Photophysical properties of nucleic acids that contain A and T bases and global analysis of steady-state fluorescence spectra in the presence of energy transfer

摘要

Abstract: We have studied the steady-state fluorescence properties of the polynucleotides poly(dA)poly(dT) and poly(dT) as well as of the 20-mers (dA)$-20$/(dT)$-20$/ and (dT)$-20$/ at room temperature for excitation at 293 nm where T is selectively excited. The fluorescence spectrum of the double-stranded 20-mer is shifted to longer wavelengths relative to that of poly(dA)poly(dT), which may be the result of reduced stacking interactions. The fluorescence spectrum of the single-stranded 20-mer is much broader than that of poly(dT); this casts doubt on the validity of the assumption made in the literature that the crystallographic results from a dimer can be used to develop a model for poly(dT). The fluorescence anisotropies of the double- stranded systems are unexpectedly lower than those of the single- stranded ones, which suggests that cooperative interactions operate in the dynamics of the former. The analysis of the results of these systems was considerably simplified because of the fact that T can be selectively excited at the long-wavelength edge of the absorption spectrum. For the more general case for which such a spectral window does not exist, we have developed a methodology that employs a global nonlinear least-squares analysis of the dependence of the fluorescence intensity on the polarization angle and have estimated, for a simulated mixture of the C and G bases, their individual fluorescence spectra in the presence of energy transfer from C to G as well as from G to C. !39