A Nanopore Phosphorylation Sensor for Single Oligonucleotides and Peptides

Abstract

The phosphorylation of oligonucleotides and peptides plays a critical role in regulating virtually all cellular processes. To fully understand these complex and fundamental regulatory pathways, the cellular phosphorylate changes of both oligonucleotides and peptides should be simultaneously identified and characterized. Here, we demonstrated a single-molecule, high-throughput, label-free, general, and one-step aerolysin nanopore method to comprehensively evaluate the phosphorylation of both oligonucleotide and peptide substrates. By virtue of electrochemically confined effects in aerolysin, our results show that the phosphorylation accelerates the traversing speed of a negatively charged substrate for about hundreds of time while significantly enhances the translocation frequency of a positively charged substrate. Thereby, the kinase/phosphatase activity could be directly measured with the aerolysin nanopore from the characteristically dose-dependent event frequency of the substrates. By using this straightforward approach, a model T₄ oligonucleotide kinase (PNK) further achieved the nanopore evaluation of its phosphatase activity and real-time monitoring of its phosphatase-catalyzed dephosphorylation at a single-molecule level. Our study provides a step forward to nanopore enzymology for analyzing the phosphorylation of both oligonucleotides and peptides with significant feasibility in fundamental biochemical researches, clinical diagnosis, and kinase/phosphatase-targeted drug discovery.

References

1. J. A. Adams, “Kinetic and catalytic mechanisms of protein kinases,” Chemical Reviews, vol. 101, no. 8, pp. 2271–2290, 2001. View at: Google Scholar
2. L. N. Johnson and R. J. Lewis, “Structural basis for control by phosphorylation,” Chemical Reviews, vol. 101, no. 8, pp. 2209–2242, 2001. View at: Google Scholar
3. G. Manning, D. B. Whyte, R. Martinez, T. Hunter, and S. Sudarsanam, “The protein kinase complement of the human genome,” Science, vol. 298, no. 5600, pp. 1912–1934, 2002. View at: Publisher Site | Google Scholar
4. S. C. Su and L. H. Tsai, “Cyclin-dependent kinases in brain development and disease,” Annual Review of Cell and Developmental Biology, vol. 27, pp. 465–491, 2011. View at: Publisher Site | Google Scholar
5. X. Liu and M. Winey, “The MPS1 family of protein kinases,” Annual Review of Biochemistry, vol. 81, pp. 561–585, 2012. View at: Publisher Site | Google Scholar
6. P. Geraldes and G. L. King, “Activation of protein kinase C isoforms and its impact on diabetic complications,” Circulation Research, vol. 106, no. 8, pp. 1319–1331, 2010. View at: Publisher Site | Google Scholar
7. V. G. Zaha and L. H. Young, “AMP-activated protein kinase regulation and biological actions in the heart,” Circulation Research, vol. 111, no. 6, pp. 800–814, 2012. View at: Publisher Site | Google Scholar
8. M. Altai, A. Orlova, and V. Tolmachev, “Radiolabeled probes targeting tyrosine-kinase receptors for personalized medicine,” Current Pharmaceutical Design, vol. 20, no. 14, pp. 2275–2292, 2014. View at: Publisher Site | Google Scholar
9. L. Lin, Y. Liu, X. Zhao, and J. Li, “Sensitive and rapid screening of T4 polynucleotide kinase activity and inhibition based on coupled exonuclease reaction and graphene oxide platform,” Analytical Chemistry, vol. 83, no. 22, pp. 8396–8402, 2011. View at: Publisher Site | Google Scholar
