Shedding light on the base-pair opening dynamics of nucleic acids in living human cells

1. Ellis, R. J. Macromolecular crowding: obvious but under- appreciated. Trends Biochem. Sci. 26, 597–604 (2001).

2. Zhou, H. X., Rivas, G. & Minton, A. P. Macromolecular crowding and confinement: biochemical, biophysical, and potential physiological consequences. Annu. Rev. Biophys. 37, 375–397 (2008).

3. Nakano, S., Miyoshi, D. & Sugimoto, N. Effects of molecular crowding on the structures, interactions, and functions of nucleic acids. Chem. Rev. 114, 2733–2758 (2014).

4. Nishida, N., Ito, Y. & Shimada, I. In situ structural biology using in- cell NMR. Biochim. Biophys. Acta Gen. Subj. 1864, 129364 (2020).

5. Kim, W. et al. Base-pair opening dynamics of primary miR156a using NMR elucidates structural determinants important for its proces- sing level and leaf number phenotype in Arabidopsis. Nucleic Acids Res. 45, 875–885 (2017).

6. Furukawa, A., Walinda, E., Arita, K.& Sugase, K. Structural dynamics of double-stranded DNA with epigenome modification. Nucleic Acids Res. 49, 1152–1162 (2021).

7. Liu, B. et al. A quantitative model predicts how m6A reshapes the kinetic landscape of nucleic acid hybridization and conformational transitions. Nat. Commun. 12, 5201 (2021).

8. Choi, S. R. et al. Base-pair opening dynamics of nucleic acids in relation to their biological function. Comput. Struct. Biotechnol. J. 17, 797–804 (2019).

9. Yamaoki, Y., Nagata, T., Sakamoto, T. & Katahira, M. Recent progress of in-cell NMR of nucleic acids in living human cells. Biophys. Rev. 12, 411–417 (2020).

10. Luchinat, E., Cremonini, M. & Banci, L. Radio signals from live cells: the coming of age of in-cell solution NMR. Chem. Rev. 122, 9267–9306 (2022).

11. Theillet, F.-X. In-cell structural biology by NMR: The benefits of the atomic scale. Chem. Rev. 122, 9497–9570 (2022).

12. Dzatko, S., Fiala, R., Hänsel-Hertsch, R., Foldynova-Trantirkova, S. & Trantirek, L. In-cell NMR spectroscopy of nucleic acids. In: Ito, Y. (ed) In-cell NMR spectroscopy: from molecular sciences to cell biology. The Royal Society of Chemistry, 272–297 (2020).

13. Yamaoki, Y. et al. The first successful observation of in-cell NMR signals of DNA and RNA in living human cells. Phys. Chem. Chem. Phys. 20, 2982–2985 (2018).

14. Dzatko, S. et al. Evaluation of the stability of DNA i-motifs in the nuclei of living mammalian cells. Angew. Chem. Int. Ed. Engl. 57, 2165–2169 (2018).

15. Cheng, M. et al. Thermal and pH stabilities of i-DNA: confronting in vitro experiments with models and in-cell NMR data. Angew. Chem. Int. Ed. Engl. 60, 10286–10294 (2021).

16. Bao, H. L., Liu, H. S. & Xu, Y. Hybrid-type and two-tetrad antiparallel telomere DNA G-quadruplex structures in living human cells. Nucleic Acids Res. 47, 4940–4947 (2019).

17. Bao, H. L.& Xu, Y. Telomeric DNA–RNA-hybrid G-quadruplex exists in environmental conditions of HeLa cells. Chem. Commun. 56, 6547–6550 (2020).

18. Bao, H. L., Masuzawa, T., Oyoshi, T. & Xu, Y. Oligonucleotides DNA containing 8-trifluoromethyl-2′-deoxyguanosine for observing Z-DNA structure. Nucleic Acids Res. 48, 7041–7051 (2020).

19. Sakamoto, T., Yamaoki, Y., Nagata, T. & Katahira, M. Detection of parallel and antiparallel DNA triplex structures in living human cells using in-cell NMR. Chem. Commun. 57, 6364–6367 (2021).

20. Krafcikova, M. et al. Monitoring DNA-ligand interactions in living human cells using NMR spectroscopy. J. Am. Chem. Soc. 141, 13281–13285 (2019).

21. Broft, P. et al. In-cell NMR spectroscopy of functional riboswitch aptamers in eukaryotic cells. Angew. Chem. Int. Ed. Engl. 60, 865–872 (2021).

22. Krafčík, D. et al. Towards profiling of the G-quadruplex targeting drugs in the living human cells using NMR spectroscopy. Int. J. Mol. Sci. 22, 6042 (2021).

23. Schlagnitweit, J. et al. Observing an antisense drug complex in intact human cells by in-cell NMR spectroscopy. ChemBioChem 20, 2474–2478 (2019).

24. Guéron, M. & Leroy, J. L. Studies of base pair kinetics by NMR measurement of proton exchange. Methods Enzymol. 261, 383–413 (1995).

25. Szulik, M. W., Voehler, M. & Stone, M. P. NMR analysis of base-pair opening kinetics in DNA. Curr. Protoc. Nucleic Acid Chem. 59, 7.20.1–18 (2014).

26. Varani, G., Cheong, C. & Tinoco, I. Jr. Structure of an unusually stable RNA hairpin. Biochemistry 30, 3280–3289 (1991).

27. Allain, F. H. T. & Varani, G. Structure of the P1 helix from group I self-splicing introns. J. Mol. Biol. 250, 333–353 (1995).

28. Woese, C. R., Winker, S. & Gutell, R. R. Architecture of ribosomal RNA: constraints on the sequence of “tetra-loops”. Proc. Natl Acad. Sci. USA 87, 8467–8471 (1990).

29. Kano, F., Nakatsu, D., Noguchi, Y., Yamamoto, A. & Murata, M. A resealed-cell system for analyzing pathogenic intracellular events: perturbation of endocytic pathways under diabetic conditions. PLoS One 7, e44127 (2012).

30. Kunishige, R., Kano, F. & Murata, M. The cell resealing technique for manipulating, visualizing, and elucidating molecular functions in living cells. Biochim. Biophys. Acta Gen. Subj. 1864, 129329 (2020).

31. Ogino, S. et al. Observation of NMR signals from proteins intro- duced into living mammalian cells by reversible membrane per- meabilization using a pore-forming toxin, streptolysin O. J. Am. Chem. Soc. 131, 10834–10835 (2009).

32. Varshney, D., Spiegel, J., Zyner, K., Tannahill, D. & Balasubramanian, S. The regulation and functions of DNA and RNA G-quadruplexes. Nat. Rev. Mol. Cell Biol. 21, 459–474 (2020).

33. Kondo, K. et al. Plastic roles of phenylalanine and tyrosine residues of TLS/FUS in complex formation with the G-quadruplexes of telomeric DNA and TERRA. Sci. Rep. 8, 2864 (2018).

34. Takahama, K. et al. Regulation of telomere length by G-quadruplex telomere DNA- and TERRA-binding protein TLS/FUS. Chem. Biol. 20, 341–350 (2013).

35. Luu, K. N., Phan, A. T., Kuryavyi, V., Lacroix, L. & Patel, D. J. Structure of the human telomere in K+ solution: an intramolecular (3 + 1) G-quadruplex scaffold. J. Am. Chem. Soc. 128, 9963–9970 (2006).

36. Every, A. E. & Russu, I. M. Opening dynamics of 8-oxoguanine in DNA. J. Mol. Recognit. 26, 175–180 (2013).

37. Huang, Y., Chen, C. & Russu, I. M. Dynamics and stability of indivi- dual base pairs in two homologous RNA−DNA hybrids. Biochemistry 48, 3988–3997 (2017).

38. Smith, A. E., Zhou, L. Z., Gorensek, A. H., Senske, M. & Pielak, G. J. In- cell thermodynamics and a new role for protein surfaces. Proc. Natl. Acad. Sci. USA 113, 1725–1730 (2016).

39. Kubo, S. et al. A gel-encapsulated bioreactor system for NMR stu- dies of protein-protein interactions in living mammalian cells. Angew. Chem. Int. Ed. Engl. 52, 1208–1211 (2013).

40. Breindel, L., DeMott, C., Burz, D. S. & Shekhtman, A. Real-time in-cell nuclear magnetic resonance: ribosome-targeted antibiotics mod- ulate quinary protein interactions. Biochemistry 57, 540–546 (2018).

