Ni-NTA bead-based real-time monitoring of PARPs inhibitor binding
A. A. Lobanova,
O. V. Saulina,
O. V. Geraskina, 
D. O. Koshkina,
N. V. Maluchenko,
A. V. Feofanov,
V. M. Studitsky
D. O. Koshkina,
N. V. Maluchenko,
A. V. Feofanov,
V. M. Studitsky https://doi.org/10.55959/MSU0137-0952-16-80-3S-11
Abstract
This study establishes a real-time fluorescence microscopy platform for visualizing ligand binding dynamics to His-tagged proteins bound to Ni-NTA agarose beads. By preserving solution-phase kinetics while enabling sub-minute temporal resolution in physiological buffers, the methodology overcomes critical limitations of surface-based techniques and gel electrophoretic methods. We applied this platform to investigate inhibitor action within a nucleosomal system, a more physiologically relevant context than free DNA. Through studies of PARP2-nucleosome interactions modulated by clinical inhibitors (talazoparib, veliparib) and by reaction of poly(ADP-ribosyl)ation in the presence of NAD+, we demonstrate direct spatial and temporal resolution of chromatin-protein dynamics. The platform’s virtually unlimited buffer compatibility, real-time monitoring capabilities, and elimination of covalent immobilization artifacts provide transformative insights into drug mechanisms and chromatin engagement processes.
Supporting agencies: This work was supported by the Russian Science Foundation, project no. 21-64-00001-p.
About the Authors
A. A. Lobanova
School of Biology, Lomonosov Moscow State University
Russian Federation
Russian Federation
1–12 Leninskie gory, Moscow, 119234
O. V. Saulina
School of Biology, Lomonosov Moscow State University
Russian Federation
Russian Federation
1–12 Leninskie gory, Moscow, 119234
O. V. Geraskina
Institute of Gene Biology, Russian Academy of Sciences
Russian Federation
Russian Federation
34/5 Vavilov Str., Moscow, 119334
D. O. Koshkina
School of Biology, Lomonosov Moscow State University
Russian Federation
Russian Federation
1–12 Leninskie gory, Moscow, 119234
N. V. Maluchenko
School of Biology, Lomonosov Moscow State University
Russian Federation
Russian Federation
1–12 Leninskie gory, Moscow, 119234
A. V. Feofanov
School of Biology, Lomonosov Moscow State University; Institute of Gene Biology, Russian Academy of Sciences
Russian Federation
Russian Federation
1–12 Leninskie gory, Moscow, 119234
34/5 Vavilov Str., Moscow, 119334
V. M. Studitsky
Fox Chase Cancer Center
United States
United States
333 Cottman Ave., Philadelphia, 19111, Pennsylvania
References
1. Luger K., Hansen J.C. Nucleosome and chromatin fiber dynamics. Curr. Opin. Struct. Biol. 2005;15(2):188–196.
2. Luger K., Dechassa M.L., Tremethick D.J. New insights into nucleosome and chromatin structure. Curr. Opin. Struct. Biol. 2012;13(7):436–447.
3. Langelier M.F., Planck J.L., Roy S., Pascal J.M. Structural basis for DNA damage-dependent poly(ADP-ribosyl) ation by human PARP-1. Science. 2012;336(6082):728–732.
4. Lakowicz J.R. Principles of fluorescence spectroscopy. 3rd ed. N.Y.: Springer; 2006. 954 pp.
5. Maluchenko N.V., Nilov D.K., Pushkarev S.V., Kotova E.Y., Gerasimova N.S., Kirpichnikov M.P., Langelier M.F., Pascal J.M., Akhtar M.S., Feofanov A.V., Studitsky V.M. Mechanisms of nucleosome reorganization by PARP1. Int. J. Mol. Sci. 2021;22(22):12127.
6. Maluchenko N., Saulina A., Geraskina O., Kotova E., Korovina A., Armeev G., Kirpichnikov M., Feofanov A., Studitsky V. Poly(ADP-ribose)polymerase 2 is zinc-dependent enzyme and nucleosome reorganizer. Cell. Mol. Life Sci. 2025;82(1):267.
7. Maluchenko N., Koshkina D., Korovina A., Studitsky V., Feofanov A. Interactions of PARP1 inhibitors with PARP1-nucleosome complexes. Cells. 2022;11(21):3343.
8. Hellman L.M., Fried M.G. Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nat. Protoc. 2007;2(8):1849–1861.
9. Pascal J.M. The comings and goings of PARP-1 in response to DNA damage. DNA Repair (Amst). 2018;71:177–182.
10. Riccio AA, Cingolani G, Pascal JM. PARP-2 domain requirements for DNA damage-dependent activation and localization to sites of DNA damage. Nucleic Acids Res. 2016;44(4):1691–1702.
