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Synthesis and site-specific incorporation of a simple fluorescent pyrimidine

Nature Protocols volume 2, pages 305–316 (2007)Cite this article

Abstract

We describe procedures for the synthesis of a fluorescent pyrimidine analog and its site-specific incorporation into a DNA oligomer. The 5′-protected and 3′-activated nucleoside 4 is synthesized in three steps with an overall yield of 40%. Site-specific incorporation into a DNA oligomer occurs with greater than 88% coupling efficiency. This isosteric fluorescent DNA analog can be used to monitor denaturation of DNA duplexes via fluorescence and can positively detect the presence of abasic sites in DNA duplexes. The total time for synthesis of the phosphoramidite 4 is about 75 h, whereas the total time for site-specific incorporation of nucleoside 2 into an oligonucleotide and purification of the corresponding oligonucleotide is about 114 hours.

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Figure 1: Scheme for the synthesis of phosphoramidite 4.
Figure 2: Key steps in solid phase DNA phosphoramidite synthesis cycle.
Figure 3: Fluorescence spectra of an oligonucleotide containing 2, hybridized to its perfect complement (red dashed line) and an oligonucleotide containing an abasic site (green solid line) at 1.0 μM.
Figure 4: Absorption spectra at 50 μM (red) and fluorescence spectra at 10 μM (blue) of nucleoside 2.
Figure 5: Typical fluorescence spectra of 5′-GCGATG2GTAGCG-3′ at 1.0 μM (red), 0.5 μM (blue) and 0.1 μM (green).

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References

  1. Daniels, M. & Hauswirth, W. Fluorescence of the purine and pyrimidine bases of the nucleic acids in neutral aqueous solution at 300 degrees K. Science 171, 675–677 (1971).

    Article  CAS  PubMed  Google Scholar 

  2. Pecourt, J.M.L., Peon, J. & Kohler, B. Ultrafast internal conversion of electronically excited RNA and DNA nucleosides in water. J. Am. Chem. Soc. 122, 9348–9349 (2000).

    Article  CAS  Google Scholar 

  3. Nir, E., Kleinermanns, K., Grace, L. & de Vries, M.S. On the photochemistry of purine nucleobases. J. Phys. Chem. A 105, 5106–5110 (2001).

    Article  CAS  Google Scholar 

  4. Coleman, R.S. & Madaras, M.L. Synthesis of a novel coumarin C-riboside as a photophysical probe of oligonucleotide dynamics. J. Org. Chem. 63, 5700–5703 (1998).

    Article  CAS  Google Scholar 

  5. Nakatani, K., Dohno, C. & Saito, I. Chemistry of sequence-dependent remote guanine oxidation: Photoreaction of duplex DNA containing cyanobenzophenone-substituted uridine. J. Am. Chem. Soc. 121, 10854–10855 (1999).

    Article  CAS  Google Scholar 

  6. Strässler, C., Davis, N.E. & Kool, E.T. Novel nucleoside analogues with fluorophores replacing the DNA base. Helv. Chim. Acta 82, 2160–2171 (1999).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Wojczewski, C., Stolze, K. & Engels, J.W. Fluorescent oligonucleotides - Versatile tools as probes and primers for DNA and RNA analysis. Synlett. 1667–1678 (1999).

  8. Seela, F., Zulauf, M., Sauer, M. & Deimel, M. 7-substituted 7-deaza-2′-deoxyadenosines and 8-aza-7-deaza-2′-deoxyadenosines: Fluorescence of DNA-base analogues induced by the 7-alkynyl side chain. Helv. Chim. Acta 83, 910–927 (2000).

    Article  CAS  Google Scholar 

  9. Hawkins, M.E. Fluorescent pteridine nucleoside analogs: a window on DNA interactions. Cell Biochem. Biophys. 34, 257–281 (2001).

    Article  CAS  PubMed  Google Scholar 

  10. Murphy, C.J. Photophysical probes of DNA sequence-directed structure and dynamics. Adv. Photochem. 26, 145–217 (2001).

    CAS  Google Scholar 

  11. Hurley, D.J., Seaman, S.E., Mazura, J.C. & Tor, Y. Fluorescent 1,10-phenanthroline-containing oligonucleotides distinguish between perfect and mismatched base pairing. Org. Lett. 4, 2305–2308 (2002).

