- Cleavable biotin probes for labeling of biomolecules via azide-alkyne cycloaddition
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The azide-alkyne cycloaddition provides a powerful tool for bio-orthogonal labeling of proteins, nucleic acids, glycans, and lipids. In some labeling experiments, e.g., in proteomic studies involving affinity purification and mass spectrometry, it is convenient to use cleavable probes that allow release of labeled biomolecules under mild conditions. Five cleavable biotin probes are described for use in labeling of proteins and other biomolecules via azide-alkyne cycloaddition. Subsequent to conjugation with metabolically labeled protein, these probes are subject to cleavage with either 50 mM Na 2S2O4, 2% HOCH2CH2SH, 10% HCO2H, 95% CF3CO2H, or irradiation at 365 nm. Most strikingly, a probe constructed around a dialkoxydiphenylsilane (DADPS) linker was found to be cleaved efficiently when treated with 10% HCO 2H for 0.5 h. A model green fluorescent protein was used to demonstrate that the DADPS probe undergoes highly selective conjugation and leaves a small (143 Da) mass tag on the labeled protein after cleavage. These features make the DADPS probe especially attractive for use in biomolecular labeling and proteomic studies.
- Szychowski, Janek,Mahdavi, Alborz,Hodas, Jennifer J. L.,Bagert, John D.,Ngo, John T.,Landgraf, Peter,Dieterich, Daniela C.,Schuman, Erin M.,Tirrell, David A.
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experimental part
p. 18351 - 18360
(2011/03/18)
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- POLYMERASE-INDEPENDENT ANALYSIS OF THE SEQUENCE OF POLYNUCLEOTIDES
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The present invention concerns methods of polymerase independent template directed elongation of polynucleotides, nucleotide building blocks used in these methods as well as the use of the methods and building blocks for the determination of nucleotide sequences, in particular for the determination of SNPs, base modifications, mutations, rearrangements and methylation patterns.
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Page/Page column 56-57
(2008/06/13)
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- Photocleavable isotope-coded affinity tags
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This invention relates to agents and conjugates that can be used to detect and isolate target components from complex mixtures such as proteins and protein fragments from biological samples from in vivo and in vitro sources. Agents comprise a detectable group bound to a photoreactive group. Conjugates comprise agents coupled to substrates by covalent bounds which can be selectively cleaved with the administration of electromagnetic radiation. Targets substances labeled with detectable molecules can be easily identified and separated from a heterologous mixture of substances. Exposure of the conjugate to radiation releases the target in a functional form and completely unaltered. Using photocleavable molecular precursors as the conjugates, label can be incorporated into macromolecules, the nascent macromolecules isolated and the label completely removed. The invention also relates to targets isolated with these conjugates which may be useful as pharmaceutical agents or compositions that can be administered to humans and other mammals.
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- Compounds and methods for detecting biomolecules
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Methods are described for synthesizing compounds useful for detecting nucleic acids, and in particular, the hybridization of nucleic acids. Photocleavable agents are described, including but not limited to photocleavable nucleotides and photocleavable phosphoramidites.
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- Methods for the detection and isolation of biomolecules
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This invention relates to agents and conjugates that can be used to detect and isolate target components from complex mixtures such as nucleic acids from biological samples, cells from bodily fluids, and nascent proteins from translation reactions. Agents comprise a detectable moiety bound to a photoreactive moiety. Conjugates comprise agents coupled to substrates by covalent bounds which can be selectively cleaved with the administration of electromagnetic radiation. Targets substances labeled with detectable molecules can be easily identified and separated from a heterologous mixture of substances. Exposure of the conjugate to radiation releases the target in a functional form and completely unaltered. Using photocleavable molecular precursors as the conjugates, label can be incorporated into macromolecules, the nascent macromolecules isolated and the label completely removed. The invention also relates to targets isolated with these conjugates which may be useful as pharmaceutical agents or compositions that can be administered to humans and other mammals. Useful compositions include biological agents such as nucleic acids, proteins, lipids and cytokines. Conjugates can also be used to monitor the pathway and half-life of pharmaceutical composition in vivo and for diagnostic, therapeutic and prophylactic purposes. The invention also relates to kits comprised of agents and conjugates that can be used for the detection of diseases, disorders and nearly any individual substance in a complex background of substances.
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- Cell-delivery agent
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A photo-cleavable compound for delivery and release of a biologically active substance to selected target cells; the compound includes a binding partner for a specific cell-surface receptor of those target cells, the biologically active substance to be delivered, and a photo-cleavable bridge between the binding partner and the biologically active substance. When the compound is exposed to a heterogeneous population of target and non-target cells, it binds selectively to the receptors on the surface of the target cells. Exposing the compound to light of selected wave length cleaves it, yielding the active substance.
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