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Spectroscopic Characterization of Fluorescein- and Tetramethylrhodamine-Labeled Oligonucleotides and Their Complexes With a DNA Template

Published

Author(s)

Lili Wang, Adolfas K. Gaigalas, J L. Blasic, Marcia J. Holden

Abstract

We measured absorption and emission spectra, fluorescence quantum yield, anisotropy, fluorescence resonance energy transfer (FRET), and melting temperature to characterize fluorescein- and tetramethyIrhodamine (TMR)-labeled oligonucleotides in solution and when hybridized to a common DNA template. Upon hybridization to the template, both the absorption and emission spectra of TMR-labeled duplexes exhibited a shift with respect to those of labeled oligonucleotides, depending on the location of the TMR on the oligonucleotide. Measurements of quantum yield, anisotropy, and melting temperature indicated that TMR interacted with nucleotides within the duplexes in the order (T1>T5>T11, T16) that the oligonucleotide with TMR labeled at the 5 end (T1) is stronger than that labeled at position 5 from the 5 end (T5), which is also stronger than those labeled further away from the 5 end (T11, T16). In the case of the duplex formed between T1 and the template, fluorescence quenching was observed, which is attributed to the interaction between the dye molecule and a guanosine at position 36 from the 5 end of the template. A two-state model was proposed to describe the conformational states of TMR in the duplex. The melting temperatures of the four FRET complexes show the same pattern as those of TMR-labeled duplexes. We infer that the interactions between TMR and guanosine persist in the FRET complexes. This interaction may bring the donor and the acceptor molecules closely together, which could cause interaction between dye molecules shown in absorbance measurements of the FET complexes
Citation
Spectrochimica ACTA Part A-Molecular and Biomolecular Spectroscopy
Volume
60
Issue
12

Keywords

absorption, anisotropy, DNA complexes, emission spectra, fluorescein, fluorescence resonance energy transfer (, oligonucleotides, quantum yield, tetramethyirhodamine

Citation

Wang, L. , Gaigalas, A. , Blasic, J. and Holden, M. (2004), Spectroscopic Characterization of Fluorescein- and Tetramethylrhodamine-Labeled Oligonucleotides and Their Complexes With a DNA Template, Spectrochimica ACTA Part A-Molecular and Biomolecular Spectroscopy (Accessed November 30, 2023)
Created October 1, 2004, Updated February 19, 2017