    Article  CAS  PubMed  Google Scholar 

  12. 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 

  13. Coleman, R.S. & Mortensen, M.A. Stereocontrolled synthesis of anthracene beta-C-ribosides: fluorescent probes for photophysical studies of DNA. Tetrahedron Lett. 44, 1215–1219 (2003).

    Article  CAS  Google Scholar 

  14. Dash, C., Rausch, J.W. & Le Grice, S.F. Using pyrrolo-deoxycytosine to probe RNA/DNA hybrids containing the human immunodeficiency virus type-1 3′ polypurine tract. Nucleic Acids Res. 32, 1539–1547 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Jiao, G.S., Kim, T.G., Topp, M.R. & Burgess, K. A blue-to-red energy-transfer thymidine analogue that functions in DNA. Org. Lett. 6, 1701–1704 (2004).

    Article  CAS  PubMed  Google Scholar 

  16. Mayer, E. et al. 1-Ethynylpyrene as a tunable and versatile molecular beacon for DNA. ChemBioChem 5, 865–868 (2004).

    Article  CAS  PubMed  Google Scholar 

  17. Okamoto, A., Kanatani, K. & Saito, I. Pyrene-labeled base-discriminating fluorescent DNA probes for homogeneous SNP typing. J. Am. Chem. Soc. 126, 4820–4827 (2004).

    Article  CAS  PubMed  Google Scholar 

  18. Gaballah, S.T., Vaught, J.D., Eaton, B.E. & Netzel, T.L. Charge-transfer excited state dynamics in DNA hairpins substituted with an ethylenylpyrenyl-dU electron source and halo-dU traps. J. Phys. Chem. B 109, 5927–5934 (2005).

    Article  CAS  PubMed  Google Scholar 

  19. Okamoto, A., Saito, Y. & Saito, I. Design of base-discriminating fluorescent nucleosides. J. Photochem. Photobiol., C 6, 108–122 (2005).

    Article  CAS  Google Scholar 

  20. Okamoto, A., Tainaka, K., Nishiza, K. & Saito, I. Monitoring DNA structures by dual fluorescence of pyrene derivatives. J. Am. Chem. Soc. 127, 13128–13129 (2005).

    Article  CAS  PubMed  Google Scholar 

  21. Okamoto, A., Tainaka, K. & Saito, I. A dielectric-sensitive fluorescent DNA probe for monitoring polarities on the interior of a DNA-binding protein. Bioconjugate Chem. 16, 1105–1111 (2005).

    Article  CAS  Google Scholar 

  22. Ranasinghe, R.T. & Brown, T. Fluorescence based strategies for genetic analysis. Chem. Commun. 5487–5502 (2005).

  23. 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  PubMed  PubMed Central  Google Scholar 

  24. Andreatta, D. et al. Ultrafast dynamics in DNA: “fraying” at the end of the helix. J. Am. Chem. Soc. 128, 6885–6892 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Asseline, U. Development and applications of fluorescent oligonucleotides. Curr. Org. Chem. 10, 491–518 (2006).

    Article  CAS  Google Scholar 

  26. Cho, Y. & Kool, E.T. Enzymatic synthesis of fluorescent oligomers assembled on a DNA backbone. ChemBioChem 7, 669–672 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Kim, S.J. & Kool, E.T. Sensing metal ions with DNA building blocks: fluorescent pyridobenzimidazole nucleosides. J. Am. Chem. Soc. 128, 6164–6171 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Silverman, A.P. & Kool, E.T. Detecting RNA and DNA with templated chemical reactions. Chem. Rev. 106, 3775–3789 (2006).

    Article  CAS  PubMed  Google Scholar 

  29. Ward, D.C., Reich, E. & Stryer, L. Fluorescence studies of nucleotides and polynucleotides. I. Formycin, 2-aminopurine riboside, 2,6-diaminopurine riboside, and their derivatives. J. Biol. Chem. 244, 1228–1237 (1969).

    CAS  PubMed  Google Scholar 

  30. Seela, F. & Becher, G. Synthesis, base pairing, and fluorescence properties of oligonucleotides containing 1H-pyrazolo[3,4-d]pyrimidin-6-amine (8-aza-7-deazapurin-2-amine) as an analogue of purin-2-amine. Helv. Chim. Acta 83, 928–942 (2000).

