Characterization of DNA/lipid complexes by fluorescence resonance energy transfer

Abstract

Fluorescence resonance energy transfer (FRET) is a potential method for the characterization of DNA-cationic lipid complexes (lipoplexes). In this work, we used FRET models assuming a multilamellar lipoplex arrangement. The application of these models allows the determination of the distance between the fluorescent intercalator on the DNA and a membrane dye on the lipid, and/or the evaluation of encapsulation efficiencies of this liposomal vehicle. The experiments were carried out in 1,2-dioleoyl-3-trimethylammonium-propane/pUC19 complexes with different charge ratios. We used 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (DPH-PC) and 2-(4,4-difluoro-5-octyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (BODIPY-PC) as membrane dyes, and ethidium bromide (EtBr) and BOBO-1 as DNA intercalators. In cationic complexes (charge ratios (+/-) >or= 2), we verified that BOBO-1 remains bound to DNA, and FRET occurs to the membrane dye. This was also confirmed by anisotropy and lifetime measurements. In complexes with all DNA bound to the lipid (charge ratio (+/-) = 4), we determined 27 A as the distance between the donor and acceptor planes (half the repeat distance for a multilamellar arrangement). In complexes with DNA unbound to the lipids (charge ratio (+/-) = 0.5 and 2), we calculated the encapsulation efficiencies. The presented FRET methodology is, to our knowledge, the first procedure allowing quantification of lipid-DNA contact.

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.

FIGURE 2

Chemical structures of fluorescently labeled lipids (DPH-PC and BODIPY-PC) and cationic lipid (DOTAP) used in this study.

FIGURE 3

Electrophoretic profile of BOBO-1/pUC19 complexes (30 mM 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 30 min incubation at room temperature. [DNA] = 0.03 μM.

FIGURE 4

Steady-state fluorescence intensity (A) and fluorescence anisotropy (B) of BOBO-1/pUC19 complexes (30 mM Tris/HCl, pH 7.4) with different dye/base ratios, at several incubation times: 10 min (⋄), 30 min (○); 60 min (*); and 90 min (•). [DNA] = 0.006 μM.

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: λ_exc = 490 nm, and λ_em = 465 nm. [DNA] = 0.006 μM. (A), fluorescence intensity; (B), anisotropy.

FIGURE 6

Electrophoresis of lipoplexes (DOTAP/pUC19) at several charge ratios (+/−) on agarose gel in 30 mM 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] = 0.02 μM.

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] = 0.007 μM. The line is a mere guide to the eye.

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).

FIGURE 9

FRET quenching ratios, I_DA/I_D = 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 = 32 Å (- - -); d = 27 Å (- - -); and d = 22 Å (·····). (B) d = 27 Å (fixed); γ = 0.20 (—); γ = 0.30 (- - -); and γ = 0.10 (·····). (C) d = 27 Å (fixed); γ = 0.50 (—); γ = 0.60 (- - -); and γ = 0.40 (·····).

FIGURE 10

FRET quenching ratios, I_DA/I_D, 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 = 27 Å (fixed); f = 0.88 (—); f = 0.82 (·····); and f = 0.91 (- - - - -). (B) d = 27 Å (fixed); f = 0.90 (—); f = 0.85 (·····); and f = 0.93 (- - - - -).