Hybridization of fluorescein-labeled DNA oligomers detected by fluorescence anisotropy with protein binding enhancement

Abstract

Fundamental aspects of the application of fluorescence anisotropy to detect the hybridization of fluorescein-labeled DNA oligomers were explored. The oligomers included a binding site for the EcoRI restriction enzyme, which binds to double-stranded DNA and is used in this work to enhance the difference between the anisotropies of the single-stranded and double-stranded oligomers by increasing the effective volume of the latter. The fluorescence anisotropy increases upon hybridization and further upon binding of EcoRI to the double strand. By varying the length of the tether used to attach the fluorescein to the 5' end of the oligonucleotide, it was found that a 6-carbon tether was optimal, providing the most dramatic increases in anisotropy in the presence of EcoRI. Dynamic fluorescence anisotropy (DFA) provided insight into the increases in steady-state anisotropy. In most cases, the best fits to the DFA data were obtained using a biexponential decay model, which describes an anisotropic rotator. Upon hybridization, the faster rotational motion is more hindered, and the contribution of the slower rotational component is increased. This effect is enhanced by binding of EcoRI to the double strand, especially when the EcoRI binding site is near the fluorescein at the 5' end and the tether length is in the optimal range. Because the rotational correlation time of the slower anisotropy decay component is much longer than the fluorescence lifetime, it is possible in some cases to reduce the anisotropic rotator model to the special limiting case of a hindered rotator.

Determination of the turnover number of the restriction endonuclease EcoRI using evanescent wave technology

“…However, in some experiments we observed a small increase of fluorescence upon EcoRI addition (indicated in Fig. 4), an enhancement effect that has been described by Kumke et al [13]. Changing to Mg buffer starts a rapid dissocia- tion, that we regard as catalytic activity of the enzyme.…”

Detection of Hybrid Formation between Peptide Nucleic Acids and DNA by Fluorescence Polarization in the Presence of Polylysine

“…Similar changes in fluorescence polarization have been observed upon hybridization of labeled DNA probes to DNA targets (19,20). These relatively small changes have been used to develop methods for the detection of DNA sequences in homogeneous phase (21), and approaches for signal enhancement have been developed, for example, by using proteins that specifically bind the doublestranded DNA hybrids (22). The advantages of such signal enhancement in terms of assay sensitivity have been discussed by Walker et al (22).…”

Toward a cancer therapy with boron-rich oligomeric phosphate diesters that target the cell nucleus

“…The carborane cage in nido-(CB) 5 is di-C-substituted and constitutes part of the backbone of the oligomer, whereas the cage in both closo-and nido-(G1) 5 is mono-C-substituted and is attached to these oligomeric structures by side chains. The fluorescein-labeled OPDs are quite stable in either moderately acidic or basic conditions, and the fluorescein group is unlikely to be released from the oligomers under physiological conditions (11).…”