    Article  CAS  Google Scholar 

  31. Jean, J.M. & Hall, K.B. 2-Aminopurine fluorescence quenching and lifetimes: role of base stacking. Proc. Natl. Acad. Sci. USA 98, 37–41 (2001).

    Article  CAS  PubMed  Google Scholar 

  32. Kawai, M., Lee, M.J., Evans, K.O. & Nordlund, T.M. Temperature and base sequence dependence of 2-aminopurine fluorescence bands in single- and double-stranded oligodeoxynucleotides. J. Fluoresc. 11, 23–32 (2001).

    Article  CAS  Google Scholar 

  33. Rachofsky, E.L., Osman, R. & Ross, J.B. Probing structure and dynamics of DNA with 2-aminopurine: effects of local environment on fluorescence. Biochemistry 40, 946–956 (2001).

    Article  CAS  PubMed  Google Scholar 

  34. Menger, M., Tuschl, T., Eckstein, F. & Porschke, D. Mg(2+)-dependent conformational changes in the hammerhead ribozyme. Biochemistry 35, 14710–14716 (1996).

    Article  CAS  PubMed  Google Scholar 

  35. Kirk, S.R., Luedtke, N.W. & Tor, Y. 2-Aminopurine as a real-time probe of enzymatic cleavage and inhibition of hammerhead ribozymes. Bioorg. Med. Chem. 9, 2295–2301 (2001).

    Article  CAS  PubMed  Google Scholar 

  36. Rist, M. & Marino, J. Association of an RNA kissing complex analyzed using 2-aminopurine fluorescence. Nucleic Acids Res. 29, 2401–2408 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Lacourciere, K.A., Stivers, J.T. & Marino, J.P. Mechanism of neomycin and Rev peptide binding to the Rev responsive element of HIV-1 as determined by fluorescence and NMR spectroscopy. Biochemistry 39, 5630–5641 (2000).

    Article  CAS  PubMed  Google Scholar 

  38. DeJong, E.S., Chang, C.E., Gilson, M.K. & Marino, J.P. Proflavine acts as a Rev inhibitor by targeting the high-affinity Rev binding site of the Rev responsive element of HIV-1. Biochemistry 42, 8035–8046 (2003).

    Article  CAS  PubMed  Google Scholar 

  39. Kaul, M., Barbieri, C.M. & Pilch, D.S. Fluorescence-based approach for detecting and characterizing antibiotic-induced conformational changes in ribosomal RNA: comparing aminoglycoside binding to prokaryotic and eukaryotic ribosomal RNA sequences. J. Am. Chem. Soc. 126, 3447–3453 (2004).

    Article  CAS  PubMed  Google Scholar 

  40. Shandrick, S. et al. Monitoring molecular recognition of the ribosomal decoding site. Angew. Chem., Int. Ed. Engl. 43, 3177–3182 (2004).

    Article  CAS  Google Scholar 

  41. Constant, J.F. et al. Design of molecules which specifically cleave abasic sites in DNA. Anti-Cancer Drug Des. 5, 59–62 (1990).

    CAS  Google Scholar 

  42. Weinfeld, M., Liuzzi, M. & Paterson, M.C. Response of phage T4 polynucleotide kinase toward dinucleotides containing apurinic sites: design of a 32P-postlabeling assay for apurinic sites in DNA. Biochemistry 29, 1737–1743 (1990).

    Article  CAS  PubMed  Google Scholar 

  43. Chen, B.X. et al. Properties of a monoclonal antibody for the detection of abasic sites, a common DNA lesion. Mutat. Res. 273, 253–261 (1992).

    Article  CAS  PubMed  Google Scholar 

  44. Kubo, K., Ide, H., Wallace, S.S. & Kow, Y.W. A novel, sensitive, and specific assay for abasic sites, the most commonly produced DNA lesion. Biochemistry 31, 3703–3708 (1992).

    Article  CAS  PubMed  Google Scholar 

  45. Fkyerat, A. et al. A new class of artificial nucleases that recognize and cleave apurinic sites in DNA with great selectivity and efficiency. J. Am. Chem. Soc. 115, 9952–9959 (1993).

    Article  CAS  Google Scholar 

  46. Ide, H. et al. Synthesis and damage specificity of a novel probe for the detection of abasic sites in DNA. Biochemistry 32, 8276–8283 (1993).

