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NIH Seeks Licensees for 'Y-Family' Polymerases for Analyzing Damaged, Ancient DNA

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By Ben Butkus

This story was originally posted May 5.

The National Institutes of Health is seeking collaborators to help it develop and commercialize a family of fungi-derived thermostable polymerases that can bypass lesions in DNA that would otherwise block replication by standard polymerases.

The new Y-family polymerases could be of interest to forensic DNA service companies and research reagent companies seeking thermophilic enzymes for use in damaged or ancient DNA analysis and for novel applications with modified nucleotides, the NIH said.

The licensing and collaborative research opportunity is being offered through the Laboratory of Genomic Integrity at the National Institute of Child Health and Human Development.

The new Y-family polymerases have characteristics that may help them succeed where commonly used high-fidelity polymerases, such as Taq polymerase, currently fail, Roger Woodgate, chief of the Laboratory of Genomic Integrity and one of the scientists that characterized the new polymerases, told PCR Insider last week.

"The main limitation for conventional PCR enzymes is that they cannot bypass damage in the template DNA and prematurely terminate synthesis," Woodgate said. "In contrast, the Y-family polymerases can bypass a variety of DNA lesions and thereby generate an undamaged DNA product that may be subsequently amplified by conventional high-fidelity PCR enzymes."

Several years ago, Woodgate, John McDonald, and colleagues at NIH identified and cloned a polymerase called Dpo4 from the archaeon Sulfolobus solfataricus, hypothesizing that enzymes from such thermostable organisms would be more stable, easier to work with, and have more utility at higher temperatures.

They discovered that Dpo4 could bypass lesions by generally inserting the correct complementary nucleotide opposite a variety of damaged bases, and could, in certain cases of damaged or ancient DNA, substitute for Taq polymerase in PCR applications.

The researchers published the crystal structure of Dpo4, developed some chimeric co-constructs of the enzyme, and made the so-called Y-family polymerases available to interested collaborators. They eventually licensed the technology to an undisclosed entity, Woodgate said.

However, the Dpo4 enzymes "have limited substrate specificity," Woodgate said, "so we started to look for thermostable enzymes from other sources. These Y-family polymerases are all over the place."

Eventually, Woodgate and McDonald identified, characterized, and cloned several other polymerases from different organisms — Thermoascus auranticus, Thermomyces lanuginosus, and Sporotrichum thermophile — that they believe are a step up from the original Dpo4 enzyme.

"In general, we believe these enzymes have more open active sites; can accommodate lesions in the active sites; and can perform translesion synthesis," Woodgate said. "We hypothesize that the newly identified fungal enzymes will have similar properties, but would be able to bypass a much broader spectrum of damage compared to the Dpo4-like enzymes."

The researchers believe that the new family of polymerases may be a good substitute for Taq polymerase in applications using fluorescent nucleoside triphosphate derivatives; or, they could be included along with a conventional thermostable polymerase in a PCR protocol designed to amplify old or damaged DNA samples, thereby greatly increasing recoverability, accuracy, and length of products.

Specific applications might include forensic analysis of compromised DNA samples; identification of the deceased using ancient DNA obtained from burial sites; or genetic analyses of previously stored formalin-fixed, paraffin-embedded tumor samples.

"To be honest about the limitations here, I don't think this enzyme is going to substitute completely for Taq," Woodgate said. "The goal here is to provide a template that you would otherwise not have that could be subsequently amplified by conventional methods.

"They don't have to do the whole workload of the PCR, but if you can now make one or 10 copies of a DNA template that you couldn't do previously, then that can be amplified by regular, conventional, high-fidelity PCR enzymes," he added.

The NIH in December filed a provisional US patent application covering the new polymerases; and Woodgate and colleagues are currently preparing a manuscript describing the enzymes for peer-reviewed publication.

In the meantime, interested parties can learn more about the collaboration and licensing opportunity on the NIH Office of Technology Transfer website.

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