The how & why of looking at individual RNAs

Many current techniques in RNA and RNP research employ increasingly large datasets to derive information on the level of RNA structures or RNA sequences: cryoEM integrates images of hundreds of thousands of particles, and next-generation sequencing, often combined with specific enrichment or crosslinking techniques, generates billions of reads from a single sequencing run. These approaches have yielded fundamental new insights on a remarkable number of structures as well as functional questions. There are however limitations to these techniques, and these become increasingly obvious when trying to obtain information in cellular context or when looking at functionally complex scenarios. In these cases, the observation and characterization of single molecules has proven as an elegant, immediate approach that can yield precise information in both spatial and temporal dimensions. The advantages of any single-molecule technique (resolving heterogeneous population compositions, real-time observation of localization or dynamics) are highlighted in the manuscripts within this special issue on single-molecule techniques, and they demonstrate their experimental powers when applied to current questions on both molecular mechanisms and spatial cellular regulation.

This Special Issue covers three general directions: the application of single-molecule approaches to the studies of RNP and RNA function, the techniques allowing for super-resolution-based localization of cellular RNA, and the experimental approaches to generate constructs and probes that enable both of the aforementioned applications.

As an area of research that remains to be of ultimate interest, studies on ligand binding RNAs, termed riboswitches, have been profiting significantly from investigations on the level of individual molecules. This is mostly due to their intricate folding landscape, but also due to a complex interplay of structures, kinetics of structure formation, and the connections between mutually exclusive functionally relevant conformations. In the case of riboswitches acting on the level of transcription, these conformations are often transient and therefore extremely difficult to detect and analyze.

In a comprehensive overview on single-molecule investigations of riboswitches, Nils Walter and colleagues [1] provide a big picture on how specific questions were and will be answered using single-molecule approaches. This is particularly highlighted in the abovementioned case of cotranscriptional folding. The variety of techniques employed and taken into consideration for this review, including single-molecule force spectroscopy, emphasizes its relevance to obtain a detailed picture of riboswitch structural dynamics and how these are responsible for efficient riboswitch-based regulatory functions.

In an elegant example on such riboswitch dynamics, the Ermolenko group describes the folding and ligand binding response of the preQ1 riboswitch using single-molecule Förster resonance energy transfer (smFRET)[2]. By dissecting structural responses to Mg²⁺ ion as well as cognate ligand binding, they show how transient preformation of a state that is competent for ligand binding enables the riboswitch to faithfully bind the ligand. This work nicely illustrates the wealth of information that is obtainable by time-dependent observation of a single FRET pair, as both the effects of Mg²⁺ ions as well as the preQ ligand would be far less well discernible in bulk experiments. The presented titration experiments also are a perfect example of how carefully designed single-molecule experiments can bridge over to characteristics usually determined in bulk experiments.

Most of the cellular interactions between two different strands of nucleic acids require the action of a protein component processing the duplex to exert its function. In this regard, Argonaut proteins have a unique role in establishing and recognizing such duplexes. In their review on single-molecule-based approaches to study the search for such targets mediated by Argonaute proteins, Chirlmin Joo and Tao Ju Cui provide an extensive overview on how such molecules scan possible targets to find the matching nucleic acid strand, and the desired target site[3]. Their examples also demonstrate how different experimental setups (such as the observation of proteins on a linear nucleic acid grid, or the combination of insights from force and fluorescence spectroscopy) can yield information on a level of detail that very few other approaches could provide.

Swinging our view to the cellular environment, Hye-Yoon Park and colleagues illustrate how approaches that use different constructs comprising different arrangements of nucleic acids, fluorescent labels, and quenchers can render individual mRNAs inside cells traceable and amenable to analysis[4]. They provide a number of available labelling approaches, an insight into state-of-the-art microscopy methods, and combine this with a complex array of cellular roles these techniques can be used to interrogate.

All of the abovementioned topics and techniques rely on the specific placement of fluorescent labels within the molecule to be analyzed, many of which are challenging to synthesize. Just as more and more derivatives of fluorescent dyes become available, the number of techniques for site-specific labelling is still growing. In our contribution, we therefore provide an up-to-date summary of different chemical, enzymatic, and chemo-enzymatic approaches that give the researcher a maximum in freedom when deciding where to place the fluorescent dye[5].

This special issue covers a wide variety of aspects; from distinct biological examples that have been successfully studied using single-molecule techniques, as well as the chemical, biochemical, and technical approaches that have made these studies possible. It is therefore precisely this combination of biological question with a suitable technical approach that has made single-molecule science on RNA so successful, and that promises to keep doing so for the coming years.

Disclosure statement

No potential conflict of interest was reported by the author.