Methods and compositions are described for immobilizing anti-ligands, such as antibodies or antigens, hormones or hormone receptors, oligonucleotides, and polysaccharides on surfaces of solid substrates for various uses. The methods provide surfaces covered with caged binding members which comprise protecting groups capable of being removed upon application of a suitable energy source. Spatially addressed irradiation of predefined regions on the surface permits immobilization of anti-ligands at the activated regions on the surface. Cycles of irradiation on different regions of the surface and immobilization of different anti-ligands allows formation of an immobilized matrix of anti-ligands at defined sites on the surface. The immobilized matrix of anti-ligands permits simultaneous screenings of a liquid sample for ligands having high affinities for certain anti-ligands of the matrix. A preferred embodiment of the invention involves attaching photoactivatable biotin derivatives to a surface. Photolytic activation of the biotin derivatives forms biotin analogs having strong binding affinity for avidin. Biotinylated anti-ligands can be immobilized on activated regions of the surface previously treated with avidin.
CROSS-REFERENCE
This application is a continuation-in-part of now-abandoned application Ser. No. 07/435,316, filed Nov. 13, 1989, which is incorporated herein by reference.
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.
A method for the direct identification of few, preferably single nucleic acid strands of a specific target sequence (10) in a test solution comprising the following steps: a) Preparation of a reference solution with a mixture (11) of different, short primers (12, 13, 14) each with a so-called antisense-sequence (a', b', c') complementary to a section (a, b, c) of the target sequence (10), and marked with one or more dye molecules, b) mixture of the reference solution with the test solution and incubation of this mixture (15) under conditions allowing hybridization of the primers (12, 1, 14) with the nucleic acid strands to be identified, and then c) identification of the target sequence (10) in the incubated solution (15) by discriminating few, preferably one of the nucleic acid strands to be identified to which one or more primers (12, 13, 14) have hybridized against the background of the non-hybridized primers (12, 13, 14).
Caged carboxyl compounds in which a 2-alkoxy-5-nitrophenyl photosensitive group blocks a carboxyl function are disclosed. Preferably the compounds are neuroactive amino acids. The compounds are photolyzable by laser pulses at wavelengths above about nm within about 3 .mu.s and provide a product quantum yield of greater than about 0.2.
The invention provides a biodegradable and biocompatible polymer article having a surface wherein a biologically active ligand is provided on said surface in a spatially controlled pattern. The pattern may be formed using a poly(dimethyl siloxane) mold. The biologically active ligand may be attached to the polymer article by a biotin-avidin-biotin linkage.
Disclosed is a method for preparing a photosensitive peptide which is capable of being activated or deactivated in a biological system, including the steps of: (a) providing an amino acid including a photolabile molecule; and (b) incorporating the amino acid into a peptide during synthesis, wherein incorporation of the amino acid into the peptide produces a photosensitive peptide. Also disclosed is a method of introducing a photosensitive cleavage site into a synthetic peptide, including synthesizing a synthetic peptide having at least one photolabile amino acid, wherein the photolabile amino acid is positioned within the synthetic peptide so that upon irradiation the synthetic peptide is cleaved.
