De novo-engineered circular DNAzymes with topological rigidity for multiplexed fluorescent-temporal barcoding

Circular DNAzyme (C-Dz) is emerging as a powerful topological catalytic tool, yet the interplay between its circular framework and cleavage kinetics remains underutilized. Here, we exploit these topological properties to develop a fluorescent-temporal (FLUO-TIME) barcoding platform termed CLOCK (CLOsed Circular DNAzyme Kit), which significantly expands multiplexing capacity without requiring additional spectral channels. CLOCK utilizes an engineered C-Dz design where topology regulates functional differentiation for synergistic encoding: the loop sequence enables traditional fluorescence encoding via hybridization, while the loop size modulates catalytic core folding through topological rigidity. This constraint regulates cleavage efficiency, allowing for the programmable kinetic control of rolling circle amplification, which is achieved by varying the loop length to tune the topological constraint of the catalytic core. By combining spectral signatures with distinct time-to-peak fluorescent signals, CLOCK generates a multidimensional FLUO-TIME barcode. Structural insights from AlphaFold3 and molecular dynamics simulations further provide new perspectives on how topological constraints affect the enzymatic activity of C-Dz. As a proof of concept, CLOCK achieved one-pot detection of four respiratory virus RNAs with a sensitivity of 1 fM. By establishing time as a programmable encoding dimension, CLOCK provides a versatile and scalable framework for next-generation biosensing and multiplexing technologies. • CLOCK (CLOsed Circular DNAzyme Kit) expands multiplexing without extra spectral channels via time encoding. • C-Dz topology enables synergistic FLUO-TIME encoding by regulating cleavage kinetics and fluorescence hybridization. • Topological C-Dz exhibits superior RNA-cleavage activity to linear DNAzyme, elucidated by AlphaFold3 and MD simulations. • CLOCK achieves 1 fM-sensitive one-pot detection of four respiratory virus RNAs for clinical diagnostics.