Strategic labelling approaches for RNA single-molecule spectroscopy

ABSTRACT

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.

KEYWORDS:

• RNA labeling
• click chemistry
• single-molecule FRET
• RNA localization
• enzymatic modification
• site-specific labeling
• orthogonal labeling

Acknowledgments

This work has been supported by the DFG, CRC 902 “Molecular Principles of RNA-based regulation”. M.H. is a member of the Cluster of Excellence EXC115 “Macromolecular complexes in action”. The authors thank Prof. Harald Schwalbe for constant support.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the Deutsche Forschungsgemeinschaft: [Grant Number SFB902].