10. X. Xu, X. Liu, Z. Nie, Y. Pan, M. Guo, and S. Yao, “Label-free fluorescent detection of protein kinase activity based on the aggregation behavior of unmodified quantum dots,” Analytical Chemistry, vol. 83, no. 1, pp. 52–59, 2011. View at: Publisher Site | Google Scholar
11. W. Tang, G. Zhu, and C. Y. Zhang, “Sensitive detection of polynucleotide kinase using rolling circle amplification-induced chemiluminescence,” Chemical Communications, vol. 50, no. 36, pp. 4733–4735, 2014. View at: Publisher Site | Google Scholar
12. Z. Wang, N. Sun, Y. He, Y. Liu, and J. Li, “DNA assembled gold nanoparticles polymeric network blocks modular highly sensitive electrochemical biosensors for protein kinase activity analysis and inhibition,” Analytical Chemistry, vol. 86, no. 12, pp. 6153–6159, 2014. View at: Publisher Site | Google Scholar
13. F. Yi, X. Huang, and J. Ren, “Simple and sensitive method for determination of protein kinase activity based on surface charge change of peptide-modified gold nanoparticles as substrates,” Analytical Chemistry, vol. 90, no. 6, pp. 3871–3877, 2018. View at: Publisher Site | Google Scholar
14. Z. Yan, Z. Wang, Z. Miao, and Y. Liu, “Dye-sensitized and localized surface plasmon resonance enhanced visible-light photoelectrochemical biosensors for highly sensitive analysis of protein kinase activity,” Analytical Chemistry, vol. 88, no. 1, pp. 922–929, 2015. View at: Publisher Site | Google Scholar
15. Z. Deng, M. Ye, Y. Bian et al., “Multiplex isotope dimethyl labeling of substrate peptides for high throughput kinase activity assay via quantitative MALDI MS,” Chemical Communications, vol. 50, no. 90, pp. 13960–13962, 2014. View at: Publisher Site | Google Scholar
16. Z. Zheng, A. Tang, Y. Guan et al., “Nanocomputed tomography imaging of bacterial alkaline phosphatase activity with an iodinated hydrogelator,” Analytical Chemistry, vol. 88, no. 24, pp. 11982–11985, 2016. View at: Publisher Site | Google Scholar
17. J. J. Kasianowicz, E. Brandin, D. Branton, and D. W. Deamer, “Characterization of individual polynucleotide molecules using a membrane channel,” Proceedings of the National Academy of Sciences of the United States of America, vol. 93, no. 24, pp. 13770–13773, 1996. View at: Publisher Site | Google Scholar
18. U. F. Keyser, B. N. Koeleman, S. van Dorp et al., “Direct force measurements on DNA in a solid-state nanopore,” Nature Physics, vol. 2, no. 7, pp. 473–477, 2006. View at: Publisher Site | Google Scholar
19. Y.-L. Ying, J. Zhang, R. Gao, and Y.-T. Long, “Nanopore-based sequencing and detection of nucleic acids,” Angewandte Chemie, International Edition, vol. 52, no. 50, pp. 13154–13161, 2013. View at: Publisher Site | Google Scholar
20. N. An, A. M. Fleming, H. S. White, and C. J. Burrows, “Crown ether-electrolyte interactions permit nanopore detection of individual DNA abasic sites in single molecules,” Proceedings of the National Academy of Sciences of the United States of America, vol. 109, no. 29, pp. 11504–11509, 2012. View at: Publisher Site | Google Scholar
21. X. Zhang, D. Zhang, C. Zhao et al., “Nanopore electric snapshots of an RNA tertiary folding pathway,” Nature Communications, vol. 8, no. 1, article 1458, 2017. View at: Publisher Site | Google Scholar
22. J. Larkin, R. Y. Henley, V. Jadhav, J. Korlach, and M. Wanunu, “Length-independent DNA packing into nanopore zero-mode waveguides for low-input DNA sequencing,” Nature Nanotechnology, vol. 12, no. 12, pp. 1169–1175, 2017. View at: Publisher Site | Google Scholar
