METHODS AND USES OF ENZYMATIC LABELING OF ADP-RIBOSE CONTAINING MOLECULES

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.

Claims

1. 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.

2. The method of claim 1 wherein the ADP-ribose is a monomer.

3. The method of claim 1 wherein the ADP-ribose is a polymer.

4. The method of claim 1 wherein the polymer of ADP-ribose has 100 or more ADP-ribose subunits.

5. The method of claim 1 wherein the polymer of ADP-ribose has less than 100 ADP-ribose subunits.

6. The method of claims 1 wherein the enzyme is 2′-5′-Oligoadenylate Synthetase (OAS).

7. The method of claim 6 wherein the enzyme is selected from the group consisting of OAS1, OAS2, OAS3, 2′-5′ Oligoadenylate Synthetase-Like protein (OASL), their sequence and structural homologues, functional part thereof or a combination thereof.

8. The method of claim 1 wherein the label is a compound having formula VII:

9. The method of claim 1 wherein the monomer or polymer of ADP-ribose labeled at the 2′OH terminus having the structure of Formula I: ##STR00010## wherein R1 is H or (ADP-ribose)n, wherein n≥1; R2 is N, a Cy3 moiety, a Cy5 moiety; and R3 is H, a Cy5, a Biotin, or an Azido moiety; and where P* is P or .sup.32P.

10. The method of claim 1 wherein the monomer or polymer of ADP-ribose labeled at the 2′OH terminus having the structure of Formula V:
ADP-ribose/PAR-X   (V) wherein X is a label of claim 8.

11. The method of claim 1 providing the monomer or polymer of ADP-ribose is in vitro

12. The method of claim 1 providing the monomer or polymer of ADP-ribose is in vivo.

13. A compound of Formula I; ##STR00011## wherein R1 is H or (ADP-ribose)n, wherein n≥1; R2 is N, a Cy3 moiety, a Cy5 moiety; and R3 is H, a Cy5, a Biotin, or an Azido moiety; and where P* is P or .sup.32P.

14. 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.

15. The method of claim 14 wherein the protein is attached to a monomer of ADP-ribose.

16. The method of claim 14 wherein the protein is attached to a polymer of ADP-ribose.

17. The method of claim 14 wherein the polymer of ADP-ribose has 100 or more ADP-ribose subunits.

18. The method of claim 14 wherein the polymer of ADP-ribose has less than 100 ADP-ribose subunits.

19. The method of claims 14 wherein the enzyme is 2′-5′-Oligoadenylate Synthetase 1 (OAS 1).

20. The method of claim 14 wherein the enzyme is selected from the group consisting of OAS1, OAS2, OAS3, 2′-5′ Oligoadenylate Synthetase-Like protein (OASL), their sequence and structural homologues, functional part thereof, or a combination thereof.

21. The method of claim 14 wherein the label is a compound having formula VII:

22. The method of claim 14 wherein the protein attached to the monomer or the polymer of ADP-ribose labeled at the 2′OH terminus having the structure of formula II, formula III, or formula IV: ##STR00012## ##STR00013## ##STR00014## wherein R1 is H or (ADP-ribose)n, wherein n≥1; R2 is N, a Cy3 moiety, a Cy5 moiety; and R3 is H, a Cy5, a Biotin, or an Azido moiety; and where P* is P or .sup.32P.

23. The method of claim 14 wherein the protein attached to the monomer or polymer of ADP-ribose labeled at the 2′OH terminus having the structure of Formula VI:
Target Protein-ADP-ribose/PAR-X   (VI) wherein X is the label of claim 21.

24. The method of claim 14 providing an in vitro protein attached to the monomer or the polymer of ADP-ribose.

25. The method of claim 14 providing an in vivo protein attached to the monomer or the polymer of ADP-ribose monomer.

26. A compound of Formula II, III, or IV; ##STR00015## ##STR00016## ##STR00017## wherein R2 is N, or one or more Cy3, Cy5, or a combination of Cy3/Cy5 moieties; and R3 is H, a Cy5, a Biotin, or an Azido moiety.

27. A compound of Formula (V):
ADP-ribose/PAR-X  (V) wherein Xis a label of claim 8.

28. A compound of Formula (VI):
Target Protein-ADP-ribose/PAR-X  (VI) wherein X is a label of claim 21.

29. 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; 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

30. 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.

