PubMed Health⌕ Search

Biomedical subjects

Stefan Jäger

Publications and source records attributed to Stefan Jäger.

10 recordsLinked to original sources
PubMed2005

A versatile toolbox for variable DNA functionalization at high density.

To broaden the applicability of chemically modified DNAs in nano- and biotechnology, material science, sensor development, and molecular recognition, strategies are required for introducing a large variety of different modifications into the same nucleic acid sequence at once. Here, we investigate the scope and limits for obtaining functionalized dsDNA by primer extension and PCR, using a broad variety of chemically modified deoxynucleotide triphosphates (dNTPs), DNA polymerases, and templates. All natural nucleobases in each strand were substituted with up to four different base-modified analogues. We studied the sequence dependence of enzymatic amplification to yield high-density functionalized DNA (fDNA) from modified dNTPs, and of fDNA templates, and found that GC-rich sequences are amplified with decreased efficiency as compared to AT-rich ones. There is also a strong dependence on the polymerase used. While family A polymerases generally performed poorly on "demanding" templates containing consecutive stretches of a particular base, family B polymerases were better suited for this purpose, in particular Pwo and Vent (exo-) DNA polymerase. A systematic analysis of fDNAs modified at increasing densities by CD spectroscopy revealed that single modified bases do not alter the overall B-type DNA structure, regardless of their chemical nature. A density of three modified bases induces conformational changes in the double helix, reflected by an inversion of the CD spectra. Our study provides a basis for establishing a generally applicable toolbox of enzymes, templates, and monomers for generating high-density functionalized DNAs for a broad range of applications.

Base Sequence↗
PubMed2005

Rapid analysis of Forster resonance energy transfer by two-color global fluorescence correlation spectroscopy: trypsin proteinase reaction.

In this study we introduce the combination of two-color global fluorescence correlation spectroscopy (2CG-FCS) and Förster resonance energy transfer (FRET) as a very powerful combination for monitoring biochemical reactions on the basis of single molecule events. 2CG-FCS, which is a new variation emerging from the family of fluorescence correlation spectroscopy, globally analyzes the simultaneously recorded auto- and cross-correlation data from two photon detectors monitoring the fluorescence emission of different colors. Overcoming the limitations inherent in mere auto- and cross-correlation analysis, 2CG-FCS is sensitive in resolving and quantifying fluorescent species that differ in their diffusion characteristics and/or their molecular brightness either in one or both detection channels. It is able to account for effects that have often been considered as sources of severe artifacts in two-color and FRET measurements, the most prominent artifacts comprising photobleaching, cross talk, or concentration variations in sample preparation. Because of its very high statistical accuracy, the combination of FRET and 2CG-FCS is suited for high-throughput applications such as drug screening. Employing beam scanning during data acquisition even further enhances this capability and allows measurement times of <2 s. The improved performance in monitoring a FRET sample was verified by following the protease cleavage reaction of a FRET-active peptide. The FRET-inactive subpopulation of uncleaved substrate could be correctly assigned, revealing a substantial portion of inactive or missing acceptor label. The results were compared to those obtained by two-dimensional fluorescence intensity distribution analysis.

Biophysics↗
PubMed2005

Mutant p53 proteins bind DNA in a DNA structure-selective mode.

Despite the loss of sequence-specific DNA binding, mutant p53 (mutp53) proteins can induce or repress transcription of mutp53-specific target genes. To date, the molecular basis for transcriptional modulation by mutp53 is not understood, but increasing evidence points to the possibility that specific interactions of mutp53 with DNA play an important role. So far, the lack of a common denominator for mutp53 DNA binding, i.e. the existence of common sequence elements, has hampered further characterization of mutp53 DNA binding. Emanating from our previous discovery that DNA structure is an important determinant of wild-type p53 (wtp53) DNA binding, we analyzed the binding of various mutp53 proteins to oligonucleotides mimicking non-B DNA structures. Using various DNA-binding assays we show that mutp53 proteins bind selectively and with high affinity to non-B DNA. In contrast to sequence-specific and DNA structure-dependent binding of wtp53, mutp53 DNA binding to non-B DNA is solely dependent on the stereo-specific configuration of the DNA, and not on DNA sequence. We propose that DNA structure-selective binding of mutp53 proteins is the basis for the well-documented interaction of mutp53 with MAR elements and for transcriptional activities mediates by mutp53.

DNA↗
PubMed2004

Confocal fluorescence detection expanded to UV excitation: the first continuous fluorimetric assay of human steroid sulfatase in nanoliter volume.

