Modular antibodies reveal DNA damage-induced mono-ADP-ribosylation as a second wave of PARP1 signaling

•A broadly applicable technology for sensitive and versatile detection of mono-ADPr•Fluorescent probes reveal serine mono-ADPr as a second wave of PARP1 signaling•Multilevel chromatin proteomics identifies histone mono-ADPr readers•RNF114 is a mono-ADPr reader in telomere maintenance and DNA repair signaling PARP1, an established anti-cancer target that regulates many cellular pathways, including DNA repair signaling, has been intensely studied for decades as a poly(ADP-ribosyl)transferase. Although recent studies have revealed the prevalence of mono-ADP-ribosylation upon DNA damage, it was unknown whether this signal plays an active role in the cell or is just a byproduct of poly-ADP-ribosylation. By engineering SpyTag-based modular antibodies for sensitive and flexible detection of mono-ADP-ribosylation, including fluorescence-based sensors for live-cell imaging, we demonstrate that serine mono-ADP-ribosylation constitutes a second wave of PARP1 signaling shaped by the cellular HPF1/PARP1 ratio. Multilevel chromatin proteomics reveals histone mono-ADP-ribosylation readers, including RNF114, a ubiquitin ligase recruited to DNA lesions through a zinc-finger domain, modulating the DNA damage response and telomere maintenance. Our work provides a technological framework for illuminating ADP-ribosylation in a wide range of applications and biological contexts and establishes mono-ADP-ribosylation by HPF1/PARP1 as an important information carrier for cell signaling. PARP1, an established anti-cancer target that regulates many cellular pathways, including DNA repair signaling, has been intensely studied for decades as a poly(ADP-ribosyl)transferase. Although recent studies have revealed the prevalence of mono-ADP-ribosylation upon DNA damage, it was unknown whether this signal plays an active role in the cell or is just a byproduct of poly-ADP-ribosylation. By engineering SpyTag-based modular antibodies for sensitive and flexible detection of mono-ADP-ribosylation, including fluorescence-based sensors for live-cell imaging, we demonstrate that serine mono-ADP-ribosylation constitutes a second wave of PARP1 signaling shaped by the cellular HPF1/PARP1 ratio. Multilevel chromatin proteomics reveals histone mono-ADP-ribosylation readers, including RNF114, a ubiquitin ligase recruited to DNA lesions through a zinc-finger domain, modulating the DNA damage response and telomere maintenance. Our work provides a technological framework for illuminating ADP-ribosylation in a wide range of applications and biological contexts and establishes mono-ADP-ribosylation by HPF1/PARP1 as an important information carrier for cell signaling. PARP1, a much-studied target for cancer therapy, plays key roles in the DNA damage response (DDR) by covalently transferring ADP-ribose from NAD+ to a target substrate, generating ADP-ribosylation (ADPr).1Gupte R. Liu Z. Kraus W.L. PARPs and ADP-ribosylation: recent advances linking molecular functions to biological outcomes.Genes Dev. 2017; 31: 101-126https://doi.org/10.1101/gad.291518.116Crossref PubMed Scopus (397) Google Scholar For more than 50 years, this enzyme has been studied exclusively in the context of poly-ADPr on aspartate and glutamate.2Cohen M.S. Chang P. Insights into the biogenesis, function, and regulation of ADP-ribosylation.Nat. Chem. Biol. 2018; 14: 236-243https://doi.org/10.1038/Nchembio.2568Crossref PubMed Scopus (0) Google Scholar,3Gibson B.A. Zhang Y. Jiang H. Hussey K.M. Shrimp J.H. Lin H. Schwede F. Yu Y. Kraus W.L. 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HPF1 dynamically controls the PARP1/2 balance between initiating and elongating ADP-ribose modifications.Nat. Commun. 