A molecular view on the escape of lipoplexed DNA from the endosome
- Groningen Biomolecular Sciences and Biotechnology Institute and Zernike Institute for Advanced Materials, University of Groningen, Netherlands
Figures
Figure 1 with 3 supplements
Schematic view of the lipoplex-membrane transfection pathways.
Unadhered lipoplex containing four dsDNAs sits above the endosomal bilayer (I). After initial stalk formation (II), a wide hemifusion diaphragm is formed (III). A pore is formed in one of the …
Figure 1—figure supplement 1
Building the small lipoplex.
First the AA geometry and force field of the dsDNA was translated into their CG counterpart (A). Then the dsDNA was stacked on top of a bilayer composed of DOTAP and DOPE in a 1:4 ratio …
Figure 1—figure supplement 2
Radius of gyration for the solvated lipoplex.
The radius of gyration for the solvated small lipoplex over time. Over the last 2 µs of the simulation the radius of gyration increases less than a quarter of an Ångstrom. We therefore concluded …
Figure 1—figure supplement 3
Coating the lipoplex.
Upon solvating the periodic lipoplex an extra layer of coating lipids was added. We used the approximate surface area of the lipoplex and divided it by the APL of the DOTAP:DOPE mixture (1:4; 0.63 …
Figure 2 with 1 supplement
Membrane and lipoplex compositions.
The membrane compositions for the fusion experiments, and the lipoplex formulations are indicated in molar ratios. All PC/PS heads were combined with all tails, resulting in six different lipids per …
Figure 2—figure supplement 1
Lipid mixing in all endosome bilayer compositions.
Snapshots of endosome bilayers after 1 µs of equilibration after being generated by insane. From left to right we show the most, more unsaturated and short lipids bilayer respectively (see Figure 2).…
Figure 3 with 5 supplements
Effect of lipid composition on transfection efficiency.
The number of successful transfections and amount of transfected DNA considering different lipid compositions. Each composition had 5 repeats with four dsDNA fragments each. Therefore the maximum …
Figure 3—figure supplement 1
Hydration/transfection, lipid mixing and orientation of the dsDNA DOPE:DPTAP + short lipid endosome bilayer.
Shown are the observables for the lipoplex-membrane fusion experiments. Each experiment was performed five times (one repeat per column). For each simulation the first 10 µs were taken into account. …
Figure 3—figure supplement 2
Hydration/transfection, lipid mixing and orientation of the DOPE:DOTAP + short lipid endosome bilayer.
Shown are the observables for the lipoplex-membrane fusion experiments. Each experiment was performed five times (one repeat per column). For each simulation the first 10 µs were taken into account. …
Figure 3—figure supplement 3
Hydration/transfection, lipid mixing and orientation of the DOPE:DLiTAP + short lipid endosome bilayer.
Shown are the observables for the lipoplex-membrane fusion experiments. Each experiment was performed five times (one repeat per column). For each simulation the first 10 µs were taken into account. …
Figure 3—figure supplement 4
Hydration/transfection, lipid mixing and orientation of the DOPE:DOTAP + DLi containing endosome bilayer.
Shown are the observables for the lipoplex-membrane fusion experiments. Each experiment was performed five times (one repeat per column). For each simulation the first 10 µs were taken into account. …
Figure 3—figure supplement 5
Hydration/transfection, lipid mixing and orientation of the DOPE:DOTAP + DA containing endosome bilayer.
Shown are the observables for the lipoplex-membrane fusion experiments. Each experiment was performed five times (one repeat per column). For each simulation the first 10 µs were taken into account. …
Figure 4 with 2 supplements
Transfection of large lipoplexes.
The large lipoplex (A) showed the same stable HII structure as the small lipoplex with dsDNA inside the aqueous channels. Small additional connecting channels are also present, as indicated. …
Figure 4—figure supplement 1
Large lipoplex channel structures with dsDNA inside.
The solvated large lipoplex contained channels: regular dsDNA/aqueous channels are indicated in peach (A); an open channel is depicted with a magenta circle (B); and aqueous connecting channels are …
Figure 4—figure supplement 2
Reorientation of the large lipoplex in the endosome.
The graph shows the lipoplex-endosome lipid contacts (<1.2 nm) over time. Within 0.5 µs the lipoplex reorients itself such that its HII-channels are roughly aligned with the membrane normal in the …
Videos
Video 1
Small lipoplex-bilayer transfection.
Fusion of a small lipoplex containing 4 fragments of dsDNA. The headgroups of the lipoplex lipids (DOTAP:1, DOPE:4) are depicted in blue and the bilayer lipids (short lipids Figure 2) are depicted …
Video 2
Large lipoplex-vesicle transfection.
Fusion of the large lipoplex from inside the endosomal vesicle. The headgroups of the endosomal and lipoplex lipids are subsequently blue and orange. The endosomal lipids have dark grey tails and …
Video 3
Reorientation of lipoplex in vesicle.
Components of the lipoplex closer than 4 nm to the endosome are colored blue, the endosomal lipids are grey and the dsDNA inside the lipoplex is yellow/pink. Within 1 µs the lipoplex reorients …
Additional files
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Supplementary file 1
DOTAP_validation.
- https://cdn.elifesciences.org/articles/52012/elife-52012-supp1-v2.docx
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Supplementary file 2
lipoplex_lipid-properties.
- https://cdn.elifesciences.org/articles/52012/elife-52012-supp2-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/52012/elife-52012-transrepform-v2.pdf
