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. 2003 Jan 21;100(2):409-13.
doi: 10.1073/pnas.242729099. Epub 2003 Jan 6.

Photocleavage of a 2-nitrobenzyl linker bridging a fluorophore to the 5' end of DNA

Xiaopeng Bai  1 , Zengmin Li, Steffen Jockusch, Nicholas J Turro, Jingyue Ju
Affiliations

Photocleavage of a 2-nitrobenzyl linker bridging a fluorophore to the 5' end of DNA

Xiaopeng Bai et al. Proc Natl Acad Sci U S A. .

Abstract

Three single-stranded DNA molecules of different lengths were synthesized and characterized, each containing a fluorescent dye (6-carboxyfluorescein) connected to the 5' end via a photocleavable 2-nitrobenzyl linker and a biotin moiety at the 3' end. UV irradiation (lambda approximately 340 nm) of solutions containing these fluorescent DNA molecules caused the complete cleavage of the nitrobenzyl linker, separating the fluorophore from the DNA. The photocleavage products were characterized by HPLC and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. Our experimental results indicated that the proximity of the chromophore 6-carboxyfluorescein to the 2-nitrobenzyl linker did not hinder the quantitative photocleavage of the linker in the DNA molecules. The biotin moiety allowed immobilization of the fluorescent DNA on streptavidin-coated glass chips. The photocleavage of the immobilized DNA was investigated directly by fluorescence spectroscopy. The results demonstrated that close to 80% of the fluorophore was removed from the immobilized DNA after UV irradiation at 340 nm. These results strongly support the application of the 2-nitrobenzyl moiety as an efficient photocleavable linker, connecting fluorescent probes to DNA molecules for a variety of biological analyses such as DNA sequencing by synthesis.

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Figures

Figure 1
Figure 1
Molecular structure of 5′-Fam-linker-(T)5,10,20-biotin-3′ and their photocleavage reaction under irradiation with near-UV light.
Figure 2
Figure 2
MALDI-MS spectrum of Fam-linker-T10-biotin: Fam-linker-T10-biotin (m/z: found, 4,417; anal. calcd, 4,423), Fam-linker (m/z: found, 799; anal. calcd, 803), and T10-biotin (m/z: found, 3,616; anal. calcd, 3,621).
Figure 3
Figure 3
HPLC analysis of the photocleavage of Fam-linker-T10-biotin (2 μM) in 1:1 (vol/vol) water/acetonitrile. Detector channels: 260 nm (Left) and 499 nm (Right). (Top) Before irradiation; (Middle) after 5 min of irradiation; and (Bottom) after 10 min of irradiation (λirr = 340 nm).
Figure 4
Figure 4
MALDI-TOF MS spectra of the photocleavage products of Fam-linker-T10-biotin in solution phase: Fam-linker (m/z: found, 795; anal. calcd, 803) and T10-biotin (m/z: found, 3,618; anal. calcd, 3,621).
Figure 5
Figure 5
Absorption spectra for Fam-linker-T10-biotin and 1-[5-(aminomethyl)-2-nitrophenyl]ethanol (2-nitrobenzyl linker) with equal molar concentration (5.2 μM) obtained in 1:1 (vol/vol) water/acetonitrile.
Figure 6
Figure 6
Fluorescence spectra (λex = 460 nm) of Fam-linker-T5-biotin immobilized on a streptavidin-coated glass chip. a, before irradiation; b–e, after 2, 7, 17, and 27 min of irradiation, respectively (λirr = 340 nm, 20 mW/cm2).
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References

    1. Pillai, V. N. R. (1980) Synthesis 1–26.
    1. Guillier F, Orain D, Bradley M. Chem Rev (Washington, DC) 2000;100:2091–2157. - PubMed
    1. Bochet C G. J Chem Soc Perkin Trans. 2002;1:125–142.
    1. Kahl J D, Greenberg M M. J Am Chem Soc. 1999;121:597–604.
    1. Walker J W, Reid G P, McCray J A, Trentham D R. J Am Chem Soc. 1988;110:7170–7177.

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