US 5,510,240 A · Method of screening a peptide library

Abstract

The instant invention provides a library of bio-oligomers of defined size and known composition, in which the library contains all of the possible sequences of the bio-oligomers, and a method of synthesis thereof. The bio-oligomers of the library may be peptides, nucleic acids, or a combination of the foregoing. The instant invention also provides methods to identify bio-oligomers from a library that demonstrate desired characteristics such as binding, bioactivity and catalytic activity. Thus the instant invention provides a unique and powerful method to identify a useful bio-oligomer sequences from a library more quickly than current state-of-the-art technology allows. Effector molecules for use in treatment or diagnosis of disease are also provided.

Claims26

Dependent claims

A method of screening a library of peptides and determining a sequence of a peptide ligand for an acceptor molecule of interest comprising the steps of:

a) introducing an acceptor molecule to a peptide library, wherein the library comprises a multiplicity of species of solid phase supports having a linker, by which analyzable quantities of deprotected peptides are covalently attached to the support, wherein the sequences of said peptides are not pre-determined and a single species of peptide is attached to each support;
b) identifying the solid-phase supports having peptides thereon that bind the acceptor molecule, wherein said identified supports represent at most about 1 part per 1,000 parts of the supports of the library;
c) isolating at least one identified support from all other species of the library;
and thereafter
d) determining the sequence of the peptide species attached to the isolated support.

2 more elements

A method of screening a peptide library and determining a sequence of a peptide ligand for an acceptor molecule of interest comprising the steps of:

a) introducing an acceptor molecule to a peptide library, wherein the library is comprised of a multiplicity of solid phase supports having a linker, wherein a single peptide species is covalently attached by the linker to each solid phase support, and in which the library is prepared by a method comprising the steps of:
(i) providing at least two aliquots of solid phase supports, said solid phase supports having an N.sup..alpha. reactive site;
(ii) separately introducing a species of subunit of the peptide to each of the aliquots of solid phase supports such that a different species of subunit is introduced into each aliquot, said subunits having a protecting group or a plurality of protecting groups;
(iii) completely coupling the subunit to substantially all the reactive sites of the solid phase supports;
(iv) thoroughly mixing and deprotecting the aliquots of solid phase supports whereby an N.sup..alpha. reactive site is provided;
and
after completing all desired executions of steps (i) through (iv), a final step of removing any remaining protecting groups such that the peptides remain covalently attached to the solid phase supports;
b) identifying the solid-phase supports having peptides thereon that bind the acceptor molecule, wherein said identified supports represent at most about 1 part per 1,000 parts of the supports of the library;
c) isolating at least one identified support from all other species of the library;
and thereafter
d) determining the sequence of the peptide species attached to the isolated support.

8 more elements

A method of screening a library of peptides and determining a sequence of a peptide ligand for an acceptor molecule of interest comprising the steps of:

a) introducing an acceptor molecule to a peptide library, wherein the library is comprised of a multiplicity of solid phase supports having a linker, wherein a single peptide species is attached covalently by the linker to each solid phase support, and in which the library is prepared by a method comprising at least three iterations of the steps of:
(i) providing at least two aliquots of solid phase supports, said solid phase supports having an N.sup..alpha. reactive site;
(ii) separately introducing a species of subunit of the peptide to each of the aliquots of solid phase supports such that a different species of subunit is introduced into each aliquot, said subunits having a protecting group or a plurality of protecting groups;
(iii) completely coupling the subunit to substantially all the reactive sites of the solid phase supports;
(iv) thoroughly mixing and deprotecting the aliquots of solid phase supports whereby an N.sup..alpha. reactive site is provided;
(v) repeating steps (i)-(iv) at least two additional times;
and
(vi) after completing all desired executions of steps (i) through (iv), a final step of removing any remaining protecting groups such that the peptides remain attached to the solid phase supports;
b) identifying the solid-phase supports having peptides thereon that bind the acceptor molecule;
c) isolating at least one identified support from all other species of the library;
and thereafter
d) determining the sequence of the peptide species attached to the isolated support.

9 more elements

A method of screening a library of peptides and determining a sequence of a peptide ligand having a biological activity of interest comprising the steps of:

a) subdividing a peptide library into a multiplicity of samples by placing each sample into a corresponding sample of solution, wherein a single peptide species is attached covalently to each solid phase support by means of a selectively cleavable linker, a plurality of selectively cleavable linkers or a combination of a non-cleavable linker and a selectively cleavable linker, and in which the library is prepared by a method comprising the steps of:
(i) providing at least two aliquots of a solid phase supports, said solid phase supports having an N.sup..alpha. reactive site;
(ii) separately introducing a species of subunit of the peptide to each of the aliquots of solid phase supports such that a different species of subunit is introduced into each aliquot, said subunits having a protecting group or a plurality of protecting groups;
(iii) completely coupling the subunits to substantially all the reactive sites of the solid phase supports;
and
(iv) thoroughly mixing and deprotecting the aliquots of solid phase supports, whereby an N.sup..alpha. reactive site is provided;
and, after completing all desired executions of steps (i) through (iv), a final step of removing any remaining protecting groups such that peptides remain linked to the solid phase supports;
b) releasing a portion of the peptides of each sample of supports into the corresponding sample of solution by cleavage of the cleavable linker;
c) detecting the biological activity of interest in the samples of solution;
d) identifying the solid phase supports having peptides thereon that are biologically active, wherein said identified supports represent at most about 1 part per 2,000 part of the supports of the library;
e) isolating at least one active support from all other species of the library;
and thereafter
f) determining the sequence of the peptide species attached to the isolated support.

10 more elements

Dependencies are inferred from the claim text.

