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α-Hemolysin nanopore studies reveal strong interactions between biogenic polyamines and DNA hairpins

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Abstract

The α-hemolysin (α-HL) nanopore is capable of analyzing DNA as it is electrophoretically driven through the pore. Respective current vs. time (i-t) traces depend on the DNA sequence, its secondary structures, or on the physical conditions of the analysis. The current study describes the analysis of a DNA hairpin with a 5′-extension by applying α-HL nanopores in the presence of the polyamines spermine (Spm), spermidine (Spd), and putrescine (Put) and revealed i-t traces characteristic of the DNA-polyamine complex. Voltage-dependent studies also revealed that the hairpin-Spm complex formed with excess Spm cannot be unzipped and translocated through the pores even if the voltage is increased to 180 mV. The DNA hairpin sample was titrated with Spm, Spd, or Put that showed a dose-dependent response in the characteristic event patterns for hairpins bound to Spm or Spd, but not for Put. Plots of the event types vs. counts were used to calculate binding constants for the Spm or Spd hairpin interactions. The titration also demonstrated that the event rate decreased ~10-fold on increasing the Spm or Spd concentrations from 0 to 4 mM. These observations impose practical limitations on the ability to use Spm or Spd for DNA studies with the α-HL nanopore.

Hairpin DNAs bearing long single-stranded tails were studied with spermine via the α-hemolysin nanopore that generated three characteristic current signatures.

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References

  1. Igarashi K, Kashiwagi K (2000) Polyamines: mysterious modulators of cellular functions. Biochem Biophys Res Commun 271:559

    Article  CAS  Google Scholar 

  2. Thomas T, Thomas TJ (2001) Polyamines in cell growth and cell death: molecular mechanisms and therapeutic applications. Cell Mol Life Sci 58:244

    Article  CAS  Google Scholar 

  3. Igarashi K, Kashiwagi K (2010) Modulation of cellular function by polyamines. Int J Biochem 42:39

    Article  CAS  Google Scholar 

  4. Minois N, Carmona-Gutierrez D, Madeo F (2011) Polyamines in aging and disease. Aging 3:1

    Google Scholar 

  5. Janne J, Alhonen L, Pietila M, Keinanen TK (2004) Genetic approaches to the cellular functions of polyamines in mammals. Eur J Biochem 271:877

    Article  CAS  Google Scholar 

  6. Tabor H (1962) The protective effect of spermine and other polyamines against heat denaturation of deoxyribonucleic acid. Biochemistry 1:496

    Article  CAS  Google Scholar 

  7. Kabir A, Suresh Kumar GS (2013) Binding of the biogenic polyamines to deoxyribonucleic acids of varying base composition: base specificity and the associated energetics of the interaction. PLoS One 8, e70510

    Article  CAS  Google Scholar 

  8. Kabir A, Hossain M, Kumar GS (2013) Thermodynamics of the DNA binding of biogenic polyamines: calorimetric and spectroscopic investigations. J Chem Thermodyn 57:445

    Article  CAS  Google Scholar 

  9. Tajmir-Riahi HA, Ahmed-Ouameur A, Hasni I, Bourassa P, Thomas TJ (2011) Probing DNA and RNA interactions with biogenic and synthetic polyamines. CRC Press, Boca Raton

    Book  Google Scholar 

  10. Ouameur AA, Tajmir-Riahi H (2004) Structural analysis of DNA Interactions with biogenic polyamines and cobalt(III)hexamine studied by fourier transform infrared and capillary electrophoresis. J Biol Chem 279:42041

    Article  CAS  Google Scholar 

  11. Deng H, Bloomfield VA, Benevides JM, Thomas GJ Jr (2000) Structural basis of polyamine–DNA recognition: spermidine and spermine interactions with genomic B-DNAs of different GC content probed by Raman spectroscopy. Nucleic Acids Res 28:3379

    Article  CAS  Google Scholar 

  12. Tari LW, Secco AS (1995) Base-pair opening and spermine binding—B-DNA features displayed in the crystal structure of a gal operon fragment: implications for protein-DNA recognition. Nucleic Acids Res 23:2065

    Article  CAS  Google Scholar 

  13. Howorka S, Siwy Z (2009) Nanopore analytics: sensing of single molecules. Chem Soc Rev 38:2360

    Article  CAS  Google Scholar 

  14. Gu LQ, Shim JW (2010) Single molecule sensing by nanopores and nanopore devices. Analyst 135:441

    Article  CAS  Google Scholar 

  15. Maglia G, Heron AJ, Stoddart D, Japrung D, Bayley H (2010) Analysis of single nucleic acid molecules with protein nanopores. Methods Enzymol 475:591

    Article  CAS  Google Scholar 

  16. Ying YL, Zhang J, Gao R, Long YT (2013) Nanopore-based sequencing and detection of nucleic acids. Angew Chem Int Ed Engl 52:13154

    Article  CAS  Google Scholar 

  17. Song L, Hobaugh M, Shustak C, Cheley S, Bayley H, Gouaux J (1996) Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 274:1859

    Article  CAS  Google Scholar 

  18. Kasianowicz JJ, Brandin E, Branton D, Deamer DW (1996) Characterization of individual polynucleotide molecules using a membrane channel. Proc Natl Acad Sci U S A 93:13770

    Article  CAS  Google Scholar 

  19. Akeson M, Branton D, Kasianowicz JJ, Brandin E, Deamer DW (1999) Microsecond time-scale discrimination among polycytidylic acid, polyadenylic acid, and polyuridylic acid as homopolymers or as segments within single RNA molecules. Biophys J 77:3227