31. The method of claim 30 wherein the polymer is selected from the group consisting of a nucleic acid, a protein a peptide, a polymer of ADP-ribose, and a combination thereof.

32. The method of claim 30 wherein the polymer is a nucleic acid.

33. The method of claim 32 wherein the wherein the nucleic acid is single stranded.

34. The method of claim 30 wherein the nucleic acid is PARylated or MARylated.

33. The method of claim 32 wherein the nucleic acid is DNA.

34. The method of claim 32 wherein the nucleic acid is RNA.

35. The method of claim 30 wherein the polymer is a protein or peptide.

36. The method of claim 35 wherein the protein is PARylated or MARylated.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG. 1A-1F illustrates (a) the general scheme of free and protein-conjugated ADP-ribose monomer and polymer, where the 2′OH terminus is indicated in green. (b) The addition of dAMP group onto ADP-ribose and NAD.sup.+, but not iso-ADP-ribose upon incubation with OAS1, poly(I:C) and dATP. (c) Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry analysis of the reaction before and after the incubation of ADP-ribose, dATP, poly(I:C) and OAS1 (d) The addition of dAMP group onto ADP-ribose and poly(ADP-ribose) of different length. (e) The addition of dAMP group onto the catalytic domain of poly(ADP-ribose) polymerase 10(PARP10.sup.CD) as an example for protein-conjugated ADP-ribose monomer or mono(ADP-ribosyl)ated proteins. (f) The addition of dAMP group onto Herpetosiphon aurantiacus PARP (haPARP) as an example for protein-conjugated ADP-ribose polymer or poly(ADP-ribosyl)ated proteins

[0051] FIG. 2A-2C illustrates (a) the structure of NAD.sup.+ and iso-ADP-ribose; (b) the separation of individual PAR chain length by high performance liquid chromatography (HPLC); (c) the SYBR GOLD staining of 6 mer, 10 mer , 15 mer, 21 mer and 25 mer of PAR (left); the labeling of these individual chain length by ELTA using .sup.32P-dATP (right); the quantitation of the signal of radioactivity (bottom).

[0052] FIG. 3A-3D illustrates (a) the labeling of a mixture of 6-23 mers, 10 mer, 15 mer, 20 mer PAR by ELTA with Cy5-dATP. (b) An example of labels that can be used with the ELTA labeling method, including radioactive, fluorescent, biotin, and clickable variations that can be modified at different positions of the dATP molecule. (c-d) An example of using ELTA-modified ADP-ribose for biophysical measurement: (c) Filter binding assay of RNF146 WWE domain binding to 10 mer and 20 mer PAR, which was radiolabeled using ELTA method. (d) Microscale Thermophoresis analysis of RNF146 WWE doming binding to 20 mer PAR, which was labeled using ELTA and Cy5-dATP.

[0053] FIG. 4A-4C illustrates (a) the labeling of a mixture of 6-23 mers, 10 mer, 15 mer, 20 mer by ELTA with .sup.32P-dATP, Cy5-dATP modified at N6 position, Cy5-dATP modified at N7 position. (b) the labeling of PAR with Cy3-dATP by ELTA. (c) the labeling of PAR with biotin-dATP by ELTA.

[0054] FIG. 5A-5E illustrates (a) the addition of dAMP group onto PARP10.sup.CD as an example for mono(ADP-ribosyl)ated proteins using ELTA with dATP conjugated at the 7 position with Cy3 (Cy3-dATP). (b-c) the addition of dAMP group onto PARP10.sup.CD (panel b) as an example for mono(ADP-ribosyl)ated proteins using ELTA with Biotin-14-dATP , a dATP analog with biotin attached at the 6-position of the purine base by a 14-atom linker, using non-ADP-ribosylated protein Bovine Serum Albumin (BSA) as a negative control (panel c). (d) ELTA reaction for labeling PARP10.sup.CD using .sup.32P-dATP with or without 1M denaturant urea. (e) ELTA reaction for labeling ADP-ribose using .sup.32P-dATP under a range of denaturing and detergent conditions.

[0055] FIG. 6A-6B illustrates the detection of ADP-ribose length using ELTA. (a) A sequencing gel of the ELTA labeling reaction of ADP-ribose monomer (lane 1), PAR (lane 2), and of ADP-ribose monomer and polymer isolated from in vitro ADP-ribosylated PARP1 by NAD.sup.+ in the presence of the PARP inhibitor Olaparib (lane 3), by NAD.sup.+ for 10 min in the absence of Olaparib (lane 4) and by NAD.sup.+ for 30 min (lane 5). As a comparison, the ADP-ribose monomer and polymer isolated from in vitro ADP-ribosylated PARP1 by NAD+ with a trace of .sup.32P- NAD+ in the presence of Olaparib for 30 min (lane 6), by NAD.sup.+ for 10 min in the absence of Olaparib (lane 7) and by NAD.sup.+ for 30 min (lane 8). (b) A sequencing gel of the ELTA labeling reaction of ADP-ribose monomer and polymer isolated from in vitro ADP-ribosylated haPARP (lane 1), from untreated cancer cells (lane 2), from cancer cells with DNA damage (lane 3), from cancer cells with DNA damage and treated with PARP inhibitor (lane 4) and from cancer cells with DNA damage and treated with PARG inhibitor (lane 5).

[0056] FIG. 7A-7E illustrates an example for the use of ELTA to enrich ADP-ribosylated substrates. (a) the schematics of the pipeline. Panel (b) demonstrates the m/z ratio of peptide (1) after ELTA labeling reaction, (2) in the unbound fraction from the solid support, (3) after elution with phosphodieaterse. Red lines indicate samples with OAS1 and black lines indicate samples without OAS1. (c) The total intensity chromatograph (TIC) of the lysate input sample (left) and of the enriched sample after elution (middle) and the mass spectrometry scan analyses at retention time of 6.36 minutes. (d) Tandem Mass spectrometry (MS/MS) spectra of ADP-ribosylated peptide with the modification site signified by the addition of a phosphoribose group and (e) Quantification of peptide-spectrum match (PSM) of the nanomole, picomole and femtomole of ADP-ribosyalted peptide dosed in milligram of cell lysate peptides.

[0057] FIG. 8A-8F illustrates the identification of endogenous ADP-ribosylated substrates. Quantification of (a) PSM and (b) MaxQuant Score of ADP-ribosylated peptide identified from 1 mg, 5 mg and 20 mg HeLa cell lysate from DNA damage condition (H.sub.2O.sub.2). The distribution of site localization probability and ADP-ribosylated amino acid are presented in panel c and d. Examples of MS/MS spectra of endogenous (e) POP1 ADP-ribosylated Ser 24 and (f) hnRNPU Lysine 814.

[0058] FIG. 9 illustrates labeling MARylated and PARylated ssDNA using ELTA. DNA-PAR-Cy5 separated using Urea-PAGE (15%) and visualized by scanning for Cy5 signal using phosphoimager. Sequence of ssDNA is TGG CGA CGG CAG CGA TCTC. Same condition was used to label MARylated and PARylated DNA.

[0059] FIG. 10 illustrates human PARP1 (hPARP1) catalyzes the ADP-ribosylation of MARylated DNA in concentration-dependent manner. Decreasing hPARP1 concentration shifts PARylation profile to shorter length of PAR as shown in on Urea-PAGE (15%). Concentration of hPARP1 is decreasing from lane 2 to 7 from 2.8 to 0.1 μM. Sequence of ssDNA is TGG CGA CGG CAG CGA TCTC. Gel is visualized using SYBR gold and all reaction contains PARylation buffer, dsDNA activator, spermine.

DETAILED DESCRIPTION OF THE INVENTION

[0060] ADP-ribosylation—the addition of one or more ADP-ribose groups onto proteins—is a therapeutically important but understudied protein modification. The attached ADP-ribose monomer or polymer, commonly known as poly(ADP-ribose) (PAR), modulate the activities of the modified substrates and/or their binding affinity to other proteins. However, there is a lack of tools to investigate this protein modification or PAR as a polynucleotide. Here, the inventors describe ELTA (Enzymatic Labeling of Terminal ADP-ribose) for the labeling of free, protein-conjugated, or nucleic acid-conjugated ADP-ribose monomer and polymers at the 2′-OH terminus. When coupled with a diverse range of chemical tags (e.g., radioactive, fluorescent, biotin-tag, clickable functional groups), ELTA can be used to adapt techniques routinely used to investigate DNA or RNA functions to explore PAR biology. Here the inventors demonstrate that ELTA enables the quantitative measurement of PAR binding to proteins in vitro, detection of endogenous PAR from cells and enrichment of ADP-ribosylated peptides from complex samples.

The Basis of ELTA: OAS1 Adds dATP onto 2′-OH Termini of ADP-Ribose Monomers and Polymers

[0061] To label ADP-ribose and its derivatives at the 2′-OH terminus (FIG. 1a, green), the inventors propose to use the enzyme 2′-5′-Oligoadenylate Synthetase 1 (OAS1). OAS1 is activated by double-stranded RNAs and oligomerizes ATP into 2′-5′ linked oligoadenylate, where the α-phosphate of the donor ATP is linked to the 2′-OH of the acceptor ATP, resulting in the addition of an AMP moiety onto the acceptor ATP in a 2′-5′ linkage. Previous data suggested that OAS1 may have the potential to be used as a labeling tool. First, OAS1 has been shown to link donor ATP not only to an acceptor ATP, but also onto 2′-OH of molecules that contain adenosine as acceptors, including NAD.sup.+20,21 and ADP-ribose.sup.22. Second, OAS1 can use other NTPs such as deoxy-ATP (dATP) as donors. We reasoned that the lack of 2′-OH group in dATP results in chain termination and, therefore, dATP would be an ideal donor substrate of OAS1 to end-label the 2′-OH terminus of free and protein-conjugated ADP-ribose monomers or polymers. However, it is unclear whether dATP can be added onto these acceptor molecules.

[0062] To test whether OAS1 can add dATP onto ADP-ribose (ADPr) in vitro, recombinant OAS1 was incubated with α-.sup.32P-dATP and ADPr (FIG. 1b). As expected, a major product running at the molecular weight of dAMP+ADPr was observed. As expected, dAMP can be added onto 2′-OH group of NAD.sup.+; however, the internal unit of PAR, iso-ADP-ribose, which does not have a free 2′-OH in the adenosine-linked ribose, cannot be served as an acceptor even though a similar concentration was used (as indicated by A260 value; FIG. 1b and 2a). To further confirm the addition of dAMP moiety onto ADP-ribose by OAS1, MALDI-TOF analyses were performed (FIG. 1c). In the absence of OAS1, dATP and ADP-ribose were observed at m/z of 492.0 and 560.0, respectively. In the presence of OAS1, an additional peak was observed at m/z of 873.0, which corresponds to the expected molecular weight of ADP-ribose-dAMP.

[0063] Given that the internal unit of PAR cannot be labeled (FIG. 1b) but each linear PAR chain has one 2′-OH terminus (cf. FIG. 1a), we next investigated whether PAR can also be end-labeled by incubating OAS1 and .sup.32P-dATP with different chain lengths of ADP-riboseshort (≤15 mers), medium (15-40 mers) and long (≥40 mers) (FIG. 1d). In each case, these PAR molecules of different lengths were effectively labeled. To test whether this OAS method can label PAR chain of different length with comparable efficiency, we optimized a purification method to efficiently separate PAR to defined chain length (FIG. 2b). The inventors then used the same .sup.32P-dATP-OAS1 method to label the defined length PAR chains. As expected, these PAR chains of different length were labeled with comparable efficiency (FIG. 2c). Taken together, OAS1 can label both ADP-ribose monomer and polymers at their 2′-OH termini using radiolabeled dATP.

OAS1 Adds dATP Analogs onto Protein-Conjugated ADP-Ribose

[0064] As ADP-ribose can be conjugated to protein at the 1″ terminus, the inventors reasoned that the free 2′-OH group of protein-conjugated ADP-ribose could also be served as substrates of OAS1. Using automodified PARP-10 catalytic domain (PARP10.sup.D) or haPARP as model substrates for mono(ADP-ribosyl)ated (MARylated) and poly(ADP-ribosyl)ated (PARylated) substrates, respectively, the inventors tested whether these ADP-ribosylated substrates can be labeled by OAS1 with .sup.32P-dATP (FIG. 1e, f). As a negative control, we pre-treated the ADP-ribosylated substrates with hsNudT16, which cleaves the pyrophosphate bond within ADP-ribose (cf. FIG. 1a) to leave a phosphoribose tag on previously ADP-ribosylated proteins. As a result, the 2′-OH containing adenosine is removed and these phosphoribosylated proteins are no longer recognized by Af1521 macrodomain (pan-ADPr reagent.sup.27) nor OAS1 for labeling. On the contrary, MARylated PARP10.sup.CD and PARylated haPARP were labeled by .sup.32P-dATP in the presence of OAS1, and such signals were quantitatively reduced in the sample pre-treated with hsNudT16 (FIG. 1e, f). Therefore, this OAS1-based method enables the inventors to effectively label both protein-conjugated ADP-ribose monomer and polymers.

