Design and characterization of two-dye and three-dye binary fluorescent probes for mRNA detection

@article{citeulike:2524532, abstract = {We report the design, synthesis, and characterization of binary oligonucleotide probes for mRNA detection. The probes were designed to avoid common problems found in standard binary probes such as direct excitation of the acceptor fluorophore and overlap between the donor and acceptor emission spectra. Two different probes were constructed that contained an array of either two or three dyes and were characterized using steady-state fluorescence spectroscopy, time-resolved fluorescence spectroscopy, and fluorescence depolarization measurements. The three-dye binary probe (BP-3d) consists of a Fam fluorophore which acts as a donor, collecting light and transferring it as energy to Tamra, which subsequently transfers energy to Cy5 when the two probes are hybridized to mRNA. This design allows the use of 488 nm excitation, which avoids the direct excitation of Cy5 and at the same time provides a good fluorescence resonance energy transfer (FRET) efficiency. The two-dye binary probe system (BP-2d) was constructed with Alexa488 and Cy5 fluorophores. Although the overlap between the fluorescence of Alexa488 and the absorption of Cy5 is relatively low, FRET still occurs due to their close physical proximity when the probes are hybridized to mRNA. This framework also decreases the direct excitation of Cy5 and reduces the fluorescence overlap between the donor and the acceptor. Picosecond time-resolved spectroscopy showed a reduction in the fluorescence lifetime of donor fluorophores after the formation of the hybrid between the probes and target mRNA. Interestingly, BP-2d in the presence of mRNA shows a slow rise in the fluorescence decay of Cy5 due to a relatively slow FRET rate, which together with the reduction in the Alexa488 lifetime provides a way to improve the signal to background ratio using time-resolved fluorescence spectra (TRES). In addition, fluorescence depolarization measurements showed complete depolarization of the acceptor dyes (Cy5) for both BP-3d (due to sequential FRET steps) and BP-2d (due to the relatively low FRET rate) in the presence of the mRNA target.}, author = {Marti, Angel A. and Li, Xiaoxu and Jockusch, Steffen and Stevens, Nathan and Li, Zengmin and Raveendra, Bindu and Kalachikov, Sergey and Morozova, Irina and Russo, James J. and Akins, Daniel L. and Ju, Jingyue and Turro, Nicholas J. }, booktitle = {Fluorescent Nucleoside Analogs: Synthesis, Properties and Applications}, citeulike-article-id = {2524532}, doi = {http://dx.doi.org/10.1016/j.tet.2006.08.109}, journal = {Tetrahedron}, keywords = {addons, addons-fluorescence, fret, origami}, month = {April}, number = {17}, pages = {3591--3600}, posted-at = {2008-03-13 09:26:03}, priority = {2}, title = {Design and characterization of two-dye and three-dye binary fluorescent probes for mRNA detection}, url = {http://dx.doi.org/10.1016/j.tet.2006.08.109}, volume = {63}, year = {2007} }

TY - JOUR ID - citeulike:2524532 L3 - citeulike-article-id:2524532 TI - Design and characterization of two-dye and three-dye binary fluorescent probes for mRNA detection T2 - Fluorescent Nucleoside Analogs: Synthesis, Properties and Applications JF - Tetrahedron VL - 63 IS - 17 SP - 3591 EP - 3600 N2 - We report the design, synthesis, and characterization of binary oligonucleotide probes for mRNA detection. The probes were designed to avoid common problems found in standard binary probes such as direct excitation of the acceptor fluorophore and overlap between the donor and acceptor emission spectra. Two different probes were constructed that contained an array of either two or three dyes and were characterized using steady-state fluorescence spectroscopy, time-resolved fluorescence spectroscopy, and fluorescence depolarization measurements. The three-dye binary probe (BP-3d) consists of a Fam fluorophore which acts as a donor, collecting light and transferring it as energy to Tamra, which subsequently transfers energy to Cy5 when the two probes are hybridized to mRNA. This design allows the use of 488 nm excitation, which avoids the direct excitation of Cy5 and at the same time provides a good fluorescence resonance energy transfer (FRET) efficiency. The two-dye binary probe system (BP-2d) was constructed with Alexa488 and Cy5 fluorophores. Although the overlap between the fluorescence of Alexa488 and the absorption of Cy5 is relatively low, FRET still occurs due to their close physical proximity when the probes are hybridized to mRNA. This framework also decreases the direct excitation of Cy5 and reduces the fluorescence overlap between the donor and the acceptor. Picosecond time-resolved spectroscopy showed a reduction in the fluorescence lifetime of donor fluorophores after the formation of the hybrid between the probes and target mRNA. Interestingly, BP-2d in the presence of mRNA shows a slow rise in the fluorescence decay of Cy5 due to a relatively slow FRET rate, which together with the reduction in the Alexa488 lifetime provides a way to improve the signal to background ratio using time-resolved fluorescence spectra (TRES). In addition, fluorescence depolarization measurements showed complete depolarization of the acceptor dyes (Cy5) for both BP-3d (due to sequential FRET steps) and BP-2d (due to the relatively low FRET rate) in the presence of the mRNA target. KW - addons KW - addons-fluorescence KW - fret KW - origami AU - Marti, Angel AU - Li, Xiaoxu AU - Jockusch, Steffen AU - Stevens, Nathan AU - Li, Zengmin AU - Raveendra, Bindu AU - Kalachikov, Sergey AU - Morozova, Irina AU - Russo, James AU - Akins, Daniel AU - Ju, Jingyue AU - Turro, Nicholas PY - 2007/04/23/ UR - http://dx.doi.org/10.1016/j.tet.2006.08.109 ER -