Life under the Microscope: Single-Molecule Fluorescence Highlights the RNA World

Abstract

The emergence of single-molecule (SM) fluorescence techniques has opened up a vast new toolbox for exploring the molecular basis of life. The ability to monitor individual biomolecules in real time enables complex, dynamic folding pathways to be interrogated without the averaging effect of ensemble measurements. In parallel, modern biology has been revolutionized by our emerging understanding of the many functions of RNA. In this comprehensive review, we survey SM fluorescence approaches and discuss how the application of these tools to RNA and RNA-containing macromolecular complexes in vitro has yielded significant insights into the underlying biology. Topics covered include the three-dimensional folding landscapes of a plethora of isolated RNA molecules, their assembly and interactions in RNA-protein complexes, and the relation of these properties to their biological functions. In all of these examples, the use of SM fluorescence methods has revealed critical information beyond the reach of ensemble averages.

A study on a telo21 G-quadruplex DNA specific binding ligand: enhancing the molecular recognition ability via the amino group interactions

“…[10][11][12] However, the sensing or detection of a particular type of targeted biomolecule selectivity in their native environment is still challenging. 13,14 In the nucleic acid research area, to discrimination of G-quadruplex (G4) structures from other type of DNA by visual methods is a big difficulty. [15][16][17] For this reason, considerable efforts are in progress for the discovery of quadruplex specic binding ligands endowed with uorescence properties.…”

Approaches for measuring the dynamics of RNA–protein interactions

“…In many cases, one fluorophor is placed on the protein, the other on the RNA (Figure b). However, both fluorophors can be strategically placed on the RNA, if the RNA structure changes upon protein binding (Ray et al, ; R. Roy et al, ). Of note, even in single stranded nucleic acids distances between two strategically placed fluorohors can change significantly upon protein binding, because unstructured RNA and DNA behave like a worm‐like chain, but adopt restricted conformations when bound to protein (Fairman‐Williams & Jankowsky, ; Murphy, Rasnik, Cheng, Lohman, & Ha, ).…”

Kinetics coming into focus: single-molecule microscopy of riboswitch dynamics

“…Strategic placement of the two fluorophores results in real-time observation of specific conformational changes of the host molecule or relative motions between two molecules in a complex. A detailed description of FRET as a technique and its adaptation for different SM experiments is found in previous reviews [46,[53][54][55]. In short, the dually labeled RNA is typically immobilized on a surface and the dynamics between the folded and the unfolded states detected upon observing the evolution of FRET states over time (Figure 2a).…”