Volume 85, Issue 5, Pages (November 2003)

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Volume 85, Issue 5, Pages 3106-3119 (November 2003) Characterization of DNA/Lipid Complexes by Fluorescence Resonance Energy Transfer Catarina Madeira, Luís M.S. Loura, M. Raquel Aires-Barros, Aleksander Fedorov, Manuel Prieto Biophysical Journal Volume 85, Issue 5, Pages 3106-3119 (November 2003) DOI: 10.1016/S0006-3495(03)74729-8 Copyright © 2003 The Biophysical Society Terms and Conditions

Figure 1 Schematic representation of the lipoplexes multilamellar structure with the fluorescent probes within the DNA and the lipid. (A) Acceptor (a) on the DNA (EtBr) and donor (d) on the lipid (DPH-PC and BODIPY-PC). This arrangement was used for DOTAP/pUC19 charge ratio (+/−)=0.5. (B) Acceptor on the lipid (BODIPY-PC) and donor on the DNA (BOBO-1). This arrangement was used for DOTAP/pUC19 charge ratio (+/−)=0.5, 2, and 4. Biophysical Journal 2003 85, 3106-3119DOI: (10.1016/S0006-3495(03)74729-8) Copyright © 2003 The Biophysical Society Terms and Conditions

Figure 2 Chemical structures of fluorescently labeled lipids (DPH-PC and BODIPY-PC) and cationic lipid (DOTAP) used in this study. Biophysical Journal 2003 85, 3106-3119DOI: (10.1016/S0006-3495(03)74729-8) Copyright © 2003 The Biophysical Society Terms and Conditions

Figure 3 Electrophoretic profile of BOBO-1/pUC19 complexes (30mM Tris/HCl, pH 7.4) at dye:DNA base (d/b) values: 0 (lane 1), 0.2 (lane 2), 0.167 (lane 3), 0.09 (lane 4), 0.06 (lane 5), 0.03 (lane 6), and 0.01 (lane 7), after 30min incubation at room temperature. [DNA]=0.03μM. Biophysical Journal 2003 85, 3106-3119DOI: (10.1016/S0006-3495(03)74729-8) Copyright © 2003 The Biophysical Society Terms and Conditions

Figure 4 Steady-state fluorescence intensity (A) and fluorescence anisotropy (B) of BOBO-1/pUC19 complexes (30mM Tris/HCl, pH 7.4) with different dye/base ratios, at several incubation times: 10min (♢), 30min (○); 60min (*); and 90min (●). [DNA]=0.006μM. Biophysical Journal 2003 85, 3106-3119DOI: (10.1016/S0006-3495(03)74729-8) Copyright © 2003 The Biophysical Society Terms and Conditions

Figure 5 Titration of BOBO/pUC19 complexes: d/b=0.06 (♢); d/b=0.03 (○); and d/b=0.01 (●) with cationic liposomes (DOTAP). BOBO-1 complexes: λexc=490nm, and λem=465nm. [DNA]=0.006μM. (A), fluorescence intensity; (B), anisotropy. Biophysical Journal 2003 85, 3106-3119DOI: (10.1016/S0006-3495(03)74729-8) Copyright © 2003 The Biophysical Society Terms and Conditions

Figure 6 Electrophoresis of lipoplexes (DOTAP/pUC19) at several charge ratios (+/−) on agarose gel in 30mM Tris/HCl, pH 7.4. Lipoplexes with charge ratios (+/−): 0 (lane 1), 0.01 (lane 2), 0.1 (lane 3), 0.5 (lane 4), 0.8 (lane 5), 1 (lane 6), 1.5 (lane 7), 2 (lane 8), 3 (lane 9), 6 (lane 10), and 10 (lane 11). [DNA]=0.02μM. Biophysical Journal 2003 85, 3106-3119DOI: (10.1016/S0006-3495(03)74729-8) Copyright © 2003 The Biophysical Society Terms and Conditions

Figure 7 Mean diameter of pUC19 without (○), and with (●), BOBO-1 at d/b=0.01, with cationic liposomes (DOTAP) at several charge ratio (+/−). [DNA]=0.007μM. The line is a mere guide to the eye. Biophysical Journal 2003 85, 3106-3119DOI: (10.1016/S0006-3495(03)74729-8) Copyright © 2003 The Biophysical Society Terms and Conditions

Figure 8 Absorption spectra (thin line) of BODIPY-PC (A) and EtBr (B and C) and emission spectra (thick line) of BOBO (A), DPH-PC (B), and BODIPY-PC (C). Biophysical Journal 2003 85, 3106-3119DOI: (10.1016/S0006-3495(03)74729-8) Copyright © 2003 The Biophysical Society Terms and Conditions

Figure 9 FRET quenching ratios, IDA/ID=1−E, for BOBO-1/BODIPY pairs in DOTAP/DNA complexes with charge ratios (+/−) of 4 (A; larger figure is a zoom of the inset), 2 (B), and 0.5 (C). Experimental data (●); Fitting curves using Eqs. 2–4 and 6. The assumed fitting parameters were: (A) γ=0 (fixed); d=32Å (- - -); d=27Å (- - -); and d=22Å (·····). (B) d=27Å (fixed); γ=0.20 (—); γ=0.30 (- - -); and γ=0.10 (·····). (C) d=27Å (fixed); γ=0.50 (—); γ=0.60 (- - -); and γ=0.40 (·····). Biophysical Journal 2003 85, 3106-3119DOI: (10.1016/S0006-3495(03)74729-8) Copyright © 2003 The Biophysical Society Terms and Conditions

Figure 10 FRET quenching ratios, IDA/ID, for DPH/EtBr (A) and BODIPY/EtBr (B) pairs in DOTAP/DNA complexes with charge ratios (+/−) of 0.5. Experimental data (●); Fitting curves using Eqs. 2, 3, 5, and 6. The assumed fitting parameters were: (A) d=27Å (fixed); f=0.88 (—); f=0.82 (·····); and f=0.91 (- - - - -). (B) d=27Å (fixed); f=0.90 (—); f=0.85 (·····); and f=0.93 (- - - - -). Biophysical Journal 2003 85, 3106-3119DOI: (10.1016/S0006-3495(03)74729-8) Copyright © 2003 The Biophysical Society Terms and Conditions