Dysregulation of the DNA damage response by phosphorothioate antisense oligonucleotides

1. Blackford, A. N. & Jackson, S. P. ATM, ATR, and DNA-PK: the trinity at the heart of the DNA damage response. Mol. Cell 66, 801–817 (2017).

   Google Scholar 

2. Ciccia, A. & Elledge, S. J. The DNA damage response: making it safe to play with knives. Mol. Cell 40, 179–204 (2010).

   Google Scholar 

3. Pandey, N. & Black, B. E. Rapid detection and signaling of DNA damage by PARP-1. Trends Biochem. Sci. 46, 744–757 (2021).

   Google Scholar 

4. Fijen, C. & Rothenberg, E. The evolving complexity of DNA damage foci: RNA, condensates and chromatin in DNA double-strand break repair. DNA Repair 105, 103170 (2021).

   Google Scholar 

5. Spegg, V. & Altmeyer, M. Biomolecular condensates at sites of DNA damage: more than just a phase. DNA Repair 106, 103179 (2021).

   Google Scholar 

6. Lin, Y. & Fang, X. Phase separation in RNA biology. J. Genet. Genomics 48, 872–880 (2021).

   Google Scholar 

7. Duan, Y. et al. PARylation regulates stress granule dynamics, phase separation, and neurotoxicity of disease-related RNA-binding proteins. Cell Res. 29, 233–247 (2019).

   Google Scholar 

8. Owen, I. & Shewmaker, F. The role of post-translational modifications in the phase transitions of intrinsically disordered proteins. Int. J. Mol. Sci. 20, 5501 (2019).

9. Soutoglou, E. & Misteli, T. Activation of the cellular DNA damage response in the absence of DNA lesions. Science 320, 1507–1510 (2008).

   Google Scholar 

10. Bonilla, C. Y., Melo, J. A. & Toczyski, D. P. Colocalization of sensors is sufficient to activate the DNA damage checkpoint in the absence of damage. Mol. Cell 30, 267–276 (2008).

    Google Scholar 

11. Shao, Z. et al. DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis. Nature 579, 291–296 (2020).

    Google Scholar 

12. Kim, D. S. et al. Activation of PARP-1 by snoRNAs controls ribosome biogenesis and cell growth via the RNA helicase DDX21. Mol. Cell 75, 1270–1285.e1214 (2019).

    Google Scholar 

13. Huang, D., Kim, D. S. & Kraus, W. L. Specific binding of snoRNAs to PARP-1 promotes NAD(+)-dependent catalytic activation. Biochemistry 59, 1559–1564 (2020).

    Google Scholar 

14. Croset, A. et al. Inhibition of DNA damage repair by artificial activation of PARP with siDNA. Nucleic Acids Res. 41, 7344–7355 (2013).

    Google Scholar 

15. Quanz, M. et al. Hyperactivation of DNA-PK by double-strand break mimicking molecules disorganizes DNA damage response. PLoS ONE 4, e6298 (2009).

    Google Scholar 

16. Dhuri, K. et al. Antisense oligonucleotides: an emerging area in drug discovery and development. J. Clin. Med. 9, 2004 (2020).

17. Egli, M. & Manoharan, M. Chemistry, structure and function of approved oligonucleotide therapeutics. Nucleic Acids Res. 51, 2529–2573 (2023).

    Google Scholar 

18. Eckstein, F. Phosphorothioates, essential components of therapeutic oligonucleotides. Nucleic Acid Ther. 24, 374–387 (2014).

    Google Scholar 

19. Crooke, S. T., Vickers, T. A. & Liang, X. H. Phosphorothioate modified oligonucleotide-protein interactions. Nucleic Acids Res. 48, 5235–5253 (2020).

    Google Scholar 

20. Shen, W. et al. Chemical modification of PS-ASO therapeutics reduces cellular protein-binding and improves the therapeutic index. Nat. Biotechnol. 37, 640–650 (2019).

    Google Scholar 

21. Vickers, T. A., Rahdar, M., Prakash, T. P. & Crooke, S. T. Kinetic and subcellular analysis of PS-ASO/protein interactions with P54nrb and RNase H1. Nucleic Acids Res. 47, 10865–10880 (2019).

