Use of Baby Spinach and Broccoli for imaging of structured cellular RNAs

Abstract

Fluorogenic RNA aptamers provide a powerful tool for study of RNA analogous to green fluorescent protein for the study of proteins. Spinach and Broccoli are RNAs selected in vitro or in vivo respectively to bind to an exogenous chromophore. They can be genetically inserted into an RNA of interest for live-cell imaging. Spinach aptamer has been altered to increase thermal stability and stabilize the desired folding. How well these fluorogenic RNA aptamers behave when inserted into structured cellular RNAs and how aptamer properties might be affected remains poorly characterized. Here, we report a study of the performance of distinct RNA Spinach and Broccoli aptamer sequences in isolation or inserted into the small subunit of the bacterial ribosome. We found that the ribosomal context helped maintaining the yield of the folded Baby Spinach aptamer; other versions of Spinach did not perform well in the context of ribosomes. In vivo, two aptamers clearly stood out. Baby Spinach and Broccoli aptamers yielded fluorescence levels markedly superior to all previous Spinach sequences including the super-folder tRNA scaffolded tSpinach2. Overall, the results suggest the use of Broccoli and Baby Spinach aptamers for live cell imaging of structured RNAs.