Raman spectroscopic detection of the T-Hg II-T base pair and the ionic characteristics of mercury

Abstract

Developing applications for metal-mediated base pairs (metallo-base-pair) has recently become a high-priority area in nucleic acid research, and physicochemical analyses are important for designing and fine-tuning molecular devices using metallo-base-pairs. In this study, we characterized the Hg(II)-mediated T-T (T-Hg(II)-T) base pair by Raman spectroscopy, which revealed the unique physical and chemical properties of Hg(II). A characteristic Raman marker band at 1586 cm(-1) was observed and assigned to the C4=O4 stretching mode. We confirmed the assignment by the isotopic shift ((18)O-labeling at O4) and density functional theory (DFT) calculations. The unusually low wavenumber of the C4=O4 stretching suggested that the bond order of the C4=O4 bond reduced from its canonical value. This reduction of the bond order can be explained if the enolate-like structure (N3=C4-O4(-)) is involved as a resonance contributor in the thymine ring of the T-Hg(II)-T pair. This resonance includes the N-Hg(II)-bonded state (Hg(II)-N3-C4=O4) and the N-Hg(II)-dissociated state (Hg(II+) N3=C4-O4(-)), and the latter contributor reduced the bond order of N-Hg(II). Consequently, the Hg(II) nucleus in the T-Hg(II)-T pair exhibited a cationic character. Natural bond orbital (NBO) analysis supports the interpretations of the Raman experiments.

Figure 1.

Sequences of DNA oligomers and the structure of the T-Hg^II-T pair. (a) The sequences of the DNA oligomers used for the NMR and Raman spectral measurements. (b) Chemical structure of thymidylyl (3′–5′) thymidine (TpT). The numbering of each carbonyl oxygens is indicated and the labeled oxygen atoms in ¹⁸O-labeled TpT are colored in red. (c) The reaction scheme for the T-Hg^II-T pair formation is shown with 2-bond ¹⁵N–¹⁵N J-coupling (²J_NN). The numbering system for thymine is also shown, and the N3 atom is colored in blue.

Figure 2.

Raman spectra of the duplex 1•2 in the absence (a) and presence (b) of Hg^II, and the difference spectrum [(b)–(a)] (c) are shown. In spectrum (b), the molar ratio (Hg^II/duplex) was 2.0. Characteristic bands are highlighted with their wavenumber and their main origins. The band at 934 cm⁻¹ is due to . Bands at 785, 831 and 1092 cm⁻¹ were mainly due to vibration from the phosphate group. The phosphate Raman band at 1092 cm⁻¹ was used as a reference for spectral intensity. Bands at 1487 and 1576 cm⁻¹ are mainly due to guanine, and their negative peaks in spectrum (c) may be ascribed to an increase in the stacking interaction of guanosine residues upon the formation of the T-Hg^II-T base pair.

Figure 3.

Raman spectra of TpT in the presence (Hg^II/TpT = 1.75) (a) and absence (b) of Hg^II. The Raman band at 934 cm⁻¹ in the spectrum of the Hg^II–TpT complex arises from .

Figure 4.

Raman spectra of TpT (650–830 cm⁻¹). (a) TpT alone in H₂O. (b) TpT alone in D₂O. (c) The Hg^II–TpT complex in H₂O. (d) The Hg^II–TpT complex in D₂O.

Figure 5.

Hg^II-titration experiments of TpT by Raman spectroscopy. The molar equivalencies represented by each color are as follows: black: 0.0 eq., indigo: 0.8 eq., blue: 1.2 eq., light blue: 1.3 eq., green: 1.5 eq. and light green: 1.75 eq.

Figure 6.

Raman spectra of (a) ¹⁸O4-labeled TpT, (b) TpT, (c) ¹⁸O4-labeled Hg^II–TpT complex (Hg^II/TpT = 1.75) and (d) Hg^II–TpT complex (Hg^II/TpT = 1.75). Normal modes for Hg^II-free 1-methylthymine (non-labeled and ¹⁸O-labeled ones) are shown in Supplementary Figure S10. As a rough assignment based on the theoretical spectra (Figure 7) and the normal mode analyses (Figure 8 and Supplementary Figure S10), the main contributors to the experimental Raman bands around 1664 cm⁻¹ were assigned as follows. (a) ¹⁸O4-labeled Hg^II-free TpT: 1660 cm⁻¹ C2=O2 stretching and C5=C6 stretching; 1630 cm⁻¹ C4=O4 stretching. (b) Hg^II-free TpT: 1685 cm⁻¹ C2=O2 stretching; 1664 cm⁻¹ C5=C6 stretching; 1655 cm⁻¹ C4=O4 stretching. (c) ¹⁸O4-labeled Hg^II–TpT complex: 1652 cm⁻¹ C2=O2 stretching and C5=C6 stretching; 1570 cm⁻¹ C4=O4 stretching. (d) Hg^II–TpT complex: 1654 cm⁻¹ C2=O2 stretching and C5=C6 stretching; 1586 cm⁻¹ C4=O4 stretching. The assignment of the Raman bands for Hg^II-free TpT was principally the same as in reference (48).

Figure 7.

The high-wavenumber range of theoretical Raman spectra. (a) ¹⁸O4-labeled 1-methylthymine; (b) non-labeled 1-methylthymine; (c) ¹⁸O4-labeled 1-methylthymine–Hg^II (2:1) complex; and (d) non-labeled 1-methylthymine–Hg^II (2:1) complex. Throughout the calculations, 1-methylthymine was used as a model of thymidine. Major contributors to the Raman bands around the C=O stretching region in the theoretical spectra are as follows: (b), 1712 cm⁻¹: C2=O2 stretching; 1686 cm⁻¹: C5=C6 stretching; 1665 cm⁻¹: C4=O4 stretching. (d), 1696 cm⁻¹, C5=C6 stretching; 1664 cm⁻¹, C2=O2 stretching; 1592 cm⁻¹ (summation of two C4=O4 stretching modes in Figure 8). Asterisks indicate an apparent wavenumber due to the band overlap.

Figure 8.

Normal modes for the experimental Raman bands around 1586 cm^-1 in the T-Hg^II-T pair. The theoretical wavenumbers (1595 and 1590 cm⁻¹) are indicated.

Figure 9.

Resonance contributors of the T-Hg^II-T pair. (a) Core resonance. (b) Further resonance associated with the anionic thymine 5. The structure of 8 is the resonance hybrid (an average structure).

Figure 10.

Results of the DFT calculations. (a) Key inter-atomic distances within the 1-methylthymine–Hg^II (2:1) complex. (b) Key inter-atomic distances within the crystal structure of the 1-methylthymine–Hg^II (2:1) complex (43). Natural charges and bond orders of (c) the T-Hg^II-T pair, (d) 1-methylthymine: 1MeThy and (e) deprotonated 1-methylthymine: [1MeThy-H⁺]⁻.