Syntheses, Photophysical Properties, and Application of Through-Bond Energy-Transfer Cassettes for Biotechnology

• Published in: Chemistry : a European journal Vol. 12; no. 30; pp. 7816 - 7826
• Main Authors: Jiao, Guan-Sheng, Thoresen, Lars H., Kim, Taeg Gyum, Haaland, Wade C., Gao, Feng, Topp, Michael R., Hochstrasser, Robin M., Metzker, Michael L., Burgess, Kevin
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 16.10.2006; WILEY‐VCH Verlag
• Subjects: Biotechnology - instrumentation; cross-coupling; DNA; dyes/pigments; Fluorescence Resonance Energy Transfer; Fluorescent Dyes - chemical synthesis; fluorescent probes; Molecular Probes - chemical synthesis; Photochemistry; Sequence Analysis, DNA - instrumentation; Sequence Analysis, DNA - methods; Spectrophotometry, Ultraviolet
• ISSN: 0947-6539; 1521-3765
• DOI: 10.1002/chem.200600197

We have designed fluorescent “through‐bond energy‐transfer cassettes” that can harvest energy of a relatively short wavelength (e.g., 490 nm), and emit it at appreciably longer wavelengths without significant loss of intensity. Probes of this type could be particularly useful in biotechnology for multiplexing experiments in which several different outputs are to be observed from a single excitation source. Cassettes 1–4 were designed, prepared, and studied as model systems to achieve this end. They were synthesized through convergent routes that feature coupling of specially prepared fluorescein‐ and rhodamine‐derived fragments. The four cassettes were shown to emit strongly, with highly efficient energy transfer. Their emission maxima cover a broad range of wavelengths (broader than the four dye cassettes currently used for most high‐throughput DNA sequencing), and they exhibit faster energy‐transfer rates than a similar through‐space energy‐transfer cassette. Specifically, energy‐transfer rates in these cassettes is around 6–7 ps, in contrast to a similar through‐space energy‐transfer system shown to have a decay time of around 35 ps. Moreover, the cassettes are considerably more stable to photobleaching than fluorescein, even though they each contain fluorescein‐derived donors. This was confirmed by bulk fluorescent measurements, and in single‐molecule‐detection studies. Modification of a commercial automated DNA‐sequencing apparatus to detect the emissions of these four energy‐transfer cassettes enabled single‐color dye–primer sequencing. Short in, long out: Through‐bond energy transfer was used in the title cassettes to produce labels that absorb strongly at around 500 nm, and emit strongly at wavelengths in the 550–650 nm range. The cassettes are relatively stable to photobleaching, even at the single‐molecule level, and are usable in DNA‐sequencing experiments.