"It also strand displaces very efficiently, and that's the basis for the isothermal method," Schoenfeld said. "Instead of using heat to denature [the target], the enzyme strand displaces it from the single-strand template."

That work represents an overall change in Lucigen's strategy moving forward as it looks to transform itself from a pure-play reagents company to a molecular diagnostics player, Schoenfeld said, adding that in general, the company's continued development of 3173 Pol is "a big part of Lucigen's overall commercial strategy."

In addition, the company has recently been awarded both European and US patents surrounding the polymerase (PCR Insider, 1/11/2012), which is expected to give potential customers and licensees additional confidence in using the polymerase for a variety of applications.

"The advantage of discovering something novel is you get a little better patent protection on it than if you just discover something like a [slightly] better use for Taq polymerase," Schoenfeld said.

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