    Google Scholar 

22. Flynn, L. L. et al. Single stranded fully modified-phosphorothioate oligonucleotides can induce structured nuclear inclusions, alter nuclear protein localization and disturb the transcriptome in vitro. Front. Genet. 13, 791416 (2022).

    Google Scholar 

23. Bailey, J. K., Shen, W., Liang, X. H. & Crooke, S. T. Nucleic acid binding proteins affect the subcellular distribution of phosphorothioate antisense oligonucleotides. Nucleic Acids Res. 45, 10649–10671 (2017).

    Google Scholar 

24. Shen, W., Liang, X. H. & Crooke, S. T. Phosphorothioate oligonucleotides can displace NEAT1 RNA and form nuclear paraspeckle-like structures. Nucleic Acids Res. 42, 8648–8662 (2014).

    Google Scholar 

25. Wang, Y., Shen, W., Liang, X. H. & Crooke, S. T. Phosphorothioate antisense oligonucleotides bind P-body proteins and mediate P-body assembly. Nucleic Acid Ther. 29, 343–358 (2019).

    Google Scholar 

26. Lorenz, P., Baker, B. F., Bennett, C. F. & Spector, D. L. Phosphorothioate antisense oligonucleotides induce the formation of nuclear bodies. Mol. Biol. Cell 9, 1007–1023 (1998).

    Google Scholar 

27. Liang, X. H., Shen, W., Sun, H., Prakash, T. P. & Crooke, S. T. TCP1 complex proteins interact with phosphorothioate oligonucleotides and can co-localize in oligonucleotide-induced nuclear bodies in mammalian cells. Nucleic Acids Res. 42, 7819–7832 (2014).

    Google Scholar 

28. Zeng, C. et al. Revisiting the chaperonin T-complex protein-1 ring complex in human health and disease: a proteostasis modulator and beyond. Clin. Transl. Med. 14, e1592 (2024).

    Google Scholar 

29. Kim, H., Park, J. & Roh, S.-H. The structural basis of eukaryotic chaperonin TRiC/CCT: action and folding. Mol. Cells 47, 100012 (2024).

    Google Scholar 

30. Crooke, S. T., Wang, S., Vickers, T. A., Shen, W. & Liang, X.-H. Cellular uptake and trafficking of antisense oligonucleotides. Nat. Biotechnol. 35, 230–237 (2017).

    Google Scholar 

31. Stein, C. A. et al. Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents. Nucleic Acids Res. 38, e3 (2010).

    Google Scholar 

32. González-Barriga, A. et al. Intracellular distribution and nuclear activity of antisense oligonucleotides after unassisted uptake in myoblasts and differentiated myotubes in vitro. Nucleic Acid Ther. 27, 144–158 (2017).

    Google Scholar 

33. Koller, E. et al. Mechanisms of single-stranded phosphorothioate modified antisense oligonucleotide accumulation in hepatocytes. Nucleic Acids Res. 39, 4795–4807 (2011).

    Google Scholar 

34. Bergstrand, S. et al. Small Cajal body-associated RNA 2 (scaRNA2) regulates DNA repair pathway choice by inhibiting DNA-PK. Nat. Commun. 13, 1015 (2022).

    Google Scholar 

35. López-Palacios, T. P. & Andersen, J. L. Kinase regulation by liquid-liquid phase separation. Trends Cell Biol. 33, 649–666 (2023).

    Google Scholar 

36. Clark, L. K. & Cullati, S. N. Activation is only the beginning: mechanisms that tune kinase substrate specificity. Biochem Soc. Trans. 27, 145–159 (2025).

    Google Scholar 

37. Pierce, A. J., Johnson, R. D., Thompson, L. H. & Jasin, M. XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes Dev. 15, 2633–2638 (1999).

    Google Scholar 

38. O’Flynn, B. G. & Mittag, T. The role of liquid-liquid phase separation in regulating enzyme activity. Curr. Opin. Cell Biol. 69, 70–79 (2021).