2021; 126675https://doi.org/10.1038/s41467-021-27043-8Crossref Scopus (16) Google Scholar,14Prokhorova E. Agnew T. Wondisford A.R. Tellier M. Kaminski N. Beijer D. Holder J. Groslambert J. Suskiewicz M.J. Zhu K. et al.Unrestrained poly-ADP-ribosylation provides insights into chromatin regulation and human disease.Mol. Cell. 2021; 81: 2640-2655.e8https://doi.org/10.1016/j.molcel.2021.04.028Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar Recent reports have shown the prevalence of cellular mono-ADPr upon DNA damage15Bonfiglio J.J. Leidecker O. Dauben H. Longarini E.J. Colby T. San Segundo-Acosta P. Perez K.A. Matic I. An HPF1/PARP1-based chemical biology strategy for exploring ADP-ribosylation.Cell. 2020; 183: 1086-1102.e23https://doi.org/10.1016/j.cell.2020.09.055Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar,16Hendriks I.A. 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Biol. 2022; 17: 810-815https://doi.org/10.1021/acschembio.2c00091Crossref PubMed Scopus (5) Google Scholar However, its cellular abundance was interpreted as an intermediate in the formation and/or degradation of poly-ADPr, and direct cellular roles of mono-ADPr by HFP1/PARP1 remained unknown. Here, we consider the possibility that monomeric ADPr is a fully fledged histone mark, written and erased by dedicated enzymes, and recognized by specific effectors. To test this hypothesis and enable sensitive, versatile mono-ADPr detection both within and beyond PARP1 signaling, we have developed modular antibodies based on SpyTag technology. Despite the clinical development of PARP inhibitors and their broad biological significance, the chemical nature of ADPr has long hampered our ability to study this PTM. Accordingly, considerable efforts have recently been invested in developing new tools for ADPr research. The Kraus laboratory has pioneered the conversion of protein domains recognizing ADPr into antibody-like reagents.19Gibson B.A. Conrad L.B. Huang D. Kraus W.L. Generation and characterization of recombinant antibody-like ADP-ribose binding proteins.Biochemistry. 2017; 56: 6305-6316https://doi.org/10.1021/acs.biochem.7b00670Crossref PubMed Scopus (58) Google Scholar Moreover, sophisticated approaches have improved the chemical synthesis of ADP-ribosylated substrates,20Liu Q. van der Marel G.A. Filippov D.V. Chemical ADP-ribosylation: mono-ADPr-peptides and oligo-ADP-ribose.Org. Biomol. Chem. 2019; 17: 5460-5474https://doi.org/10.1039/c9ob00501cCrossref PubMed Scopus (11) Google Scholar and our phospho-guided enzymatic strategy has provided antigens for generating broad- and site-specific antibodies.15Bonfiglio J.J. Leidecker O. Dauben H. Longarini E.J. Colby T. San Segundo-Acosta P. Perez K.A. Matic I. An HPF1/PARP1-based chemical biology strategy for exploring ADP-ribosylation.Cell. 2020; 183: 1086-1102.e23https://doi.org/10.1016/j.cell.2020.09.055Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar However, detection of ADPr in cellular contexts is still in its infancy compared with other PTMs. Although it is becoming clear that most PARPs and other transferases conjugate monomeric ADPr,21Vyas S. Matic I. Uchima L. Rood J. Zaja R. Hay R.T. Ahel I. Chang P. Family-wide analysis of poly(ADP-ribose) polymerase activity.Nat. Commun. 