Steroid sulfatase is an enzyme that currently enjoys considerable interest as a potential drug target in the treatment of estrogen- and androgen-dependent diseases, in particular breast cancer. We have purified human steroid sulfatase to apparent homogeneity from recombinant Chinese hamster ovary cells, and we established an assay with a new fluorogenic substrate, 3,4-benzocoumarin-7-O-sulfate (1). Substrate 1 features a K(m) value of 22.5 microM, which is close to the value for the natural substrate dehydroepiandrosterone sulfate (26 microM) and much lower than the K(m) values of other synthetic substrates (276-736 microM). Importantly, the cleavage of substrate 1 can be monitored continuously during the enzymatic cleavage, since a change in fluorescence intensity is detectable at the pH where the enzyme is active; in contrast, all other synthetic substrates described so far require alkalization to reveal a measurable absorbance or fluorescence signal. The adaptation of the assay to the 96-well format allows continuous monitoring of multiple wells in a microplate fluorescence reader. Applications of the assay for the determination of IC(50) and K(i) values of novel steroid sulfatase inhibitors are presented. Most importantly the assay was transferred to the nanoscale format (1-microl assay volume) in 2080-well plates with confocal fluorescence detection. This miniaturization will permit screening with a minimum throughput of 20000 compounds per day. The system presented demonstrates that the confocal detection platform used for nanoscreening can be successfully adapted to assays for which conventional ultraviolet dyes like coumarins are necessary. This strongly broadens the application range of confocal readers in drug screening.

Algorithms↗
PubMed2003

Highly sensitive fluorescence detection technology currently available for HTS.

Homogeneous fluorescence methods are providing an important tool for HTS technologies. A wide range of different techniques have been established on the market, with read-outs ranging from total fluorescence intensity to statistical analysis of fluorescence fluctuations for biochemical assays or fluorescence imaging techniques for cellular systems. Each method has its own advantages and limitations, which have to be accounted for when designing a specific assay. Here, recently developed fluorescence techniques and some of their applications, with a particular focus on sensitivity, are summarized and their principles are presented.

Fluorescence Polarization↗
PubMed2003

Functional tuning of nucleic acids by chemical modifications: tailored oligonucleotides as drugs, devices, and diagnostics.

Chemical modifications of nucleic acids present vast opportunities for extending the functions and properties of these biomolecules. In general, efforts invested in this direction pertain to the introduction of reactive functional groups for further derivatizations of oligonucleotides with numerous reporter groups and for equipping nucleic acids with catalytic chemical moieties. This review deals with representative chemical modifications in the nucleobases, sugars, and the phosphate ester backbone and their application from novel catalytic RNA selection to nucleic acid-based biosensors.

Biosensing Techniques↗
PubMed2003

A modular, fully integrated ultra-high-throughput screening system based on confocal fluorescence analysis techniques.

The rapid increase in size of compound libraries, as well as new targets emerging from the Human Genome Project, require progress in ultra-high-throughput screening (uHTS) systems. In a joint effort with scientists and engineers from the biotech and the pharmaceutical industry, a modular, fully integrated system for miniaturized uHTS was developed. The goal was to achieve high data quality in small assay volumes (1-4 microL) combined with reliable and unattended operation. Two new confocal fluorescence readers have been designed. One of the instruments is a 4-channel confocal fluorescence reader, measuring with 4 objectives in parallel. The fluorescence readout is based on single-molecule detection methods, allowing high sensitivity at low tracer concentrations and delivering an information-rich output. The other instrument is a confocal fluorescence imaging reader, where the images are analyzed in terms of generic patterns and quantified in units of intensity per pixel. Both readers are spanning the application range from assays with isolated targets in homogenous solution or membrane vesicle-based assays (4-channel reader) to cell-based assays (imaging reader). Results from a comprehensive test on these assay types demonstrate the high quality and robustness of this screening system.

Anti-Bacterial Agents↗
PubMed2002

Fluorescence intensity and lifetime distribution analysis: toward higher accuracy in fluorescence fluctuation spectroscopy.

Fluorescence fluctuation methods such as fluorescence correlation spectroscopy and fluorescence intensity distribution analysis (FIDA) have proven to be versatile tools for studying molecular interactions with single molecule sensitivity. Another well-known fluorescence technique is the measurement of the fluorescence lifetime. Here, we introduce a method that combines the benefits of both FIDA and fluorescence lifetime analysis. It is based on fitting the two-dimensional histogram of the number of photons detected in counting time intervals of given width and the sum of excitation to detection delay times of these photons. Referred to as fluorescence intensity and lifetime distribution analysis (FILDA), the technique distinguishes fluorescence species on the basis of both their specific molecular brightness and the lifetime of the excited state and is also able to determine absolute fluorophore concentrations. The combined information yielded by FILDA results in significantly increased accuracy compared to that of FIDA or fluorescence lifetime analysis alone. In this paper, the theory of FILDA is elaborated and applied to both simulated and experimental data. The outstanding power of this technique in resolving different species is shown by quantifying the binding of calmodulin to a peptide ligand, thus indicating the potential for application of FILDA to similar problems in the life sciences.

Algorithms↗