Rolling circle transcription amplification-directed construction of tandem spinach-based fluorescent light-up...

β-glucosyltransferase (β-GT) can specifically catalyze the conversion of 5-hydroxymethylcytosine (5-hmC) to 5-glucosylhydroxy methylcytosine (5-ghmC), and it is associated with the control of phage-specific gene expression by affecting transcription process in vivo and in vitro. The current strategies for β-GT assay usually involve expensive equipment, laborious treatment, radioactive hazard, and poor sensitivity. Here, we report a Spinach-based fluorescent light-up biosensor for label-free measurement of β-GT activity by utilizing 5-hmC glucosylation-initiated rolling circle transcription amplification (RCTA). We design a 5-hmC-modified multifunctional circular detection probe (5-hmC-MCDP) that integrates the functions of target-recognition, signal transduction, and transcription amplification in one probe. The introduction of β-GT catalyzes 5-hmC glucosylation of 5-hmC-MCDP probe, protecting the glucosylated 5-mC-MCDP probe from the cleavage by MspI. The remaining 5-hmC-MCDP probe can initiate RCTA reaction with the aid of T7 RNA polymerase, generating tandem Spinach RNA aptamers. The tandem Spinach RNA aptamers can be lightened up by fluorophore 3,5-difluoro-4-hydroxybenzylidene imidazolinone, facilitating label-free measurement of β-GT activity. Notably, the high specificity of MspI-catalyzed cleavage of nonglucosylated probe can efficiently inhibit nonspecific amplification, endowing this assay with a low background. Due to the higher efficiency of RCTA than the canonical promoter-initiated RNA synthesis, the signal-to-noise ratio of RCTA is 4.6-fold higher than that of linear template-based transcription amplification. This method is capable of sensitively detecting β-GT activity with a limit of detection of 2.03 × 10^-5 U/mL, and it can be used for the screening of inhibitors and determination of kinetic parameters, with great potential in epigenetic research and drug discovery.