Unique Thermal Stability of Unnatural Hydrophobic Ds Bases in Double-Stranded DNAs

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Unique Thermal Stability of Unnatural Hydrophobic Ds Bases in Double-Stranded DNAs
Unique Thermal Stability of Unnatural Hydrophobic Ds Bases in Double-Stranded DNAs
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ACS Synthetic Biology
Publication Date:
26 July 2017
ACS Synth. Biol. 2017, 6, 10, 1944-1951
Genetic alphabet expansion technology, the introduction of unnatural bases or base pairs into replicable DNA, has rapidly advanced as a new synthetic biology area. A hydrophobic unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) exhibited high fidelity as a third base pair in PCR. SELEX methods using the Ds–Px pair enabled high-affinity DNA aptamer generation, and introducing a few Ds bases into DNA aptamers extremely augmented their affinities and selectivities to target proteins. Here, to further scrutinize the functions of this highly hydrophobic Ds base, the thermal stabilities of double-stranded DNAs (dsDNA) containing a non-cognate Ds–Ds or G–Ds pair were examined. The thermal stability of the Ds–Ds self-pair was as high as that of the natural G–C pair, and apart from the generally higher stability of the G–C pair than that of the A–T pair, most of the 5′-pyrimidine-Ds-purine-3′ sequences, such as CDsA and TDsA, exhibited higher stability than the 5′-purine-Ds-pyrimidine-3′ sequences, such as GDsC and ADsC, in dsDNAs. This trait enabled the GC-content-independent control of the thermal stability of the designed dsDNA fragments. The melting temperatures of dsDNA fragments containing the Ds–Ds pair can be predicted from the nearest-neighbor parameters including the Ds base. In addition, the non-cognate G–Ds pair can efficiently distinguish its neighboring cognate natural base pairs from non-cognate pairs. We demonstrated that real-time PCR using primers containing Ds accurately detected a single-nucleotide mismatch in target DNAs. These unique properties of the Ds base that affects the stabilities of the neighboring base pairs could impart new functions to DNA molecules and technologies.
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Funding Info:
This work was supported by a Grant-in-Aid for Scientific Research [KAKENHI 26248043] from the Ministry of Education, Culture, Sports, Science and Technology (I.H.) and by the Japan Science and Technology Agency (JST) Precursory Research for Embryonic Science and Technology (PRESTO) (M.K.).
This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Synthetic Biology, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acssynbio.7b00165
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