2014; 54426https://doi.org/10.1038/ncomms5426Crossref PubMed Scopus (312) Google Scholar researchers still lack a widely applicable toolbox for sensitive detection of this modification in cellular contexts. Given the difficulties in generating conventional anti-ADPr antibodies, most currently available tools are recombinant, either domain-based reagents or phage-display antibodies.15Bonfiglio J.J. Leidecker O. Dauben H. Longarini E.J. Colby T. San Segundo-Acosta P. Perez K.A. Matic I. An HPF1/PARP1-based chemical biology strategy for exploring ADP-ribosylation.Cell. 2020; 183: 1086-1102.e23https://doi.org/10.1016/j.cell.2020.09.055Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar,19Gibson B.A. Conrad L.B. Huang D. Kraus W.L. Generation and characterization of recombinant antibody-like ADP-ribose binding proteins.Biochemistry. 2017; 56: 6305-6316https://doi.org/10.1021/acs.biochem.7b00670Crossref PubMed Scopus (58) Google Scholar,22Nowak K. Rosenthal F. Karlberg T. Bütepage M. Thorsell A.G. Dreier B. Grossmann J. Sobek J. Imhof R. Lüscher B. et al.Engineering Af1521 improves ADP-ribose binding and identification of ADP-ribosylated proteins.Nat. Commun. 2020; 115199https://doi.org/10.1038/s41467-020-18981-wCrossref Scopus (32) Google Scholar We aimed to exploit the recombinant nature of these tools to expand their functionality by applying SpyTag technology.23Zakeri B. Fierer J.O. Celik E. Chittock E.C. Schwarz-Linek U. Moy V.T. Howarth M. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin.Proc. Natl. Acad. Sci. USA. 2012; 109: E690-E697https://doi.org/10.1073/pnas.1115485109Crossref PubMed Scopus (858) Google Scholar In this protein ligation system, the SpyTag peptide and the SpyCatcher domain covalently bond when brought together; hence, antibodies generated as SpyTagged antigen-binding fragments (Fabs) can be ligated to various domains and chemical labels via the SpyCatcher, yielding an expandable library of antibody formats.24Hentrich C. Kellmann S.J. Putyrski M. Cavada M. Hanuschka H. Knappik A. Ylera F. Periplasmic expression of SpyTagged antibody fragments enables rapid modular antibody assembly.Cell Chem. Biol. 2021; 28: 813-824.e6https://doi.org/10.1016/j.chembiol.2021.01.011Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar The availability of the SpyCatcher reagents allows the implementation of this strategy in any biological laboratory. Here, we have developed an entire toolkit for flexible and sensitive detection of mono-ADPr (Figure 1A) by applying affinity maturation and the SpyTag/SpyCatcher system to our previously generated antibodies15Bonfiglio J.J. Leidecker O. Dauben H. Longarini E.J. Colby T. San Segundo-Acosta P. Perez K.A. Matic I. An HPF1/PARP1-based chemical biology strategy for exploring ADP-ribosylation.Cell. 2020; 183: 1086-1102.e23https://doi.org/10.1016/j.cell.2020.09.055Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar and show the utility of these modular antibodies in immunoblotting, immunofluorescence, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), and live-cell imaging. Although detection of ADPr has been significantly advanced by recent introduction of specific tools,15Bonfiglio J.J. Leidecker O. Dauben H. Longarini E.J. Colby T. San Segundo-Acosta P. Perez K.A. Matic I. An HPF1/PARP1-based chemical biology strategy for exploring ADP-ribosylation.Cell. 2020; 183: 1086-1102.e23https://doi.org/10.1016/j.cell.2020.09.055Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar,19Gibson B.A. Conrad L.B. Huang D. Kraus W.L. Generation and characterization of recombinant antibody-like ADP-ribose binding proteins.Biochemistry. 2017; 56: 6305-6316https://doi.org/10.1021/acs.biochem.7b00670Crossref PubMed Scopus (58) Google Scholar,22Nowak K. Rosenthal F. Karlberg T. Bütepage M. Thorsell A.G. Dreier B. Grossmann J. Sobek J. Imhof R. Lüscher B. et al.Engineering Af1521 improves ADP-ribose binding and identification of ADP-ribosylated proteins.Nat. Commun. 2020; 115199https://doi.org/10.1038/s41467-020-18981-wCrossref Scopus (32) Google Scholar,25Challa S. Ryu K.W. Whitaker A.L. Abshier J.C. Camacho C.V. Kraus W.L. Development and characterization of new tools for detecting poly(ADP-ribose) in vitro and in vivo.eLife. 