Chip Electrophoresis of viral RNA transfer through membranes

The current project proposal concerns the development of an in vitro model system for virus infection. The system will be based on chip electrophoresis of molecular beacons (MBs) encapsulated within liposomes (cell membrane substitutes). Upon MB binding to an RNA target, spatial proximity between a fluorophore and a quencher modification of the MB is lost resulting in increased fluorescence. Successful transfer of viral RNA through the liposomal membrane as measure of infection will therefore be detected by increased fluorescence of liposomes. The developed model system will be compared to other methods determining virus infectivity. Experiments will be carried out with human Rhinovirus serotype 2 (HRV2), a picornavirus. HRVs are responsible for the majority of common cold infections. Especially HRV2 has been well characterized, nevertheless, still some major questions concerning the transfer of viral RNA though lipid membranes during cell infection remain open. However, a modular design of the developed model system will allow future application of the setup in the analysis of a larger number of different viruses. Chip electrophoresis will be carried out on a commercial instrument equipped with a red laser and a blue LED enabling for dual detection at 470 / 525 and 635 / 685 nm excitation / emission wavelength, respectively. The applicability of this separation system for the analysis of HRV2 / liposome adducts as detected in vitro during early viral infection processes was already demonstrated in a series of papers (e.g. Weiss, V. U. et al, Electrophoresis 2009). However, in vitro transfer of viral RNA through lipid membranes was so far only shown via encapsulation of a reverse transcription (RT) PCR kit (Bilek, G. et al, Journal of Virology 2011). Substitution of RT PCR by MBs was promising in first experiments. Application of liposome encapsulated MBs offers several advantages over RT PCR liposomes like (i) fast analysis, (ii) simultaneous detection of RNA released into the liposomal lumen as well as to the surrounding medium due to electrophoretic separation of analytes and (iii) potential application of size markers within liposomes to monitor vesicle tightness during viral RNA transfer. The sensitivity of the developed assay will be dependent on the initial MB design as well as the identification of suitable reaction conditions (buffer type, ionic strength, etc.). Therefore, appropriate time is dedicated to these far- reaching development steps. The application of nano electrospray gas phase electrophoretic mobility molecular analysis (nES GEMMA) as well as nESI (electrospray ionization) ion mobility mass spectrometry (nESI IM MS) in the development of the in vitro model system will be demonstrated as orthogonal methods to verify the mode of operation of the MB.

The project Chip electrophoresis of viral RNA transfer through membranes aimed to develop a model system for viral cell infection. This model was based on electrophoresis in miniaturized form on a commercially available single-use chip to allow for analyte separation in an electrolyte solution upon application of an electric field. Liposomes were intended as cell membrane analogues as liposome vesicles likewise consist of a lipid bilayer encapsulating an aqueous interior. Transfer of the viral genome as key step of viral cell infection was intended to be monitored via fluorescent probes (molecular beacons, MBs), lighting up as soon as the RNA of the employed virus (human Rhinovirus serotype 2, HRV2) enters the liposome interior. In the first part of the project detection of genomic material via MBs (in the absence of liposomes) could be demonstrated via chip electrophoresis. Antioxidant addition to the electrolyte lead to a significant increase in fluorescence. Application of two internal standards detected at a different wavelength allowed for a better comparison of electropherograms not only from one but from several chips. Application of the developed method to HRV2 samples allowed us to follow the release of RNA from virus particles to the surrounding medium. In a next step liposome preparation was established at TU Wien. Vesicle characterization was based on gas- and liquid-phase electrophoresis in order to check for vesicle size, homogeneity and occurrence of undesired smaller fragments. MBs were encapsulated within liposomes and their reactivity was subsequently assessed via chip electrophoresis. In addition, in order to assess vesicle integrity fluorescent size standards were encapsulated within vesicles. These standards (polysaccharides) were likewise characterized via gas-phase electrophoresis. Subsequently, HRV2 RNA transfer experiments to liposomes were carried out. As alternative to chip electrophoresis, a spectroscopic approach was investigated after gas-phase electrophoresis based collection of vesicles. Limitations of the spectroscopic instrumentation resulted, however, only in a general proof of principle. No information concerning the viral infection process was obtained. Thus research on preparation and purification of HRV2 was intensified to carry on chip electrophoresis. Two virus purification protocols (lipase digestion, anion exchange chromatography) were tested. Highly pure batches enabled us to determine the molecular weight of corresponding large intact protein assemblies via mass spectrometry. However, naturally occuring HRV2 heterogeneity impaired RNA transfer experiments. Therefore, a follow up study is suggested dealing with preparation and in-depth characterization of HRV2 in order to reduce naturally occurring virus heterogeneity and to repeat RNA transfer measurements with well-defined material. Despite the intended virus cell infection model could not be setup in last consequence, implications of the project concern the characterization of (bio-) nanoparticle material, especially liposomes and polysaccharides. Polishing of HRV2 was significantly improved as was the chip electrophoretic assay.