TY  - GEN
AV  - public
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/26675/
N2  - Förster resonance energy transfer (FRET) is a quantum effect of energy transfer from the donor chromophore to the acceptor one via non-radiative dipole-dipole coupling when those chromophores are positioned close enough and in the right orientation to each other. Genetically encoded FRET sensors consist of donor and acceptor fluorescent protein pair (usually CFP/YFP) and a protein based sensing domain in between which responds to the presence or the activity of desired molecule via conformational change resulting in change of FRET efficiency. In this thesis, I developed a genetically encoded FRET sensor for detecting double stranded RNA (dsRNA) during viral infection. The response of eukaryotic cells to infection by RNA viruses is based to a large extent on the regulation by protein kinase R. Protein kinase R (PKR, RNA-regulated protein kinase, eIF2? kinase 2) becomes activated via homodimerisation in the presence of double-stranded RNA produced during replication of RNA viruses (or if endogenously present). Active PKR phosphorylates its target ? mostly ? subunit of eIF2 ? and inhibits the translation of viral proteins. PKR is a two domain protein which consists of an N-terminal double-stranded RNA binding domain (RBD) and a C terminal catalytic domain separated by a 100-amino acid unstructured region. The idea exploited in the current project is based on the ability of the N-terminal PKR domain to undergo a conformational change when binding double-stranded RNA, hence functioning as a sensor for double-stranded RNA. The fluorescent sensor is completed by the addition of the fluorescent proteins mTurquoise and cp173Venus forming a FRET pair. I successfully tested the sensor termed KPR1 in vitro as well as in HeLa Kyoto cells against double-stranded RNA and found a high FRET increase upon binding. The sensor responded well to the presence of self-replicating subgenomic Hepatitis C Virus RNA replicon in Huh7 cells. The data on detection of full Hepatitis C infection was inconclusive.
CY  - Heidelberg, Germany
A1  - Bolbat, Andrey
ID  - heidok26675
Y1  - 2019///
TI  - Genetically encoded FRET sensor to detect double-stranded RNA during viral infection
ER  -