Abstract
It is clear that a crowded environment influences the structure, dynamics, and interactions of biological molecules, but the complexity of this phenomenon demands the development of new experimental and theoretical approaches. Here we use two complementary single-molecule FRET techniques to show that the kinetics of DNA base pairing and unpairing, which are fundamental to both the biological role of DNA and its technological applications, are strongly modulated by a crowded environment. We directly observed single DNA hairpins, which are excellent model systems for studying hybridization, either freely diffusing in solution or immobilized on a surface under crowding conditions. The hairpins followed two-state folding dynamics with a closing rate increasing by 4-fold and the opening rate decreasing 2-fold, for only modest concentrations of crowder [10% (w/w) polyethylene glycol (PEG)]. These experiments serve both to unambiguously highlight the impact of a crowded environment on a fundamental biological process, DNA base pairing, and to illustrate the benefits of single-molecule approaches to probing the structure and dynamics of complex biomolecular systems.
Highlights
The iconic DNA double helix continues to inspire fundamental research into its mechanical properties,[1,2] its interactions with the cellular machinery,[3] and the development of new nanotechnologies.[4]
It is widely recognized that changes to biomolecular structure and dynamics under crowding conditions can be attributed to several other factors, including water and ion activity, dielectric constant, preferential interactions between crowder and biomolecule, viscosity, and diffusion.[10−12] the various contributions can often support or counteract each other, resulting in a difficulty in assessing and quantifying the effects, and significance, of crowding, and in developing a theoretical framework that is generally applicable.[13]
A number of single-molecule Förster resonance energy transfer[17] studies have recently appeared concerning the structural dynamics of RNA structures in the presence of polyethylene glycol (PEG), as a model macromolecular crowder,[18,19] in addition to related work on RNA and DNA in the presence of small solutes such as urea.[20]
Summary
The iconic DNA double helix continues to inspire fundamental research into its mechanical properties,[1,2] its interactions with the cellular machinery,[3] and the development of new nanotechnologies.[4]. Much has been learned about the role of crowding on DNA using ensemble methods, there are many unanswered questions, regarding the kinetics of base pairing.[16] A number of single-molecule Förster resonance energy transfer (smFRET)[17] studies have recently appeared concerning the structural dynamics of RNA structures in the presence of polyethylene glycol (PEG), as a model macromolecular crowder,[18,19] in addition to related work on RNA and DNA in the presence of small solutes (osmolytes) such as urea.[20]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
