11 research outputs found
Azo-Sulforhodamine Dyes: A Novel Class of Broad Spectrum Dark Quenchers
A rapid access to
a novel class of water-soluble dark quencher
dyes was achieved using an azo-coupling reaction between a fluorescent
primary arylamine derived from a sulforhodamine 101 scaffold and a
tertiary aniline equipped with different bioconjugatable groups. The
thus obtained nonfluorescent azo-sulforhodamine hybrids display a
broad quenching range spanning the visible to NIR regions. This was
demonstrated through the preparation and enzymatic activation of FRET-based
fluorogenic substrates of urokinase
Universal Dark Quencher Based on āClickedā Spectrally Distinct Azo Dyes
The first synthesis of an heterotrifunctional molecular scaffold
derived from the popular DABCYL azo dye quencher has been achieved.
The sequential derviatization of this trivalent azobenzene derivative
with two other nonfluorescent azo dyes (Black Hole Quencher BHQ-1
and BHQ-3) and through effective reactions from the ābioconjugation
chemistryā repertoire has led to an universal dark quencher
(UDQ). This āclickedā poly azo dye is able to turn off
an array of fluorophores covering the UV/NIR (300ā750 nm) spectral
range
Surface DNA content analysis by TdT labeling of 3ā²-OH using Cy5-ddNTP Visual Genetics sequencer trace recorded for disulfide-T2 template (80mer) grafted on BTA glass, labeled with 500 nM ddNTP-Cy5
<p><b>Copyright information:</b></p><p>Taken from "BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies"</p><p>Nucleic Acids Research 2006;34(3):e22-e22.</p><p>Published online 9 Feb 2006</p><p>PMCID:PMC1363783.</p><p>Ā© The Author 2006. Published by Oxford University Press. All rights reserved</p>0 at 3ā² end (TdT) and cleaved from the surface in 50 mM DTT/Tris solution pH 8.5 (1 h). Grafting conditions: 130 nM 5ā²-amino-SS-T2 template, 10 mM EDC/10 mM 1-Methyl-Imidazole (50Ā°C)/1 h. Terminal Deoxynucleotidyl Transferase (TdT), 20 Āµ/ml, NEB buffer4, CoCl (250 mM). Sequencer (Visible Genetics, VG90008), SureFill 6% Sequencing Gel (Visible Genetics, Ref. #VG40006), Stop Loading Dye (Amersham). The upper traces represent DNA size markers labeled with Cy5.5. In the lower trace, the Cy5 labelled oligonucleotide extracted from the surface migrates as a single peak with an approximate apparent size of 87 bases
() Simplified diagram of steps required for glass functionalization with amino (ATS) and carboxyl (BTA) groups
<p><b>Copyright information:</b></p><p>Taken from "BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies"</p><p>Nucleic Acids Research 2006;34(3):e22-e22.</p><p>Published online 9 Feb 2006</p><p>PMCID:PMC1363783.</p><p>Ā© The Author 2006. Published by Oxford University Press. All rights reserved</p> Staining of derivatized glass surfaces using NBD reagents of () aminated (ATS) and () carboxylated (BTA) glass slides
Probe concentration and time dependence for the attachment of aminated oligonucleotides to BTA-derivatized slides monitored by hybridization using a TR-labeled oligonucleotide
<p><b>Copyright information:</b></p><p>Taken from "BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies"</p><p>Nucleic Acids Research 2006;34(3):e22-e22.</p><p>Published online 9 Feb 2006</p><p>PMCID:PMC1363783.</p><p>Ā© The Author 2006. Published by Oxford University Press. All rights reserved</p> () Concentration dependence for the grafting of 5ā²-amino-10T-P primer (34mer) on BTA glass. Grafting conditions: 10 mM EDC/10 mM 1-Methyl-imidazole (50Ā°C)/1 h. Hybridization: 500 nM reverse-P-Texas Red in TMN buffer. The solid line is to guide the eye. Background fluorescence is subtracted. () Time dependence for the grafting of 5ā²amino-10T-SS-P primer on BTA glass. Grafting conditions: 1 ĀµM primer, 10 mM EDC/10 mM 1-Methyl-Imidazole (50Ā°C at different incubation times. Hybridization (circles), 500 nM reverse-P-Texas Red in TMN buffer. Denaturation (triangle): three times, 5 min at 80Ā°C in 50% formamide (v/v in HO). Experimental data points are background subtracted
Yield of DNA attachment using different cross-linkers between aminated slides (ATS) and modified oligonucleotides
<p><b>Copyright information:</b></p><p>Taken from "BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies"</p><p>Nucleic Acids Research 2006;34(3):e22-e22.</p><p>Published online 9 Feb 2006</p><p>PMCID:PMC1363783.</p><p>Ā© The Author 2006. Published by Oxford University Press. All rights reserved</p> Experiment is performed in one glass channel chip. The channels differ by the cross-linker used: First 3 channels are treated with s-MBS. Channels 4 to 6 are treated with TMA and 7ā8 with BTA. The surfaces are then grafted and hybridized with the appropriate DNA material (see text for grafting and hybridization conditions)
