A universally applicable process for preparing stoichiometrically 1:1 labelled functional proteins.

A universally applicable labelling and purification process was established to prepare biologically active proteins with a stoichiometric 1:1 ratio of attached dye-label. The dye-label is linked to a specific DNA sequence, which acts as a barcode-like tag for affinity purification. The DNA-dye tag is covalently bound to the target protein, which is present in excess to assure the binding of not more than one dye per molecule. Affinity purification occurs at magnetic beads that are functionalized with oligonucleotides that are complementary to the DNA-tag of the labelled proteins but for one or two mismatches. Washing removes all unbound, unlabelled molecules. The labelled protein is subsequently released by the addition of a fully complementary oligonucleotide. This process allows a gentle purification of a protein fraction that has exactly one label attached to each molecule under conditions that preserve protein structure.

Single-molecule detection on a protein-array assay platform for the exposure of a tuberculosis antigen.

Based on a single-molecule sensitive fluorescence-linked immunosorbent assay, an analytical platform for the detection of lipoarabinomannan (LAM), a lipopolysaccharide marker of tuberculosis, was established that is about 3 orders of magnitude more sensitive than comparable current ELISA assays. No amplification step was required. Also, no particular sample preparation had to be done. Since individual binding events are detected, true quantification was possible simply by counting individual signals. Utilizing a total internal reflection configuration, unprocessed biological samples (human urine and plasma) to which LAM was added could be analyzed without the requirement of sample purification or washing steps during analysis. Samples containing about 600 antigen molecules per microliter produced a distinct signal. The methodology developed can be employed for any set of target molecules for which appropriate antibodies exist.

Sensitive bioanalysis: combining single-molecule spectroscopy with mono-labeled self-quenching probes.

Fluorescence single-molecule spectroscopy is an appropriate tool for modern bioanalysis. This technique enables the development of ultra sensitive assays, especially when combined with self-quenching probes. In this review we report novel DNA, enzyme, and antibody assays based on mono-labeled fluorescent probes that are quenched by photoinduced electron transfer (PET).

DNA-probes for the highly sensitive identification of single nucleotide polymorphism using single-molecule spectroscopy.

This article presents a new, highly sensitive method for the identification of single nucleotide polymorphisms (SNPs) in homogeneous solutions using fluorescently labeled hairpin-structured oligonucleotides (smart probes) and fluorescence single-molecule spectroscopy. While the hairpin probe is closed, fluorescence intensity is quenched due to close contact between the chromophore and several guanosine residues. Upon hybridization to the respective target SNP sequence, contact is lost and the fluorescence intensity increases significantly. High specificity is achieved by blocking sequences containing mismatch with unlabeled oligonucleotides. Time-resolved single-molecule fluorescence spectroscopy enables the detection of individual smart probes passing a small detection volume. This method leads to a subnanomolar sensitivity for this single nucleotide specific DNA assay technique.

Recent patents on self-quenching DNA probes.

In this review, we report on patents concerning self-quenching DNA probes for assaying DNA during or after amplification as well as for direct assaying DNA or RNA, for example in living cells. Usually the probes consist of fluorescently labeled oligonucleotides whose fluorescence is quenched in the absence of the matching target DNA. Thereby the fluorescence quenching is based on fluorescence resonance energy transfer (FRET), photoinduced electron transfer (PET), or electronically interactions between dye and quencher. However, upon hybridization to the target or after the degradation during a PCR, the fluorescence of the dye is restored. Although the presented probes were originally developed for use in homogeneous assay formats, most of them are also appropriate to improve surface-based assay methods. In particular we describe patents for self-quenching primers, self-quenching probes for TaqMan assays, probes based on G-quartets, Molecular Beacons, Smart Probes, and Pleiades Probes.

Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy.

We demonstrate the specific identification of single nucleotide polymorphism (SNP) responsible for rifampicin resistance of Mycobacterium tuberculosis applying fluorescently labeled DNA-hairpin structures (smart probes) in combination with single-molecule fluorescence spectroscopy. Smart probes are singly labeled hairpin-shaped oligonucleotides bearing a fluorescent dye at the 5' end that is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs, reflected in a strong increase in fluorescence intensity. An excess of unlabeled ('cold') oligonucleotides was used to prevent the formation of secondary structures in the target sequence and thus facilitates hybridization of smart probes. Applying standard ensemble fluorescence spectroscopy we demonstrate the identification of SNPs in PCR amplicons of mycobacterial rpoB gene fragments with a detection sensitivity of 10(-8) M. To increase the detection sensitivity, confocal fluorescence microscopy was used to observe fluorescence bursts of individual smart probes freely diffusing through the detection volume. By measuring burst size, burst duration and fluorescence lifetime for each fluorescence burst the discrimination accuracy between closed and open (hybridized) smart probes could be substantially increased. The developed technique enables the identification of SNPs in 10(-11) M solutions of PCR amplicons from M.tuberculosis in only 100 s.

Inter- and intramolecular fluorescence quenching of organic dyes by tryptophan.

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.

Detection and identification of single molecules in living cells using spectrally resolved fluorescence lifetime imaging microscopy.

The detection of single mRNA molecules tagged by microinjected, singly fluorescently labeled oligo(dT) 43-mer molecules in living cells in quasi-natural surrounding, that is, cell culture medium, is demonstrated. Single-stranded oligonucleotides were labeled at the 5'-end with a red-absorbing oxazine derivative (MR121) and excited by a pulsed laser diode emitting at 635 nm with a repetition rate of 64 MHz. Spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) on untreated living 3T3 mouse fibroblast cells reveals autofluorescence signals found predominately in the cytoplasm with fluorescence lifetimes of approximately 1.3 ns and emission maximums of approximately 665-670 nm. Hence, fluorescence signals of single MR121-labeled oligonucleotide molecules that exhibit a fluorescence lifetime of 2.8 ns and a fluorescence emission maximum of 685 nm can be easily discriminated against autofluorescence. MR121-labeled oligonucleotides were microinjected into the cytoplasm or nucleus of living 3T3 mouse fibroblast cells using a micropipet. Since the micropipet exhibits an inner diameter of 500 +/- 200 nm at the very end of the tip-comparable to the diameter of the detection volume applied-the number of molecules delivered into the cell via the micropipet can be counted. Furthermore, the presented technique enables the quantitative detection and time-resolved identification of single molecules in living cells as a result of their characteristic emission maximums and fluorescence lifetime. The results obtained from single-molecule studies demonstrate for the first time that 10-30% of the microinjected oligo(dT) 43-mer molecules cannot diffuse freely inside of the nucleus but, rather, are tethered to immobile elements of the transcriptional, splicing, or polyadenylation machinery.