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Spectroscopic and photophysical properties of dUTP and internally DNA bound fluorophores for optimized signal detection in biological formats

  • 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 dyeEfficient 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.zeige mehrzeige weniger

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Metadaten
Autor*innen:Lena Linck, P. Kapusta, Ute Resch-GengerORCiD
Dokumenttyp:Zeitschriftenartikel
Veröffentlichungsform:Verlagsliteratur
Sprache:Englisch
Titel des übergeordneten Werkes (Englisch):Photochemistry and photobiology
Jahr der Erstveröffentlichung:2012
Verlag:American Society for Photobiology
Verlagsort:Augusta, Ga.
Jahrgang/Band:88
Ausgabe/Heft:4
Erste Seite:867
Letzte Seite:875
Freie Schlagwörter:Cyanine; DNA; Fluorescence lifetime; Fluorescence quantum yield; Fluorescent dye; Guanosine quenching; Rhodamine
DOI:10.1111/j.1751-1097.2012.01119.x
ISSN:0031-8655
ISSN:1751-1097
Verfügbarkeit des Dokuments:Physisches Exemplar in der Bibliothek der BAM vorhanden ("Hardcopy Access")
Bibliotheksstandort:Sonderstandort: Publica-Schrank
Datum der Freischaltung:19.02.2016
Referierte Publikation:Ja
Datum der Eintragung als referierte Publikation:22.11.2012
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