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New Method Pairs Artificial Nucleotide, Mutant Polymerase to Detect DNA Adducts

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NEW YORK (GenomeWeb) – Alkylating substances such as tobacco smoke and environmental toxins can create mutagenic alkylguanine DNA adducts. This type of DNA damage is often a first step in carcinogenesis, but information on these rare, site-specific modifications is hard to come by.

Researchers at the Swiss Federal Institute of Technology, Zurich, and the University of Konstanz in Germany have now developed a method that uses a mutant polymerase to selectively incorporate an artificial nucleic acid, both marking and bypassing DNA adducts, allowing for linear amplification of damaged DNA.

The new method was published last week in The Journal of the American Chemical Society.

Adducts are rare, typically forming at concentrations of about three per 100,000 bases. They are also non-random. A carcinogen-induced adduct in a tumor-suppressor gene can have dire consequences.

Most techniques to analyze adducts ― including radioactive labeling, mass spectrometry, and antibody-based assays ― have drawbacks, and no method to date preserves sequence information.

"Normally if you have a DNA adduct in your template, either the polymerase will be stalled, or there will be an error where the polymerase will mis-incorporate a base and you'll get an incorrect identification of what is at that position," ETH Zurich researcher Shana Sturla, corresponding author on the study, explained in an interview.

The new method uses a mutated KlenTaq DNA polymerase, KTqM747K, made by Sturla's co-authors at Konstanz.

But the key to the work, she said, is the combination of that mutant enzyme and a small molecule her group developed called BenziTP.

"What we've done that is special is we've actually given [the polymerase] the choice of a special synthetic nucleotide triphosphate that it can choose as a partner for the DNA adduct," she explained.

Sturla's group designed a first generation of artificial nucleotides by modeling compounds that could potentially bind DNA adducts yet also fit the active zone of polymerase. They then began a process of synthesizing and testing each of these molecules over the course of a few years.

"When we design them, we're usually focusing on … how they interact with DNA adducts just in a DNA duplex, and then we took some candidates from our duplex studies and tested them with enzymes," Sturla said. While the duplex studies give some insight, the polymerase active site also influences the process. "It's really a modulation of both things at the same time," she said, because "you're trying to go against what the polymerases have evolved to do, and fit in these modifications into the active site."

The BenziTP and KTqM747K combination "was our lucky hit," Sturla said.

The amplification uses a standard thermocycler, but "it's just a linear primer extension, so we only use one primer," Sturla explained. Her lab uses mass spectrometry to determine adduct burden as well, but she noted that instrumentation and expertise can be limiting for that technique, while "PCR-based technologies are much more widely accessible."

Importantly, mass spec also requires digesting a DNA sample down to its constituent bases, yielding information about total number of modifications that lacks any sequence context. Guided by proper primers, the JACS method would now allow querying intact strands of DNA for adducts.

A person's DNA adduct burden is impacted by environmental toxin exposure, diet, and behaviors such as smoking, so "measuring DNA adducts from a fundamental perspective is important in trying to understand cancer risk, and trying to make a link between particular chemicals that we're exposed to in our lives and the risk for developing cancer," Sturla said.

Adducts can also be very individualized, and adduct burden may vary depending on an individual's metabolic and DNA repair capacities. Measuring them might then be "a kind of exposure monitoring" that takes into account all of the particular biochemistries of an individual controlling his or her susceptibility to a toxin, Sturla said, adding, "That's really important in understanding the development of cancer."

Some cancer drugs also work by forming DNA adducts, so the new method could be used for in vitro sensitivity testing of conventional DNA alkylating therapeutics. Alkylating chemotherapy drugs are used to treat leukemia, lymphoma, Hodgkin disease, multiple myeloma, and sarcoma, as well as lung, breast, and ovarian cancer, according to the American Cancer Society.

And, although understanding the connection between exposure and disease may sound basic, making DNA adduct measurement accessible means it might ultimately be developed into a sort of "pre-prognostic" for cancer risk, Sturla said. "You could really use it as a tool to modify your behaviors or exposures, or to implement different prevention strategies." That application is forward looking, but knowledge gained by using this tool in basic cancer research is a prerequisite.

The group decided against patenting the method itself, but does have IP around hybridization probes that incorporate its modified bases. The KlenTaq mutant is commercialized by co-author Andreas Marx at a company called MyPols. As previously covered in GenomeWeb, this company will also commercialize a high-discrimination polymerase called HiDi.

The group is now seeking to collaborate with companies that develop gene enrichment strategies, or that can help develop the probes, and would also like to collaborate to test the method on primary samples from patients.