Steady-state and time-resolved fluorescence measurements were performed to elucidate the fluorescence quenching of oxazine, rhodamine, carbocyanine, and bora-diaza-indacene dyes by amino acids. Among the natural amino acids, tryptophan exhibits the most pronounced quenching efficiency. Especially, the red-absorbing dyes ATTO 655, ATTO 680, and the oxazine derivative MR 121 are strongly quenched almost exclusively by tryptophan due to the formation of weak or nonfluorescent ground-state complexes with association constants, K(ass.), ranging from 96 to 206 M(-1). Rhodamine, fluorescein, and bora-diaza-indacene derivatives that absorb at shorter wavelengths are also quenched substantially by tyrosine residues. The quenching of carbocyanine dyes, such as Cy5, and Alexa 647 by amino acids can be almost neglected. While quenching of ATTO 655, ATTO 680, and the oxazine derivative MR121 by tryptophan is dominated by static quenching, dynamic quenching is more efficient for the two bora-diaza-indacene dyes Bodipy-FL and Bodipy630/650. Labeling of the dyes to tryptophan, tryptophan-containing peptides, and proteins (streptavidin) demonstrates that knowledge of these fluorescence quenching processes is crucial for the development of fluorescence-based diagnostic assays. Changes in the fluorescence quantum yield of dye-labeled peptides and proteins might be used advantageously for the quantification of proteases and specific binding partners.

Nucleobase-specific quenching interactions of fluorescent dyes can be used in singly labeled hairpin-shaped oligonucleotides to detect hybridization to specific target DNA sequences. In these DNA-hairpins, the dye is attached at the 5′-end and quenched by guanosine residues in the complementary stem. Upon hybridization, a conformational reorganization occurs reflected in an increase in fluorescence intensity. To gain a better insight into the underlying quenching mechanism, we have performed intermolecular quenching experiments with different dyes (rhodamine and oxazine derivatives) and DNA nucleotides. The bimolecular dynamic quenching rate constants k q,dyn of ∼1.0-2.0 × 10 9 M -1 s -1 are relatively small for all dyes investigated though the measured decrease in fluorescence intensity indicates strong static fluorescence quenching. The data give evidence for the formation of weak or nonfluorescent (fluorescence lifetime, τ < 40 ps) groundstate complexes between the fluorophores and guanosine residues. Only within these complexes, that is, upon contact formation, efficient fluorescence quenching via electron transfer occurs. Using a model DNA-hairpin labeled at the 5′-end with the oxazine derivative MR121, we varied the position of guanosine in the complementary stem sequence to reveal the distance dependence of fluorescence quenching. Qualitatively, it is apparent that the double-stranded stem of the DNA-hairpin facilitates efficient electron transfer from the guanosine residue to MR121 with a shallow distance dependence. This result strongly supports the idea that an end-capped conformation with stacking interactions and subsequent DNA mediated electron transfer is required for efficient fluorescence quenching. Our data show that the quenching efficiency can be increased substantially by the attachment of additional overhanging single-stranded nucleotides at the 3′-end and the substitution of guanosine by stronger electron-donating nucleotides, such as 7-deazaguanosine residues. Consideration of the data obtained in this study enables the synthesis of DNA-hairpins solely quenched by guanosine residues and its analogous with a 20-fold increase in fluorescence intensity upon specific binding to the target sequence.

A method has been developed for highly sensitive detection of specific DNA sequences in a homogeneous assay using labeled oligonucleotide molecules in combination with single-molecule photon burst counting and identification. The fluorescently labeled oligonucleotides are called smart probes because they report the presence of complementary target sequences by a strong increase in fluorescence intensity. The smart probes consist of a fluorescent dye attached at the terminus of a hairpin oligonucleotide. The presented technique takes advantage of the fact that the used oxazine dye JA242 is efficiently quenched by complementary guanosine residues. Upon specific hybridization to the target DNA, the smart probe undergoes a conformational change that forces the fluorescent dye and the guanosine residues apart, thereby increasing the fluorescence intensity about six fold in ensemble measurements. To increase the detection sensitivity below the nanomolar range, a confocal fluorescence microscope was used to observe the fluorescence bursts from individual smart probes in the presence and absence of target DNA as they passed through the focused laser beam. Smart probes were excited by a pulsed diode laser emitting at 635 nm with a repetition rate of 64 MHz. Each fluorescence burst was identified by three independent parameters: (a) the burst size, (b) the burst duration, and (c) the fluorescence lifetime. Through the use of this multiparameter analysis, higher discrimination accuracies between smart probes and hybridized probe-target duplexes were achieved. The presented multiparameter detection technique permits the identification of picomolar target DNA concentrations in a homogeneous assay, i.e., the detection of specific DNA sequences in a 200-fold excess of labeled probe molecules.

We describe a method for detection of sub-picomolar concentrations of DNA or RNA sequences using novel surface-immobilized DNA hairpins. Within the DNA hairpins a fluorophore is specifically quenched by guanosine residues in the complementary stem sequence via photoinduced intramolecular electron transfer. Upon hybridization to the target sequence, fluorescence is restored due to a conformational reorganization that forces the stem apart. Proper immobilization of the DNA hairpins using biotin/streptavidin binding with minimal perturbation of the surface is required to ensure efficient quenching in the closed state.