TY - JOUR A1 - Linck, Lena A1 - Kapusta, P. A1 - Resch-Genger, Ute T1 - Spectroscopic and photophysical properties of dUTP and internally DNA bound fluorophores for optimized signal detection in biological formats JF - Photochemistry and photobiology N2 - Efficient signal generation in DNA-based assays requires understanding of the influence of fluorophore’s interactions on the spectroscopic properties. The resulting changes in fluorescence intensity, quantum yield, emission anisotropy, and fluorescence lifetime provide straightforward tools for the study of molecular dynamics and interaction between labels and nucleic acids. Searching for bright fluorescent reporters for rolling circle amplification (RCA) as efficient signal enhancement strategy for biological formats, we investigated the spectroscopic properties of seven dyes: cyanines, rhodamines, and BODIPYs. They spectrally resemble Cy3, the most frequently used fluorophore in biodetection formats, and are measured in six samples (free dye, dye-dUTP, internally labeled ssDNA and dsDNA—single- and triple-labeled) using steady-state and time-resolved fluorometry. Special emphasis was dedicated to characterizing the nature of the interaction of these fluorophores differing in dye class, charge, and rigidity. Our results suggest dye charge and structure as main factors governing the dye’s interactions, with DY-555 and Cy3B presenting the best candidates for our envisaged signal amplification strategy. This label comparison underlines the importance of a proper understanding of structure-property relations and dye–biomolecule interactions for reporter choice and presents a road map towards the design and interpretation of experiments using these labels on DNA of known sequence. KW - DNA KW - Fluorescent dye KW - Fluorescence quantum yield KW - Cyanine KW - Rhodamine KW - Fluorescence lifetime KW - Guanosine quenching PY - 2012 DO - https://doi.org/10.1111/j.1751-1097.2012.01119.x SN - 0031-8655 SN - 1751-1097 VL - 88 IS - 4 SP - 867 EP - 875 PB - American Society for Photobiology CY - Augusta, Ga. AN - OPUS4-27080 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -