Determination of Escherichia coli Ligase Activity by Carbon Nanotube Fluorescence Anisotropy

Abstract

The characterization of nucleic acid ligase activity is of great significance for the study of physiological processes. In the present work, a new method for the detection of E. coli DNA ligase is designed based on the non-covalent interaction of carbon nanotubes with dyes and ssDNA and DNA ligation reactions. When no E. coli DNA ligase is present, the ssDNA probe and dye SG I adsorb to the surface of carbon nanotubes. After centrifugation, the carbon nanotubes with DNA and dye SG I have larger molecular weights, resulting in higher fluorescence anisotropy. When E. coli DNA ligase is present, a double-stranded structure is formed, with SG I embedded in the groove of the DNA double strand. Due to the weak interaction between the DNA double-strand structure and the carbon nanotubes, after centrifugation, the supernatant does not contain the carbon nanotubes, resulting in lower fluorescence anisotropy. The feasibility of the assay was confirmed by optimizing the conditions. This method improves the accuracy of the detection with a relative error within 1%. The increase of fluorescence anisotropy in samples is small compared with in the buffer solution. Therefore, the system has good selectivity and is suitable for the characterization of enzyme activity in biological samples. Moreover, the method does not require the labeling of fluorescent molecules or a special design of nucleic acid sequences, which avoids complex probe modification.