Tandem Invasive Signal Amplification for Sensitive, Fluorescence Polarization-Based Terminal Transferase Activity Assay

초록

Terminal transferase (TdT) has been regarded as a promising biomarker for diagnosis and prognosis of leukemia. For TdT, previous fluorescent assays showed reasonable sensitivities. However, they had complications in workflow including preparation of signaling materials, assay time and cost. They were also exposed to external interferences (i.e. ambient light, electronics, oscillation) arising from the fluorescence readout, which discouraged robust signal detection. As an endeavor to overcome these drawbacks, we herein describe a simple, sensitive TdT activity assay, termed ‘TELLER’ (TdT ELongation-LEd tandem invasive signal amplification Reaction), on the basis of a fluorescence polarization (FP) signaling modality. In TELLER, target-triggered elongation of polythymine sequences spurs intensive invasive signal amplification, forming tandem repeats of a junction structure with detection probes (lever probe and reporter probe (RP)). Flap endonuclease I (FEN1) with a specific, high-turnover cleavage activity to the junction structure induces cyclic cleavage reactions on RPs, liberating a large number of fluorophores (Fs) with a single nucleotide. This incurs drastic molecular weight reduction of Fs to accelerate their tumbling, accompanying huge variation of FP signal. This assay scheme detected the TdT activity down to 0.17 U/mL in 50 min. In addition, TELLER verified its practical applicability by fulfilling the recovery test for TdT activities in biological matrices (plasma and urine) with high precision and reproducibility. The FP modality offered further advantages. It employed a quencher-free RP, allowing for simple, affordable preparation of signaling materials. Its readout is ratiometric, so it is intrinsically tolerant to the environment interferences. Thus, it is proficient at catching subtle signals by traces of target, assuming high sensitivity. Overall, the idea of TELLER would be tailored to develop biosensors for other enzymes with activities of elongating certain sequences or those adopting TdT as a mediating tool for signal amplification.

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