Controlling the Depth of Hybridization Chain Reaction by Extended Dangling Ends and Its Analytical Applications

Hybridization chain reaction (HCR) is a powerful enzyme-free nucleic acid amplification strategy. Triggered by an initiator strand, it yields nicked double helices analogous to alternating copolymers. However, there is no effective way to regulate the HCR reaction, and the most apparent phenomenon is the uncontrollable polymerization of product after introducing an initiator. Here we explore controlling the depth of the HCR reaction by extended dangling ends on hairpin monomers and report that sequence length, nucleotide composition, and secondary structure can alter HCR polymerization and can be utilized for the desired regulation. Interaction dynamics between initiator and hairpin monomers simulated by oxDNA are in good accordance with experimental results. Such a controlling effect can be utilized for new analytical applications that HCR cannot previously achieve, such as analyzing strand-extension enzymes and identifying short-sequence structures. The finding provides a concise but effective way for controlling the depth of HCR reaction and opens the application scope of HCR to more fields.