NDLI: Vesicle Encapsulation Studies Reveal that Single Molecule Ribozyme Heterogeneities Are Intrinsic

Content Provider	PubMed Central
Author	Burak, Okumus Wilson, Timothy J. Lilley, David M. J. Ha, Taekjip
Copyright Year	2004
Abstract	Single-molecule measurements have revealed that what were assumed to be identical molecules can differ significantly in their static and dynamic properties. One of the most striking examples is the hairpin ribozyme, which was shown to exhibit two to three orders of magnitude variation in folding kinetics between molecules. Although averaged behavior of single molecules matched the bulk solution data, it was not possible to exclude rigorously the possibility that the variations around the mean values arose from different ways of interacting with the surface environment. To test this, we minimized the molecules' interaction with the surface by encapsulating DNA or RNA molecules inside 100- to 200-nm diameter unilamellar vesicles, following the procedures described by Haran and coworkers. Vesicles were immobilized on a supported lipid bilayer via biotin-streptavidin linkages. We observed no direct binding of DNA or RNA on the supported bilayer even at concentrations exceeding 100 nM, indicating that these molecules do not bind stably on the membrane. Since the vesicle diameter is smaller than the resolution of optical microscopy, the lateral mobility of the molecules is severely constrained, allowing long observation periods. We used fluorescence correlation spectroscopy, nuclease digestion, and external buffer exchange to show that the molecules were indeed encapsulated within the vesicles. When contained within vesicles, the natural form of the hairpin ribozyme exhibited 50-fold variation in both folding and unfolding rates in 0.5 mM Mg2+, which is identical to what was observed from the molecules tethered directly on the surface. This strongly indicates that the observed heterogeneity in dynamic properties does not arise as an artifact of surface attachment, but is intrinsic to the nature of the molecules.
Related Links	http://dx.doi.org/10.1529/biophysj.104.045971
Ending Page	2806
Page Count	9
Starting Page	2798
File Format	PDF
ISSN	00063495
e-ISSN	15420086
Journal	Biophysical Journal
Issue Number	4
Volume Number	87
Language	English
Publisher	Biophysical Society
Publisher Date	2004-10-01
Access Restriction	Open
Rights Holder	Biophysical Society
Subject Keyword	Biophysics Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Biophysics

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