Jump to Main Content
Characterization and use of an unprecedentedly bright and structurally non-perturbing fluorescent DNA base analogue
- Sandin, Peter, Börjesson, Karl, Li, Hong, Mårtensson, Jerker, Brown, Tom, Wilhelmsson, L. Marcus, Albinsson, Bo
- Nucleic acids research 2008 v.36 no.1 pp. 157-167
- DNA, DNA probes, absorption, energy transfer, fluorescence, guanine, nucleosides, oligonucleotides, protein tertiary structure, proteins
- This article presents the first evidence that the DNA base analogue 1,3-diaza-2-oxophenoxazine, tCO, is highly fluorescent, both as free nucleoside and incorporated in an arbitrary DNA structure. tCO 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 (ε = 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, tCO shows only minor variations in fluorescence intensity and lifetime with neighbouring bases, and the average quantum yield is 0.22. These features make tCO, 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 tCO 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).