Photoinduced electron transfer between fluorescent dyes and guanosine residues in DNA-hairpins

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 similar to1.0-2.0 x 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, tau < 40 ps) ground-state 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.