    Google Scholar 

39. Luo, J., Feng, Z.-S. & Tang, J.-X. The essential role of CCT2 in the regulation of aggrephagy. Front. Aging Neurosci. 16, 1491001 (2024).

40. Menolfi, D. & Zha, S. ATM, ATR and DNA-PKcs kinases—the lessons from the mouse models: inhibition ≠ deletion. Cell Biosci. 10, 8 (2020).

    Google Scholar 

41. Wu, Y. et al. RNA phosphorothioate modification in prokaryotes and eukaryotes. ACS Chem. Biol. 15, 1301–1305 (2020).

    Google Scholar 

42. Wang, L. et al. Phosphorothioation of DNA in bacteria by dnd genes. Nat. Chem. Biol. 3, 709–710 (2007).

    Google Scholar 

43. Tong, T. et al. Occurrence, evolution, and functions of DNA phosphorothioate epigenetics in bacteria. Proc. Natl. Acad. Sci. USA 115, E2988–e2996 (2018).

    Google Scholar 

44. Jie, J. et al. Sulfur-centered hemi-bond radicals as active intermediates in S-DNA phosphorothioate oxidation. Nucleic Acids Res. 47, 11514–11526 (2019).

    Google Scholar 

45. Wang, L., Jiang, S., Deng, Z., Dedon, P. C. & Chen, S. DNA phosphorothioate modification—a new multi-functional epigenetic system in bacteria. FEMS Microbiol. Rev. 43, 109–122 (2019).

    Google Scholar 

46. Kellner, S. et al. Oxidation of phosphorothioate DNA modifications leads to lethal genomic instability. Nat. Chem. Biol. 13, 888–894 (2017).

    Google Scholar 

47. Kilic, S. et al. Phase separation of 53BP1 determines liquid-like behavior of DNA repair compartments. EMBO J. 38, e101379 (2019).

    Google Scholar 

48. Pessina, F. et al. Functional transcription promoters at DNA double-strand breaks mediate RNA-driven phase separation of damage-response factors. Nat. Cell Biol. 21, 1286–1299 (2019).

    Google Scholar 

49. Altmeyer, M. et al. Liquid demixing of intrinsically disordered proteins is seeded by poly(ADP-ribose). Nat. Commun. 6, 8088 (2015).

    Google Scholar 

50. Oshidari, R. et al. DNA repair by Rad52 liquid droplets. Nat. Commun. 11, 695 (2020).

    Google Scholar 

51. Chappidi, N. et al. PARP1-DNA co-condensation drives DNA repair site assembly to prevent disjunction of broken DNA ends. Cell 187, 945–961.e918 (2024).

    Google Scholar 

52. Han, C. et al. Chromatin-associated orphan snoRNA regulates DNA damage-mediated differentiation via a non-canonical complex. Cell Rep. 38, 110421 (2022).

    Google Scholar 

53. Stirling, D. R. et al. CellProfiler 4: improvements in speed, utility and usability. BMC Bioinforma. 22, 433 (2021).

    Google Scholar 

54. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

    Google Scholar 

55. Tang, J. et al. Acetylation limits 53BP1 association with damaged chromatin to promote homologous recombination. Nat. Struct. Mol. Biol. 20, 317–325 (2013).

    Google Scholar 

56. Barbe, L. et al. AutoComet: a fully automated algorithm to quickly and accurately analyze comet assays. Redox Biol. 62, 102680 (2023).

    Google Scholar 

57. Hagemann-Jensen, M. et al. Single-cell RNA counting at allele and isoform resolution using Smart-seq3. Nat. Biotechnol. 38, 708–714 (2020).

    Google Scholar 

58. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    Google Scholar 

59. Hagey, D. W. et al. CYCLIN-B1/2 and -D1 act in opposition to coordinate cortical progenitor self-renewal and lineage commitment. Nat. Commun. 11, 2898 (2020).

    Google Scholar 

60. Hagey, D. W. et al. Myeloid cells from Langerhans cell histiocytosis patients exhibit increased vesicle trafficking and an altered secretome capable of activating NK cells. Haematologica 108, 2422–2434 (2023).

    Google Scholar 

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