In this article we present a method for the highly specific identification of single nucleotide polymorphism (SNP)
responsible for rifampicin resistance of <i>Mycobacterium tuberculosis</i>. This approach applies fluorescently labeled
hairpin-structured oligonucleotides (smart probes) and confocal single-molecule fluorescence spectroscopy. Smart
probes are fluorescently labeled at the 5'-end. The dye's fluorescence is quenched in the closed hairpin conformation due
to close proximity of the guanosine residues located at the 3'-end. As a result of the hybridization to the complementary
target sequence the hairpin structure and thus fluorescence quenching gets lost and a strong fluorescence increase
appears. To enhance the specificity of the SNP detection unlabeled "blocking oligonucleotides" were added to the
sample. These oligonucleotides hybridizes to the DNA sequence containing the mismatch thus masking this sequence
and hereby preventing the smart probe from hybridizing to the mismatched sequence.
Avoiding nonspecific surface adsorption is a crucial and often challenging issue in many single-molecule
studies and analytical applications. In this work, we investigated glass surfaces coated with
cross-linking star-shaped polyethylene glycol (4-arm PEG) and demonstrated that this coating can be
used for effective suppression of nonspecific protein binding, such as streptavidin. Single-molecule
fluorescence images show that only a few molecules remain nonspecifically bound to surfaces
treated with protein after sufficient rinsing, i.e. less than to a state-of-the-art BSA coating.
Furthermore, different applications for star-shaped PEG-passivated surfaces are shown.
In this article we report on two different classes of self-quenching hairpin-structured DNA probes that can be used as
alternatives to Molecular Beacons. Compared to other hairpin-structured DNA probes, the so-called smart probes are
labeled with only one extrinsic dye. The fluorescence of this dye is efficiently quenched by intrinsic guanine bases via a
photo-induced electron transfer reaction in the closed hairpin. After hybridization to a target DNA, the distance between
dye and the guanines is enlarged and the fluorescence is restored. The working mechanism of the second class of hairpin
DNA probes is similar, but the probe oligonucleotide is labeled at both ends with an identical chromophore and thus the
fluorescence of the closed hairpin is reduced due to formation of non-fluorescent dye dimers. Both types of probes are
appropriate for the identification of single nucleotide polymorphisms and in combination with confocal single-molecule
spectroscopy sensitivities in the picomolar range can be achieved.
In this paper we applied the efficient fluorescence quenching of the red-absorbing oxazine derivative MR121 by the amino acid tryptophan to develop a new fluorescence based enzyme assay that can be used for detection of exopeptidases and endopeptidases. Therefore, we developed peptide substrates labeled with only one chromophore, which is quenched by a neighbored tryptophan residue via photoinduced electron transfer. The specific cleavage site for the target enzyme is located between the chromophore and the tryptophan residue. After digestion of the substrate the contact formation between tryptophan and fluorescent dye is precluded and a significant increase in fluorescence intensity occurs. To demonstrate the new assay technique for exopeptidases, a substrate for the Carboxypeptidase A was designed and a detection limit below the picomolar range (~10<sup>-13</sup> M) was achieved with standard fluorescence spectrometry. The primary objective was the detection of the HIV-protease, which is an endopeptidase digesting substrates containing seven specific amino acids in the cleavage site. We designed a substrate, which enables the detection of 10<sup>-9</sup> M HIV-protease, whereas the continuous monitoring of the fluorescence signal also allows kinetic studies.
Here we present a novel class of self-quenching, double-labeled DNA probes based on the formation of non fluorescent H-type dye dimers. We therefore investigated the aggregation behavior of the red-absorbing oxazine derivative MR121 and found a dimerization constant of about 3000 M-1. This dye was successfully used to develop hairpin-structured as well as linear self-quenching DNA probes that report the presence of the target DNA by an increase of the fluorescence intensity by a factor of 3 to 12. Generally fluorescence quenching of the hairpin-structure probes is more efficient compared to the linear probes, whereas the kinetic of the fluorescence increase is significantly slower. The new probes were used for the identification of different mycobacteria and their antibiotic resistant species. As a test system a probe for the identification of a DNA sequence specific for the Mycobacterium xenopi was synthesized differing from the sequence of the Mycobacterium fortuitum by 6 nucleotides. Furthermore we developed a method for the discrimination between the sequences of the wild type and an antibiotic resistant species of Mycobacterium tuberculosis. Both sequences differ by just 2 nucleotides and were detected specifically by the use of competing olignonucleotides.