US 2002/0055104 A1 · Nucleic acid binding site assay and selection method

Abstract

The invention provides a novel nucleic acid binding assay, which is useful for assessing the sequence-dependence of the binding of ligands to nucleic acid molecules, as well as for determining the affinity of the binding interaction. The invention also provides a selection method based on this nucleic acid binding assay. This selection method allows co-selection of ligands and the nucleic acid molecules that they bind. Additionally, the invention provides kits that can be used for carrying out the methods of the invention.

Claims61

Dependent claims

A method for screening a ligand for its ability to bind to a nucleotide sequence in a nucleic acid molecule, comprising:

(a) providing a nucleic acid molecule having a first region and second region, adjacent to said first region, wherein said first region comprises a first site that is susceptible to modification by a modifying agent, wherein such modification is effective to block strand-directed duplication by a polymerase enzyme at said first site;
(b) providing a ligand;
(c) contacting said nucleic acid molecule with said ligand, under conditions suitable for sequence-dependent binding of said ligand to said nucleic acid molecule, to form a reaction mixture;
(d) treating the reaction mixture of (c) with said modifying agent, under conditions wherein nucleic acid molecules comprising a ligand bound to said second region are substantially protected from modification at said first site, and nucleic acid molecules wherein a ligand is not bound to said second region are substantially modified at said first site;
(e) after said treatment, contacting said nucleic acid molecule with said polymerase enzyme, under conditions wherein only unmodified nucleic acid strands are amplified, wherein said amplification indicates that said ligand binds to a nucleotide sequence in said second region.

2 more elements

A method for screening ligands for the ability to bind to nucleotide sequence(s) in nucleic acid molecules, comprising:

(a) providing a set of nucleic acid molecules, each having a first region and a second region, adjacent to said first region, wherein:
(i) in said first region, said nucleic acid molecules comprise a first site that is susceptible to modification by a modifying agent, wherein such modification is effective to block strand-directed duplication by a polymerase enzyme at said first site;
and
(ii) said nucleic acid molecules comprise different nucleotide sequences in said second region;
(b) providing a set of different ligands;
(c) contacting said set of nucleic acid molecules with said set of different ligands, under conditions suitable for sequence-dependent binding of one or more ligands to one or more nucleic acid molecules, to form a reaction mixture;
(d) treating the reaction mixture of (c) with said modifying agent, under conditions wherein nucleic acid molecules comprising a ligand bound to said second region are substantially protected from modification at said first site, and nucleic acid molecules wherein a ligand is not bound to said second region are substantially modified at said first site;
(e) after said treatment, removing or inactivating said modifying agent;
(f) after said removal or inactivation, separating ligands that bind to said nucleic acid molecules from unbound ligands, and recovering the ligands that bind to said nucleic acid molecules;
and
(g) also after the removal or inactivation of (e), contacting said set of nucleic acid molecules with said polymerase enzyme, under conditions wherein only unmodified nucleic acid strands are amplified;
thereby obtaining a pool of selected ligands that bind in a sequence-dependent manner to one or more nucleotide sequences present in a pool of selected nucleic acid molecules.

9 more elements

A method for screening a ligand for its ability to bind to one or more nucleotide sequences in a set of double-stranded DNA molecules, comprising:

(a) providing a set of double-stranded DNA molecules, each having a first duplex region and a second duplex region, adjacent to said first duplex region, wherein:
(i) said first duplex region comprises, on a first strand of said DNA molecules, a first site comprising a non-standard nucleotide that is susceptible to modification by a modifying agent, wherein said modifying agent is a prokaryotic or eukaryotic repair enzyme that binds to the minor groove of duplex DNA and removes said non-standard nucleotide from duplex DNA;
(ii) said DNA molecules comprise different nucleotide sequences in said second duplex region;
and
(iii) said DNA molecules comprise a second strand comprising one or more non-standard nucleotides that can be modified by the modifying agent;
(b) providing a ligand;
(c) contacting said set of DNA molecules with said ligand, under conditions suitable for sequence-dependent binding of said ligand to said nucleic acid molecule, to form a reaction mixture;
(d) treating the reaction mixture of (c) with said modifying agent, under conditions wherein non-standard nucleotides in said DNA molecules are substantially modified, except for non-standard nucleotides at said first site in DNA molecules comprising ligand bound to said second duplex region;
(e) after said treatment, removing or inactivating said modifying agent;
(f) after said removal or inactivation, contacting the DNA molecules with said polymerase enzyme, under conditions wherein only unmodified first strands are amplified by primer-directed PCR amplification, wherein said amplification indicates that the ligand binds to a nucleotide sequence in said second duplex region;
and
(g) determining the nucleotide sequence of the amplification product in the second duplex region to determine whether the ligand shows binding specificity for one or more nucleotide sequences in said second duplex region.