Characterization of DNA colonies generated using BTA chemistry by Sybr-Green I staining
<p><b>Copyright information:</b></p><p>Taken from "BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies"</p><p>Nucleic Acids Research 2006;34(3):e22-e22.</p><p>Published online 9 Feb 2006</p><p>PMCID:PMC1363783.</p><p>Ā© The Author 2006. Published by Oxford University Press. All rights reserved</p> Histograms of colonies average intensity () Before BbvI digestion colonies are double stranded and their length is 347 bp. () After BbvI digestion, colonies are still double stranded and their length is reduced to 43 bp, showing consequently a lower average intensity
Thiocarbamate-Linked PolysulfonateāPeptide Conjugates As Selective Hepatocyte Growth Factor Receptor Binders
The capacity of many proteins to
interact with natural or synthetic
polyanions has been exploited for modulating their biological action.
However, the polydispersity of these macromolecular polyanions as
well as their poor specificity is a severe limitation to their use
as drugs. An emerging trend in this field is the synthesis of homogeneous
and well-defined polyanionāpeptide conjugates, which act as
bivalent ligands, with the peptide part bringing the selectivity of
the scaffold. Alternately, this strategy can be used for improving
the binding of short peptides to polyanion-binding protein targets.
This work describes the design and first synthesis of homogeneous
polysulfonateāpeptide conjugates using thiocarbamate ligation
for binding to the extracellular domain of MET tyrosine kinase receptor
for hepatocyte growth factor
A Novel Bio-Orthogonal Cross-Linker for Improved Protein/Protein Interaction Analysis
The
variety of protein cross-linkers developed in recent years illustrates
the current requirement for efficient reagents optimized for mass
spectrometry (MS) analysis. To date, the most widely used strategy
relies on commercial cross-linkers that bear an isotopically labeled
tag and <i>N</i>-hydroxysuccinimid-ester (NHS-ester) moieties.
Moreover, an enrichment step using liquid chromatography is usually
performed after enzymatic digestion of the cross-linked proteins.
Unfortunately, this approach suffers from several limitations. First,
it requires large amounts of proteins. Second, NHS-ester cross-linkers
are poorly efficient because of their fast hydrolysis in water. Finally,
data analysis is complicated because of uneven fragmentation of complex
isotopic cross-linked peptide mixtures. We therefore synthesized a
new type of trifunctional cross-linker to overrule these limitations.
This reagent, named NNP9, comprises a rigid core and bears two activated
carbamate moieties and an azido group. NNP9 was used to establish
intra- and intermolecular cross-links within creatine kinase, then
to map the interaction surfaces between Ī±-Synuclein (Ī±-Syn),
the aggregation of which leads to Parkinsonās disease, and
the molecular chaperone Hsc70. We show that NNP9 cross-linking efficiency
is significantly higher than that of NHS-ester commercial cross-linkers.
The number of cross-linked peptides identified was increased, and
a high quality of MS/MS spectra leading to high sequence coverage
was observed. Our data demonstrate the potential of NNP9 for an efficient
and straightforward characterization of proteināprotein interfaces
and illustrate the power of using different cross-linkers to map thoroughly
the surface interfaces within protein complexes
Kondratāeva Ligation: DielsāAlder-Based Irreversible Reaction for Bioconjugation
Diversification of existing chemoselective
ligations is required
to efficiently access complex and well-defined biomolecular assemblies
with unique and valuable properties. The development and bioconjugation
applications of a novel DielsāAlder-based irreversible site-specific
ligation are reported. The strategy is based on a Kondratāeva
cycloaddition between bioinert and readily functionalizable 5-alkoxyoxazoles
and maleimides that readily react together under mild and easily tunable
reaction conditions to afford a fully stable pyridine scaffold. The
potential of this novel bioconjugation is demonstrated through the
preparation of fluorescent conjugates of biomolecules and a novel
FoĢrster resonance energy transfer (FRET)-based probe suitable
for the in vivo detection and imaging of urokinase-like plasminogen
activator (uPA), which is a key protease involved in cancer invasion
and metastasis