23. A. Asandei, M. Chinappi, H. K. Kang et al., “Acidity-mediated, electrostatic tuning of asymmetrically charged peptides interactions with protein nanopores,” ACS Applied Materials & Interfaces, vol. 7, no. 30, pp. 16706–16714, 2015. View at: Publisher Site | Google Scholar
24. F. Piguet, H. Ouldali, M. Pastoriza-Gallego, P. Manivet, J. Pelta, and A. Oukhaled, “Identification of single amino acid differences in uniformly charged homopolymeric peptides with aerolysin nanopore,” Nature Communications, vol. 9, no. 1, p. 966, 2018. View at: Publisher Site | Google Scholar
25. S. Li, C. Cao, J. Yang, and Y. T. Long, “Detection of peptides with different charges and lengths by using the aerolysin nanopore,” ChemElectroChem, vol. 6, no. 1, pp. 126–129, 2019. View at: Publisher Site | Google Scholar
26. P. Waduge, R. Hu, P. Bandarkar et al., “Nanopore-based measurements of protein size, fluctuations, and conformational changes,” ACS Nano, vol. 11, no. 6, pp. 5706–5716, 2017. View at: Publisher Site | Google Scholar
27. S. Zhou, L. Wang, X. Chen, and X. Guan, “Label-free nanopore single-molecule measurement of trypsin activity,” ACS Sensors, vol. 1, no. 5, pp. 607–613, 2016. View at: Publisher Site | Google Scholar
28. M. A. Fahie and M. Chen, “Electrostatic interactions between OmpG nanopore and analyte protein surface can distinguish between glycosylated isoforms,” The Journal of Physical Chemistry B, vol. 119, no. 32, pp. 10198–10206, 2015. View at: Publisher Site | Google Scholar
29. R. Hu, J. Diao, J. Li et al., “Intrinsic and membrane-facilitated α-synuclein oligomerization revealed by label-free detection through solid-state nanopores,” Scientific Reports, vol. 6, no. 1, article 20776, 2016. View at: Publisher Site | Google Scholar
30. Q. Zhao, R. S. S. de Zoysa, D. Wang, D. A. Jayawardhana, and X. Guan, “Real-time monitoring of peptide cleavage using a nanopore probe,” Journal of the American Chemical Society, vol. 131, no. 18, pp. 6324-6325, 2009. View at: Publisher Site | Google Scholar
31. K. Willems, V. Van Meervelt, C. Wloka, and G. Maglia, “Single-molecule nanopore enzymology,” Philosophical Transactions of the Royal Society B, vol. 372, no. 1726, 2017. View at: Publisher Site | Google Scholar
32. F. N. Meng, X. Yao, Y.-L. Ying, J. Zhang, H. Tian, and Y.-T. Long, “Single-molecule analysis of the self-assembly process facilitated by host–guest interactions,” Chemical Communications, vol. 51, no. 7, pp. 1202–1205, 2015. View at: Publisher Site | Google Scholar
33. T. Li, L. Liu, Y. Li, J. Xie, and H. C. Wu, “A universal strategy for aptamer‐based nanopore sensing through host–guest interactions inside α‐hemolysin,” Angewandte Chemie, International Edition, vol. 54, no. 26, pp. 7568–7571, 2015. View at: Publisher Site | Google Scholar
34. L. Harrington, L. T. Alexander, S. Knapp, and H. Bayley, “Pim kinase inhibitors evaluated with a single‐molecule engineered nanopore sensor,” Angewandte Chemie, International Edition, vol. 54, no. 28, pp. 8154–8159, 2015. View at: Publisher Site | Google Scholar
35. L. Harrington, S. Cheley, L. T. Alexander, S. Knapp, and H. Bayley, “Stochastic detection of Pim protein kinases reveals electrostatically enhanced association of a peptide substrate,” Proceedings of the National Academy of Sciences of the United States of America, vol. 110, no. 47, pp. E4417–E4426, 2013. View at: Publisher Site | Google Scholar
