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Synthesis, purification and sample experiment for fluorescent pteridine-containing DNA: tools for studying DNA interactive systems

Nature Protocols volume 2, pages 1013–1021 (2007)Cite this article

Abstract

Fluorescent nucleoside analogs provide a means to study DNA interactive systems through direct measurement of fluorescence properties. As integrated parts of DNA, these probes provide opportunities for monitoring subtle changes in DNA structure as it meets and reacts with other molecules. This protocol describes modifications to standard DNA synthesis to efficiently use smaller volumes of the probe phosphoramidite, purification of pteridine-containing sequences and a deprotection procedure specific for 6MI-containing sequences. Yields for probe incorporation in DNA synthesis are comparable to those for standard phosphoramidites. Examples of the fluorescence signals one can expect are described. Automated synthesis, which is dependent on the length of the sequence, takes about 4–5 h for a 20-mer. The deprotection of 6MI-containing sequences takes approximately 6–7 h before the standard ammonium hydroxide overnight incubation. Purification through polyacrylamide gels, electroelution and ethanol precipitation can be accomplished in 6–8 h.

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Figure 1: Schematic representation of the structures of 3MI, 6MI and 6MAP.
Figure 2: The absorption, fluorescence excitation and fluorescence emission scans of 6MI are representative of those of the other pteridines.
Figure 3: Photograph showing the small-volume vial inserted into the standard-size phosphoramidite bottle in place on the oligonucleotide synthesizer.
Figure 4
Figure 5: The real-time increase in fluorescence intensity displayed here is directly related to the activity of the P1 nuclease enzyme as it digests the sequence containing the probe (6MAP).
Figure 6: The scans shown here display the quantitative difference between the starting material (intact 6MAP-containing sequence) and the digested products of the same sequence after an overnight incubation at 37 °C.

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References

  1. Lakowicz, J.R. Principles of Fluorescence Spectroscopy. (Plenum Publishers, New York, 1999).

    Book  Google Scholar 

  2. Rist, M.J. & Marino, J.P. Fluorescent nucleotide base analogs as probes of nucleic acid structure, dynamics and interactions. Curr. Org. Chem. 6, 775–793 (2002).

    Article  CAS  Google Scholar 

  3. Guest, C.R., Hochstrasser, R.A., Sowers, L.C. & Millar, D.P. Dynamics of mismatched base pairs in DNA. Biochemistry 30, 3271–3279 (1991).

    Article  CAS  Google Scholar 

  4. Hochstrasser, R.A., Carver, T.E., Sowers, L.C. & Millar, D.P. Melting of a DNA helix terminus within the active site of a DNA polymerase. Biochemistry 33, 11971–11979 (1994).

    Article  CAS  Google Scholar 

  5. Nordlund, T.M. et al. Structural dynamics of DNA sensed by fluorescence from chemically-modified bases. SPIE Time-Resolved Laser Spectroscopy in Biochemistry II 1204, 344–353 (1990).

    Article  CAS  Google Scholar 

  6. Raney, K.D., Sowers, L.C., Millar, D.P. & Benkovic, S.J. A fluorescence-based assay for monitoring helicase activity. Proc. Natl. Acad. Sci. USA 91, 6644–6648 (1994).

    Article  CAS  Google Scholar 

  7. Sandin, P. et al. Fluorescent properties of DNA base analogue tC upon incorporation into DNA—negligible influence of neighbouring bases on fluorescence quantum yield. Nucleic Acids Res. 33, 5019–5025 (2005).

    Article  CAS  Google Scholar 

  8. Berry, D.A. et al. Pyrrolo-dC and pyrrolo-C: fluorescent analogs of cytidine and 2′-deoxycytidine for the study of oligonucleotides. Tetrahedron Lett. 45(11), 2457–2461 (2004).

    Article  Google Scholar 

  9. Augustyn, K.E., Wojtuszewski, K., Hawkins, M.E., Knutson, J.R. & Mukerji, I. Examination of the premelting transition of DNA A-tracts using a fluorescent adenosine analogue. Biochemistry 45, 5039–5047 (2006).

    Article  CAS  Google Scholar 

  10. Beecham, J.M. et al. Exonuclease-polymerase active site partitioning of primer-template DNA strands and equilibrium Mg2+ binding properties of bacteriophage T4 DNA polymerase. Biochemistry 37, 10144–10155 (1998).

    Article  Google Scholar 

  11. Bloom, L.B., Otto, M.R., Beechem, J.M. & Goodman, M.F. Influence of 5′-nearest neighbors on the insertion kinetics of the fluorescent nucleotide analog 2-aminopurine by Klenow fragment. Biochemistry 32, 11247–11258 (1993).

    Article  CAS  Google Scholar 

  12. Frey, M.W., Sowers, L.C., Millar, D.P. & Benkovic, S.J. The nucleotide analog 2-aminopurine as a spectroscopic probe of nucleotide incorporation by the Klenow fragment of Escherichia coli polymerase I and bacteriophage T4 DNA polymerase. Biochemistry 34, 9185–9192 (1995).

