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Nucleic Acids Research 2005 33(11):e101; doi:10.1093/nar/gni101
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Published online 24 June 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Methods Online

Immobilized stem–loop structured probes as conformational switches for enzymatic detection of microbial 16S rRNA

Benjamin Bockisch, Thomas Grunwald1, Edzard Spillner and Reinhard Bredehorst*

Institut für Biochemie und Lebensmittelchemie, Abteilung für Biochemie und Molekularbiologie, Universität Hamburg Martin-Luther-King-Platz 6, 20146 Hamburg, Germany 1PLS-Design GmbH Eichenstrasse 42, 20255 Hamburg, Germany

*To whom correspondence should be addressed. Tel: +49 40 42838 2840; Fax: +49 40 42838 7255; Email: bredehorst{at}chemie.uni-hamburg.de

Received February 11, 2005. Revised May 26, 2005. Accepted June 9, 2005.

We have designed and evaluated novel DNA stem–loop structured probes for enzymatic detection of nucleic acid targets. These probes constitute a novel class of conformational switches for enzymatic activity, which in the absence of a target sterically shield an affinity label and upon hybridization of the target to the recognition sequence that forms the loop of the probe restore accessibility of the label for the binding of a reporter enzyme. Analysis of probe characteristics revealed stem stability as the most important parameter governing detection functionality, while other factors such as the length of linker molecules attaching the label to the stem–loop structure and the nature of the solid support proved to be less critical. Apparently, the bulky nature of the reporter enzyme facilitates shielding of the label in the absence of the target, thereby conferring considerable structural tolerance to the conformational switch system. The stem–loop structured probes allow sensitive detection of unlabeled nucleic acid targets. Employing a microtiter assay format, 4 ng of bacterial 16S ribosomal RNA corresponding to 8 fmol could be detected, which can be compared favorably with current immobilized molecular beacon concepts based on fluorescence detection.


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