2022; 11e72464https://doi.org/10.7554/eLife.72464Crossref PubMed Scopus (6) Google Scholar important ADPr events have remained undetectable. A striking illustration is that histone mono-ADPr was undetectable in the absence of DNA damage, although the high levels detected in undamaged cells lacking the serine mono-ADP-ribose hydrolase ARH314Prokhorova E. Agnew T. Wondisford A.R. Tellier M. Kaminski N. Beijer D. Holder J. Groslambert J. Suskiewicz M.J. Zhu K. et al.Unrestrained poly-ADP-ribosylation provides insights into chromatin regulation and human disease.Mol. Cell. 2021; 81: 2640-2655.e8https://doi.org/10.1016/j.molcel.2021.04.028Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar,15Bonfiglio J.J. Leidecker O. Dauben H. Longarini E.J. Colby T. San Segundo-Acosta P. Perez K.A. Matic I. An HPF1/PARP1-based chemical biology strategy for exploring ADP-ribosylation.Cell. 2020; 183: 1086-1102.e23https://doi.org/10.1016/j.cell.2020.09.055Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar,26Hanzlikova H. Prokhorova E. Krejcikova K. Cihlarova Z. Kalasova I. Kubovciak J. Sachova J. Hailstone R. Brazina J. Ghosh S. et al.Pathogenic ARH3 mutations result in ADP-ribose chromatin scars during DNA strand break repair.Nat. Commun. 2020; 113391https://doi.org/10.1038/s41467-020-17069-9Crossref PubMed Scopus (16) Google Scholar indicate that it is constantly formed. For broader investigations of mono-ADPr, we have re-engineered the recombinant antibodies generated with our HPF1/PARP1-based chemical biology approach.15Bonfiglio J.J. Leidecker O. Dauben H. Longarini E.J. Colby T. San Segundo-Acosta P. Perez K.A. Matic I. An HPF1/PARP1-based chemical biology strategy for exploring ADP-ribosylation.Cell. 2020; 183: 1086-1102.e23https://doi.org/10.1016/j.cell.2020.09.055Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar To increase their sensitivity and versatility, we applied a modular platform24Hentrich C. Kellmann S.J. Putyrski M. Cavada M. Hanuschka H. Knappik A. Ylera F. Periplasmic expression of SpyTagged antibody fragments enables rapid modular antibody assembly.Cell Chem. Biol. 2021; 28: 813-824.e6https://doi.org/10.1016/j.chembiol.2021.01.011Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar based on the SpyTag system23Zakeri B. Fierer J.O. Celik E. Chittock E.C. Schwarz-Linek U. Moy V.T. Howarth M. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin.Proc. Natl. Acad. Sci. USA. 2012; 109: E690-E697https://doi.org/10.1073/pnas.1115485109Crossref PubMed Scopus (858) Google Scholar to couple multiple features onto Fabs (Figure 1A). Among the formats we obtained for all our antibodies,15Bonfiglio J.J. Leidecker O. Dauben H. Longarini E.J. Colby T. San Segundo-Acosta P. Perez K.A. Matic I. An HPF1/PARP1-based chemical biology strategy for exploring ADP-ribosylation.Cell. 2020; 183: 1086-1102.e23https://doi.org/10.1016/j.cell.2020.09.055Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar site-directed labeling with three copies of horseradish peroxidase (HRP) through SpyTag coupling24Hentrich C. Kellmann S.J. Putyrski M. Cavada M. Hanuschka H. Knappik A. Ylera F. Periplasmic expression of SpyTagged antibody fragments enables rapid modular antibody assembly.Cell Chem. Biol. 2021; 28: 813-824.e6https://doi.org/10.1016/j.chembiol.2021.01.011Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar increased the immunoblotting sensitivity dramatically compared with the conventional IgG format (Figure S1A; Methods S1). We further improved the detection of mono-ADPr by using our serine mono-ADP-ribosylated peptides15Bonfiglio J.J. Leidecker