Catalytic Hairpin Assembly-Assisted Rolling Circle Amplification for High-Sensitive Telomerase Activity Detection

Abstract

Telomerase is a promising biomarker and a potential therapeutic target of malignant tumors. Reliable, facile, and sensitive telomerase activity analysis is of vital importance for both early diagnosis and therapy of malignant tumors. Herein, we proposed a novel fluorescent assay termed catalytic hairpin assembly-assisted rolling circle amplification (CAR) for both in vitro and in situ high-sensitive telomerase activity detection. In the presence of active telomerase, the extension of a designed telomerase primer was limited to five bases (GGGTT), thus forming short telomerase products. Afterward, the obtained telomerase extension products cyclized Padlock and subsequently initiated the rolling circle amplification (RCA). In order to maintain a higher sensitivity, an ingeniously designed catalytic hairpin assembly (CHA) was attached for both signal amplification and result readout. The highlights of the CAR method were concluded as follows: (i) dual signal amplification from CHA and RCA ensures high sensitivity and (ii) the CAR method has the potential for both in vitro and intracellular imaging of telomerase activity. We believe that the CAR method would be of great potential for the diagnosis and therapy of various human diseases.

Scheme 1. Working Principle of the Proposed Telomerase Activity-Sensing Platform

Scheme 2. Feasibility of the CAR Method; (a) PAGE Electrophoresis Result of TEPL Sequence-Triggered RCA; (b) Fluorescence Intensity of the CAR Method When Free from Part of Components

Data are represented as the means ± SD (n = 3).

Scheme 3. Optimization of Experimental Conditions; (a) Fluorescence Spectra of the CAR Method When dATP Utilized in the Telomerase-Catalyzed Primer Extension Step or Not; Control Refers to the Initial Fluorescence of H2 Probes; (b) Fluorescence Intensity of the Different Padlock Probe in the Presence of Active Telomerase; Three Telomerase Samples Duplicates Were Used for Three Padlock Optimizations; (c) Fluorescence Spectra of the CHA Process with RCA Products Existed or Not; Inset shows a Histogram of the Fluorescence Intensity of the Corresponding Group at E_m = 560 nm

Data are represented as the means ± SD (n = 3).

Scheme 4. Sensitivity of CAR for Telomerase Activity Detection; (a) Fluorescence Spectra of the CAR Method When Incubated with Different Concentrations of Telomerase Extracted from 0, 50, 100, 200, 400, 800, 1000, 1500, 2000, 2500, 3000, 3500 HeLa Cells μL^–1; (b) Linear Relationship between the Fluorescence Intensity and Concentrations of Cell Extracts; (c) Correlation of Telomerase Activity Detection through the CAR Method and ELISA

Scheme 5. Confocal Images of Telomerase Activity in HeLa Cells When EGCG Existed or Not

Scale bar = 10 μm. From left to right, Hoechst (blue), Cy3 (green), and merged images.