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Issue 10, 2010
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Designed thiazole orange nucleotides for the synthesis of single labelled oligonucleotides that fluoresce upon matched hybridization

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Abstract

Probe molecules that enable the detection of specific DNA sequences are used in diagnostic and basic research. Most methods rely on the specificity of hybridization reactions, which complicates the detection of single base mutations at low temperature. Significant efforts have been devoted to the development of oligonucleotides that allow discrimination of single base mutations at temperatures where both the match and the mismatch probe–target complexes coexist. Oligonucleotides that contain environmentally sensitive fluorescence dyes such as thiazole orange (TO) provide single nucleotide specific fluorescence. However, most previously reported dyeDNA conjugates showed only little if any difference between the fluorescence of the single and the double stranded state. Here, we introduce a TO-containing acyclic nucleotide, which is coupled during automated oligonucleotide synthesis and provides for the desired fluorescence-up properties. The study reveals the conjugation mode as the most important issue. We show a design that leads to low fluorescence of the unbound probe (background) yet permits TO to adopt fluorescent binding modes after the probe–target complex has formed. In these probes, TO replaces a canonical nucleobase. Of note, the fluorescence of the “TO–base” remains low when a base mismatch is positioned in immediate vicinity.

Graphical abstract: Designed thiazole orange nucleotides for the synthesis of single labelled oligonucleotides that fluoresce upon matched hybridization

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Publication details

The article was received on 15 Jan 2010, accepted on 12 Feb 2010 and first published on 23 Mar 2010


Article type: Paper
DOI: 10.1039/C000697A
Citation: Org. Biomol. Chem., 2010,8, 2439-2448
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    Designed thiazole orange nucleotides for the synthesis of single labelled oligonucleotides that fluoresce upon matched hybridization

    L. Bethge, I. Singh and O. Seitz, Org. Biomol. Chem., 2010, 8, 2439
    DOI: 10.1039/C000697A

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