Research / 2019 / Article

Research Article | Open Access

Volume 2019 |Article ID 1050735 | https://doi.org/10.34133/2019/1050735

Yi-Lun Ying, Jie Yang, Fu-Na Meng, Shuang Li, Meng-Ying Li, Yi-Tao Long, "A Nanopore Phosphorylation Sensor for Single Oligonucleotides and Peptides", Research, vol. 2019, Article ID 1050735, 8 pages, 2019. https://doi.org/10.34133/2019/1050735

A Nanopore Phosphorylation Sensor for Single Oligonucleotides and Peptides

Received27 May 2019
Accepted07 Oct 2019
Published29 Oct 2019

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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