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Nucleic Acids Research 2006 34(6):e50; doi:10.1093/nar/gkl134
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Published online 4 April 2006

© The Author 2006. 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@oxfordjournals.org

Methods Online

Molecular beacons with intrinsically fluorescent nucleotides

Angel A. Martí1, Steffen Jockusch1, Zengmin Li2,3, Jingyue Ju2,3 and Nicholas J. Turro1,2,*

1 Department of Chemistry, Columbia University New York, NY 10027, USA 2 Department of Chemical Engineering, Columbia University New York, NY 10027, USA 3 Columbia Genome Center, Columbia University College of Physicians and Surgeons New York, NY, 10032, USA

*To whom correspondence should be addressed. Tel: +1 212 854 2175; Fax: +1 212 932 1289; Email: njt3{at}columbia.edu

Received September 1, 2005. Revised November 23, 2005. Accepted March 14, 2006.

We report the design, synthesis and characterization of a novel molecular beacon (MB-FB) which uses the fluorescent bases (FB) 2-aminopurine (AP) and pyrrolo-dC (P-dC) as fluorophores. Because the quantum yield of these FB depend on hybridization with complementary target, the fluorescent properties of MB-FB were tuned by placing the FB site specifically within the MB such that hybridization with complementary sequence switches from single strand to double strand for AP and vice versa for P-dC. The MB-FB produces a ratiometric fluorescence increase (the fluorescence emission of P-dC over that of AP in the presence and absence of complementary sequence) of 8.5 when excited at 310 nm, the maximum absorption of AP. This ratiometric fluorescence is increased to 14 by further optimizing excitation (325 nm). The fluorescence lifetime is also affected by the addition of target, producing a change in the long-lived component from 6.5 to 8.7 ns (Exc. 310 nm, Em. 450 nm). Thermal denaturation profiles monitored at 450 nm (P-dC emission) show a cooperative denaturation of the MB-FB with a melting temperature of 53°C. The thermal denaturation profile of MB-FB hybridized with its target shows a marked fluorescence reduction at 53°C, consistent with a transition from double stranded helix to random coil DNA.


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