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Single-molecule Chemistry and Biology Special Feature
SINGLE-MOLECULE CHEMISTRY AND BIOLOGY SPECIAL FEATURE / BIOLOGICAL SCIENCES / RESEARCH ARTICLES / BIOPHYSICS
Fueling protein–DNA interactions inside porous nanocontainers
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*Department of Physics, Center for Biophysics and Computational Biology, Howard Hughes Medical Institute, University of Illinois at Urbana–Champaign, Urbana, IL 61801
Edited by Robert J. Silbey, Massachusetts Institute of Technology, Cambridge, MA, and approved April 25, 2007 (received for review December 5, 2006)
Vesicle encapsulation offers a biologically relevant environment for many soluble proteins and nucleic acids and an optimal immobilization medium for single-molecule fluorescence assays. Furthermore, the confinement of biomolecules within small volumes opens up new avenues to unique experimental configurations. Nevertheless, the vesicles' impermeability, even toward ions and other small molecules such as ATP, hinders more general applications. We therefore developed methods to induce pores into vesicles. Porous vesicles were then used to modulate the interaction between Escherichia coli RecA proteins and ssDNA by changing the extravesicular nucleotides. Repetitive binding and dissociation of the same RecA filament on the DNA was observed with a rebinding rate two orders of magnitude greater than in the absence of confinement, suggesting a previously unreported nucleation pathway for RecA filament. This method provides a biofriendly and simple alternative to surface tethering that is ideal for the study of transient and weakly interacting biological complexes.
RecA | single molecule | vesicle encapsulation
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0610673104/DC1.
¶ The interior volume of a 100-nm diameter vesicle is 
0.5 attoliters, and the effective concentration of one molecule within that volume is 3 µM.
|| Buffer A is composed of 1 mM 2-mercaptoethanol, 10 mM Mg(CH3COO)2, 100 mM Na(CH3COO), 25 mM Tris·CH3COOH (pH 7.5), and 1 mM ATP. To increase the photostability of the fluorescent molecules, we include 1 mg/ml glucose oxidase/0.04 mg/ml catalase/1% (v/v) 2-mercaptoethanol/0.4% glucose.
** Monomeric dissociation, should it occur, would be detected in the 100-ms resolution.
When the PEG surface was not covered with neutravidin, essentially no binding was observed. In sharp contrast, even a lower dilution of the sample yielded a good coverage on the surface after neutravidin treatment. The binding was therefore specific to only the biotinylated molecules, which would be the vesicles.
To whom correspondence should be addressed. E-mail: tjha{at}uiuc.edu
© 2007 by The National Academy of Sciences of the USA
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