Rie Yamashige

Generation of high-affinity DNA aptamers using an expanded genetic alphabet

DNA aptamers produced with natural or modified natural nucleotides often lack the desired binding affinity and specificity to target proteins. Here we describe a method for selecting DNA aptamers containing the four natural nucleotides and an unnatural nucleotide with the hydrophobic base 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds). We incorporated up to three Ds nucleotides in a random sequence library, which is expected to increase the chemical and structural diversity of the DNA molecules. Selection experiments against two human target proteins, vascular endothelial cell growth factor-165 (VEGF-165) and interferon-γ (IFN-γ), yielded DNA aptamers that bind with KD values of 0.65 pM and 0.038 nM, respectively, affinities that are >100-fold improved over those of aptamers containing only natural bases. These results show that incorporation of unnatural bases can yield aptamers with greatly augmented affinities, suggesting the potential of genetic alphabet expansion as a powerful tool for creating highly functional nucleic acids.

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

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.

PCR amplification and transcription for site-specific labeling of large RNA molecules by a two-unnatural-base-pair system.

For the site-specific labeling and modification of RNA by genetic alphabet expansion, we developed a PCR and transcription system using two hydrophobic unnatural base pairs: 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) as a third pair for PCR amplification and Ds and pyrrole-2-carbaldehyde (Pa) for the incorporation of functional components as modified Pa bases into RNA by T7 transcription. To prepare Ds-containing DNA templates with long chains, the Ds-Px pair was utilized in a fusion PCR method, by which we demonstrated the synthesis of 282-bp DNA templates containing Ds at specific positions. Using these Ds-containing DNA templates and a biotin-linked Pa substrate (Biotin-PaTP) as a modified Pa base, 260-mer RNA transcripts containing Biotin-Pa at a specific position were generated by T7 RNA polymerase. This two-unnatural-base-pair system, combining the Ds-Px and Ds-Pa pairs with modified Pa substrates, provides a powerful tool for the site-specific labeling and modification of desired positions in large RNA molecules.

Visual Detection of Amplified DNA by Polymerase Chain Reaction Using a Genetic Alphabet Expansion System

Visual DNA amplification using a simple polymerase chain reaction (PCR) device is useful for field tests to detect target DNA and RNA. We hereby describe a detection system involving PCR amplification visualized with the naked eye, by genetic alphabet expansion. The system employs fluorescence resonance energy transfer (FRET) between unnatural base combinations: self-quenched dinucleotides of 2-amino-6-(2-thienyl)purine (s) as a donor and Cy3-conjugated 2-nitro-4-propynylpyrrole (Cy3-hx-Px) as an acceptor. During PCR, the triphosphate substrate of Cy3-hx-Px (Cy3-hx-dPxTP) is incorporated into DNA opposite its pairing partner, 7-(2-thienyl)-imidazo[4,5- b]pyridine (Ds), in the primer, which also contains the dinucleotides of s. Thus, the amplified DNA can be visualized by the Cy3 fluorescence resulting from the FRET between the s-dinucleotides and the incorporated Cy3-hx-Px upon 365 nm irradiation. Using this system, we demonstrated the visual single nucleotide polymorphism detection of a series of quinolone-resistant bacteria genes.