OAS1 is a Versatile Enzyme that Accepts Different Modified dATP Analogs for Labeling

[0065] Next, the inventors investigated whether the OAS1 enzymatic labeling reaction can be extended to the labeling of ADP-ribose with modified dATP analogues (e.g., fluorescent, biotin-tag, clickable functional groups). Following a similar labeling protocol, we used Cy5-dATP attached at two different positions on the adenine base (6′ and 7′; FIG. 3a,b and 4a). The inventors were able to efficiently label 3 different PAR chains lengths (10 mer, 15 mer, and 20 mer) and a mixture of 6-23 mer PAR chains with the two Cy5 labels (FIG. 4a). OAS1 labeling can also be performed with other fluorescent derivatives such as Cy3 (FIG. 4b) and a biotinylated dATP modified on the 6′ position via a 14-carbon linker (FIG. 4c). Similarly, this labeling reaction can also be performed on protein-conjugated ADP-ribose using modified dATP as well as performed in a range of detergent and denaturant conditions (FIG. 5).

[0066] Given the versatility of OAS1 to label free and protein-conjugated ADP-ribose using a range of modified dATP analogues, the inventors named this general method to enzymatically label terminal ADP-ribose as ELTA. Using several proof-of-concept applications, the inventors will illustrate below how to use ELTA for measurement, detection and enrichment in ADP-ribosylation biology.

Labeled PAR Chain Can be Used to Measure the Affinity of PAR Binding to Protein In Vitro

[0067] Poly(ADP-ribosyl)ation (PARylation) of target proteins regulates many biological processes. For example, PAR recognition by WWE domain of RNF146 is required for the poly-ubiquitination and subsequent degradation of axina key regulator of Wnt signaling pathway. PAR-protein binding affinity measurement for RNF146 WWE has been evaluated either with a mixed population of PAR or with the internal unit iso-ADP-ribose as a proxy using isothermal titration calorimetry (ITC), which requires a significant amount of materials (typically at μM). PAR-binding affinity has also been assayed using nanomolar amounts of fluorescently labeled PAR by fluorescent polarization, or biotinylated PAR by electrophoretic mobility shift assay or surface plasmon resonance. However, both methods require PAR synthesis and conjugation through chemical methods, which are not readily accessible to most molecular biology laboratories. Here, by combining ELTA with the inventors' improved single chain PAR purification, the inventors were able to measure the affinity of a single length PAR chain to the WWE domain of RNF146. First, the inventors used a filter-binding assay and measured the affinity of radiolabeled 10 and 20 mer PAR to RNF146 (FIG. 3c). Both PAR chains bind tightly to RNF146 WWE domain with affinities of 75nM (10 mer) and 67 nM (20 mer), which are comparable with the affinity reported for the RNF146 WWEiso-ADP-ribose binding. The inventors further extended the repertoire of biophysical measurement of the PAR binding assays by using the commonly used Cy5 fluorescence dye and measured the affinity Cy5-PAR to RNF146 using Microscale Thermophoresis (MST). The binding affinity was similar to the affinity measured using the filter binding assays (FIG. 3d).

