| 1. |
|
|
| 2. |
- Dabkowska, Aleksandra, et al.
(författare)
-
Assembly of RNA nanostructures on supported lipid bilayers.
- 2015
-
Ingår i: Nanoscale. - : Royal Society of Chemistry (RSC). - 2040-3372 .- 2040-3364. ; 7:2, s. 583-596
-
Tidskriftsartikel (refereegranskat)abstract
- The assembly of nucleic acid nanostructures with controlled size and shape has large impact in the fields of nanotechnology, nanomedicine and synthetic biology. The directed arrangement of nano-structures at interfaces is important for many applications. In spite of this, the use of laterally mobile lipid bilayers to control RNA three-dimensional nanostructure formation on surfaces remains largely unexplored. Here, we direct the self-assembly of RNA building blocks into three-dimensional structures of RNA on fluid lipid bilayers composed of cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or mixtures of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) and cationic sphingosine. We demonstrate the stepwise supramolecular assembly of discrete building blocks through specific and selective RNA-RNA interactions, based on results from quartz crystal microbalance with dissipation (QCM-D), ellipsometry, fluorescence recovery after photobleaching (FRAP) and total internal reflection fluorescence microscopy (TIRF) experiments. The assembly can be controlled to give a densely packed single layer of RNA polyhedrons at the fluid lipid bilayer surface. We show that assembly of the 3D structure can be modulated by sequence specific interactions, surface charge and changes in the salt composition and concentration. In addition, the tertiary structure of the RNA polyhedron can be controllably switched from an extended structure to one that is dense and compact. The versatile approach to building up three-dimensional structures of RNA does not require modification of the surface or the RNA molecules, and can be used as a bottom-up means of nanofabrication of functionalized bio-mimicking surfaces.
|
|
| 3. |
- Dabkowska, Aleksandra P., et al.
(författare)
-
Supported fluid lipid bilayer as a scaffold to direct assembly of RNA nanostructures
- 2017
-
Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1064-3745. ; 1632, s. 107-122
-
Bokkapitel (refereegranskat)abstract
- RNA architectonics offers the possibility to design and assemble RNA into specific shapes, such as nanoscale 3D solids or nanogrids. Combining the minute size of these programmable shapes with precise positioning on a surface further enhances their potential as building blocks in nanotechnology and nanomedicine. Here we describe a bottom-up approach to direct the arrangement of nucleic acid nanostructures by using a supported fluid lipid bilayer as a surface scaffold. The strong attractive electrostatic interactions between RNA polyanions and cationic lipids promote RNA adsorption and self-assembly. Protocol steps for the characterization of assembled RNA complexes via several complementary methods (QCM-D, ellipsometry, confocal fluorescence microscopy, AFM) are also provided. Due to their tunable nature, lipid bilayers can be used to organize RNA laterally on the micrometer scale and thus facilitate the building of more complex 3D structures. The bilayer-based approach can be extended to other programmable RNA or DNA objects to construct intricate structures, such as 2D grids or 3D cages, with high precision.
|
|
| 4. |
- Michanek, Agnes, et al.
(författare)
-
RNA and DNA Association to Zwitterionic and Charged Monolayers at the Air-Liquid Interface
- 2012
-
Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 28:25, s. 9621-9633
-
Tidskriftsartikel (refereegranskat)abstract
- The objective of this work is to establish under which conditions short RNA molecules (similar to miRNA) associate with zwitterionic phospholipids and how this differs from the association with cationic surfactants. We study how the base pairing (i.e., single stranded versus double stranded nucleic acids) and the length of the nucleic acid and the charge of the lipid/surfactant monolayer affect the association behavior. For this purpose, we study the adsorption of nucleic acids to monolayers composed of dipalmitoyl phosphatidylcholine (DPPC) or dioctadecyl-dimethyl-ammoniumbromide (DODAB) using the surface film balance, neutron reflectometry, and fluorescence microscopy. The monolayer studies with the surface film balance suggested that short single-stranded ssRNA associates with liquid expanded zwitterionic phospholipid monolayers, whereas less or no association is detected for double-stranded dsRNA and dsDNA In order to quantify the interaction and to determine the location of the nucleic acid in the lipid/surfactant monolayer we performed neutron reflectometry measurements. It was shown that ssRNA adsorbs to and penetrates the liquid expanded monolayers, whereas there is no penetration of nucleic acids into the liquid condensed monolayer. No adsorption was detected for dsDNA to zwitterionic monolayers. On the basis of these results, we propose that the association is driven by the hydrophobic interactions between the exposed hydrophobic bases of the ssRNA and the hydrocarbon chains of the phospholipids. The addition of ssRNA also influences domain formation in the DPPC monolayer, leading to fractal-like interconnected domains. The experimental results are discussed in terms of the implication for biological processes and new leads for applications in medicine and biotechnology.
