Intrinsic fluorescence of B and Z forms of poly d(G-m5C)·poly d(G-m5C), a synthetic double-stranded DNA: spectra and lifetimes by the maximum entropy method

Abstract

A study has been made of the fluorescence of poly d(G-m⁵C)·poly d(G-m⁵C), a synthetic double-stranded DNA, in buffered neutral aqueous solution at room temperature, excited by synchrotron radiation at 280 nm and 250 nm and by a frequency-doubled pulse dye laser at 290 nm. Exciting at 280 nm, the B form shows a uni-modal UV spectrum with λ_f(max) ≈ 340 nm. The Z form has in addition a visible emission λ_f(max) at 450 nm. The spectral positions remain unchanged on exciting at 250 nm but the relative intensities change considerably. Decay profiles have been obtained at 360 nm and 450 nm for both the B and Z forms and have been analyzed by fitting to a pseudo-continuous distribution of 100 (and occasionally 200) exponentials, ranging from 10 ps to 20 ns, by optimizing the ‘entropy’ of the signal (the method of maximum entropy). We find the mean lifetimes for both wavelengths of emission and for both structural forms fall into three well-separated regions in the ranges indicated τ₁ ≈ 0.04–0.21 ns, τ₂ ≈ 0.9–1.26 ns, and τ₃ ≈ 5.1–6.5 ns. The UV emission, from its spectral position and half-width, correlates with monomeric emission from m⁵C (and from C for poly d(G-C)). However the lifetime τ₁ is ≈ 2 orders of magnitude longer than the monomers and points to an involvement of protonated guanosine (GH⁺, τ_f ≈ 200 ps) in the overall absorption/emission sequence. In the UV the τ₃ emission is predominant, with fractional time-integrated emission ≈86% for B DNA and ≈64% for Z. We suggest it results from exciton (stacked) absorption followed by dissociative emission. For Z DNA the visible (450 nm) emission is dominated by a τ₃ species (≈91%) with a lifetime of 6.5 ns and we suggest it represents a hetero-excimer emission consequent upon absorption by the strongly overlapped base-stacking, which differs from that in B DNA. The weak emission corresponding to τ₂ is made more apparent by scanned gated detection of the emission from laser excitation (290 nm) of single-crystal d(m⁵C-G)₃. A central role is attributed to the tight stacking of the bases in the Z form which correlates with enhanced hypochromism at 250 nm vs. 280 nm and with the reversal of the fluorescence intensity ratios UV-visible between these wavelengths.