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.

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.

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To obtain detailed information about the three-dimensional (3D) organization of small biomolecular assemblies with a size of less than 100 nanometers, advanced techniques are required that enable the determination of absolute 3D positions and distances between individual fluorophores well below the resolution limit of conventional light microscopy. We show how spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) can provide significant contributions and allow us to determine distances between conventional individual fluorophores (Bodipy 630/650 and Cy5.5) that are less than 20 nm apart. We take advantage of fluorescent dyes (here Cy5.5 and Bodipy 630/650) that can be efficiently excited by a single pulsed diode laser emitting at 635 nm but differ in their fluorescence lifetime and emission maxima. The potential of the method for ultrahigh colocalization studies is demonstrated by measuring the end-to-end distance between single fluorophores separated by double-stranded DNA of various lengths. Combining SFLIM with polarization-modulated excitation allows us to obtain, simultaneously, information about the relative orientation of fluorophores. Furthermore, we show that the environment-dependent photophysics of conventional fluorophores, that is, photostability, blinking pattern, and the tendency to enter irreversible nonfluorescent states, sets certain limitations to their in vitro and in vivo applications.