Autonomous Exponential Amplification via Rolling Circle–DNAzyme Feedback Programming
Jinhua Shang, Mengdi Yu, Yifei Wang, Shanshan Yu, Xian‐Zheng Zhang, Fuan Wang, Yingfu Li
Abstract
Rolling circle amplification (RCA) is a powerful isothermal strategy for nucleic acid detection, but its linear kinetics and dependence on externally supplied primers limit its sensitivity and programmability. Here, we report an exponential RCA (E-RCA) platform that integrates primer regeneration and signal amplification into a single DNA-encoded system. The design uses a circular DNA template encoding the I-R3 self-cleaving DNAzyme sequence; during RCA, tandem I-R3 units are generated within the DNA amplicons, which then catalyze site-specific cleavage to release new primers. This self-sustained (RCA ↔ DNAzyme) amplification circuit enables robust exponential signal growth using only a circular DNA probe and a DNA polymerase without requiring external primers or protein enzymes. We elucidate the mechanism through biochemical experiments and kinetic modeling and validate the system in multiplexed intracellular microRNA imaging and quantitative dual-marker profiling of clinical breast cancer tissues. The E-RCA strategy achieved high diagnostic accuracy (AUC = 0.914; specificity = 100%; sensitivity = 81.3%), demonstrating its potential for sensitive, programmable, and autonomous molecular analysis in both biological and clinical contexts.