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.