Article

Supported lipid bilayers and DNA curtains for high-throughput single-molecule studies.

Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2011; 745:447-61. DOI: 10.1007/978-1-61779-129-1_26
Source: PubMed

ABSTRACT Single-molecule studies of protein-DNA interactions continue to yield new information on numerous DNA processing pathways. For example, optical microscopy-based techniques permit the real-time observation of proteins that interact with DNA substrates, which in turn allows direct insight into reaction mechanisms. However, these experiments remain technically challenging and are limited by the paucity of stable chromophores and the difficulty of acquiring statistically significant observations. In this protocol, we describe a novel, high-throughput, nanofabricated experimental platform enabling real-time imaging of hundreds of individual protein-DNA complexes over hour timescales.

0 Bookmarks
 · 
142 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: DNA nanotechnology enables the precise construction of nanoscale devices that mimic aspects of natural biomolecular systems yet exhibit robustly programmable behavior. While many important biological processes involve dynamic interactions between components associated with phospholipid membranes, little progress has been made towards creating synthetic mimics of such interfacial systems. We report the assembly and characterization of cholesterol-labeled DNA origami "barges" capable of reversible association with and lateral diffusion on supported lipid bilayers. Using single-particle fluorescence microscopy, we show that these DNA barges rapidly and stably embed in lipid bilayers and exhibit Brownian diffusion in a manner dependent on both cholesterol labeling and bilayer composition. Tracking of individual barges rapidly generates super-resolution maps of the contiguous regions of a membrane. Addition of appropriate command oligonucleotides enables membrane-associated barges to reversibly exchange fluorescent cargo with bulk solution, dissociate from the membrane, or form oligomers within the membrane, opening up new possibilities for programmable membrane-bound molecular devices.
    ACS Nano 05/2014; · 12.03 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Ring NTPases represent a large and diverse group of proteins that couple their nucleotide hydrolysis activity to a mechanical task involving force generation and some type of transport process in the cell. Because of their shape, these enzymes often operate as gates that separate distinct cellular compartments to control and regulate the passage of chemical species across them. In this manner, ions and small molecules are moved across membranes, biopolymer substrates are segregated between cells or moved into confined spaces, double-stranded nucleic acids are separated into single strands to provide access to the genetic information, and polypeptides are unfolded and processed for recycling. Here we review the recent advances in the characterization of these motors using single-molecule manipulation and detection approaches. We describe the various mechanisms by which ring motors convert chemical energy to mechanical force or torque and coordinate the activities of individual subunits that constitute the ring. We also examine how single-molecule studies have contributed to a better understanding of the structural elements involved in motor-substrate interaction, mechanochemical coupling, and intersubunit coordination. Finally, we discuss how these molecular motors tailor their operation-often through regulation by other cofactors-to suit their unique biological functions.
    Biophysical Journal 05/2014; 106(9):1844-1858. · 3.83 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Fluorescence and force-based single-molecule studies of protein-nucleic acid interactions continue to shed critical insights into many aspects of DNA and RNA processing. As single-molecule assays are inherently low-throughput, obtaining statistically relevant datasets remains a major challenge. Additionally, most fluorescence-based singlemolecule particle-tracking assays are limited to observing fluorescent proteins that are in the low-nanomolar range, as spurious background signals predominate at higher fluorophore concentrations. These technical limitations have traditionally limited the types of questions that could be addressed via single-molecule methods. In this review, we describe new approaches for high-throughput and high-concentration single-molecule biochemical studies. We conclude with a discussion of outstanding challenges for the single-molecule biologist and how these challenges can be tackled to further approach the biochemical complexity of the cell.
    FEBS Letters 10/2014; · 3.34 Impact Factor

Preview

Download
3 Downloads
Available from