Ultra-specific fluorescence detection of DNA modifying enzymes by dissipation system

Abnormal expression of DNA modifying enzymes (DMEs) is linked to a variety of diseases including cancers. It is desirable to develop accurate methods for DME detection. However, the substrate-based probe for target DMEs is disturbed by various non-target DMEs that have similar activity resulting in a loss of specificity. Here we utilized dissipative DNA networks to develop an ultra-specific fluorescence assay for DME, absolutely distinguishing between target and non-target enzymes. Unlike the conventional sensors in which the discrimination of target and non-target relies on signal intensity, in our system, target DMEs exhibit featured fluorescence oscillatory signals, while non-target DMEs show irreversible 'one-way' fluorescence increase. These dissipation-enabled probes (DEPs) exhibit excellent generality for various types of DMEs including DNA repair enzyme apurinic/apyrimidinic endonuclease 1 (APE1), polynucleotide kinase (T4 PNK), and methyltransferase (Dam). DEPs provide a novel quantification mode based on area under curve which is more robust than those intensity-based quantifications. The detection limits of APE1, T4 PNK, and Dam reach 0.025 U/mL, 0.44 U/mL, and 0.113 U/mL, respectively. DEPs can accurately identify their corresponding DMEs with excellent specificity in cell extracts. Fluorescence sensors based on DEPs herein represent a conceptually new class of methods for enzyme detection, which can be easily adapted to other sensing platforms such as electrochemical sensors.