Rapid Signal Amplification Based on Planetary Cross-Catalytic Hairpin Assembly Reactions

Non-enzymatic nucleic acid catalytic systems based on branch migration have been developed, with applications ranging from biological sensing to molecular computation. A scalable planetary cross-catalytic (PCC) system is built up in this work by cross-cascading three planetary catalytic hairpin assembly (CHA) reactions with a central three-arm-branched CHA reaction. With the bottom-up hierarchy strategy, we designed four levels of catalytic reactions, simple CHA reactions, two-layered linear cascades, conventional one-planetary PCC reactions, and two- and three-planetary PCC reactions, and examined the reaction products and intermediates in each level via native polyacrylamide gel electrophoresis. The gel shift assay optimized the designs of hairpin strands to keep the leaking reactions at a manageable level and protect against signal attenuation during serial signal transduction in nucleic acid circuits. The reaction kinetics, measured via fluorescence, are strongly dependent on the number of planetary reactions. As a result, the three-planetary PCC system achieved an exponential amplification factor of about 3, while the conventional one-planetary cross-catalytic system has an amplification factor of 2 (k represents the cycling number). Finally, we demonstrated the rapid detection of a cancer biomarker, microRNA141, used as the catalyst in a two-planetary PCC system. We envision that the PCC systems could be applied in biological signal transduction, biocomputing, rapid detection of single- and multi-target nucleic acid probes, etc.