Figure 4.

Fluorescence spectra of 2AP nucleotide and an unfolded, substituted oligonucleotide. (A) Fluorescence excitation and emission spectra of 0.5 µM 2′-deoxy-2-aminopurine-3′,5′-diphosphate mononucleotide. An aqueous solution (blue) of the nucleotide, pH ~5.5, in a 1.5 ml stirred luminescence cuvette was converted to TMK buffer, pH 7.5, by consecutive addition of buffer components. First, Tris, pH 7.5, was added to a concentration of 100 mM (red), then KCl to concentrations of 5, 10, 20 (light green), 40 and 80 mM (dark green), and finally MgCl₂ (black) to a concentration of 10 mM. Instrument parameters were as in Materials and Methods, apart from the spectral bandwidths, which were set at 5 (excitation) and 6.5 nm (emission). (B) Temperature dependence of the fluorescence emission spectrum of the 2AP derivative –7TR. The emission spectra are shown of 0.5 µM –7TR in water or in TMK buffer, pH 7.5, at 25 (blue) and 75°C (red). Solid and dashed lines represent fluorescence emission in water and TMK, respectively. The 75°C curves are superimposed. Fluorescence measurements were taken using a Photon Technology International (PTI) spectrofluorimeter controlled by the programme FeliX, fitted with a PE60 Linkam thermo-controller and PTI temperature sensor accessory. Emission spectra were scanned from 330 to 500 nm at 240 nm min^–1, exciting at 306 nm. Spectra were generated from an average of three scans and the baseline subtracted.