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Proceedings Paper

Time-resolved spectroscopy of the intrinsic fluorescence of nucleic acid species
Author(s): Malcolm Daniels; Lucas P. Hart; Paul Shing Ho; Jean-Pierre Ballini; Paul Vigny
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Paper Abstract

Polarization and lifetime studies have shown that the fluorescence from nucleic acid species is complex, both at the individual chromophore level and because of the effect of stacking interactions on the electronic states. Recent work aimed at elucidating some aspects of this behavior by decay analysis and time-resolved spectroscopy is surveyed. Experimental work has been carried out using the ACO synchrotron at LURE (France) with time-correlated single photon counting, or a frequency-doubled N2-pumped dye laser, pulse width 700 ps, with fast-gated (100 ps width) analog detection and signal averaging. Decay curves are treated by global analysis using the Marquardt non-linear least-squares algorithm (synchrotron data) or the SPLMOD program (EMBO), which carries out a non-linear leastsquares minimization using cubic splines, for the laser data. Resolution of the decay data gives a model-based estimate of the number of components and their lifetimes. This information is then used to deconvolute timewindowed spectra (time-delayed spectra) into the time-resolved spectra. It is a particular feature of the combination of delayed photon counting with the continuous wavelength distribution of pulsed synchrotron radiation that excitation spectra correlating with emissions of different lifetimes can be obtained by uninterrupted repetitive scanning over a wide range of exciting wavelengths, in the present work from 230 nm to 354 urn. Such time-delayed excitation spectra can also be deconvoluted into components corresponding to the various time-resolved emission spectra. Examples of these three types of information viz resolved lifetimes, time-resolved emission spectra and their excitation spectra are presented and discussed for the following systems. I. adenosine; 6N, 6N-dimethyladenosine; protonated adenosine; this work shows the role of rotamers in the excited state behavior of this chromophore and demonstrates the forbidden nature of the lowest excited state. II. d(AT); d(TA); we observe the sequence dependence of emission from the stacked state which has been observed previously in polarization studies and an unusual excitation spectrum. III. d(CG); poly d(CG), "B"-DNA structure; single crystal duplex d(CG)3, "Z"-DNA structure. Distinctive differences are observed between the stacked emissions from the "B" and "Z" structures which we attribute to different overlapping of the stacked bases.

Paper Details

Date Published: 1 May 1990
PDF: 10 pages
Proc. SPIE 1204, Time-Resolved Laser Spectroscopy in Biochemistry II, (1 May 1990); doi: 10.1117/12.17696
Show Author Affiliations
Malcolm Daniels, Oregon State Univ. (United States)
LURE/Univ. Paris-Sud (France)
Lucas P. Hart, Oregon State Univ. (United States)
Paul Shing Ho, Oregon State Univ. (United States)
Jean-Pierre Ballini, Univ. Pierre et Marie Curie (France)
Paul Vigny, Univ. Pierre et Marie Curie (France)

Published in SPIE Proceedings Vol. 1204:
Time-Resolved Laser Spectroscopy in Biochemistry II
Joseph R. Lakowicz, Editor(s)

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