ELTA Labels PAR Isolated In Vitro and from Cells for Assessing Polymer Length

[0068] The number of ADP-ribose units attached to proteins is dynamically regulated within cells and notably, protein binding to PAR is dependent on the length of PAR chain. However, it is not possible to examine the length of PAR from modified substrates without prior labeling (e.g., using radioactive NAD.sup.+). Given that ELTA can effectively label pre-made PAR, the inventors tested whether this labeling technique can be used to assess the chain length of PAR isolated from modified substrates and compared with the existing technique that requires prior labeling. To examine the labeling efficiency of PAR isolated from PARylated PARP1, the inventors modified PARP1 with 1 mM NAD.sup.+ with or without a trace of .sup.32P-NAD.sup.+for either 0, 10, 30 min. PAR was extracted from both non-radioactive and radioactive samples. The non-radioactive samples were further labeled with OAS1 and .sup.32P-dATP. All samples were then run on a 15% denaturing urea gel for autoradiography. As shown in FIG. 6a, the patterns of isolated PAR sample labeled by .sup.32P-NAD.sup.+ and .sup.32P-dATP are comparable in length distribution. However, the inventors note that ELTA is more sensitive: though the .sup.32P-dATP and .sup.32P-NAD.sup.+ labeled PAR appear similar in intensity, the inventors loaded 50-fold less PAR in lanes 3, 4 and 5. Therefore, ELTA provides a sensitive approach to assess the polymer length of a PARylated protein without prior labeling.

[0069] Next, the inventors tested whether ELTA can be used to examine PAR isolated from cells (FIG. 6b). PAR was retrieved from cells using a solid-phase extraction method that allows for the high recovery of PAR without any bias of different chain length. HaCaT cells were either untreated, H.sub.2O.sub.2-treated to induce PARylation, pre-treated with PARP inhibitor Olaparib prior to H.sub.2O.sub.2 induction of PAR, or pre-treated with inhibitor against the PAR-degrading enzyme PARG (PDD 00017273) prior to H.sub.2O.sub.2 induction of PAR. As a control, the inventors also isolated PAR from in vitro modified Herpetosiphon aurantiacus PARP (haPARP), which usually generates PAR of 2-25 mer (lane 1), using the same solid-phase extraction method. As expected, we observed significant PAR signals in H.sub.2O.sub.2-treated cell samples (FIG. 6b; lane 3), which is longer from the one isolated from haPARP (lane 1). PARP inhibitor Olaparib (lane 4) reduced the signal to background level as in the untreated cells (lane 2). On the contrary, PARG inhibitor resulted in the enrichment of overall signals and, intriguingly, stronger signals were observed at a shorter range at the tested conditions (lane 5). Taken together, ELTA can assess the length of PAR isolated in vitro or from cells.

ELTA Allows for Selective Labeling and Enrichment of ADP-Ribosylated Peptides

[0070] Building on our findings that ELTA can be used to label protein-conjugated ADP-ribose (c.f. FIG. 1e, f); we sought to develop a workflow for selective labeling and enrichment of ADP-ribosylated peptides from cell lysates for downstream proteomic analysis. The inventors propose a pipeline composed of three steps (FIG. 7a): 1) the labeling of ADP-ribosylated peptides with the “clickable” dATP analogue, N.sup.6-(N-azido)hexyl-dATP, by OAS1, 2) conjugation of OAS1-labeled peptides to Dibenzocyclooctyne (DBCO)-agarose through ring-strain promoted copper-free click chemistry, followed by subsequent pulldown, and 3) treatment of agarose-conjugated peptides with hsNudT16 phosphodiesterase to release peptides, now possessing a phosphoribose tag at former site of ADP-ribosylation, for downstream analysis.

[0071] First, the inventors tested whether OAS1 can label an ADP-ribosylated peptide, HK533, with N.sup.6-(N-azido)hexyl-dATP and analyzed the reaction product by MALDI-TOF. As expected, a peak shift of 438.9 Da was observed (FIG. 7b, panel 1), which corresponds to N.sup.6-(N-azido)hexyl-dAMP. Next, we incubated this reaction with DBCO-agarose to allow for the conjugation of OAS1-labeled HK533 to the resin. The inventors observed that there are no peaks in the mass range of OAS1-labeled HK533 in the unbound fraction, suggesting that a large proportion of the OAS1-labeled HK533 was conjugated to and pulled down by the DBCO-agarose (FIG. 7b, panel 2). Consistently, the unlabeled HK533 peptide were present in the unbound fraction of the sample not treated with OAS1, but absent in the OAS1-treated samples. These data indicate that unlabeled HK533 is not significantly retained by the DBCO-agarose (FIG. 7b, panel 2). To confirm the conjugation of OAS1-labeled HK533 peptide with the resin, the inventors treated the peptide-conjugated DBCO-agarose resin with hsNudT16 phosphodiesterase (cf. FIG. 1e, f; FIG. 7a). The inventors observed a defined peak at the expected m/z for phosphoribosylated HK533 (observed m/z 1530.0; expected m/z 1529.8) in the eluent of the peptide sample treated with OAS1 and no significant peaks in the sample without OAS1 (FIG. 7b, panel 3). Together these results demonstrate the feasibility of this workflow for the labeling and enrichment of ADP-ribosylated peptides.

