Self-assembly of cholesterol-modified nucleosides into microtubes: Characterization, functionalization, loading and release of model substances
Final Report Abstract
We have studied the self-assembly of a cholesteryl nucleoside conjugates, cholesterylaminouridine (CholAU) and it’s analogous. In particular, we were interested in pseudo-one dimensional tubular structures. Two distinct types of tubules were observed, first allocated by their outer diameter: nano-sized and micro-sized tubular structures were observed using light microscopy in aqueous suspensions of CholAU and phosphatidylcholines (PC) while slowly cooling from 70 °C to room temperature. In the course of the project, we showed that the two tubules types have different morphologies: cryo scanning electron microscopy revealed that nano-tubules were densely and micro-tubules were loosely rolled. This explains the difference in stability to air-drying: nano-tubules could be dried, whereas micro-tubules collapsed upon drying. We have tested different phosphatidylcholines: both micro- and nano-tubules formed in the mixtures of CholAU with unsaturated PC having the acyl chains of 14 to 18 carbons, whereas in mixtures of CholAU with partially or fully saturated PC only nano-tubules had formed. Presumably, the higher miscibility of CholAU with unsaturated phospholipids allowed formation of the micro-tubules. Nano-tubules had outer diameters in the range of 50 - 300 nm and the length of 1 - 15 µm. Micro-tubules had outer diameters in the range of 1 - 3 µm; occasionally tubules with a diameter of 4 - 6 µm were observed. The length of the micro-tubules was about 10 µm. Additionally, helices and open sheets were observed. The assembly was similar in pure water and buffer solutions and depended on neither osmolality nor pH of the solutions. Our results indicate that the micro-tubules are formed via helical ribbons, whereas the nano-tubules are rolled from micrometer large sheets. From solid-state NMR and AFM results we could conclude that the CholAU in nano-tubules was in crystalline phase, whereas the phospholipids were in liquid-crystalline state. Loading PC giant unilamellar vesicles with CholAU from a complex with mβcyclodextrin, we showed that nano-tubule assembly occurred even at room temperature. Assembly at room temperature is advantageous if working with biological molecules. We suggest that the PC membrane serves as a platform for the assembly. First, incorporation of CholAU into the PC membrane prevents precipitation of the pure conjugate. Second, the bilayers serves as a platform (two-dimensional phase) for the initial de-mixing of CholAU. Third, vesicles changing upon formation of the intermediate aggregates, provide the necessary environment for tubular formation, which otherwise often can be seen only in organic solvents. Fourth, the assembled tubules are covered by a fluid layer of lipids providing a hydrophilic surface and allowing an easy functionalization using lipophilic conjugates. Fluorescent lipid analogs and lipophilic conjugates, for example lipophilic nucleic acids (LiNAs), were incorporated into the outer layer of the tubules. DNA origamis with complementary sticky ends were subsequently attached to the tubules. Alignment of the tubules using lipophilic nucleic acids with the two anchors at the end was not successful yet. However, the lipophilic DNA with 2 tocopherol anchors was used to mimic the action of tetherin, an unusual transmembrane protein which blocks spreading of virus infection. Using another LiNA system we showed a hybridization and enzymatic cleavage controlled lateral re-distribution of functional moieties. Self-assembled tubules functionalized with functional lipophilic moieties can be further developed for virus diagnostics and biosensor platforms, and also as depot-scaffold for tissue regeneration.
Publications
- Remote control of lipophilic nucleic acids domain partitioning by DNA hybridization and enzymatic cleavage. J. Am. Chem. Soc., 2012, 134, 20490–20497
Schade, M., Knoll, A., Vogel, A., Seitz, O., Liebscher, J., Huster, D., Herrmann, A., and Arbuzova, A.
(See online at https://doi.org/10.1021/ja309256t) - Cholesterol modified nucleosides as precursors for microtube self-assembly. Process. Isot. Mol., 2013, 243–245
Petran, A., Losensky, L., Arbuzova, A., and Liebscher, J.
- DNA-controlled aggregation of virus like particles – mimicking a tetherin-like mechanism. New J. Chem., 2014, 38, 5181–5185
Serien, D., Grimm, C., Liebscher, J., Herrmann, A., and Arbuzova A.
(See online at https://doi.org/10.1039/c4nj00724g) - Lipophilic nucleic acids – a flexible construction kit for organization and functionalization of surfaces. Adv. Colloid Interface Sci., 2014, 208, 235–251
Schade, M., Berti, D., Huster, D., Herrmann, A., and Arbuzova A.
(See online at https://doi.org/10.1016/j.cis.2014.02.019) - Phys. Chem. Chem. Phys., 2015, Accepted
Losensky, L., Goldenbogen, Holland, G., Laue, M., Petran, A., Liebscher, Vogel, A., Scheidt, H., Huster, D., J. Klipp, E., and Arbuzova, A.
(See online at https://doi.org/10.1039/c5cp06084b) - Self-assembly of a cholesteryl-modified nucleoside into tubular structures from giant unilamellar vesicles. RSC Adv., 2015, 5, 4502–4510
Losensky, L., Chiantia, S., Holland, G., Laue, M., Petran, A., Liebscher, J. and Arbuzova, A.
(See online at https://doi.org/10.1039/c4ra11289j)
