Luciferase gene expression and DNA accessibility as a function of <i>r</i>_charge using pre-casted RNA gels.

<p>(A) G4 dendrimers and (B) CTAB. The synthesized amounts of RNA are displayed and samples were not pretreated with Dnase I. References are displayed in B where lane 1 shows the sample consisting only of DNA and without any compacting agent or transcriptional activity. Lane 2 shows the control sample containing DNA and the <i>in vitro</i> transcription mixture in the absence of compacting agents. Gels were post-stained using GelStar.</p

Cryo-TEM images of DNA (0.1 mg mL⁻¹) complexes.

<p>(A, C) G4/DNA of <i>r</i>_charge  = 0.5, and (B, D) CTAB/DNA of <i>r</i>_charge  = 7.5. All samples were prepared in aqueous solutions of 10 mM NaBr, but (A) and (B) display G4/DNA and CTAB/DNA complexes, respectively, after being transferred to the tRB used for <i>in vitro</i> transcription/translation experiments. (C) and (D) are the reference samples in the 10 mM NaBr solution used for DNA compaction. Scale bars are 100 nm, the arrow indicates frost (artifact), the white stars indicate the carbon film and the black star shows a fracture of the vitreous film.</p

DNA condensation using G4 dendrimers and CTAB surfactants.

<p>(A) The fluorescence intensity of GelStar bound to DNA, shown as a function of <i>r</i>_charge in solutions containing 10 mM NaBr for G4 dendrimers (▵) and CTAB (•). Data are normalized to the amounts produced in the samples only containing DNA (in the absence of dendrimer or surfactant) and the <i>I</i>_max value is linearly dependent on the amount of DNA that is available to bind GelStar. The DNA concentration is 2 μg mL⁻¹ and error bars are smaller or equal to the size of the markers. (B) The mean number of G4 per compacted DNA chain at varying <i>r</i>_charge, calculated as described in the text. Note that below charge neutralization the amount of bound G4 per DNA strand is constant, that is each complex contains the same number of G4. Once the neutralization point is reached, the solution only contains compacted DNA and the number of G4 per compacted DNA increases. The results from the electrophoreses study - DNA condensation by G4 dendrimers and CTAB surfactants - are shown in (C) (D), respectively. Lane 1 in both C and D displays free linearized plasmid DNA in the absence of any compacting agent (control, 4331 bp). Samples in lanes 2–11 contain increasing amounts of the compacting agent and the corresponding <i>r</i>_charge values are indicated. The DNA concentration was 25 μg mL⁻¹ and the gels were stained with Ethidium Bromide (EtBr).</p

Protection of DNA against Dnase I digestion using gels stained with GelStar.

<p>(A) G4/DNA complexes with <i>r</i>_charge  = 0.4 are used and the untreated complex is loaded on lane 1. The 2^nd lane displays the dissociated complex after treatment with 10 μg mL⁻¹ heparin for 30 min. All other samples (lanes 3–7) are treated with 1 unit of Dnase I for the indicated time periods. To the samples in lanes 4–7, heparin was added after the Dnase I enzyme was heat inactivated. (B) Linearized plasmid DNA only is loaded onto lane 1 and the sample loaded onto lane 2 contains DNA, treated with Dnase I for 30 min. Samples loaded onto lanes 3–7 contain DNA and CTAB (<i>r</i>_charge  = 7.5). Samples loaded onto lanes 4–7 are treated with Dnase I for the time periods indicated.</p

Inhibition of <i>in vitro</i> transcription and translation as a consequence of DNA compaction.

<p>(A) G4 dendrimers and (B) CTAB. The (SYTO RNASelect) fluorescence intensity (▵) corresponds to the produced amount of RNA and the GelStar exclusion data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092692#pone-0092692-g001" target="_blank">Figure 1A</a> (•) is added as a reference. The chemiluminescence intensities corresponding to the produced amount of luciferase (□) for both the G4/DNA and the CTAB/DNA systems are also shown. The concentration of linearized plasmid DNA is 2 μg mL⁻¹ in the GelStar exclusion experiments and 10 μg mL⁻¹ in the <i>in vitro</i> transcription and translation experiments. Data are normalized to the amounts produced in the samples only containing DNA (without compacting agent).</p

DNA condensation using G4 dendrimers and CTAB surfactants.

