Visualization of the Delivery and Release of Small RNAs Using Genetic Code Expansion and Unnatural RNA-Binding Proteins
journal contributionposted on 2018-11-09, 00:00 authored by Noreen Ahmed, Joanna Fréderique De Graaf, Nadine Ahmed, Dana V. Foss, Julie Delcorde, Peter G. Schultz, John Paul Pezacki
Endogenously expressed noncoding RNAs are regulators of mRNA translation and affect diverse biological pathways spanning embryogenesis to cholesterol and fatty acid metabolism. Recently, microRNAs have become an important therapeutic target with strategies that employ oligonucleotides as both mimics and inhibitors of target microRNAs, successfully altering gene expression and cellular pathways in relevant contexts. However, delivery of these exogenous effectors remains a major challenge. Here, we present a method for evaluating noncoding RNA delivery using the viral suppressor of RNA silencing (VSRS) protein p19, optimized for cellular delivery of small RNAs. Using genetic code expansion technology, p-azidophenylalanine (AzF) was incorporated into a recombinant p19 protein and used to develop a fluorescence resonance energy transfer (FRET) sensor. AzF was used to attach FRET acceptor moieties using bioorthogonal chemistry. We show that this strategy not only gives rise to FRET signals that report on small RNA binding, but also allows for fluorescence quenching as well, convenient for measuring RNA release. We demonstrate the successful use of a modified version of the probe to track the delivery and release of small RNAs into mammalian cells. The results provide a basis for a further development of vehicles for small RNA delivery and release for intervening in noncoding RNA biology.
Unnatural RNA-Binding Proteins Endogenouslyexogenous effectorsFRET signalscode expansion technologyRNA bindingnoncoding RNA biologyRNA deliverygene expressiontarget microRNAsacid metabolismp 19 proteinnoncoding RNAsbioorthogonal chemistryGenetic Code Expansionfluorescence resonance energy transferfluorescence quenchingnoncoding RNA deliveryRNA releaseFRET acceptor moietiesVSRSSmall RNAsmRNA translation