[0072] Next, the inventors assessed the robustness of this workflow in cell lysate to determine if it could be applied to enrich for and identify endogenous ADP-ribosylated substrates by liquid chromatography tandem mass spectrometry (LC-MS/MS). The inventors spiked 1 nmol of HK533 into 1 mg of a tryptic digest of HeLa cell lysate and used this complex peptide mixture as the input for the workflow. LC-MS/MS analysis of the input and eluent demonstrated that the workflow resulted in the robust enrichment of molecular species with a retention time of approximately 6.5 min (FIG. 7c, panels 1 and 2). MS analysis of this peak demonstrated that it was predominantly composed of two species that matched the expected m/z for phosphoribosylated HK533 in the +2 and +3 charge states (FIG. 7c, panel 3). Subsequent MS/MS analyses confirmed the sequence and site localization of HK533 (FIG. 7d). The inventors then applied this workflow to identify endogenous sites of ADP-ribosylation from cells. The inventors induced ADP-ribosylation by treatment of HeLa cells with H.sub.2O.sub.2, followed by cell lysis, protein isolation and protease digestion to peptides. After desalting, the inventors used 5 mgs of peptides as input for the ELTA-based enrichment workflow and analyzed the eluents by high-resolution LC-MS/MS for ADP-ribosylated peptide. The inventors' MS analyses resulted in the identification of 180 endogenous ADP-ribosylated sites, where many of these ADP-ribosylated proteins have been identified in previous proteomics studies (FIG. 8)

[0073] The inventors discovered a novel enzymatic approach to label the 2′ OH terminus of ADP-ribose monomer and polymers. Though it is possible to label ADP-ribose at 1″ aldehyde.sup.16, the current method requires a large amount of materials for good yield, a special setup for chemical reactions and takes multiple days compared with 2 hours for ELTA. In addition, ELTA has now made it possible for labeling ADP-ribose that is conjugated to protein at 1″ position (cf. FIG. 1a). Given OAS1 can use a range of dATP analogs, ELTA can serve as a research platform being applied to various techniques commonly used in molecular biology labs that require the derivation of the assayed molecule (e.g., radioactive, fluorescent, biotinylated, etc.). The inventors have demonstrated several proof-of-concept applications: (1) the use of fluorescently labeled PAR of defined chain length for biophysical measurements; (2) radiolabelling of PAR isolated from cells allows the assessment of polymer length; (3) the use of an azide tag to directly conjugate ADP-ribosylated peptides onto agarose bead for enrichment of ADP-ribosylated substrates for mass spectrometry identification. The inventors noted that the azido labeling also allows us to tap into the versatility of click chemistry for bioconjugation to any compatible substrates. Given that the synthetic routes of various ATP derivatives have already been established.sup.44, it is foreseeable that similar dATP analogs can be made and applied to ELTA for new applications. For example, adding fluorine-19 for making an NMR probe or a diazirine group for a photoaffinity probe. On the other hand, an additional use of ATP analog may allow signal amplification. When combined with the existing chemical approach to label PAR at 1″ position, we can now attach functionality at both termini. Such dual labeling approach has been transformative for investigating DNA and RNA biology, and, therefore, ELTA may now open the possibility for PAR to serve as a building block in synthetic biology (e.g., creating model substrates with a defined length of PAR). In addition, given that ELTA labels 2′-OH terminus and that some PARPs create branchpoints resulting in additional 2′-OH groups from a single 1″ terminus, labeling at both 1″ and 2′ ends may determine the branching frequency of intact PAR polymer isolated in vitro or from cells.

