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Nucleic Acids Research 2005 33(20):e177; doi:10.1093/nar/gni174
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Published online 27 November 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}oxfordjournals.org

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Bis-aptazyme sensors for hepatitis C virus replicase and helicase without blank signal

Suhyung Cho1, Ji-Eun Kim2, Bo-Rahm Lee2, June-Hyung Kim2 and Byung-Gee Kim1,2,*

1Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University Shinlim-dong, Kwanak-Gu, Seoul 151-744, Korea 2School of Chemical and Biological Engineering, Seoul National University Shinlim-dong, Kwanak-Gu, Seoul 151-744, Korea

*To whom correspondence should be addressed at Laboratory of Molecular Biotechnology and Biomaterials, School of Chemical and Biological Engineering, Seoul National University, Shinlim-dong, Kwanak-Gu, Seoul 151-744, Korea. Tel: +82 2 880 6774; Fax: +82 2 883 6020; Email: byungkim{at}snu.ac.kr

Received May 31, 2005. Revised August 27, 2005. Accepted October 18, 2005.

The fusion molecule (i.e. aptazyme) of aptamer and hammerhead ribozyme was developed as in situ sensor. Previously, the hammerhead ribozyme conjugated with aptamer through its stem II module showed a significant blank signal by self-cleavage. To reduce or remove its self-cleavage activity in the absence of target molecule, rational designs were attempted by reducing the binding affinity of the aptazyme to its RNA substrate, while maintaining the ribonuclease activity of the aptazyme. Interestingly, the bis-aptazymes which comprise the two aptamer-binding sites at both stem I and stem III of the hammerhead ribozyme showed very low blank signals, and their ratios of reaction rate constants, i.e. signal to noise ratios, were several tens to hundred times higher than those of the stem II-conjugated bis-aptazymes. The reduction in the blank signals seems to be caused by a higher dissociation constant between the main strand of the bis-aptazyme and its substrate arising from multi-point base-pairing of the bis-aptazymes. The bis-aptazymes for HCV replicase and helicase showed high selectivity against other proteins, and a linear relationship existed between their ribozyme activities and the target concentrations. In addition, a bis-aptazyme of dual functions was designed by inserting both aptamers for HCV replicase and helicase into the stem I and stem III of hammerhead ribozyme, respectively, and it also showed greater sensitivity and specificity for both proteins without blank signal.


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