O. Dauben H. Longarini E.J. Colby T. San Segundo-Acosta P. Perez K.A. Matic I. An HPF1/PARP1-based chemical biology strategy for exploring ADP-ribosylation.Cell. 2020; 183: 1086-1102.e23https://doi.org/10.1016/j.cell.2020.09.055Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar for phage-display-based affinity maturation of AbD33204, whose binding affinity was ∼4.4 μM,15Bonfiglio J.J. Leidecker O. Dauben H. Longarini E.J. Colby T. San Segundo-Acosta P. Perez K.A. Matic I. An HPF1/PARP1-based chemical biology strategy for exploring ADP-ribosylation.Cell. 2020; 183: 1086-1102.e23https://doi.org/10.1016/j.cell.2020.09.055Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar to obtain AbD43647, a mono-ADPr-specific SpyTag antibody with a binding affinity of 248 nM and very minor unspecific binding (Figures 1B and S1B–S1K). Even when the cellular levels of poly-ADPr were massively boosted by inhibiting the poly-ADPr eraser PARG, AbD43647 did not show any cross-reactivity toward poly-ADPr (Figures 1C and S2A). An ELISA competition assay indicates that binding of AbD43647 to ADP-ribose requires adenine and a free 2′-hydroxyl group on the adenine-proximal ribose (Figure S2B). The high affinity of AbD43647 together with the HRP-conjugated format for immunoblotting rendered HPF1-dependent histone mono-ADPr detectable in WT cells in the absence of exogenous DNA damage (Figure 1D). The signal was significantly improved when immunoblotting was preceded by immunoprecipitation with AbD43647 IgG, illustrating the synergy of multiple conjugate formats of an affinity-matured antibody to maximize mono-ADPr detection (Figures 1D, S2C, and S2D). Upon H2O2 treatment, mono-ADPr was dramatically reduced in HPF1-KO cells, compared with WT cells (Figures 1E, S2E, and S2F), and restored by HPF1-WT expression in HPF1-KO cells, but not HPF1-E284A (Figure S2G), a catalytically inactive mutant that still interacts with PARP1.12Suskiewicz M.J. Zobel F. Ogden T.E.H. Fontana P. Ariza A. Yang J.C. Zhu K. Bracken L. Hawthorne W.J. Ahel D. et al.HPF1 completes the PARP active site for DNA damage-induced ADP-ribosylation.Nature. 2020; 579: 598-602https://doi.org/10.1038/s41586-020-2013-6Crossref PubMed Scopus (114) Google Scholar Recently, ARH3 has been implicated in ALT-mediated telomere maintenance,14Prokhorova E. Agnew T. Wondisford A.R. Tellier M. Kaminski N. Beijer D. Holder J. Groslambert J. Suskiewicz M.J. Zhu K. et al.Unrestrained poly-ADP-ribosylation provides insights into chromatin regulation and human disease.Mol. Cell. 2021; 81: 2640-2655.e8https://doi.org/10.1016/j.molcel.2021.04.028Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar suggesting a role for mono-ADPr at telomeres. To test this, we used the TRF1-FokI system, which specifically cleaves telomeric DNA to induce telomere DDR.27Cho N.W. Dilley R.L. Lampson M.A. Greenberg R.A. Interchromosomal homology searches drive directional ALT telomere movement and synapsis.Cell. 2014; 159: 108-121https://doi.org/10.1016/j.cell.2014.08.030Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar Our antibodies reveal the formation of mono-ADPr foci at telomeres with WT-TRF1-FokI, but not with the catalytically dead (D450A) mutant. Compared with WT cells, this mono-ADPr was increased in ARH3-KO cells and abrogated in HPF1-KO cells (Figures 1F and S2H). To demonstrate the broad value of AbD43647, we have gone beyond PARP1 signaling and protein ADPr. In untreated cells, AbD43647 gives a clear mitochondrial signal,28Hopp A.K. Teloni F. Bisceglie L. Gondrand C. Raith F. Nowak K. Muskalla L. Howald A. Pedrioli P.G.A. Johnsson K. et al.Mitochondrial NAD(+) controls nuclear ARTD1-induced ADP-ribosylation.Mol. Cell. 2021; 81: 340-354.e5https://doi.org/10.1016/j.molcel.2020.12.034Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar independent of HPF1 or ARH3 and undetectable by AbD33205 (Figures 1G and S2I). Moreover, we tested our antibodies on DNA ADPr.29Schuller M. Butler R.E. Ariza A. Tromans-Coia C. Jankevicius G. Claridge T.D.W. Kendall S.L. Goh S. Stewart G.R. Ahel I. Molecular basis for DarT ADP-ribosylation of a DNA base.Nature. 