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Nucleic Acids Research 2005 33(3):e28; doi:10.1093/nar/gni024
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Published online 17 February 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

Methods Online

Identification of antisense nucleic acid hybridization sites in mRNA molecules with self-quenching fluorescent reporter molecules

Lida K. Gifford1, Joanna B. Opalinska2, David Jordan1, Vikram Pattanayak1,2, Paul Greenham1, Anna Kalota2, Michelle Robbins1,2, Kathy Vernovsky1, Lesbeth C. Rodriguez1, Bao T. Do1, Ponzy Lu1 and Alan M. Gewirtz2,*

1 Department of Chemistry, School of Arts and Sciences 231 South 34th Street, Philadelphia, PA 19104, USA 2 Division of Hematology/Oncology, Department of Medicine, School of Medicine, University of Pennsylvania Room 713, BRB II/III 421 Curie Boulevard, Philadelphia, PA 19104, USA

*To whom correspondence should be addressed. Tel: +1 215 898 4499; Fax: +1 215 573 2078; Email: gewirtz{at}mail.med.upenn.edu

Received January 18, 2005. Accepted January 19, 2005.

We describe a physical mRNA mapping strategy employing fluorescent self-quenching reporter molecules (SQRMs) that facilitates the identification of mRNA sequence accessible for hybridization with antisense nucleic acids in vitro and in vivo, real time. SQRMs are 20–30 base oligodeoxynucleotides with 5–6 bp complementary ends to which a 5' fluorophore and 3' quenching group are attached. Alone, the SQRM complementary ends form a stem that holds the fluorophore and quencher in contact. When the SQRM forms base pairs with its target, the structure separates the fluorophore from the quencher. This event can be reported by fluorescence emission when the fluorophore is excited. The stem–loop of the SQRM suggests that SQRM be made to target natural stem–loop structures formed during mRNA synthesis. The general utility of this method is demonstrated by SQRM identification of targetable sequence within c-myb and bcl-6 mRNA. Corresponding antisense oligonucleotides reduce these gene products in cells.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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