9 more elements

A method for screening peptide ligands for the ability to bind to one or more nucleotide sequences in a set of double-stranded DNA molecules, comprising:

(a) providing a set of double-stranded DNA molecules linked to a substrate, each having a first duplex region and a second duplex region, adjacent to said first duplex region, wherein:
(i) said first duplex region comprises, on a first strand of said DNA molecules, a first site comprising a non-standard nucleotide that is susceptible to modification by a modifying agent, wherein said modifying agent is a prokaryotic or eukaryotic repair enzyme that binds to the minor groove of duplex DNA and removes said non-standard nucleotide from duplex DNA;
(ii) said DNA molecules comprise different nucleotide sequences in said second duplex region;
and
(iii) said DNA molecules comprise a second strand comprising one or more non-standard nucleotides that can be modified by the modifying agent;
(b) providing a set of different phage particles, wherein each phage particle expresses a heterologous peptide on its surface and comprises a nucleic acid encoding said heterologous peptide;
(c) contacting said set of DNA molecules with said set of phage particles, under conditions suitable for specific binding of one or more heterologous peptides to one or more DNA molecules, to form a reaction mixture;
(d) treating the reaction mixture of (c) with said modifying agent, under conditions wherein non-standard nucleotides in said DNA molecules are substantially modified, except for non-standard nucleotides at said first site in DNA molecules comprising heterologous peptide bound to said second duplex region;
(e) after said treatment, removing or inactivating said modifying agent;
(f) after said removal or inactivation, washing the substrate to separate phage particles that bind to said DNA molecules from unbound phage particles, and recovering the phage particles that bind to said DNA by elution;
and
(g) also after the removal or inactivation of (e), contacting the DNA molecules with said polymerase enzyme, under conditions wherein only unmodified first strands are amplified by primer-directed PCR amplification;
thereby obtaining a pool of selected phage particles expressing heterologous peptides that bind in a sequence-dependent manner to one or more nucleotide sequences present in a pool of selected DNA molecules.

10 more elements

Dependencies are inferred from the claim text.

Abstract

Claims61

Dependent claims

A method for screening a ligand for its ability to bind to a nucleotide sequence in a nucleic acid molecule, comprising:

(a) providing a nucleic acid molecule having a first region and second region, adjacent to said first region, wherein said first region comprises a first site that is susceptible to modification by a modifying agent, wherein such modification is effective to block strand-directed duplication by a polymerase enzyme at said first site;
(b) providing a ligand;
(c) contacting said nucleic acid molecule with said ligand, under conditions suitable for sequence-dependent binding of said ligand to said nucleic acid molecule, to form a reaction mixture;
(d) treating the reaction mixture of (c) with said modifying agent, under conditions wherein nucleic acid molecules comprising a ligand bound to said second region are substantially protected from modification at said first site, and nucleic acid molecules wherein a ligand is not bound to said second region are substantially modified at said first site;
(e) after said treatment, contacting said nucleic acid molecule with said polymerase enzyme, under conditions wherein only unmodified nucleic acid strands are amplified, wherein said amplification indicates that said ligand binds to a nucleotide sequence in said second region.

2 more elements

A method for screening ligands for the ability to bind to nucleotide sequence(s) in nucleic acid molecules, comprising:

(a) providing a set of nucleic acid molecules, each having a first region and a second region, adjacent to said first region, wherein:
(i) in said first region, said nucleic acid molecules comprise a first site that is susceptible to modification by a modifying agent, wherein such modification is effective to block strand-directed duplication by a polymerase enzyme at said first site;
and
(ii) said nucleic acid molecules comprise different nucleotide sequences in said second region;
(b) providing a set of different ligands;
(c) contacting said set of nucleic acid molecules with said set of different ligands, under conditions suitable for sequence-dependent binding of one or more ligands to one or more nucleic acid molecules, to form a reaction mixture;
(d) treating the reaction mixture of (c) with said modifying agent, under conditions wherein nucleic acid molecules comprising a ligand bound to said second region are substantially protected from modification at said first site, and nucleic acid molecules wherein a ligand is not bound to said second region are substantially modified at said first site;
(e) after said treatment, removing or inactivating said modifying agent;
(f) after said removal or inactivation, separating ligands that bind to said nucleic acid molecules from unbound ligands, and recovering the ligands that bind to said nucleic acid molecules;
and
(g) also after the removal or inactivation of (e), contacting said set of nucleic acid molecules with said polymerase enzyme, under conditions wherein only unmodified nucleic acid strands are amplified;
thereby obtaining a pool of selected ligands that bind in a sequence-dependent manner to one or more nucleotide sequences present in a pool of selected nucleic acid molecules.