    Article  CAS  PubMed  Google Scholar 

  47. Berthet, N. et al. Design of molecules that specifically recognize and cleave apurinic sites in DNA. J. Mol. Recognit. 7, 99–107 (1994).

    Article  CAS  PubMed  Google Scholar 

  48. Belmont, P. et al. Efficient and versatile chemical tools for cleavage of abasic sites in DNA. New J. Chem. 21, 47–54 (1997).

    CAS  Google Scholar 

  49. Berthet, N. et al. Search for DNA repair inhibitors: selective binding of nucleic bases-acridine conjugates to a DNA duplex containing an abasic site. J. Med. Chem. 40, 3346–3352 (1997).

    Article  CAS  PubMed  Google Scholar 

  50. Boturyn, D. et al. Synthesis of fluorescent probes for the detection of abasic sites in DNA. Tetrahedron 53, 5485–5492 (1997).

    Article  CAS  Google Scholar 

  51. Boturyn, D. et al. Quantitative one step derivatization of oligonucleotides by a fluorescent label through abasic site formation. Nucleosides Nucleotides 16, 2069–2077 (1997).

    Article  CAS  Google Scholar 

  52. Adamczyk, M., Mattingly, P.G. & Moore, J.A. O-(fluoresceinylmethyl)hydroxylamine (OFMHA): a fluorescent reagent for detection of damaged nucleic acids. Bioorg. Med. Chem. Lett. 8, 3599–3602 (1998).

    Article  CAS  PubMed  Google Scholar 

  53. Asaeda, A. et al. Highly sensitive assay of DNA abasic sites in mammalian cells optimization of the aldehyde reactive probe method. Anal. Chim. Acta 365, 35–41 (1998).

    Article  CAS  Google Scholar 

  54. Makrigiorgos, G.M., Chakrabarti, S. & Mahmood, A. Fluorescent labelling of abasic sites: a novel methodology to detect closely-spaced damage sites in DNA. Int. J. Radiat. Biol. 74, 99–109 (1998).

    Article  CAS  PubMed  Google Scholar 

  55. Berthet, N. et al. Recognition of abasic sites in DNA by a cyclobisacridine molecule. Chem. Eur. J. 5, 3625–3630 (1999).

    Article  CAS  Google Scholar 

  56. Boturyn, D. et al. A simple and sensitive method for in vitro quantitation of abasic sites in DNA. Chem. Res. Toxicol. 12, 476–482 (1999).

    Article  CAS  PubMed  Google Scholar 

  57. Lhomme, J., Constant, J.F. & Demeunynck, M. Abasic DNA structure, reactivity, and recognition. Biopolymers 52, 65–83 (1999).

    Article  CAS  PubMed  Google Scholar 

  58. Teulade-Fichou, M.P. et al. Specific recognition and stabilization of an abasic site-containing DNA duplex by a macrocyclic bisacridine. Nucleosides Nucleotides 18, 1351–1353 (1999).

    Article  CAS  Google Scholar 

  59. Atamna, H., Cheung, I. & Ames, B.N. A method for detecting abasic sites in living cells: age-dependent changes in base excision repair. Proc. Natl. Acad. Sci. USA 97, 686–691 (2000).

    Article  CAS  PubMed  Google Scholar 

  60. Bowman, K.J. et al. Evaluation of phosphodiesterase I-based protocols for the detection of multiply damaged sites in DNA: the detection of abasic, oxidative and alkylative tandem damage in DNA oligonucleotides. Nucleic Acids Res. 29, E101 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Sun, H.B., Qian, L. & Yokota, H. Detection of abasic sites on individual DNA molecules using atomic force microscopy. Anal. Chem. 73, 2229–2232 (2001).

    Article  CAS  PubMed  Google Scholar 

  62. Zhang, L.K., Rempel, D. & Gross, M.L. Matrix-assisted laser desorption/ionization mass spectrometry for locating abasic sites and determining the rates of enzymatic hydrolysis of model oligodeoxynucleotides. Anal. Chem. 73, 3263–3273 (2001).

