Methods and uses of enzymatic labeling of ADP-ribose containing molecules
AssigneesThe Johns Hopkins University, Cold Spring Harbor Laboratory
InventorsAnthony K. L. Leung, Yoshinari Ando, Robert L McPherson, Mohsen Badiee, Elad Elkayam
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Abstract
Described is ELTA (Enzymatic Labeling of Terminal ADP-ribose) to label free, protein-conjugated, or nucleic acid-conjugated ADP-ribose monomer and polymers at their 2′-OH termini. When coupled with different chemical analogs, ELTA can be used for various applications including fluorescence-based biophysical measurement of PAR-protein interaction, detection of PAR length from cells, and enrichment of ADP-ribosylated peptides for mass spectrometry identification.
Claims28
A method of labeling ADP-ribose comprising:
- providing a monomer or polymer of ADP-ribose;
- and
- incubating the monomer or polymer of ADP-ribose with an enzyme and a label;
- forming a monomer or polymer of ADP-ribose labeled at the 2′OH terminus,
- wherein the enzyme is a 2′-5′-Oligoadenylate Synthetase (OAS) selected from the group consisting of OAS1, OAS2, OAS3, and 2′-5′ Oligoadenylate Synthetase-Like protein (OASL), and
- wherein the label comprises a Cy3 moiety, a Cy5 moiety, a Biotin moiety, an Azido moiety, and/or a 32P moiety.
A method of labeling ADP-ribose comprising:
- providing a protein attached to a monomer or a polymer of ADP-ribose;
- incubating the protein attached to a monomer or a polymer of ADP-ribose with an enzyme and a label;
- forming a protein attached to the monomer or polymer of ADP-ribose labeled at the 2′OH terminus,
- wherein the enzyme is a 2′-5′-Oligoadenylate Synthetase (OAS) selected from the group consisting of OAS1, OAS2, OAS3, and 2′-5′ Oligoadenylate Synthetase-Like protein (OASL), and
- wherein the label comprises a Cy3 moiety, a Cy5 moiety, a Biotin moiety, an Azido moiety, and/or a 32P moiety.
A method of identifying a protein ADP-ribose conjugation site comprising the steps of:
- providing a protein attached to a monomer or polymer of ADP-ribose;
- incubating the cellular proteins with an enzyme that attaches a label at the 2′OH terminus of the monomer or polymer of ADP-ribose wherein the label binds to a separation agent,
- wherein the enzyme is a 2′-5′-Oligoadenylate Synthetase (OAS) selected from the group consisting of OAS1, OAS2, OAS3, and 2′-5′ Oligoadenylate Synthetase-Like protein (OASL), and
- wherein the label comprises a Cy3 moiety, a Cy5 moiety, a Biotin moiety, an Azido moiety, and/or a 32P moiety;
- forming a labeled protein;
- binding the labeled protein to a separation agent;
- purifying the labeled protein;
- identifying a protein ADP-ribose binding site on the labeled protein.
A method of labeling a polymer comprising the steps of:
- providing a polymer comprising an ADP-ribose;
- incubating the polymer with an enzyme and a label;
- and
- forming a polymer comprising an ADP-ribose labeled at the 2′OH terminus,
- wherein the enzyme is a 2′-5′-Oligoadenylate Synthetase (OAS) selected from the group consisting of OAS1, OAS2, OAS3, and 2′-5′ Oligoadenylate Synthetase-Like protein (OASL), and
- wherein the label comprises a Cy3 moiety, a Cy5 moiety, a Biotin moiety, an Azido moiety, and/or a 32P moiety.
Dependencies are inferred from the claim text.