9 more elements

A method for screening a ligand for its ability to bind to one or more nucleotide sequences in a set of double-stranded DNA molecules, comprising:

(a) providing a set of double-stranded DNA molecules, each having a first duplex region and a second duplex region, adjacent to said first duplex region, wherein:
(i) said first duplex region comprises, on a first strand of said DNA molecules, a first site comprising a non-standard nucleotide that is susceptible to modification by a modifying agent, wherein said modifying agent is a prokaryotic or eukaryotic repair enzyme that binds to the minor groove of duplex DNA and removes said non-standard nucleotide from duplex DNA;
(ii) said DNA molecules comprise different nucleotide sequences in said second duplex region;
and
(iii) said DNA molecules comprise a second strand comprising one or more non-standard nucleotides that can be modified by the modifying agent;
(b) providing a ligand;
(c) contacting said set of DNA molecules with said ligand, under conditions suitable for sequence-dependent binding of said ligand to said nucleic acid molecule, to form a reaction mixture;
(d) treating the reaction mixture of (c) with said modifying agent, under conditions wherein non-standard nucleotides in said DNA molecules are substantially modified, except for non-standard nucleotides at said first site in DNA molecules comprising ligand bound to said second duplex region;
(e) after said treatment, removing or inactivating said modifying agent;
(f) after said removal or inactivation, contacting the DNA molecules with said polymerase enzyme, under conditions wherein only unmodified first strands are amplified by primer-directed PCR amplification, wherein said amplification indicates that the ligand binds to a nucleotide sequence in said second duplex region;
and
(g) determining the nucleotide sequence of the amplification product in the second duplex region to determine whether the ligand shows binding specificity for one or more nucleotide sequences in said second duplex region.

9 more elements

A method for screening peptide ligands for the ability to bind to one or more nucleotide sequences in a set of double-stranded DNA molecules, comprising:

(a) providing a set of double-stranded DNA molecules linked to a substrate, each having a first duplex region and a second duplex region, adjacent to said first duplex region, wherein:
(i) said first duplex region comprises, on a first strand of said DNA molecules, a first site comprising a non-standard nucleotide that is susceptible to modification by a modifying agent, wherein said modifying agent is a prokaryotic or eukaryotic repair enzyme that binds to the minor groove of duplex DNA and removes said non-standard nucleotide from duplex DNA;
(ii) said DNA molecules comprise different nucleotide sequences in said second duplex region;
and
(iii) said DNA molecules comprise a second strand comprising one or more non-standard nucleotides that can be modified by the modifying agent;
(b) providing a set of different phage particles, wherein each phage particle expresses a heterologous peptide on its surface and comprises a nucleic acid encoding said heterologous peptide;
(c) contacting said set of DNA molecules with said set of phage particles, under conditions suitable for specific binding of one or more heterologous peptides to one or more DNA molecules, to form a reaction mixture;
(d) treating the reaction mixture of (c) with said modifying agent, under conditions wherein non-standard nucleotides in said DNA molecules are substantially modified, except for non-standard nucleotides at said first site in DNA molecules comprising heterologous peptide bound to said second duplex region;
(e) after said treatment, removing or inactivating said modifying agent;
(f) after said removal or inactivation, washing the substrate to separate phage particles that bind to said DNA molecules from unbound phage particles, and recovering the phage particles that bind to said DNA by elution;
and
(g) also after the removal or inactivation of (e), contacting the DNA molecules with said polymerase enzyme, under conditions wherein only unmodified first strands are amplified by primer-directed PCR amplification;
thereby obtaining a pool of selected phage particles expressing heterologous peptides that bind in a sequence-dependent manner to one or more nucleotide sequences present in a pool of selected DNA molecules.

10 more elements

Dependencies are inferred from the claim text.

Nucleic acid binding site assay and selection method

Life of this patent

Abstract

Claims61

Nucleic acid binding site assay and selection method

Life of this patent

Abstract

Claims61