    Article  CAS  PubMed  Google Scholar 

  63. Georgakilas, A.G., Bennett, P.V. & Sutherland, B.M. High efficiency detection of bi-stranded abasic clusters in gamma-irradiated DNA by putrescine. Nucleic Acids Res. 30, 2800–2808 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Hirose, T., Ohtani, T., Muramatsu, H. & Tanaka, A. Direct visualization of abasic sites on a single DNA molecule using fluorescence microscopy. Photochem. Photobiol. 76, 123–126 (2002).

    Article  CAS  PubMed  Google Scholar 

  65. Zhang, L.K. & Gross, M.L. Location of abasic sites in oligodeoxynucleotides by tandem mass spectrometry and by a chemical cleavage initiated by an unusual reaction of the ODN with MALDI matrix. J. Am. Soc. Mass. Spectrom. 13, 1418–1426 (2002).

    Article  CAS  PubMed  Google Scholar 

  66. Ide, H. et al. Detection of DNA damage by the aldehyde reactive probe (ARP) assay: principles and its application. Recent Res. Devel. Nucleosides Nucleotides 1, 33–47 (2003).

    CAS  Google Scholar 

  67. Kim, J., Muramatsu, H., Lee, H. & Kawai, T. Near-field optical imaging of abasic sites on a single DNA molecule. FEBS Lett. 555, 611–615 (2003).

    Article  CAS  PubMed  Google Scholar 

  68. Yanagisawa, H., Hirano, A. & Sugawara, M. A dot-blot method for quantification of apurinic/apyrimidinic sites in DNA using an avidin plate and liposomes encapsulating a fluorescence dye. Anal. biochem. 332, 358–367 (2004).

    Article  CAS  PubMed  Google Scholar 

  69. Chakravarti, D. et al. Improved measurement of dibenzo[a,l]pyrene-induced abasic sites by the aldehyde-reactive probe assay. Mutat. Res. 588, 158–165 (2005).

    Article  CAS  PubMed  Google Scholar 

  70. Sato, K. & Greenberg, M.M. Selective detection of 2-deoxyribonolactone in DNA. J. Am. Chem. Soc. 127, 2806–2807 (2005).

    Article  CAS  PubMed  Google Scholar 

  71. Zhou, X. et al. Quantification of DNA strand breaks and abasic sites by oxime derivatization and accelerator mass spectrometry: application to gamma-radiation and peroxynitrite. Anal. biochem. 343, 84–92 (2005).

    Article  CAS  PubMed  Google Scholar 

  72. Dolinnaya, N.G. et al. Electrochemical detection of abasic site-containing DNA. Electroanalysis 18, 399–404 (2006).

    Article  CAS  Google Scholar 

  73. Huang, W. et al. Electrochemical detection at low temperature for a specific nucleobase of target nucleic acids by an abasic site-containing DNA binding ligand. Electrochem. Commun. 8, 395–398 (2006).

    Article  CAS  Google Scholar 

  74. Martelli, A. et al. Photoreactive threading agent that specifically binds to abasic sites in DNA. Bioorg. Med. Chem. Lett. 16, 154–157 (2006).

    Article  CAS  PubMed  Google Scholar 

  75. Morita, K. et al. Electrochemical SNPs detection using an abasic site-containing DNA on a gold electrode. Chem. Commun. 2376–2378 (2006).

  76. Roberts, K.P. et al. Determination of apurinic/apyrimidinic lesions in DNA with high-performance liquid chromatography and tandem mass spectrometry. Chem. Res. Toxicol. 19, 300–309 (2006).

    Article  CAS  PubMed  Google Scholar 

  77. Fukui, K. et al. Synthesis and properties of an oligonucleotide modified with an acridine derivative at the artificial abasic site. Bioconjugate Chem. 7, 349–355 (1996).

    Article  CAS  Google Scholar 

  78. Fukui, K. & Tanaka, K. The acridine ring selectively intercalated into a DNA helix at various types of abasic sites: double strand formation and photophysical properties. Nucleic Acids Res. 24, 3962–3967 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Fukui, K.I., Kazunori, Shimidzu, T. & Tanaka, K. Oligonucleotides covalently linked to an acridine at artificial abasic site: influence of linker length and the base-sequence. Tetrahedron Lett. 37, 4983–4986 (1996).

    Article  CAS  Google Scholar 

  80. Belmont, P., Alarcon, K., Demeunynck, M. & Lhomme, J. Synthesis of an imidazo[1,2-e]purine-acridine heterodimer for targeting abasic sites in DNA. Bioorg. Med. Chem. Lett. 9, 233–236 (1999).