36. C. B. Rosen, D. Rodriguez-Larrea, and H. Bayley, “Single-molecule site-specific detection of protein phosphorylation with a nanopore,” Nature Biotechnology, vol. 32, no. 2, pp. 179–181, 2014. View at: Publisher Site | Google Scholar
37. L. Harrington, L. T. Alexander, S. Knapp, and H. Bayley, “Single-molecule protein phosphorylation and dephosphorylation by nanopore enzymology,” ACS Nano, vol. 13, no. 1, pp. 633–641, 2019. View at: Publisher Site | Google Scholar
38. C. Cao, Y.-L. Ying, Z. L. Hu, D. F. Liao, H. Tian, and Y.-T. Long, “Discrimination of oligonucleotides of different lengths with a wild-type aerolysin nanopore,” Nature Nanotechnology, vol. 11, no. 8, pp. 713–718, 2016. View at: Publisher Site | Google Scholar
39. C. Cao, M. Y. Li, N. Cirauqui et al., “Mapping the sensing spots of aerolysin for single oligonucleotides analysis,” Nature Communications, vol. 9, no. 1, article 2823, 2018. View at: Publisher Site | Google Scholar
40. Y. Q. Wang, M. Y. Li, H. Qiu et al., “Identification of essential sensitive regions of the aerolysin nanopore for single oligonucleotide analysis,” Analytical Chemistry, vol. 90, no. 13, pp. 7790–7794, 2018. View at: Publisher Site | Google Scholar
41. L. K. Wang, C. D. Lima, and S. Shuman, “Structure and mechanism of T4 polynucleotide kinase: an RNA repair enzyme,” The EMBO Journal, vol. 21, no. 14, pp. 3873–3880, 2002. View at: Publisher Site | Google Scholar
42. M. Amitsur, R. Levitz, and G. Kaufmann, “Bacteriophage T4 anticodon nuclease, polynucleotide kinase and RNA ligase reprocess the host lysine tRNA,” The EMBO Journal, vol. 6, no. 8, pp. 2499–2503, 1987. View at: Google Scholar
43. L. J. Wang, Q. Zhang, B. Tang, and C. Y. Zhang, “Single-molecule detection of polynucleotide kinase based on phosphorylation-directed recovery of fluorescence quenched by au nanoparticles,” Analytical Chemistry, vol. 89, no. 13, pp. 7255–7261, 2017. View at: Publisher Site | Google Scholar
44. C. Cao, J. Yu, M. Y. Li, Y. Q. Wang, H. Tian, and Y.-T. Long, “Direct readout of single nucleobase variations in an oligonucleotide,” Small, vol. 13, no. 44, article 1702011, 2017. View at: Publisher Site | Google Scholar
45. V. Cameron and O. C. Uhlenbeck, “3-Phosphatase activity in T4 polynucleotide kinase,” Biochemistry, vol. 16, no. 23, pp. 5120–5126, 1977. View at: Publisher Site | Google Scholar
46. M. Wanunu, W. Morrison, Y. Rabin, A. Y. Grosberg, and A. Meller, “Electrostatic focusing of unlabelled DNA into nanoscale pores using a salt gradient,” Nature Nanotechnology, vol. 5, no. 2, pp. 160–165, 2010. View at: Publisher Site | Google Scholar
47. Z. L. Hu, M. Y. Li, S. C. Liu, Y.-L. Ying, and Y.-T. Long, “A lithium-ion-active aerolysin nanopore for effectively trapping long single-stranded DNA,” Chemical Science, vol. 10, no. 2, pp. 354–358, 2019. View at: Publisher Site | Google Scholar
48. Y.-L. Ying, C. Cao, Y. X. Hu, and Y.-T. Long, “A single biomolecule interface for advancing the sensitivity, selectivity and accuracy of sensors,” National Science Review, vol. 5, no. 4, pp. 450–452, 2018. View at: Publisher Site | Google Scholar
49. J. Yu, C. Cao, and Y.-T. Long, “Selective and Sensitive Detection of Methylcytosine by Aerolysin Nanopore under Serum Condition,” Analytical Chemistry, vol. 89, no. 21, pp. 11685–11689, 2017. View at: Publisher Site | Google Scholar

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