Development and establishment of photoswitchable systems on the surface of plant virus particles
- Entwicklung und Etablierung von lichtkontrollierbaren Systemen auf der Oberfläche von pflanzlichen Viruspartikeln
Kauth, Louisa; van Dongen, Joost Thomas (Thesis advisor); Prüfer, Dirk (Thesis advisor)
Aachen : RWTH Aachen University (2022)
Dissertation, RWTH Aachen University, 2022
Light-switchable proteins can reversibly interact upon light excitation and are gaining increasing interest in the biomedical and material sciences. Light represents an effective stimulus that is easy to regulate and can be applied with high spatial and temporal control. The plant viruses potato virus X (PVX) and tobacco mosaic virus (TMV) are frequently used particles for the presentation of foreign proteins or epitopes. The use of plant virus particles has already shown promising results in biomedical applications such as preferential accumulation in tumor tissue. Displaying one light-switchable protein on the viral surface and fusing a cargo to the protein partners creates the possibility of developing release systems of substances from the nanoparticle surface. In this work, the optogenetic protein systems, LOVTRAP, Dronpa145N and BphP1/QPAS1 were chosen, which cover the range from violet to infrared light by different excitation and reversion wavelengths. First, the in vitro functionality of all tested systems could be confirmed by native gel electrophoresis. The LOVTRAP and the BphP1/QPAS1 system each showed a shift of the signal for the complex upon irradiation with the corresponding wavelength. The switch between the monomeric and tetrameric forms of Dronpa145N was also demonstrated in vitro. Presentation of all systems on the surface of PVX and TMV was achieved using SpyTag/SpyCatcher (ST/SC). The successful coupling of the proteins to the coat protein (CP) was verified by SDS-PAGE and western blot analysis. Coupling efficiencies for PVX of up to 63% and for TMV of up to 54% could be achieved, which is comparable to the values from the literature. Dense decoration of particles with gold in electron micrographs confirmed the modification of the nanoparticles with the SC-fusion proteins. PVX particle loading and unloading with the optogenetic systems were also verified by electron microscopy and gold labeling. The addition of the protein partner to the modified virus particles upon irradiation with the excitation wavelength and subsequent gold decoration of the virus particles by detection of the protein partner on the surface confirmed protein binding. This labeling was not elicited upon illumination with the reversion wavelength. For TMV, a particle loading with a photoswitchable system could not be achieved, which might be due to the rigid composition of the virus leading to steric hindrance regarding protein binding. The results confirm the functionality of light switchable systems on the PVX nanoparticle surface. Especially, the red light-switchable system BphP1/QPAS1 is of great interest, as longer wavelengths enable significantly deeper tissue penetration and non-hazardous application. The size limitations that can occur with BphP1 are circumvented with the LOVTRAP and Dronpa145N systems by a significantly smaller protein size. Thus, each system offers unique advantages for subsequent applications. The present study confirms the development of a new release system based on the light-switchable systems LOVTRAP, BphP1/QPAS1 and Dronpa145N. Thereby, the results form the basis for establishing novel nanoparticle-based controllable release systems.
- Fachgruppe Biologie 
- Lehrstuhl für Molekulare Botanik