Brightness enhanced DNA FIT-Probes for wash-free RNA imaging in tissue. Hövelmann, F., Gaspar, I., Ephrussi, A., & Seitz, O. Journal of the American Chemical Society, December, 2013. ![Brightness enhanced DNA FIT-Probes for wash-free RNA imaging in tissue. [link]](https://bibbase.org/img/filetypes/link.svg "link")
Paper doi abstract bibtex Fluorogenic oligonucleotides enable RNA imaging in cells and tissues. A high responsiveness of fluorescence is required when unbound probes cannot be washed away. Furthermore, emission should be bright in order to enable detection against autofluorescent background. The development of fluorescence-quenched hybridization probes has led to remarkable improvement of fluorescence responsiveness. Yet, comparably little attention has been paid to the brightness of smart probes. We describe hybridization probes that combine responsiveness with a high brightness of the measured signal. The method relies upon quencher-free DNA forced intercalation (FIT)-probes, in which two (or more) intercalator dyes of the thiazole orange (TO) family serve as nucleobase surrogates. Initial experiments on multi-TO-labeled probes led to improvements of responsiveness, but self-quenching limited their brightness. To enhance both brightness and responsiveness an energy gradient was introduced by combining the highly responsive TO nucleoside and the highly emissive oxazolopyridine analogue JO. Single-stranded TO/JO FIT-probes are dark.. In the probe-target duplex, quenching caused by torsional twisting and dye-dye contact is prevented. The TO nucleoside serves as a light harvester that increases the extinction coefficient and transfers energy to the JO emitter. This leads to very bright JO emission upon hybridization (F/F0 = 23, brightness = 43 mL∙mol-1∙cm-1 at 516 nm excitation). TO/JO FIT-probes allowed the direct fluorescence microscopic imaging of oskar mRNA within a complex tissue. Of note, RNA imaging was feasible under wide-field excitation conditions. The described protocol enables rapid RNA imaging in tissue without the need for cutting-edge equipment, time consuming washing or signal amplification.
@article{hovelmann_brightness_2013,
title = {Brightness enhanced {DNA} {FIT}-{Probes} for wash-free {RNA} imaging in tissue.},
issn = {1520-5126},
url = {http://www.ncbi.nlm.nih.gov/pubmed/24295172},
doi = {10.1021/ja410674h},
abstract = {Fluorogenic oligonucleotides enable RNA imaging in cells and tissues. A high responsiveness of fluorescence is required when unbound probes cannot be washed away. Furthermore, emission should be bright in order to enable detection against autofluorescent background. The development of fluorescence-quenched hybridization probes has led to remarkable improvement of fluorescence responsiveness. Yet, comparably little attention has been paid to the brightness of smart probes. We describe hybridization probes that combine responsiveness with a high brightness of the measured signal. The method relies upon quencher-free DNA forced intercalation (FIT)-probes, in which two (or more) intercalator dyes of the thiazole orange (TO) family serve as nucleobase surrogates. Initial experiments on multi-TO-labeled probes led to improvements of responsiveness, but self-quenching limited their brightness. To enhance both brightness and responsiveness an energy gradient was introduced by combining the highly responsive TO nucleoside and the highly emissive oxazolopyridine analogue JO. Single-stranded TO/JO FIT-probes are dark.. In the probe-target duplex, quenching caused by torsional twisting and dye-dye contact is prevented. The TO nucleoside serves as a light harvester that increases the extinction coefficient and transfers energy to the JO emitter. This leads to very bright JO emission upon hybridization (F/F0 = 23, brightness = 43 mL∙mol-1∙cm-1 at 516 nm excitation). TO/JO FIT-probes allowed the direct fluorescence microscopic imaging of oskar mRNA within a complex tissue. Of note, RNA imaging was feasible under wide-field excitation conditions. The described protocol enables rapid RNA imaging in tissue without the need for cutting-edge equipment, time consuming washing or signal amplification.},
journal = {Journal of the American Chemical Society},
author = {Hövelmann, Felix and Gaspar, Imre and Ephrussi, Anne and Seitz, Oliver},
month = dec,
year = {2013},
pmid = {24295172},
keywords = {\#nosource},
}
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We describe hybridization probes that combine responsiveness with a high brightness of the measured signal. The method relies upon quencher-free DNA forced intercalation (FIT)-probes, in which two (or more) intercalator dyes of the thiazole orange (TO) family serve as nucleobase surrogates. Initial experiments on multi-TO-labeled probes led to improvements of responsiveness, but self-quenching limited their brightness. To enhance both brightness and responsiveness an energy gradient was introduced by combining the highly responsive TO nucleoside and the highly emissive oxazolopyridine analogue JO. Single-stranded TO/JO FIT-probes are dark.. In the probe-target duplex, quenching caused by torsional twisting and dye-dye contact is prevented. The TO nucleoside serves as a light harvester that increases the extinction coefficient and transfers energy to the JO emitter. This leads to very bright JO emission upon hybridization (F/F0 = 23, brightness = 43 mL∙mol-1∙cm-1 at 516 nm excitation). TO/JO FIT-probes allowed the direct fluorescence microscopic imaging of oskar mRNA within a complex tissue. Of note, RNA imaging was feasible under wide-field excitation conditions. 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To enhance both brightness and responsiveness an energy gradient was introduced by combining the highly responsive TO nucleoside and the highly emissive oxazolopyridine analogue JO. Single-stranded TO/JO FIT-probes are dark.. In the probe-target duplex, quenching caused by torsional twisting and dye-dye contact is prevented. The TO nucleoside serves as a light harvester that increases the extinction coefficient and transfers energy to the JO emitter. This leads to very bright JO emission upon hybridization (F/F0 = 23, brightness = 43 mL∙mol-1∙cm-1 at 516 nm excitation). TO/JO FIT-probes allowed the direct fluorescence microscopic imaging of oskar mRNA within a complex tissue. Of note, RNA imaging was feasible under wide-field excitation conditions. 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