Helicase-mediated changes in RNA structure at the single-molecule level

Abstract

RNA helicases are a diverse group of RNA-dependent ATPases known to play a large number of biological roles inside the cell, such as RNA unwinding, remodeling, export and degradation. Understanding how helicases mediate changes in RNA structure is therefore of fundamental interest. The advent of single-molecule spectroscopic techniques has unveiled with unprecedented detail the interplay of RNA helicases with their substrates. In this review, we describe the characterization of helicase-RNA interactions by single-molecule approaches. State-of-the-art techniques are presented, followed by a discussion of recent advancements in this exciting field.

Kinetic Subpopulations Detected by Single-molecule Spectroscopy: Fundamental Property of Functional Nucleic Acids or Experimental Artefact?

“…by proteins, but they may accumulate in the artificial environment of the single-molecule studies that are frequently carried out under non-physiological conditions. [2] It is believed that kinetic heterogeneity is caused by the co-existence of several structures and a number of culprits have been proposed to this end: (i) Surface tethering. Immobilisation of biomolecules might alter their structure and hence their functionality.…”

“…[1] FRET is the distance-dependent, non-radiative energy transfer between two dipoles, typically the transition dipole moments of two fluorophores that are referred to as donor and acceptor. [2] By estimating the interdye distance in real time, single-molecule FRET (smFRET) therefore allows the detection of intermediates along the folding pathway of biologically relevant molecules, as well as misfolded structures. As a consequence, it has been frequently used to study folding and function of catalytically active RNA molecules (ribozymes), regulatory elements of translation (riboswitches), and functional DNAs.…”

Single-molecule tools for enzymology, structural biology, systems biology and nanotechnology: an update

“…Depending on signal-to-noise ratio and excitation power, setups utilizing these cameras can readily achieve time resolutions on the order of tens of milliseconds. If one is concerned with achieving the highest-possible time resolution, typically down to tens of microseconds (König et al 2013; Phelps et al 2013), one may sacrifice the advantages of a large field of view and instead image one molecule at a time onto a single-photon counting avalanche photodiode (APD). This is accomplished by placing a pinhole in the emission path to allow only the signal from one molecule to reach the detector.…”

Single-molecule FRET for Ultrasensitive Detection of Biomolecules

“…The application of smFRET in RNA study focuses mainly on the dynamics of RNA folding and catalysis [60]. Recently, Hengesbach et al employed smFRET to reveal the folding dynamics of human telomerase RNA (hTR) pseudoknot domain [61].…”