DNA base flipping by a base pair-mimic nucleoside

Abstract

On the basis of non-covalent bond interactions in nucleic acids, we synthesized the deoxyadenosine derivatives tethering a phenyl group (X) and a naphthyl group (Z) by an amide linker, which mimic a Watson-Crick base pair. Circular dichroism spectra indicated that the duplexes containing X and Z formed a similar conformation regardless of the opposite nucleotide species (A, G, C, T and an abasic site analogue F), which was not observed for the natural duplexes. The values among the natural duplexes containing the A/A, A/G, A/C, A/T and A/F pairs differed by 5.2 kcal mol(-1) while that among the duplexes containing X or Z in place of the adenine differed by only 1.9 or 2.8 kcal mol(-1), respectively. Fluorescence quenching experiments confirmed that 2-amino purine opposite X adopted an unstacked conformation. The structural and thermodynamic analyses suggest that the aromatic hydrocarbon group of X and Z intercalates into a double helix, resulting in the opposite nucleotide base flipping into an unstacked position regardless of the nucleotide species. This observation implies that modifications at the aromatic hydrocarbon group and the amide linker may expand the application of the base pair-mimic nucleosides for molecular biology and biotechnology.

Scheme 1

Chemical structures of the deoxyadenosine derivatives used in this study.

Figure 1

The sequences of (A) the A-series, (B) the X-series and (C) the Z-series DNA duplexes forming the A/W₂, X/W₂ or Z/W₂ pair, respectively, where W₂ is A, G, C, T or F.

Figure 2

CD spectra of the (A) A-series, (B) X-series and (C) Z-series duplexes [W₂ = A (red), G (purple), C (blue), T (green) or F (orange)]. All measurements were done in the 1 M NaCl-phosphate buffer at 4°C.

Figure 3

Melting curves of the (A) A-series, (B) X-series and (C) Z-series duplexes monitored at 260 nm [W₂ = A (red), G (purple), C (blue), T (green) or F (orange)]. All measurements were carried out at ∼40 µM in the 1 M NaCl-phosphate buffer. The T_m values are as follows: A/A (42.5°C), A/G (51.3°C), A/C (44.5°C), A/T (55.4°C), A/F (38.5°C), X/A (46.6°C), X/G (48.0°C), X/C (49.3°C), X/T (52.2°C), X/F (50.8°C), Z/A (53.4°C), Z/G (53.2°C), Z/C (58.5°C), Z/T (57.4°C) and Z/F (58.7°C). (D) Melting curves of the Z-series duplexes monitored at 325 nm. The fits to a two-state model are indicated in a black line.

Figure 4

The duplexes containing the 2-amino purine nucleotide (P) opposite W₁ (A, T, X or •), forming the A/P, T/P or X/P pair or a single P-bulge, respectively. The 2-amino purine nucleotide is adjacent to (A) two cytosines or (B) two guanines.

Figure 5

Fluorescence emission spectra of the duplexes containing 2-amino purine adjacent to two cytosines (A) or two guanines (B), opposite A (red), T (green) or X (blue) in a duplex, in addition to a duplex forming a 2-amino purine bulge (orange) and the single-stranded DNA (black). All measurements were carried out in the 1 M NaCl-phosphate buffer at 4°C.

Figure 6

Comparison of the $\Delta G_{37}^0$ values for the DNA duplex formations. A model of the base flipping is also indicated, containing the W₁ (red) and the W₂ (blue) nucleotides.