title: High Fidelity, Efficiency and Functionalization of Ds-Px Unnatural Base Pairs in PCR Amplification for a Genetic Alphabet Expansion System.
abstract: Genetic alphabet expansion of DNA using an artificial extra base pair (unnatural base pair) could augment nucleic acid and protein functionalities by increasing their components. We previously developed an unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px), which exhibits high fidelity as a third base pair in PCR amplification. Here, the fidelity and efficiency of Ds-Px pairing using modified Px bases with functional groups, such as diol, azide, ethynyl and biotin, were evaluated by an improved method with optimized PCR conditions. The results revealed that all of the base pairs between Ds and either one of the modified Px bases functioned with high amplification efficiency (0.76-0.81), high selectivity (≥99.96% per doubling), and less sequence dependency, in PCR using 3'-exonuclease-proficient Deep Vent DNA polymerase. We also demonstrated that the azide-Px in PCR-amplified DNA was efficiently modified with any functional groups by copper-free click reaction. This genetic alphabet expansion system could endow nucleic acids with a wide variety of increased functionalities by the site-specific incorporation of modified Px bases at desired positions in DNA.
authors.affiliation: Institute of Bioengineering and Nanotechnology , 31 Biopolis Way, The Nanos, #04-01, Singapore 138669.|RIKEN Center for Life Science Technologies , 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
authors.forename: Itaru
authors.initials: I
authors.lastname: Okamoto
authors.affiliation: RIKEN Center for Life Science Technologies , 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.|Department of Life Science, Tokyo Institute of Technology , 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 226-8501, Japan.
authors.forename: Yuya
authors.initials: Y
authors.lastname: Miyatake
authors.affiliation: Institute of Bioengineering and Nanotechnology , 31 Biopolis Way, The Nanos, #04-01, Singapore 138669.|RIKEN Center for Life Science Technologies , 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.|PRESTO, JST, Honcho , Kawaguchi-shi, Saitama 332-0012, Japan.
authors.forename: Michiko
authors.initials: M
authors.lastname: Kimoto
authors.affiliation: Institute of Bioengineering and Nanotechnology , 31 Biopolis Way, The Nanos, #04-01, Singapore 138669.|RIKEN Center for Life Science Technologies , 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
authors.forename: Ichiro
authors.initials: I
authors.lastname: Hirao
mesh_terms: D053938:DNA Fragmentation; D042622:DNA Repeat Expansion; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D007093:Imidazoles / Q000737:chemistry*; D009711:Nucleotides / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011725:Pyridines / Q000737:chemistry*; D011758:Pyrroles / Q000737:chemistry; D017422:Sequence Analysis, DNA; D058615:Synthetic Biology; D014158:Transcription, Genetic
chemical_list: C000625585:2-nitro-4-propynylpyrrole; C528341:7-(2-thienyl)imidazo(4,5-b)pyridine; D007093:Imidazoles; D009711:Nucleotides; D011725:Pyridines; D011758:Pyrroles; C088944:Deep Vent DNA polymerase; D004259:DNA-Directed DNA Polymerase
keywords: PCR; copper-free click chemistry; expansion of genetic alphabet; synthetic biology; unnatural base pair; xenobiology