Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization

Background: State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests. Methodology and Principal Findings: The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique. Conclusions and Significance: The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution fo

J. Mol. Biol.

Bivalent peptidic thrombin inhibitors consisting of an N- terminal D-cyclohexylalanine-Pro-N-alpha(Me)Arg active-site fragment, a flexible polyglycine linker, and a C-terminal hirugen-like segment directed towards the fibrinogen recognition exosite inhibit thrombin with K-i values in the pico-molar range, remaining stable in buffered solution at pH 7.8 for at least 15 hours. In order to investigate the structural basis of this increased, stability, the most potent of these inhibitors, I-11 (K-i = 37pM), containing an N- alpha(Me)Arg-Thr bond, was crystallized in complex with human a-thrombin. X-ray data were collected to 1.8 Angstrom resolution and the crystal structure of this complex was determined. The Fourier map displays clear electron density for the N-terminal fragment and for the exosite binding segment. It indicates, however, that in agreement with Edman sequencing, the peptide had been cleaved in the crystal, presumably due to the long incubation time of 14 days needed for crystallization and data collection. The N-alpha(Me) group is directed toward the carbonyl oxygen atom of Ser214, pushing the Ser195 O-gamma atom out of its normal site. This structure suggests that upon thrombin binding, the scissile peptide bond of the intact peptide and the Ser195 O-gamma are separated from each other, impairing the nucleophilic attack of the Ser195 07 toward the N-chi(Me)Arg carbonyl group. In the time-scale of two weeks, however, cleavage geometries favoured by the crystal allow catalysis at a slow rate. (C) 2002 Elsevier Science Ltd

The methyl group of N-alpha(Me)Arg-containing peptides disturbs the active-site geometry of thrombin, impairing efficient cleavage

Bivalent peptidic thrombin inhibitors consisting of an N- terminal D-cyclohexylalanine-Pro-N-alpha(Me)Arg active-site fragment, a flexible polyglycine linker, and a C-terminal hirugen-like segment directed towards the fibrinogen recognition exosite inhibit thrombin with K-i values in the pico-molar range, remaining stable in buffered solution at pH 7.8 for at least 15 hours. In order to investigate the structural basis of this increased, stability, the most potent of these inhibitors, I-11 (K-i = 37pM), containing an N- alpha(Me)Arg-Thr bond, was crystallized in complex with human a-thrombin. X-ray data were collected to 1.8 Angstrom resolution and the crystal structure of this complex was determined. The Fourier map displays clear electron density for the N-terminal fragment and for the exosite binding segment. It indicates, however, that in agreement with Edman sequencing, the peptide had been cleaved in the crystal, presumably due to the long incubation time of 14 days needed for crystallization and data collection. The N-alpha(Me) group is directed toward the carbonyl oxygen atom of Ser214, pushing the Ser195 O-gamma atom out of its normal site. This structure suggests that upon thrombin binding, the scissile peptide bond of the intact peptide and the Ser195 O-gamma are separated from each other, impairing the nucleophilic attack of the Ser195 07 toward the N-chi(Me)Arg carbonyl group. In the time-scale of two weeks, however, cleavage geometries favoured by the crystal allow catalysis at a slow rate. (C) 2002 Elsevier Science Ltd

Excited-State Switching Frustrates the Tuning of Properties in Triphenylamine-Donor-Ligand Rhenium(I) and Platinum(II) Complexes

The photophysical properties of a series of rhenium(I) tricarbonyl and platinum(II) bis(acetylide) complexes containing a triphenylamine (TPA)-substituted 1,10-phenanthroline ligand have been examined. The complexes possess both metal-to-ligand charge-transfer (MLCT) and intraligand charge-transfer (ILCT) transitions that absorb in the visible region. The relative energies and ordering of the absorbing CT states have been successfully controlled by changing the metal center and modulating the donating ability of the TPA group through the addition of electron-donating methoxy and electron-withdrawing cyano groups. The ground-state properties behave in a predictable manner as a function of the TPA substituent and are characterized with a suite of techniques including electronic absorption spectroscopy, resonance Raman spectroscopy, electrochemistry, and time-dependent density functional theory calculations. However, systematic control over the ground-state properties of the complexes does not extend to their excited-state behavior. Unexpectedly, despite variation of both the MLCT and ILCT state energies, all of the luminescent complexes displayed near-isoenergetic emission at 298 K, yet the emissive lifetimes of the complexes vary from 290 ns to 3.9 μs. Excited-state techniques including transient absorption and transient resonance Raman, combined with a suite of quantum-chemical calculations, including scalar relativistic effects to elucidate competitive excited-state relaxation pathways, have been utilized to aid in assignment of the long-lived state in the complexes, which was shown to possess differing 3MLCT and 3ILCT contributions across the series