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Nucleic Acids Research 2005 33(6):e64; doi:10.1093/nar/gni063
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Published online 7 April 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

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Proximity extension of circular DNA aptamers with real-time protein detection

Daniel A. Di Giusto, Wjatschesslaw A. Wlassoff, J. Justin Gooding1, Barbara A. Messerle1 and Garry C. King*

School of Biotechnology and Biomolecular Sciences, The University of New South Wales Sydney NSW 2052, Australia 1School of Chemistry, The University of New South Wales Sydney NSW 2052, Australia

*To whom correspondence should be addressed. Tel: +61 2 9385 2021; Fax: +61 2 9385 1483; Email: garry{at}kinglab.unsw.edu.au

Received January 23, 2005. Revised March 16, 2005. Accepted March 16, 2005.

Multivalent circular aptamers or ‘captamers’ have recently been introduced through the merger of aptameric recognition functions with the basic principles of DNA nanotechnology. Aptamers have strong utility as protein-binding motifs for diagnostic applications, where their ease of discovery, thermal stability and low cost make them ideal components for incorporation into targeted protein assays. Here we report upon a property specific to circular DNA aptamers: their intrinsic compatibility with a highly sensitive protein detection method termed the ‘proximity extension’ assay. The circular DNA architecture facilitates the integration of multiple functional elements into a single molecule: aptameric target recognition, nucleic acid hybridization specificity and rolling circle amplification. Successful exploitation of these properties is demonstrated for the molecular analysis of thrombin, with the assay delivering a detection limit nearly three orders of magnitude below the dissociation constants of the two contributing aptamer–thrombin interactions. Real-time signal amplification and detection under isothermal conditions points towards potential clinical applications, with both fluorescent and bioelectronic methods of detection achieved. This application elaborates the pleiotropic properties of circular DNA aptamers beyond the stability, potency and multitargeting characteristics described earlier.


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