Photoelectrochemical biosensor for HEN1 RNA methyltransferase detection using peroxidase mimics PtCu NFs and poly(U) polymerase-mediated RNA extension

2'-O-methyl group on the 3' terminal nucleotide in plant microRNAs, as one kind of RNA methylations, is caused by HEN1 RNA methyltransferase (HENMT1), which is thought to be crucial for ribosome biogenesis and function. Herein, a simple and label-free PEC biosensing method was proposed for assay of HENMT1 activity and inhibitor screening based on peroxidase mimic PtCu nanoframes (PtCu NFs) catalytic signal amplification. In this work, MoS₂@Graphene quantum dots/Phosphorus-doped rodlike carbon nitride (MoS₂@GQDs/P-RCN) heterojunction was used as photoactive materials. With the doping of GQDs and the formation of heterojunction, the photoactivity of MoS₂ is greatly improved. After the double-stranded RNA (dsRNA) with 2 nt 3' overhangs was treated with HENMT1 in the presence of S-adenosyl-L-methionine, the 3' terminal nucleotide of the unmethylated dsRNA could be extended under the catalysis of the poly(U) polymerase in the existence of UTP. Poly(A) nucleotide chain modified with carboxyl group was captured on the electrode surface through hybridization reaction and acted as a bridge for the immobilization of reticular DNA-functionalized PtCu NFs (PtCu@DNA). Under the catalysis effect of peroxidase mimics PtCu@DNA towards hydrogen peroxide, O^2- was in situ generated as electron donor and a strong photocurrent was obtained. The proposed PEC bioassay exhibited high selectivity and low detection limit of 3.36ng/mL for HENMT1 activity assay. Furthermore, the inhibition research indicated that chlorpyrifos could inhibit the HENMT1 activity with the IC₅₀ value of 48.32nM.