|
|
| 5. |
- Michanek, Agnes
(författare)
-
RNA in model lipid membranes - interactions in bulk and at surfaces
- 2011
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The work presented in the thesis “RNA in model lipid membranes- interactions in bulk and at surfaces” aims to characterize the interaction between RNA and model membranes with different properties as well as to determine if ssRNA associate with model membranes in different ways. Several techniques have been used to characterize these systems, including quartz crystal microbalance with dissipation monitoring (QCM-D), Langmuir film balance in combination with neutron reflectometry and fluorescence microscopy as well as differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). Understanding RNA-lipid interactions is important for many research areas, for example for understanding self-assembly in natural systems as well as for biotechnological development and for the development of new delivery systems for RNA based drugs. Association of RNA to model membranes was indicated in the Langmuir monolayer studies and confirmed with neutron reflectometry and QCM-D. The Langmuir monolayer experiments indicated that ssRNA was present at the interface of both zwitterionic and cationic fluid monolayers and with neutron reflectometry we were able to confirm that the ssRNA associated with the monolayer. From the neutron reflectometry studies it was also concluded that ssRNA penetrates into fluid monolayers. With QCM-D we confirmed that ssRNA associates to supported bilayers and that the orientation of the adsorbed ssRNA depends on the lipid phase behaviour. The utilization of model membranes as scaffolds for assembly of nucleic acids has also been studied, using QCM-D and null ellipsometry. The results show that it is possible to perform base pairing of simple RNA oligonucleotides as well as RNA assemblies with well-defined 3D shapes at the bilayer scaffold. From the calorimetric studies of RNA-lipid interactions in bulk solution, it was shown that RNA induces increased Tm as well as splitting of the transition peak in mixed lipid membranes, indicating segregation. We have been able to shown that the model membrane phase behaviour is essential for the association of ssRNA. The interpretation of these results is that the presence of hydrophobic moieties at the surface of fluid membranes is of significant importance for the association of ssRNA indicating that hydrophobic interaction between the exposed RNA bases and the lipid hydrocarbon region occurs. Our studies also showed that the bilayer is suitable to us as scaffolds for building both simple and more complex RNA constructs.
|
|
| 6. |
- Michanek, Agnes, et al.
(författare)
-
ssRNA base pairing at a bilayer interface can be controlled by the acyl chain order
- 2012
-
Ingår i: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 8:40, s. 10428-10438
-
Tidskriftsartikel (refereegranskat)abstract
- RNA–lipid interactions are central to structure and function in biological systems as well as to the development of new applications in medicine and biotechnology. We have studied adsorption and base pairing of short RNA oligonucleotides at model lipid membranes with different compositions by means of QCM-D, confocal microscopy and ITC. The major finding is that base pairing of short complementary RNA strands can be controlled by the acyl-chain chain order, i.e. chains in the solid vs. liquid state, in a deposited bilayer. It was shown that the base pairing with a complementary strand ssRNA takes place at the bilayer when the first strand is pre-adsorbed to a bilayer with solid chains, but not when the first strand is pre-adsorbed to a liquid crystalline bilayer with fluid chains. The results imply that the ssRNA hydrophobic bases are not accessible to the complementary strand bases when RNA is adsorbed to a fluid bilayer, which can be due to hydrophobic interactions with the apolar layer in the fluid bilayer. It is also likely that the difference in lipid phase behaviour affects the kinetics for the base pair reaction at the surface. The corresponding base pairing experiment at the interface of a soft cationic polymer layer consisting of poly(amido amine) PAMAM dendrimers of generation 4 yielded similar results. RNA and DNA were found to adsorb to mixed bilayers that contain the naturally occurring cationic lipid sphingosine as well as to bilayers that contain more conventional surfactants, and it is found that the surface with adsorbed nucleic acid is close to electroneutral at saturation.
|
|
