Fluorescence characteristics of 5-carboxytetramethylrhodamine linked covalently to the 5' end of oligonucleotides: Multiple conformers of single- stranded and double-stranded dye-DNA complexes

G. Vámosi, Christoph Gohlke, Robert M. Clegg

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Abstract

Fluorescence steady-state and lifetime experiments have been carried out on duplex and single-stranded DNA molecules labeled at the 5' ends with 5- carboxytetramethylrhodamine (TMRh). The temperature and ionic strength of the solutions were varied over large ranges. The results reveal at least three well-defined states of the TMRh-DNA molecules for the single-stranded as well as for the double-stranded DNA molecules. Two states are fluorescent, with lifetimes in the range of 0.5-1 ns and 2.5-3 ns. A third state of TMRh-DNA does not fluoresce (a dark species of TMRh-DNA). The distribution of the TMRh-DNA molecules among these three states is strongly temperature and ionic strength dependent. Estimates are made of some reaction parameters of the multistate model. The results are discussed in terms of the photophysics of TMRh, and consequences of the multiple conformers of TMRh-DNA for studies involving fluorescence studies with TMRh-labeled DNA are considered.

Original languageEnglish
Pages (from-to)972-994
Number of pages23
JournalBiophysical Journal
Volume71
Issue number2
Publication statusPublished - Aug 1996

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Oligonucleotides
Coloring Agents
Fluorescence
DNA
Single-Stranded DNA
Osmolar Concentration
Temperature
5-carboxytetramethylrhodamine

ASJC Scopus subject areas

  • Biophysics

Cite this

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