11. Murai J., Huang S.Y., Das B.B., Renaud A., Zhang Y., Doroshow J.H., Ji J., Takeda S., Pommier Y. Trapping of PARP1 and PARP2 by clinical PARP inhibitors. Cancer Res. 2012;72(21):5588–5599.
12. Krüger A., Bürkle A., Hauser K., Mangerich A. Real-time monitoring of PARP1-dependent PARylation by ATR-FTIR spectroscopy. Nat. Commun. 2020;11(1):2174.
13. Kurgina T.A., Anarbaev R.O., Sukhanova M.V., Lavrik O.I. A rapid fluorescent method for the real-time measurement of poly(ADP-ribose) polymerase 1 activity. Anal. Biochem. 2018;545:91–97.
14. Langelier M.F., Riccio A.A., Pascal J.M. PARP-2 and PARP-3 are selectively activated by 5′ phosphorylated DNA breaks through an allosteric regulatory mechanism shared with PARP-1. Nucleic Acids Res. 2014;42(12):7762–7775.
15. Luger K., Mäder A.W., Richmond R.K., Sargent D.F., Richmond T.J. Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature. 1997;389(6648):251–260.
16. Richmond T.J., Davey C.A. The structure of DNA in the nucleosome core. Nature. 2003;423(6936):145–150.
17. Luger K., Rechsteiner T.J., Richmond T.J. Preparation of nucleosome core particle from recombinant histones. Chromatin. Methods in Enzymology, vol. 304. Eds. P.M. Wassarman and A.P. Wolffe. Academic Press; 1999:3–19.
18. Wasserberg D., Cabanas-Danés J., Prangsma J., O’Mahony S., Cazade P.A., Tromp E., Blum C., Thompson D., Huskens J., Subramaniam V., Jonkheijm P. Controlling protein surface orientation by strategic placement of His-tags. ACS Nano. 2017;11(9):9350–9361.
19. Wu C.C., Reinhoudt D.N., Otto C., Velders A.H., Subramaniam V. Protein immobilization on Ni(II) ion patterns prepared by microcontact printing and dip-pen nanolithography. ACS Nano. 2010;4(2):1083–1091.
20. Castro-Hinojosa C., Del Sol-Fernández S., Moreno-Antolín E., Martín-Gracia B., Ovejero J.G., de la Fuente J.M., Grazú V., Fratila R.M., Moros M. A simple and versatile strategy for oriented immobilization of Histagged proteins on magnetic nanoparticles. Bioconjug. Chem. 2023;34(12):2275–2292.
21. Zandarashvili L., Langelier M.F., Velagapudi U.K., Hancock M.A., Steffen J.D., Billur R., Hannan Z.M., Wicks A.J., Krastev D.B., Pettitt S.J., Lord C.J., Talele T.T., Pascal J.M., Black B.E. Structural basis for allosteric PARP-1 retention on DNA breaks. Science. 2020;368(6486):eaax6367.
22. Lobanova A.A., Korovina A.N., Koshkina D.O., Chernikova P.A., Feofanov A.V., Studitsky V.M., Nilov D.K., Maluchenko N.V. Gel electrophoresis as a method for classifying inhibitors of poly(ADP-ribose) polymerases 1 and 2. Mosc. Univ. Biol. Sci. Bull. 2024;79(4):239–245.
23. Langelier M.F., Steffen J.D., Riccio A.A., McCauley M., Pascal J.M. Purification of DNA damage-dependent PARPs from E. coli for structural and biochemical analysis. Poly(ADP-Ribose) Polymerase. Methods in Molecular Biology, vol 1608. N.Y.: Humana Press; 2017:431–444.
24. Koshkina D., Maluchenko N., Nilov D., Lyubitelev A., Korovina A., Pushkarev S., Armeev G., Kirpichnikov M., Studitsky V., Feofanov A. Non-classical H1-like PARP1 binding to chromatosome. Cells. 2025;14(17):1309.
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Lobanova A.A., Saulina O.V., Geraskina O.V., Koshkina D.O., Maluchenko N.V., Feofanov A.V., Studitsky V.M. Ni-NTA bead-based real-time monitoring of PARPs inhibitor binding. Vestnik Moskovskogo universiteta. Seriya 16. Biologiya. 2025;80(3):73–80. (In Russ.) https://doi.org/10.55959/MSU0137-0952-16-80-3S-11
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