An Efficient Unnatural Base Pair for PCR Amplification

Abstract

Expansion of the genetic alphabet by an unnatural base pair system provides a powerful tool for modern biotechnology. As an alternative to previous unnatural base pairs, we have developed a new pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitropyrrole (Pn), which functions in DNA amplification. Pn more selectively pairs with Ds in replication than another previously reported pairing partner, pyrrole-2-carbaldehyde (Pa). The nitro group of Pn efficiently prevented the mispairing with A. High efficiency and selectivity of the Ds-Pn pair in PCR amplification were achieved by using a substrate mixture of the gamma-amidotriphosphate of Ds and the usual triphosphates of Pn and the natural bases, with Vent DNA polymerase as a 3' to 5' exonuclease-proficient polymerase. After 20 cycles of PCR, the total mutation rate of the Ds-Pn site in an amplified DNA fragment was approximately 1%. PCR amplification of DNA fragments containing the unnatural Ds-Pn pair would be useful for expanded genetic systems in DNA-based biotechnology.

Site-Specific Incorporation of Functional Components into RNA by an Unnatural Base Pair Transcription System

“…In the course of our research, we developed several unnatural base pairs, such as those between 2-amino-6-thienylpurine (s) and 2-oxopyridine (y) [13,[20][21][22], 7-(2-thienyl)-imidazo [4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) [6], Ds and 2-nitropyrrole (Pn) [23], and Ds and 2-nitro-4-propynylpyrrole (Px) [7,8], toward practical use in DNA/RNA-based biotechnologies. The Ds-Px pair exhibits the highest selectivity and efficiency in PCR amplification, and the Ds-Pa pair is suitable for the site-specific incorporation of Pa and its modified bases, as well as Ds, into RNA by transcription.…”

Metal‐Ion‐Mediated Base Pairs in Nucleic Acids

“…This challenge will certainly have to be addressed in the near future, possibly also in combination with the more general question to what extent artificial nucleotides can be handled by polymerases. [138] Addition of a ligand-based nucleoside to the end of an otherwise self-complementary oligonucleotide enables the formation of higher-order structures in the presence of appropriate metal ions. [54] Although the resulting complexes cannot necessarily be considered metal-ion-mediated base pairs, they still represent interesting building blocks that might have an impact on DNA-based nanoarchitecture.…”

Generation of Synthetic Copolymer Libraries by Combinatorial Assembly on Nucleic Acid Templates

“…Further optimization yielded dPn which replaced the aldehyde in dPa with a nitro group. 83 This removed the need for γ-amidotriphosphates. Addition of an alkynylamine group to the dPn group yielded dPx, which further increased the fidelity of the copolymerization.…”