Nucleobase-Specific Quenching of Fluorescent Dyes. 1. Nucleobase One-Electron Redox Potentials and Their Correlation with Static and Dynamic Quenching Efficiencies

Abstract

Intermolecular static and dynamic fluorescence quenching constants of eight coumarin derivatives by nucleobase derivatives have been determined in aqueous media. One common sequence of the quenching efficiency has been found for the nucleobases. The feasibility of a photoinduced electron transfer reaction for the nucleobase-specific quenching of fluorescent dyes is investigated by the calculation of the standard free energy changes with the Rehm−Weller equation. A complete set of one-electron redox potential data for the nucleobases are determined electrochemically in aprotic solvents for the first time, which are compared with values obtained by various other methods. Depending on the redox properties of the fluorescent dyes, the sequences of the quenching efficiencies can be rationalized by the orders of electrochemical oxidation potentials (vs NHE) of nucleosides (dG (+1.47 V) < dA < dC ≈ dT < U (≥ +2.39 V)) and reduction potentials (dG (< −2.76 V) < dA < dC < dT < U (−2.07 V)). The correlation between the intermolecular dynamic quenching constants and the standard free energy of photoinduced electron transfer according to the classical Marcus equation indicates that photoinduced electron transfer is the rate-limiting step. However, an additional, water-specific gain of free energy between −0.5 and −0.9 eV shows that additional effects, like a coupled proton transfer and a hydrophobic effect, have to be considered, too. Furthermore, the capability of the nucleobases to form ground state complexes with fluorescent dyes is influenced by their redox potentials. The relevance of these observations to current efforts for DNA sequencing with a detection by laser-induced fluorescence and their application to other dyes are discussed.

Effects of strand and directional asymmetry on base–base coupling and charge transfer in double-helical DNA

“…Likewise, the reorganization energy in analogous assemblies is also expected to be essentially constant. The driving force for HT between Ap* and G is estimated to be Ϸ200 mV based on the redox potentials of the free base (Ap) and nucleotide (G) in solution (7,53,54). Within DNA it is likely that the driving force differs somewhat, and it is also possible that the precise HT energetics depend on the bases adjacent to the redox participants.…”

Chapter 12 Using Fluorophore-Labeled Oligonucleotides to Measure Affinities of Protein–DNA Interactions

“…Time-resolved fluorescence studies of various tetramethylrhodamine (TMR)-or TAMRA-labeled oligonucleotides demonstrate that the fluorophore can have multiple fluorescent lifetimes, suggesting that the probe exists in multiple environments (Edman et al, 1996;Eggeling et al, 1998;Harley et al, 2002;Vamosi et al, 1996). One of these lifetimes may involve an interaction between fluorophore and DNA bases, which can potentially quench fluorescence emission or otherwise affect the measured values (Sauer et al, 1995;Seidel et al, 1996;Sevenich et al, 1998). Fluorophore-nucleic acid interactions have also been proposed for Cy3 and fluorescein (Nazarenko et al, 2002;Nelson et al, 1993).…”

DNA repair glycosylases with a [4Fe–4S] cluster: A redox cofactor for DNA-mediated charge transport?

“…This reaction would provide a positive signal for electron trapping from the DNA-bound protein. Significantly, the midpoint potentials of the BER enzymes (~0.1 V versus NHE) render them incapable of reducing the intervening bases [73].…”