    Article  CAS  Google Scholar 

  20. Vercoutere W, Winters-Hilt S, Olsen H, Deamer D, Haussler D, Akeson M (2001) Rapid discrimination among individual DNA hairpin molecules at single-nucleotide resolution using an ion channel. Nat Biotechnol 19:248

    Article  CAS  Google Scholar 

  21. Wolna AH, Fleming AM, Burrows CJ (2014) Single-molecule analysis of thymine dimer-containing G-quadruplexes formed from the human telomere sequence. Biochemistry 53:7484

    Article  CAS  Google Scholar 

  22. An N, Fleming AM, Middleton EG, Burrows CJ (2014) Single-molecule investigation of G-quadruplex folds of the human telomere sequence in a protein nanocavity. Proc Natl Acad Sci U S A 111:14325

    Article  CAS  Google Scholar 

  23. An N, Fleming AM, Burrows CJ (2013) Interactions of the human telomere sequence with the nanocavity of the α-Hemolysin ion channel reveal structure-dependent electrical signatures for hybrid folds. J Am Chem Soc 135:8562

    Article  CAS  Google Scholar 

  24. Shim J, Gu LQ (2012) Single-molecule investigation of G-quadruplex using a nanopore sensor. Methods 57:40

    Article  CAS  Google Scholar 

  25. Ding Y, Fleming AM, White HS, Burrows CJ (2014) Internal vs fishhook hairpin DNA: unzipping locations and mechanisms in the alpha-hemolysin nanopore. J Phys Chem B 118:12873

    Article  CAS  Google Scholar 

  26. Jin Q, Fleming AM, Burrows CJ, White HS (2012) Unzipping kinetics of duplex DNA containing oxidized lesions in an alpha-hemolysin nanopore. J Am Chem Soc 134:11006

    Article  CAS  Google Scholar 

  27. Schibel AEP, Fleming AM, Jin Q, An N, Liu J, Blakemore CP, White HS, Burrows CJ (2011) Sequence-specific single-molecule analysis of 8-oxo-7,8-dihydroguanine lesions in DNA based on unzipping kinetics of complementary probes in ion channel recordings. J Am Chem Soc 133:14778

    Article  CAS  Google Scholar 

  28. Winters-Hilt S, Vercoutere W, DeGuzman VS, Deamer D, Akeson M, Haussler D (2003) Highly accurate classification of Watson-Crick basepairs on termini of single DNA molecules. Biophys J 84:967

    Article  CAS  Google Scholar 

  29. Mathé J, Visram H, Viasnoff V, Rabin Y, Meller A (2004) Nanopore unzipping of individual DNA hairpin molecules. Biophys J 87:3205

    Article  Google Scholar 

  30. Deamer DW, Vercoutere WA, DeGuzman VS, Lee CC (2006) Sequence-dependent gating of an ion channel by DNA hairpin molecules. Nucleic Acids Res 34:6425

    Article  Google Scholar 

  31. Vercoutere WA, Winters-Hilt S, DeGuzman VS, Deamer D, Ridino SE, Rodgers JT, Olsen HE, Marziali A, Akeson M (2003) Discrimination among individual Watson-Crick base pairs at the termini of single DNA hairpin molecules. Nucleic Acids Res 31:1311

    Article  CAS  Google Scholar 

  32. Wanunu M, Bhattacharya S, Xie Y, Tor Y, Aksimentiev A, Drndic M (2011) Nanopore analysis of individual RNA/antibiotic complexes. ACS Nano 5:9345

    Article  CAS  Google Scholar 

  33. Yao F, Duan J, Wang Y, Zhang Y, Guo Y, Guo H, Kang X (2015) Nanopore single-molecule analysis of DNA-doxorubicin interactions. Anal Chem 87:338

    Article  CAS  Google Scholar 

  34. Zhang B, Galusha J, Shiozawa PG, Wang G, Bergren AJ, Jones RM, White RJ, Ervin EN, Cauley CC, White HS (2007) Bench-top method for fabricating glass-sealed nanodisk electrodes, glass nanopore electrodes, and glass nanopore membranes of controlled size. Anal Chem 79:4778

    Article  CAS  Google Scholar 

  35. White RJ, Ervin EN, Yang T, Chen X, Daniel S, Cremer PS, White HS (2007) Single ion-channel recordings using glass nanopore membranes. J Am Chem Soc 129:11766

    Article  CAS  Google Scholar 

  36. van Dongen MJP, Mooren MMW, Willems EFA, van der Marel GA, van Boom JH, Wijmenga SS, Hilbers CW (1997) Structural features of the DNA hairpin d(ATCCTA-GTTA-TAGGAT): formation of a G-A Base pair in the loop. Nucleic Acids Res 25:1537

    Article  Google Scholar 

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Acknowledgments

This work was supported by a grant from the National Institutes of Health (R01GM093099). The nanopore instrument and software were generously donated by Electronic BioSciences, San Diego, CA (http://electronicbio.com). Discussions with Prof. H. S. White (University of Utah) are gratefully acknowledged.

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Authors and Affiliations

  1. Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT, 84112-0850, USA

    Yun Ding, Aaron M. Fleming & Cynthia J. Burrows

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  1. Yun Ding
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  2. Aaron M. Fleming
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  3. Cynthia J. Burrows
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Correspondence to Cynthia J. Burrows.

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ESM 1

T m data, PAGE data, sample i − t traces, and event duration histograms are available in the supporting information. (DOCX 1150 kb)

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Ding, Y., Fleming, A.M. & Burrows, C.J. α-Hemolysin nanopore studies reveal strong interactions between biogenic polyamines and DNA hairpins. Microchim Acta 183, 973–979 (2016). https://doi.org/10.1007/s00604-015-1516-6

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  • DOI: https://doi.org/10.1007/s00604-015-1516-6

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