    Article  CAS  PubMed  Google Scholar 

  81. Matray, T.J. & Kool, E.T. A specific partner for abasic damage in DNA. Nature 399, 704–708 (1999).

    Article  CAS  PubMed  Google Scholar 

  82. Alarcon, K. et al. Diaminopurine-acridine heterodimers for specific recognition of abasic site containing DNA. Influence on the biological activity of the position of the linker on the purine ring. Bioorg. Med. Chem. Lett. 11, 1855–1858 (2001).

    Article  CAS  PubMed  Google Scholar 

  83. Alarcon, K. et al. Potentiation of BCNU cytotoxicity by molecules targeting abasic lesions in DNA. Bioorg. Med. Chem. 9, 1901–1910 (2001).

    Article  CAS  PubMed  Google Scholar 

  84. Brotschi, C., Häberli, A. & Leumann, C.J. A stable DNA duplex containing a non-hydrogen-bonding and non-shape-complementary base couple: interstrand stacking as the stability determining factor. Angew. Chem. Int. Ed. Engl. 40, 3012–3014 (2001).

    Article  CAS  PubMed  Google Scholar 

  85. Yoshimoto, K., Nishizawa, S., Minagawa, M. & Teramae, N. Use of abasic site-containing DNA strands for nucleobase recognition in water. J. Am. Chem. Soc. 125, 8982–8983 (2003).

    Article  CAS  PubMed  Google Scholar 

  86. Langenegger, S.M. & Haener, R. A simple, non-nucleosidic base surrogate increases the duplex stability of DNA containing an abasic site. Chem. Biodiversity 1, 259–264 (2004).

    Article  CAS  Google Scholar 

  87. Singh, I. et al. Abasic site stabilization by aromatic DNA base surrogates: High-affinity binding to a baseflipping DNA-methyltransferase. Pure Appl. Chem. 76, 1563–1570 (2004).

    Article  CAS  Google Scholar 

  88. Langenegger, S.M. & Haener, R. Remarkable stabilization of duplex DNA containing an abasic site by non-nucleosidic phenanthroline and pyrene building blocks. ChemBioChem 6, 848–851 (2005).

    Article  CAS  PubMed  Google Scholar 

  89. Valis, L., Amann, N. & Wagenknecht, H.A. Detection of single base mismatches and abasic sites using phenanthridinium as an artificial DNA base and charge donor. Org. Biomol. Chem. 3, 36–38 (2005).

    Article  CAS  PubMed  Google Scholar 

  90. 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  PubMed  PubMed Central  Google Scholar 

  91. Pompizi, I., Häberli, A. & Leumann, C.J. Oligodeoxynucleotides containing conformationally constrained abasic sites: a UV and fluorescence spectroscopic investigation on duplex stability and structure. Nucleic Acids Res. 28, 2702–2708 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Rachofsky, E.L. et al. Conformation and dynamics of abasic sites in DNA investigated by time-resolved fluorescence of 2-aminopurine. Biochemistry 40, 957–967 (2001).

    Article  CAS  PubMed  Google Scholar 

  93. Greco, N.J. & Tor, Y. Simple fluorescent pyrimidine analogs detect the presence of DNA abasic sites. J. Am. Chem. Soc. 127, 10784–10785 (2005).

    Article  CAS  PubMed  Google Scholar 

  94. Gait, M.J. Oligonucleotide Synthesis: a Practical Approach. (IRL Press, Washington, DC, 1984).

    Google Scholar 

  95. Eckstein, F. Oligonucleotides and Analogues: a Practical Approach. (IRL Press at Oxford University Press, Oxford, New York, 1991).

    Google Scholar 

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Acknowledgements

This work is supported by the US National Institutes of Health (GM069773).

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  1. Department of Chemistry and Biochemistry, University of California, 9500 Gilman Drive, MC 0358, La Jolla, San Diego, 92093, California, USA

    Nicholas J Greco & Yitzhak Tor

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Greco, N., Tor, Y. Synthesis and site-specific incorporation of a simple fluorescent pyrimidine. Nat Protoc 2, 305–316 (2007). https://doi.org/10.1038/nprot.2006.464

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