<p>(A) The fluorescence intensity of GelStar bound to DNA, shown as a function of <i>r</i>_charge in solutions containing 10 mM NaBr for G4 dendrimers (▵) and CTAB (•). Data are normalized to the amounts produced in the samples only containing DNA (in the absence of dendrimer or surfactant) and the <i>I</i>_max value is linearly dependent on the amount of DNA that is available to bind GelStar. The DNA concentration is 2 μg mL⁻¹ and error bars are smaller or equal to the size of the markers. (B) The mean number of G4 per compacted DNA chain at varying <i>r</i>_charge, calculated as described in the text. Note that below charge neutralization the amount of bound G4 per DNA strand is constant, that is each complex contains the same number of G4. Once the neutralization point is reached, the solution only contains compacted DNA and the number of G4 per compacted DNA increases. The results from the electrophoreses study - DNA condensation by G4 dendrimers and CTAB surfactants - are shown in (C) (D), respectively. Lane 1 in both C and D displays free linearized plasmid DNA in the absence of any compacting agent (control, 4331 bp). Samples in lanes 2–11 contain increasing amounts of the compacting agent and the corresponding <i>r</i>_charge values are indicated. The DNA concentration was 25 μg mL⁻¹ and the gels were stained with Ethidium Bromide (EtBr).</p

Dissociation of CTAB/DNA complexes using C₁₂E₅.

<p>(A) Gel stained with EtBr where lane 1 shows the linearized plasmid DNA only (control). The CTAB/DNA samples loaded onto the other lanes are of <i>r</i>_charge  = 1.5 (0.11 mM) and 7.5 (0.57 mM), and were treated with the indicated amounts of C₁₂E₅. The DNA concentration is 25 μg mL⁻¹ (75.8 μM). (B) Fluorescence intensities are measured by fluorescence spectroscopy as a function of C₁₂E₅ concentration. The GelStar intensities (•) reflects the amount of free DNA and the SYTO RNASelect intensity (▵) reflects the amount of RNA produced by <i>in vitro</i> transcription. Error bars are smaller or equal to the size of the markers. The concentrations of DNA and CTAB are 2 μg mL⁻¹ and 60.6 μM, respectively, (<i>r</i>_charge  = 10) in the GelStar exclusion experiments and 10 μg mL⁻¹ and 0.30 mM, respectively, (<i>r</i>_charge  = 10) in the <i>in vitro</i> transcription experiments. The intensity was normalized to the sample only containing DNA (in the absence of CTAB).</p

Decompaction of G4/DNA complexes using the polysaccharide heparin.

<p>(A) GelStar-stained gel showing the degree of dissociation of G4/DNA complexes (<i>r</i>_charge  = 0.90) for the indicated concentrations of heparin. The 1^st lane shows the linearized plasmid DNA only (25 μg mL⁻¹, control). (B) GelStar fluorescence measured by steady state fluorescence spectroscopy as a function of heparin concentration. Measurements are performed for DNA (2 μg mL⁻¹) with complexes of <i>r</i>_charge  = 0.35 (•), 0.55 (□) and 0.90 (▵). The intensity is normalized to that of free DNA (in the absence of G4) and error bars are smaller or equal to the marker size. Inset shows the concentration ratios of heparin and DNA.</p

Cryo-TEM of DNA dissolved in the tRB used for <i>in vitro</i> transcription and translation experiments.

<p>Coexisting domains of free DNA (A) and tightly packed clusters of DNA (B) are shown. Scale bars are 100 nm and the DNA concentration is 0.1 mg mL⁻¹.</p

The solvent effect on DNA compaction using steady state fluorescence spectroscopy.

<p>Columns marked as NaBr correspond to samples in aqueous solutions of 10<i>in vitro</i> gene expression kit. Data are normalized to the emitted intensity in the sample only containing DNA (without compacting agent) in 10 mM NaBr solution (1^st column). The <i>r</i>_charge values reported for the samples containing compacted DNA are 7.5 for CTAB/DNA and 0.5 for G4/DNA. The DNA concentration is 2 μg mL⁻¹ and the dye used is GelStar.</p