    Article  CAS  Google Scholar 

  13. Purohit, V., Grindley, N.D. & Joyce, C.M. Use of 2-aminopurine fluorescence to examine conformational changes during nucleotide incorporation by DNA polymerase I (Klenow fragment). Biochemistry 42, 10200–10211 (2003).

    Article  CAS  Google Scholar 

  14. Roy, S., Semsey, S., Liu, M., Gussin, G.N. & Adhya, S. GalR represses galP1 by inhibiting the rate-determining open complex formation through RNA polymerase contact: a GalR negative control mutant. J. Mol. Biol. 344, 609–618 (2004).

    Article  CAS  Google Scholar 

  15. Stivers, J.T. 2-Aminopurine fluorescence studies of base stacking interactions at abasic sites in DNA: metal-ion and base sequence effects. Nucleic Acids Res. 26, 3837–3844 (1998).

    Article  CAS  Google Scholar 

  16. Walker, R.K., McCullough, A.K. & Lloyd, R.S. Uncoupling of nucleotide flipping and DNA bending by the t4 pyrimidine dimer DNA glycosylase. Biochemistry 45, 14192–14200 (2006).

    Article  CAS  Google Scholar 

  17. Xu, Y. & Sugiyama, H. Formation of the G-quadruplex and i-motif structures in retinoblastoma susceptibility genes (Rb). Nucleic Acids Res. 34, 949–954 (2005).

    Article  Google Scholar 

  18. Wilson, J.N. & Kool, E.T. Fluorescent DNA base replacements: reporters and sensors for biological systems. Org. Biomol. Chem. 4, 4265–4274 (2006).

    Article  CAS  Google Scholar 

  19. Virta, P. et al. Synthesis, characterisation and theoretical calculations of 2,6-diaminopurine etheno derivatives. Org. Biomol. Chem. 3, 2924–2929 (2005).

    Article  CAS  Google Scholar 

  20. Hawkins, M.E., Pfleiderer, W., Balis, F.M., Porter, D. & Knutson, J.R. Fluorescence properties of pteridine nucleoside analogs as monomers and incorporated into oligonucleotides. Anal. Biochem. 244, 86–95 (1997).

    Article  CAS  Google Scholar 

  21. Hawkins, M.E., Pfleiderer, W., Mazumder, A., Pommier, Y.G. & Balis, F.M. Incorporation of a fluorescent guanosine analog into oligonucleotides and its application to a real time assay for the HIV-1 integrase 3′-processing reaction. Nucleic Acids Res. 23, 2872–2880 (1995).

    Article  CAS  Google Scholar 

  22. Turingan, R.S., Liu, C., Hawkins, M.E. & Martin, C.T. Structural confirmation of a bent and open model for the initiation complex of T7 RNA polymerase. Biochemistry 46(7), 1714–1723 (2007).

    Article  Google Scholar 

  23. Wojtuszewski, K., Hawkins, M., Cole, J.L. & Mukerji, I. HU binding to DNA: evidence for multiple complex formation and DNA bending. Biochemistry 40, 2588–2598 (2001).

    Article  CAS  Google Scholar 

  24. Myers, J.C., Moore, S.A. & Shamoo, Y. Structure-based incorporation of 6-methyl-8-(2-deoxy-beta-ribofuranosyl)isoxanthopteridine into the human telomeric repeat DNA as a probe for UP1 binding and destabilization of G-tetrad structures. J. Biol. Chem. 278, 42300–42306 (2003).

    Article  CAS  Google Scholar 

  25. Hawkins, M.E. Fluorescent nucleoside analogues as DNA probes. (ed. Lakowicz, J.P.) (Kluwer Academic/Plenum Publishers, New York, 2003).

  26. Hawkins, M.E., Pfleiderer, W., Jungmann, O. & Balis, F.M. Synthesis and fluorescence characterization of pteridine adenosine nucleoside analogs for DNA incorporation. Anal. Biochem. 298, 231–240 (2001).

    Article  CAS  Google Scholar 

  27. Sanabia, J.E., Goldner, L.S., Lacaze, P.A. & Hawkins, M.E. On the feasibility of single-molecule detection of the guanosine-analogue 3-MI. J. Phys. Chem. B 108, 15293–15300 (2004).

    Article  CAS  Google Scholar 

  28. Stanley, R.J., Hou, Z., Yang, A. & Hawkins, M.E. The two-photon excitation cross section of 6MAP, a fluorescent adenine analogue. J. Phys. Chem. B 109, 3690–3695 (2005).

    Article  CAS  Google Scholar 

  29. Roca, A.I. & Singleton, S.F. Direct evaluation of a mechanism for activation of the RecA nucleoprotein filament. J. Am. Chem. Soc. 125, 15366–15375 (2003).

    Article  CAS  Google Scholar 

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  1. National Cancer Institute, NIH, Pediatric Oncology Branch, 10 Center Drive, CRC 1-3872, Bethesda, 20854, Maryland, USA

    Mary E Hawkins

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Correspondence to Mary E Hawkins.

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The National Cancer Institute holds patents on the materials described in this paper.

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Hawkins, M. Synthesis, purification and sample experiment for fluorescent pteridine-containing DNA: tools for studying DNA interactive systems. Nat Protoc 2, 1013–1021 (2007). https://doi.org/10.1038/nprot.2007.150

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