This story has been updated from a previous version to include comments from New England Biolabs and to correct data regarding the synthetic base pair amplification.
By Ben Butkus
Scientists from Japan's Riken and spinout company TagCyx Biotechnologies have published research demonstrating a synthetic base pair system with high enough sensitivity, fidelity, and efficiency to be consistently used as an artificial third base pair for PCR amplification.
As such, TagCyx is currently exploring developing and commercializing the synthetic base pairs for a variety of life science applications, including as DNA and RNA aptamers and nucleic acid ribozymes for basic research, diagnostics, and therapeutics, TagCyx CEO Ichiro Hirao said this week.
In addition, TagCyx and New England Biolabs have entered into a collaboration to discern why the base pair system seems to work better with a specific NEB DNA polymerase than with other commercial polymerases, Hirao said.
In a research paper published online last week in Nucleic Acids Research, a group led by Hirao, who is also a researcher in Riken's Systems and Structural Biology Center, described the development and proof of principle for its hydrophobic unnatural base pair system, 7-(2-thienyl)imidazo[4,5-b]pyridine and 2-nitro-4-propynylpyrrole, abbreviated to Ds and Px.
As described in the paper, the researchers fine-tuned the Ds-Px pair by assessing the amplification efficiency and fidelity using different DNA polymerases and template sequences. More specifically, they developed a variety of methods to determine the fidelity of both the incorporation selectivity of the Ds-Px pairing and misincorporation rates when replacing natural bases with the synthetic bases in DNA templates during PCR.
They also gauged the amplification efficiency by repeating 10 cycles of amplification of DNA templates incorporating the bases to stay within exponential amplification.
Hirao and colleagues found that their Ds-Px pair survived through 100 cycles of PCR (10 cycles of PCR repeated 10 times). In addition, after 100 PCR cycles under optimized conditions, a DNA fragment containing the synthetic base pair was amplified 1028-fold with 97 percent of the Ds-Px pair retained in the amplified products, resulting in a base pair selectivity as high as 99.97 percent per replication.
Further, the group found that attaching certain functional groups to the Px base reduced the misincorporation rates of the unnatural base substrate opposite the natural bases in templates without reducing the amplification efficiency and the incorporation selectivity of the unnatural base pairing between Ds and the modified Px.
The bottom line, Hirao told PCR Insider in an email, is that the group's Ds-Px pair exhibits the highest fidelity — and particularly the lowest misincorporation rate — of any unnatural base pair developed to date. In fact, the fidelity and efficiency are as high as those of natural A-T or G-C base pairings, "indicating that a six-base system as genetic information material is possible."
For example, current DNA recombination technology involves only the four natural bases found in nucleic acids or 20 standard amino acids in proteins, Hirao explained.
In contrast, "the expansion of the genetic alphabet by unnatural base pairs enables the site-specific incorporation of extra components into nucleic acids and proteins by replication, transcription, and translation via the unnatural base pairs," he said. "In particular, standard nucleic acids comprise only four different base nucleotides [that] have similar chemical and physical properties," compared to the variety offered by the 20 amino acids in proteins.
"Thus, introducing our unnatural bases, which are more hydrophobic relative to those of the natural bases, could increase the functionality of nucleic acids for making DNA and RNA aptamers [such as] nucleic acid antibodies; and nucleic acid enzymes, such as ribozymes," Hirao added. "In addition, our … modified Px bases can also be incorporated into DNA by PCR, making various functional DNA molecules."
When developing the Ds-Px pair, Hirao and colleagues examined a variety of PCR amplification conditions such as the polymerases used and the natural and unnatural base substrate concentrations. Among several DNA polymerase tested, the group found that New England Biolabs' DeepVent DNA polymerase and Life Technologies' AccuPrime Pfx DNA polymerase exhibited high fidelity and efficiency for the Ds-Px pair under conventional base substrate concentration conditions.
The NEB polymerase proved to be particularly optimal, spurring the group to look into the matter further.
"We still do not know why DeepVent DNA polymerase is good for the Ds-Px pair," he said. "The only thing that we know is that the exonuclease activity of the polymerase has an important role for the base pairing fidelity in PCR. We are now scrutinizing this by collaborating with New England Biolabs."
In an email to PCR Insider, Bill Jack, research director at NEB, confirmed that the company has worked with Hirao and colleagues at Riken and TagCyx and "shares their fascination with the synergy between Deep Vent DNA exo- polymerase and TagCyx unnatural base pairs."
Jack noted that Deep Vent DNA polymerase shares considerable sequence homology with other hyperthermophilic archaeal DNA polymerases typically used for PCR and other thermophilic applications.
"It is fascinating that despite 80 to 95 percent sequence similarities within this group, distinct differences in substrate reactivity and biochemical parameters exist," Jack added. "We at NEB are committed to exploring the biochemical basis for observed differences, both independently and working together with fellow scientists at Riken and TagCyx. We anticipate an increased understanding will help to enable not only TagCyx, but also a variety of other technologies."
In the meantime, Hirao's company TagCyx is investigating ways to further improve the synthetic base pair and use it in a variety of applications.
"We are now functionalizing the Ds-Px pair, such as [with] fluorescently active modifications and modifications with several active functional groups for diagnostic and therapeutic applications," Hirao said. The group is also developing an in vitro selection method called systematic evolution of ligands by exponential enrichment, or SELEX, which involves using the unnatural base pair to generate nucleic acid aptamers and enzymes with increased functionality.
TagCyx hopes to disseminate the unnatural base pair system to researchers for additional application development, and will also develop its own reagents geared toward basic research uses and diagnostic and therapeutic applications. "In addition, TagCyx is providing new platforms, such as DNA authentication (DNA ink), visible PCR amplification systems, and DNA aptamers," he said.
Riken and TagCyx have filed approximately 20 patents surrounding the technology. When it was founded in 2007, TagCyx received an undisclosed amount of venture capital from various sources; and in January it entered into a strategic business alliance with the life sciences group of Japanese technology commercialization firm Nagase.
As part of this deal, which included an undisclosed investment from Nagase, the partners are further developing TagCyx's technology in nucleic acid-based therapeutics and diagnostics; and seeking pharmaceutical collaborators in the same areas.
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