Modulating fluorescence anisotropy of dye-labeled DNA without involving mass amplification

Fluorescence anisotropy, known as a simple, homogeneous and cost-effective analytical technology, is an invaluable technique for studying the micro -environmental changes of the dye associated with the molecular interactions. An in-depth understanding of the variables affecting the fluorescence anisotropy signal can facilitate better experimental designs to effectively improve the analytical performance. This work is a follow-up effort in evaluating the factors that can significantly influence fluorescence anisotropy. We systematically studied fluorescence anisotropy of dsDNA with the changing length based on dye-DNA interactions, with the fluorophores in the end -labeling, the middle -site -labeling, and multiple number of labeling manners. The fluorescence anisotropy value and the base -pair response dynamic range could be expanded by labeling the fluorophores in the middle of dsDNA and increasing the number of labels on dsDNA. The C overhang configuration in the end -labeling manner could enhance the fluorescence anisotropy signal but not expand the base -pair response range. Results from all the labeling fluorophores reinforced the leveling -off effect, i.e., the fluorescence anisotropy signal does not response to the increased length of the DNA duplex when the length is larger than a critical number of base pairs. These findings provide perspectives about choosing appropriate fluorescent dyes and labeling sites for simple and universal fluorescence anisotropy designs in various applications. (C) 2016 Elsevier B.V. All rights reserved.