Highly specific unnatural base pair systems as a third base pair for PCR amplification

Abstract

Toward the expansion of the genetic alphabet of DNA, we present highly efficient unnatural base pair systems as an artificial third base pair for PCR. Hydrophobic unnatural base pair systems between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) were fine-tuned for efficient PCR, by assessing the amplification efficiency and fidelity using different polymerases and template sequence contexts and modified Px bases. Then, we found that some modifications of the Px base reduced the misincorporation rate of the unnatural base substrates opposite the natural bases in templates without reducing the Ds–Px pairing selectivity. Under optimized conditions using Deep Vent DNA polymerase, the misincorporation rate was extremely low (0.005%/bp/replication), which is close to that of the natural base mispairings by the polymerase. DNA fragments with different sequence contexts were amplified ∼1010-fold by 40 cycles of PCR, and the selectivity of the Ds–Px pairing was >99.9%/replication, except for 99.77%/replication for unfavorable purine-Ds-purine motifs. Furthermore, >97% of the Ds–Px pair in DNA survived in the 1028-fold amplified products after 100-cycle PCR (10 cycles repeated 10 times). This highly specific Ds–Px pair system provides a framework for new biotechnology.

MOLECULAR DYNAMICS STUDY OF NON-HYDROGEN-BONDING BASE-PAIR DNA DUPLEX d(GTCDNAM GCGCCGTGGC). d(GCCACGGCGCD5SICSGAC)

“…(ii) The nucleobases paired in an intercalated manner, similar to their pairing in free duplex DNA and the mode of pairing depends on the length of the single-stranded template, and (iii) more importantly, the mode of intercalation appeared to depend mostly on sequencespecific interactions of the flanking nucleotides, with the specific packing interactions between the intercalating nucleobases of secondary importance. These conclusions are drawn on the basis of the few crystallographic and experimental results [10][11][12][13]. And are limited to an insertion of DNAM and 5SICS in XACCAGGGCGCY (12-mer primer, and corresponding templet and 'X' represents purines and 'Y' represents DNAM or D5SICS) in different crystal and NMR experiments.…”

Unnatural Base Pairs for Synthetic Biology

“…The low misincorporation rate of the recently developed Ds : diol1-Px pair (5×10 −5 errors/bp) is close to the mispairing error rate of natural WC pairs (2×10 −5 errors/bp). 34,35) By making use of this superior property of the Ds : diol1-Px pair, they succeeded in obtaining a DNA aptamer containing the Ds base against human protein target, vascular endothelial cell growth factor-165 (VEGF-165). 36) Because the affinities of aptamers that have Ds bases are >100-fold improved over those of aptamers containing only natural bases, the potential of genetic alphabet expansion as a powerful tool for creating highly functional nucleic acids is demonstrated.…”

Chemical biology: DNA's new alphabet

“…Hirao and his colleagues found that they could reduce mispairing by designing shapes that fit awkwardly with natural bases, and by adding negatively charged or electron-rich chemical groups that repel the natural bases' corresponding parts. In 2011, Hirao's team reported that DNA containing an unnatural hydrophobic base pair, called Ds and Diol1-Px, could be copied with 99.77-99.92% fidelity per replication 9 . The same year, Benner and his colleagues showed that another unnatural base pair -P and Z, which join using hydrogen bonds -achieved fidelity of 99.8% per replication 10 .…”