[0074] Currently, ELTA can effectively label femtomole levels of ADP-ribose in a complex background, thereby allowing for efficient labeling of limited materials for downstream analyses. The inventors have demonstrated the use of fluorescent PAR of defined length for measurement of the equilibrium dissociation constant with a PAR-binding module using ITC and MST, but these labeled molecules can also be used for single molecule-based measurement.sup.45 or intracellular tracking of PAR. Several methods have been developed to measure the length of PAR chain from cells; however, the majority of them requires the digestion of PAR into monomeric nucleosides prior to analyses, thereby losing the information of length distribution of intact PAR chain. Alternatively, it is possible to measure the length of intact PAR chain from cells by feeding radiolabeled adenine. However, this approach suffers several drawbacks, including non-specific labeling of other polynucleotides (DNA and RNA) and potential induction of PAR signals by the radiodamage of DNA. Using ETLA, we found that PAR length distribution reduced significantly in H.sub.2O.sub.2-treated cells by PARP inhibitors and, unexpectedly, the accumulation of shorter PAR upon PARG inhibition, which might have therapeutic implications. Notably, FDA-approved PARP inhibitors Niraparib and Olaparib accumulate DNA damage only when PAR level is reduced down to >90%. One possible interpretation is that a longer PAR chain can still result in DNA repair, but not with a shorter one, consistent with the previous observation that DNA repair factors bind to PAR in a length-selective manner. Therefore, it will be interesting to explore whether PAR length distribution could be an important biomarker for clinical effectiveness of these inhibitors.

[0075] The inventors demonstrated that ELTA labels free and protein-conjugated ADP-ribose as well as several examples of its applications. However, this novel technique may have broader applications on ADP-ribose metabolism or adenosine-containing molecules that possess free 2′OH terminus. Besides protein-conjugated ADP-ribose, studies in prokaryotes and eukaryotes have revealed several ADP-ribose derivatives, including O-acetyl-ADP-ribose by the sirtuin deacetylase family, ADP-ribose-1″-phosphate from tRNA splicing. ADP-ribosylation of the antibiotics rifamycin, as well as the recently discovered DNA ADP-ribosylation. In humans, ADP-ribosylation is catalyzed by poly(ADP-ribose) polymerases (PARPs), which consists of 17 members PARPs covalently attach the ADP-ribose (ADPR) unit to all polar residues, including asparate, glutamate, serine, cysteine, lysine, arginine, histidine and tyrosine. However, most of them are only able to transfer single mono(ADP-ribose) (MAR) group onto their target proteins. To date PARP1, 2, and 3 have been identified to catalyze PARylation during DNA damage response (DDR). In addition, tankyrases including tankyrase-1 (PARP5a) and lanky rase-2 (PARP5b) have also been shown to contribute to genomic stability. Among these PARPs, PARP1 is the founding member of PARP family for the synthesis of PAR chains. The mechanism of PARP1 activation by single-strand and double-strand DNA breaks (SSBs and DSBs) is well established. Using NAD.sup.30 as substrate, PARPs repeatedly catalyze the transfer of successive units of ADPR moieties via a unique 2′1″-O-glycosidic ribose-ribose bond to target proteins, finally producing PAR chain. Several reports have demonstrated that PAR chains can comprise up to 200 ADPR units in length. In addition, PARP1 can introduce branching into PAR chains through the 2′, 1″-glycosidic bond.

[0076] To illustrate the ability of ELTA to labeled MARylated and PARylated DNA, the inventor made MARylated DNA using the bacterial toxin DarT, which modifies the second thymidine in a single stranded DNA containing a TNTC motif. The inventor also developed a novel protocol to make PARylated DNA by incubating MARylated DNA with the human enzyme PARP1. NAD.sup.+, double-stranded DNA and spermine. The MARylated and PARylated DNA were both labeled by ELTA (FIG. 9 and 10). ELTA may, therefore, provide a timely tool for discovering the functions of these various forms of ADP-ribosylation in antibiotic resistance, as well as NAD.sup.+, RNA and DNA metabolism.

Kits

[0077] Any of the compositions described herein may be comprised in a kit. In a non-limiting example, labeled ADP-ribose/PAR, 2′OH labelling enzyme such as OAS, and/or a label, may be comprised in a kit.

[0078] The kits may comprise a suitably aliquoted of these components and, in some cases, one or more additional agents. The component(s) of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the compositions of the present invention and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

[0079] When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. The composition(s) of the present invention may be formulated into a syringeable composition. In which case, the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and/or even applied to and/or mixed with the other components of the kit.

[0080] However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.

[0081] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0082] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0083] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.