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. 2008 Jan;36(1):157-67.
doi: 10.1093/nar/gkm1006. Epub 2007 Nov 14.

Characterization and use of an unprecedentedly bright and structurally non-perturbing fluorescent DNA base analogue

Peter Sandin  1 , Karl Börjesson, Hong Li, Jerker Mårtensson, Tom Brown, L Marcus Wilhelmsson, Bo Albinsson
Affiliations

Characterization and use of an unprecedentedly bright and structurally non-perturbing fluorescent DNA base analogue

Peter Sandin et al. Nucleic Acids Res. 2008 Jan.

Abstract

This article presents the first evidence that the DNA base analogue 1,3-diaza-2-oxophenoxazine, tC(O), is highly fluorescent, both as free nucleoside and incorporated in an arbitrary DNA structure. tC(O) is thoroughly characterized with respect to its photophysical properties and structural performance in single- and double-stranded oligonucleotides. The lowest energy absorption band at 360 nm (epsilon = 9000 M(-1) cm(-1)) is dominated by a single in-plane polarized electronic transition and the fluorescence, centred at 465 nm, has a quantum yield of 0.3. When incorporated into double-stranded DNA, tC(O) shows only minor variations in fluorescence intensity and lifetime with neighbouring bases, and the average quantum yield is 0.22. These features make tC(O), on average, the brightest DNA-incorporated base analogue so far reported. Furthermore, it base pairs exclusively with guanine and causes minimal perturbations to the native structure of DNA. These properties make tC(O) a promising base analogue that is perfectly suited for e.g. photophysical studies of DNA interacting with macromolecules (proteins) or for determining size and shape of DNA tertiary structures using techniques such as fluorescence anisotropy and fluorescence resonance energy transfer (FRET).

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Figures

Figure 1.
Figure 1.
Structure of the G-tCO base-pair (left) and the derivatives of tCO used in the measurements (right) (R = CH2COO− K+ for the potassium salt of 1,3-diaza-2-oxophenoxazin-3-yl acetic acid (KtCO) and deoxyribose for the 2-deoxyribonucleoside of tCO). δ is an in-plane angle relative the molecular orientation axis (z) that is parallel to the molecular long axis. μ indicates the polarization of the lowest energy electronic transition moment.
Figure 2.
Figure 2.
Isotropic absorption and emission spectra of the tCO nucleoside (blue), tCO-containing single strand (AA, red) and tCO-containing double strand (AA, black). The spectra have been normalized at the maxima of the lowest energy absorption band (left) and of the emission (right), respectively.
Figure 3.
Figure 3.
(a) Isotropic absorption (Aiso, black) and excitation anisotropy spectra (r, red) of the tCO nucleoside and reduced linear dichroism spectra (LDr, blue) of KtCO. Aiso was measured in a phosphate buffer (50 mM Na+, pH 7.5), r in a solid H2O/ethylene glycol (1:2 mixture) matrix at −100°C and LDr in a stretched PVA film. (b) Magnetic circular dichroism (MCD) of KtCO in a phosphate buffer (50 mM Na+, pH 7.5). (c) Electronic transitions of the tCO chromophore and their oscillator strengths calculated using the ZINDO/S method on an AM1 geometry optimized structure. Note that the calculated wavenumbers have been multiplied by 0.9 to facilitate comparison.
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References

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