Facts

Application

08/014,979Filed 8 Feb 1993 · division

Priority

2 Jul 1990

Granted

23 Apr 1996

Term ends

Not estimatedThe source records don’t support an estimate.

Classification31 codes

C12N 15/1075
Isolating an individual clone by screening libraries: by coupling phenotype to genotype, not provided for in other groups of this subclass
C07K 1/047
On carriers: simultaneous synthesis of different peptide species; Peptide libraries
C07K 14/70
Derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin: enkephalins
C07K 14/82
Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof: translation products from oncogenes

Abstract

Claims26

Dependent claims

A method of screening a library of peptides and determining a sequence of a peptide ligand for an acceptor molecule of interest comprising the steps of:

a) introducing an acceptor molecule to a peptide library, wherein the library comprises a multiplicity of species of solid phase supports having a linker, by which analyzable quantities of deprotected peptides are covalently attached to the support, wherein the sequences of said peptides are not pre-determined and a single species of peptide is attached to each support;
b) identifying the solid-phase supports having peptides thereon that bind the acceptor molecule, wherein said identified supports represent at most about 1 part per 1,000 parts of the supports of the library;
c) isolating at least one identified support from all other species of the library;
and thereafter
d) determining the sequence of the peptide species attached to the isolated support.

2 more elements

A method of screening a peptide library and determining a sequence of a peptide ligand for an acceptor molecule of interest comprising the steps of:

a) introducing an acceptor molecule to a peptide library, wherein the library is comprised of a multiplicity of solid phase supports having a linker, wherein a single peptide species is covalently attached by the linker to each solid phase support, and in which the library is prepared by a method comprising the steps of:
(i) providing at least two aliquots of solid phase supports, said solid phase supports having an N.sup..alpha. reactive site;
(ii) separately introducing a species of subunit of the peptide to each of the aliquots of solid phase supports such that a different species of subunit is introduced into each aliquot, said subunits having a protecting group or a plurality of protecting groups;
(iii) completely coupling the subunit to substantially all the reactive sites of the solid phase supports;
(iv) thoroughly mixing and deprotecting the aliquots of solid phase supports whereby an N.sup..alpha. reactive site is provided;
and
after completing all desired executions of steps (i) through (iv), a final step of removing any remaining protecting groups such that the peptides remain covalently attached to the solid phase supports;
b) identifying the solid-phase supports having peptides thereon that bind the acceptor molecule, wherein said identified supports represent at most about 1 part per 1,000 parts of the supports of the library;
c) isolating at least one identified support from all other species of the library;
and thereafter
d) determining the sequence of the peptide species attached to the isolated support.

8 more elements

A method of screening a library of peptides and determining a sequence of a peptide ligand for an acceptor molecule of interest comprising the steps of:

a) introducing an acceptor molecule to a peptide library, wherein the library is comprised of a multiplicity of solid phase supports having a linker, wherein a single peptide species is attached covalently by the linker to each solid phase support, and in which the library is prepared by a method comprising at least three iterations of the steps of:
(i) providing at least two aliquots of solid phase supports, said solid phase supports having an N.sup..alpha. reactive site;
(ii) separately introducing a species of subunit of the peptide to each of the aliquots of solid phase supports such that a different species of subunit is introduced into each aliquot, said subunits having a protecting group or a plurality of protecting groups;
(iii) completely coupling the subunit to substantially all the reactive sites of the solid phase supports;
(iv) thoroughly mixing and deprotecting the aliquots of solid phase supports whereby an N.sup..alpha. reactive site is provided;
(v) repeating steps (i)-(iv) at least two additional times;
and
(vi) after completing all desired executions of steps (i) through (iv), a final step of removing any remaining protecting groups such that the peptides remain attached to the solid phase supports;
b) identifying the solid-phase supports having peptides thereon that bind the acceptor molecule;
c) isolating at least one identified support from all other species of the library;
and thereafter
d) determining the sequence of the peptide species attached to the isolated support.

9 more elements

A method of screening a library of peptides and determining a sequence of a peptide ligand having a biological activity of interest comprising the steps of:

a) subdividing a peptide library into a multiplicity of samples by placing each sample into a corresponding sample of solution, wherein a single peptide species is attached covalently to each solid phase support by means of a selectively cleavable linker, a plurality of selectively cleavable linkers or a combination of a non-cleavable linker and a selectively cleavable linker, and in which the library is prepared by a method comprising the steps of:
(i) providing at least two aliquots of a solid phase supports, said solid phase supports having an N.sup..alpha. reactive site;
(ii) separately introducing a species of subunit of the peptide to each of the aliquots of solid phase supports such that a different species of subunit is introduced into each aliquot, said subunits having a protecting group or a plurality of protecting groups;
(iii) completely coupling the subunits to substantially all the reactive sites of the solid phase supports;
and
(iv) thoroughly mixing and deprotecting the aliquots of solid phase supports, whereby an N.sup..alpha. reactive site is provided;
and, after completing all desired executions of steps (i) through (iv), a final step of removing any remaining protecting groups such that peptides remain linked to the solid phase supports;
b) releasing a portion of the peptides of each sample of supports into the corresponding sample of solution by cleavage of the cleavable linker;
c) detecting the biological activity of interest in the samples of solution;
d) identifying the solid phase supports having peptides thereon that are biologically active, wherein said identified supports represent at most about 1 part per 2,000 part of the supports of the library;
e) isolating at least one active support from all other species of the library;
and thereafter
f) determining the sequence of the peptide species attached to the isolated support.

10 more elements

Dependencies are inferred from the claim text.

Method of screening a peptide library

Life of this patent

Abstract

Claims26

Method of screening a peptide library

Life of this patent

Abstract

Claims26