Intrinsic time- and wavelength-resolved fluorescence of oligonucleotides: A systematic investigation using a novel picosecond laser approach

A novel picosecond laser approach is used to investigate the intrinsic fluo rescence of several oligonucleotides. All biomolecules are excited at 283 n m with laser pulses of typically 80 ps duration and an energy of 250 mu J; concentrations were on the order of 10(-5) M. Detection of the resulting fl uorescence behind a spectrometer with a streak camera permits the simultane ous acquisition of spectral and lifetime information in two-dimensional ima ges. In a systematic study, the fluorescence spectra and the associated tem poral decays are analyzed with respect to monomer and potential excimer com ponents. For this, the nucleotides AMP, CMP, GMP, and TMP are studied as we ll as homo-oligonucleotides of the type d(X)(n) with variable sequence leng th of n = 2-15, enabling a comparison of the emission characteristics of th ese single-stranded compounds under physiologic conditions in solution at r oom temperature. Also, the influence of conformational changes on the fluor escence response is investigated using mixtures of complementary oligonucle otides d(X)(15)xd(Y)(15) With the combinations X = A, Y = T and X = G, Y = C. These structures, which form double helices, differ in flexibility and s tacking geometry from the single-stranded compounds. From experiments with self-complementary variants with alternating base sequences of the type d(X Y)(8) with XY = AT and GC, information on exciplex formation tendencies is obtained for these compounds, which also form double helices in solution. P reliminary results of time-dependent fluorescence anisotropy measurements w ith this direct picosecond laser approach are discussed.