Fluorescence resonance energy transfer (FRET) for DNA biosensors: FRET pairs and Forster distances for various dye-DNA conjugates

@article{citeulike:1178489, abstract = {Fluorescence resonance energy transfer (FRET) between the extrinsic dye labels Cyanine 3 (Cy3), Cyanine 5 (Cy5), Carboxytetramethyl Rhodamine (TAMRA), Iowa Black Fluorescence Quencher (IabFQ), and Iowa Black RQ (IabRQ) has been studied. The Forster distances for these FRET-pairs in single- and double-stranded DNA conjugates have been determined. In particular, it should be noted that the quantum yield of the donors Cy3 and TAMRA varies between single- and double-stranded DNA. While this alters the Forster distance for a donor-acceptor pair, this also allows for detection of thermal denaturation events with a single non-intercalating fluorophore. The utility of FRET in the development of nucleic acid biosensor technology is illustrated by using TAMRA and IabRQ as a FRET pair in selectivity experiments. The differential quenching of TAMRA fluorescence by IabRQ in solution has been used to discriminate between 0 and 3 base pair mismatches at 60 [degree sign]C for a 19 base sequence. At room temperature, the quenching of TAMRA fluorescence was not an effective indicator of the degree of base pair mismatch. There appears to be a threshold of duplex stability at room temperature which occurs beyond two base pair mismatches and reverses the observed trend in TAMRA fluorescence prior to that degree of mismatch. When this experimental system is transferred to a glass surface through covalent coupling and organosilane chemistry, the observed trend in TAMRA fluorescence at room temperature is similar to that obtained in bulk solution, but without a threshold of duplex stability. In addition to quenching of fluorescence by FRET, it is believed that several other quenching mechanisms are occurring at the surface.}, author = {Massey, Melissa and Algar, Russ W. and Krull, Ulrich J. }, booktitle = {Molecular Electronics and Analytical Chemistry}, citeulike-article-id = {1178489}, doi = {10.1016/j.aca.2005.12.050}, journal = {Analytica Chimica Acta}, keywords = {fret, origami}, month = {May}, number = {1-2}, pages = {181--189}, posted-at = {2008-03-13 10:44:22}, priority = {2}, title = {Fluorescence resonance energy transfer (FRET) for DNA biosensors: FRET pairs and Forster distances for various dye-DNA conjugates}, url = {http://dx.doi.org/10.1016/j.aca.2005.12.050}, volume = {568}, year = {2006} }

TY - JOUR ID - citeulike:1178489 L3 - citeulike-article-id:1178489 TI - Fluorescence resonance energy transfer (FRET) for DNA biosensors: FRET pairs and Forster distances for various dye-DNA conjugates T2 - Molecular Electronics and Analytical Chemistry JF - Analytica Chimica Acta VL - 568 IS - 1-2 SP - 181 EP - 189 N2 - Fluorescence resonance energy transfer (FRET) between the extrinsic dye labels Cyanine 3 (Cy3), Cyanine 5 (Cy5), Carboxytetramethyl Rhodamine (TAMRA), Iowa Black Fluorescence Quencher (IabFQ), and Iowa Black RQ (IabRQ) has been studied. The Forster distances for these FRET-pairs in single- and double-stranded DNA conjugates have been determined. In particular, it should be noted that the quantum yield of the donors Cy3 and TAMRA varies between single- and double-stranded DNA. While this alters the Forster distance for a donor-acceptor pair, this also allows for detection of thermal denaturation events with a single non-intercalating fluorophore. The utility of FRET in the development of nucleic acid biosensor technology is illustrated by using TAMRA and IabRQ as a FRET pair in selectivity experiments. The differential quenching of TAMRA fluorescence by IabRQ in solution has been used to discriminate between 0 and 3 base pair mismatches at 60 [degree sign]C for a 19 base sequence. At room temperature, the quenching of TAMRA fluorescence was not an effective indicator of the degree of base pair mismatch. There appears to be a threshold of duplex stability at room temperature which occurs beyond two base pair mismatches and reverses the observed trend in TAMRA fluorescence prior to that degree of mismatch. When this experimental system is transferred to a glass surface through covalent coupling and organosilane chemistry, the observed trend in TAMRA fluorescence at room temperature is similar to that obtained in bulk solution, but without a threshold of duplex stability. In addition to quenching of fluorescence by FRET, it is believed that several other quenching mechanisms are occurring at the surface. KW - fret KW - origami AU - Massey, Melissa AU - Algar, Russ AU - Krull, Ulrich PY - 2006/05/24/ UR - http://dx.doi.org/10.1016/j.aca.2005.12.050 ER -