2021; 596: 597-602https://doi.org/10.1038/s41586-021-03825-4Crossref PubMed Scopus (20) Google Scholar Although AbD33205 detects ADP-ribosylated DNA weakly, AbD43647 gives a strong signal, especially in the HRP-coupled format (Figures 1H and S2J). Thus, the range of applications for our new high-affinity antibody AbD43647 is much wider than for the “anti-protein mono-ADPr” AbD33205.15Bonfiglio J.J. Leidecker O. Dauben H. Longarini E.J. Colby T. San Segundo-Acosta P. Perez K.A. Matic I. An HPF1/PARP1-based chemical biology strategy for exploring ADP-ribosylation.Cell. 2020; 183: 1086-1102.e23https://doi.org/10.1016/j.cell.2020.09.055Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar With these tools for sensitive detection of endogenous mono-ADPr in various applications (Figure 1), we set out to investigate mono-ADPr dynamics in the DDR. To visualize mono-ADPr in living cells, we took advantage of the multiple formats of our SpyTagged recombinant antibodies (Figure 1A) and combined laser irradiation with bead loading, a simple method for introducing proteins into the cells.30Hayashi-Takanaka Y. Yamagata K. Wakayama T. Stasevich T.J. Kainuma T. Tsurimoto T. Tachibana M. Shinkai Y. Kurumizaka H. Nozaki N. et al.Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling.Nucleic Acids Res. 2011; 39: 6475-6488https://doi.org/10.1093/nar/gkr343Crossref PubMed Scopus (175) Google Scholar Reasoning that the recruitment kinetics of mono-ADPr-specific, fluorescence-based probes should reflect the dynamics of DNA damage-induced mono-ADPr in living cells, we conjugated the Fab version of AbD33205 to a fluorescent dye (Figure S3A). A key feature of the Fab format is its small size, allowing the sensor to enter the nucleus after bead loading into the cytoplasm (Figures 2A, S3B, and S3C). To test the specificity of our Fab-based probe, we treated the cells with olaparib and observed that PARP1 inhibition abolished the accumulation of the mono-ADPr sensor at DNA damage sites (Figure S3D). Recruitment of the probe was also abolished in HPF1-KO cells (Figure 2B), corroborating the dependence of serine mono-ADPr on HPF1 in cells (Figure 1E). PARP1 signaling is one of the earliest pathways activated during the DDR, as illustrated by the rapid formation of poly-ADPr upon DNA damage.31Smith R. Lebeaupin T. Juhász S. Chapuis C. D'Augustin O. Dutertre S. Burkovics P. Biertümpfel C. Timinszky G. Huet S. Poly(ADP-ribose)-dependent chromatin unfolding facilitates the association of DNA-binding proteins with DNA at sites of damage.Nucleic Acids Res. 2019; 47: 11250-11267https://doi.org/10.1093/nar/gkz820Crossref PubMed Scopus (31) Google Scholar,32Smith R. Zentout S. Chapuis C. Timinszky G. Huet S. HPF1-dependent histone ADP-ribosylation triggers chromatin relaxation to promote the recruitment of repair factors at sites of DNA damage.2021https://doi.org/10.1101/2021.08.27.457930Crossref Google Scholar Surprisingly, we observed a more gradual mono-ADPr response (Figures 2B and S3D). To directly compare the dynamics of these two forms of ADPr in living cells, we bead loaded the anti-mono-ADPr fluorescent Fab and expressed GFP-WWE, an established probe for poly-ADPr.25Challa S. Ryu K.W. Whitaker A.L. Abshier J.C. Camacho C.V. Kraus W.L. 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