Super-resolution imaging of RNA structures and processes

In addition to carrying genetic information, the flexibility and dynamics of RNA molecules allows them to fold into specific structures that dictate their fate and role in many crucial biological processes. Traditional structure determination methods such as X-ray, NMR or cryo-EM generate exquisitely resolved static 3D-structures, but they cannot be applied to study the structure of long and dynamic RNA sequences. The first step to determine the structure of long RNA molecules is to be able to discriminate stretches of single-stranded RNA from secondary and tertiary folded segments. Super-resolution optical imaging of long DNA sequences using fluorescence intercalators or fluorescently labelled DNA-binding proteins has been recently demonstrated as a tool to reveal the structure of the duplex DNA. In contrast, the application of super-resolution imaging of RNA sequences has been limited to detecting their presence, but not their structure, using in-situ hybridization or the incorporation of fluorescent aptamers within the RNA sequence. Current intercalator-based methods used commonly for DNA staining cannot discriminate between single- or duplex nucleic acid sequences, therefore they have limited application to image RNA structure.
Our aim is to demonstrate super-resolution structural imaging of i) fully transcribed RNA sequences ii) and co-transcriptionally emerging RNA molecules, using a novel approach combining fluorescently labelled single-strand binding proteins (SSBs) and STED imaging. As part of our current BBSRC funding to investigate DNA repair pathways, we have identified a monomeric single-strand binding protein from an archaeal organism that binds single-strand RNA with ~1000-fold higher affinity (KD~ 4 nM) than duplex RNA (KD ~ 5 M). The 14 kDa SSB protein binds only four RNA nucleotides and can be tagged with indocarbocyanine derivatives that have been shown to behave as excellent probes to STED imaging. Importantly, single-molecule FRET microscopy has shown a fast binding and unbinding dynamics of the SSB proteins to the nucleic acid single stranded region and this can be further modulated by the ionic strength of the medium [Morten et al, 2017]. The stochastic binding of fluorescently labelled SSBs to the nucleic acid single strand sequences can be considered as a point accumulation for imaging nanoscale topography (PAINT) method that uses a protein-based reporter instead of a short DNA sequence (DNA-PAINT). Because the interaction of the SSB protein with the RNA is not sequence specific, SSB stochastic binding will light up the entire single-strand RNA sequence which is desirable for many applications. Thus, the combination of SSB-PAINT and STED will allow, for the first time, to discriminate entire single-strand RNA regions from duplex RNA segments, which is currently impossible, and monitor transcriptional processes with an unprecedented spatial resolution. We expect the fast exchange of labelled SSB monomers on the nucleic acid strand to minimize the impact of sample photobleaching by the STED depletion laser and thus increase resolution. SSB-PAINT will be an invaluable tool to researchers working in a wide range of RNA-related processes within BBSRC remit including transcription, RNA-protein interactions, splicing and the structure of viral RNAs, both in vitro and in vivo.