RESUMEN
The thymidine analogue 1 is a blue-to-red energy transfer cassette designed to absorb UV light in the 300 nm region and emit it as fluorescence at around 520 nm. When incorporated into DNA, the fluorescence intensity of this modified nucleobase is not significantly reduced by the surrounding structure in the oligonucleotides. [structure--see text]
Asunto(s)
ADN/química , Timidina/análogos & derivados , Timidina/química , Secuencia de Bases , ADN/genética , Transferencia de Energía , Estructura Molecular , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/genética , Espectrometría de FluorescenciaRESUMEN
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.
Asunto(s)
Biotecnología/instrumentación , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes , Sondas Moleculares , Análisis de Secuencia de ADN/instrumentación , Colorantes Fluorescentes/síntesis química , Sondas Moleculares/síntesis química , Fotoquímica , Análisis de Secuencia de ADN/métodos , Espectrofotometría UltravioletaRESUMEN
The Sn-->S0 ultraviolet fluorescence spectra (270-380 nm) of 4-aminophthalimide (4AP) and its N-methyl derivative 4-amino-N-methylphthalimide (4ANMP) are reported, following the absorption of two laser pulses. In polar but non-hydrogen-bonding solvents, both molecules exhibit a principal emission maximum near 290 nm, whereas solutions in hydrogen-bonding solvents display two prominent emission bands, near 300 and 350 nm. The relative intensities of these bands depend on solvent type and, in a pump-probe experiment, on the wavelengths and temporal spacing of two ultrashort pulses. Experiments covering the range approximately 0.1-100 ps showed evidence for at least two distinct ultrashort relaxation processes, the rates of which depend on solvent. Fluorescence upconversion experiments at < 0.2 ps resolution have shown that the longer-duration process correlates with the fluorescence Stokes shift, and provide evidence that the solvent-dependent shift of the S1-->S0 fluorescence spectrum is reflected in the fluorescence quantum efficiency of a nearby electronic state.
Asunto(s)
Ftalimidas/química , Soluciones , Espectrometría de Fluorescencia , Espectrofotometría UltravioletaRESUMEN
Compounds based on the 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY) framework are excellent fluorescent markers. When BODIPY dyes of this type are conjugated to functionalities that absorb at relatively short wavelengths, those functionalities can, in some molecules, transmit the absorbed energy to the BODIPY which then fluoresces. In such cases the BODIPY fragment acts as an acceptor while the other group serves as a donor. Energy transfer efficiencies in such donor-acceptor cassette systems must vary with the relative orientation of the two components, and with the structure of the linkers that attach them. This study was designed to probe these issues for the special case in which the linkers between the donor and acceptor fragments are conjugated. To do this, the cassettes 3-10 were prepared. Electrochemical studies were performed to provide insight into the degree of donor-acceptor conjugation in these systems. X-ray Crystallographic studies on single crystals of compounds 7 and 9 revealed the favored conformations of the donor and acceptor fragments in the solid state. Absorption, fluorescence, and time-resolved fluorescence spectra of the compounds were recorded, and quantum yields for the cassettes excited at the donor lambda(max) were measured. Fluorescence steady-state anisotropy data were determined for cassettes 3 and 9 to provide information about the mutual direction of the transition dipole moments.