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Nucleic Acids Research 2006 34(10):3161-3168; doi:10.1093/nar/gkl406
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Published online 12 June 2006

© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.

Article

Pyrene binary probes for unambiguous detection of mRNA using time-resolved fluorescence spectroscopy

Angel A. Martí1, Xiaoxu Li3, Steffen Jockusch1, Zengmin Li2,3, Bindu Raveendra3, Sergey Kalachikov3, James J. Russo3, Irina Morozova3, Sathyanarayanan V. Puthanveettil4, 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 4 Center for Neurobiology & Behavior, 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 December 27, 2005. Revised March 1, 2006. Accepted April 27, 2006.

We report here the design, synthesis and application of pyrene binary oligonucleotide probes for selective detection of cellular mRNA. The detection strategy is based on the formation of a fluorescent excimer when two pyrene groups are brought into close proximity upon hybridization of the probes with the target mRNA. The pyrene excimer has a long fluorescence lifetime (>40 ns) compared with that of cellular extracts (~7 ns), allowing selective detection of the excimer using time-resolved emission spectra (TRES). Optimized probes were used to target a specific region of sensorin mRNA yielding a strong excimer emission peak at 485 nm in the presence of the target and no excimer emission in the absence of the target in buffer solution. While direct fluorescence measurement of neuronal extracts showed a strong fluorescent background, obscuring the detection of the excimer signal, time-resolved emission measurements indicated that the emission decay of the cellular extracts is ~8 times faster than that of the pyrene excimer probes. Thus, using TRES of the pyrene probes, we are able to selectively detect mRNA in the presence of cellular extracts, demonstrating the potential for application of pyrene excimer probes for imaging mRNAs in cellular environments that have background fluorescence.


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