41. Hwang, T. L., van Zijl, P. C. & Mori, S. Accurate quantitation of water- amide proton exchange rates using the phase-modulated CLEAN chemical EXchange (CLEANEX-PM) approach with a Fast-HSQC (FHSQC) detection scheme. J. Biomol. NMR 11, 221–226 (1998).

42. Majumder, S. et al. Probing protein quinary interactions by in-cell nuclear magnetic resonance spectroscopy. Biochemistry 54, 2727–2738 (2015).

43. Selenko, P. Quo vadis biomolecular NMR spectroscopy? Int. J. Mol. Sci. 20, 1278 (2019).

44. Cohen, R. D. & Pielak, G. J. Electrostatic contributions to protein quinary structure. J. Am. Chem. Soc. 138, 13139–13142 (2016).

45. Cohen, R. D. & Pielak, G. J. Quinary interactions with an unfolded state ensemble. Protein Sci. 26, 1698–1703 (2017).

46. Sukenik, S., Ren, P. & Gruebele, M. Weak protein-protein interac- tions in live cells are quantified by cell-volume modulation. Proc. Natl Acad. Sci. USA 114, 6776–6781 (2017).

47. Rabouille, C. & Alberti, S. Cell adaptation upon stress: The emerging role of membrane-less compartments. Curr. Opin. Cell Biol. 47, 34–42 (2017).

48. Monteith, W. B., Cohen, R. D., Smith, A. E., Guzman-Cisneros, E. & Pielak, G. J. Quinary structure modulates protein stability in cells. Proc. Natl Acad. Sci. USA 112, 1739–1742 (2015).

49. Eladl, A. et al. Investigation of the interaction of human origin recognition complex subunit 1 with G-quadruplex DNAs of human c-myc promoter and telomere regions. Int. J. Mol. Sci. 22, 3481 (2021).

50. Robinson, J., Raguseo, F., Nuccio, S. P., Liano, D. & Di Antonio, M. DNA G-quadruplex structures: more than simple roadblocks to transcription? Nucleic Acids Res. 49, 8419–8431 (2021).

51. Kanoh, Y. et al. Rif1 binds to G quadruplexes and suppresses repli- cation over long distances. Nat. Struct. Mol. Biol. 22, 889–897 (2015).

52. Di Antonio, M. et al. Single-molecule visualization of DNA G-quadruplex formation in live cells. Nat. Chem. 12, 832–837 (2020).

53. Balasubramanian, S., Hurley, L. H.& Neidle, S. Targeting G-quadruplexes in gene promoters: a novel anticancer strategy? Nat. Rev. Drug Discov. 10, 261–275 (2011).

54. Hänsel-Hertsch, R., Di Antonio, M.& Balasubramanian, S. DNA G-quadruplexes in the human genome: detection, functions and therapeutic potential. Nat. Rev. Mol. Cell Biol. 18, 279–284 (2017).

55. Dickerhoff, J., Dai, J. & Yang, D. Structural recognition of the MYC promoter G-quadruplex by a quinoline derivative: insights into molecular targeting of parallel G-quadruplexes. Nucleic Acids Res. 49, 5905–5915 (2021).

56. Wang, K. B., Dickerhoff, J. & Yang, D. Solution structure of ternary complex of berberine bound to a dGMP-fill-in vacancy G-quadruplex formed in the PDGFR-β promoter. J. Am. Chem. Soc. 143, 16549–16555 (2021).

57. Shibata, T. et al. Small molecule targeting r(UGGAA)n disrupts RNA foci and alleviates disease phenotype in Drosophila model. Nat. Commun. 12, 236 (2021).

58. Howe, J. A. et al. Selective small-molecule inhibition of an RNA structural element. Nature 526, 672–677 (2015).

59. Schanda, P., Kupce, E. & Brutscher, B. SOFAST-HMQC experiments for recording two-dimensional heteronuclear correlation spectra of proteins within a few seconds. J. Biomol. NMR 33, 199–211 (2005).

60. Schanda, P., Forge, V. & Brutscher, B. HET-SOFAST NMR for fast detection of structural compactness and heterogeneity along polypeptide chains. Magn. Reson. Chem. 44, S177–S184 (2006).