Assay for TET1 activity and its inhibitors screening with signal amplification by both nanoparticles and Ru(III) redox recycling

Abstract

Ten-eleven translocation protein 1 (TET1) is one member of TET proteins family which plays a key role in dynamic DNA methylation-demethylation process. Herein, a novel biosensor was constructed for TET1 detection and its inhibitors screening utilizing restriction digestion of endonuclease enzyme MspI. Half-methylated oligonucleotide (5mC DNA) was used as target and Ru(NH₃)₆³⁺ as electrochemical signal probe. After the treatment by TET1 and T4 β-glucosyltransferase (T4 β-GT), target oligonucleotide would not be recognized and digested. If there was no TET1, the target would be digested and the response of biosensor decreased greatly. The current difference of biosensor with and without the incubation with TET1 was therefore dependent on the concentration of TET1. To increase sensitivity of the biosensor, nanostructured film at electrode surface and nanoparticles modified oligonucleotides were employed as signal amplification elements for Ru(NH₃)₆³⁺ recycling. Finally, this biosensor showed high performance with a wide linear range of TET1 concentration from 3.5-21 ng/μL and a low detection limit of 0.33 ng/μL, which is superior to other existing methods. The inhibition effects of Bobcat339 on TET1 was successfully proved by our biosensor with an IC₅₀ of 38 μM. Not only that, but the feasibility of the biosensor for inhibitors screening was evaluated and further confirmed by other compounds including two anticancer drugs and three active ingredients of traditional Chinese medicine.

Keywords: Demethylation; Electrochemical biosensor; Inhibitors screening; Nanoparticles; Ten-eleven translocation protein 1.