Strategic labelling approaches for RNA single-molecule spectroscopy.

Most single-molecule techniques observing RNA in vitro or in vivo require fluorescent labels that have to be connected to the RNA of interest. In recent years, a plethora of methods has been developed to achieve site-specific labelling, in many cases under near-native conditions. Here, we review chemical as well as enzymatic labelling methods that are compatible with single-molecule fluorescence spectroscopy or microscopy and show how these can be combined to offer a large variety of options to site-specifically place one or more labels in an RNA of interest. By either chemically forming a covalent bond or non-covalent hybridization, these techniques are prerequisites to perform state-of-the-art single-molecule experiments.