Biosensing by Tandem Reactions of Structure Switching, Nucleolytic Digestion, and DNA Amplification of a DNA Assembly

Abstract ϕ29 DNA polymerase (ϕ29DP) is able to carry out repetitive rounds of DNA synthesis using a circular DNA template by rolling circle amplification (RCA). It also has the ability to execute 3′–5′ digestion of single‐stranded but not double‐stranded DNA. A biosensor engineering strategy is presented that takes advantage of these two properties of ϕ29DP coupled with structure‐switching DNA aptamers. The design employs a DNA assembly made of a circular DNA template, a DNA aptamer, and a pre‐primer. The DNA assembly is unable to undergo RCA in the absence of cognate target owing to the formation of duplex structures. The presence of the target, however, triggers a structure‐switching event that causes nucleolytic conversion of the pre‐primer by ϕ29DP into a mature primer to facilitate RCA. This method relays target detection by the aptamer to the production of massive DNA amplicons, giving rise to dramatically enhanced detection sensitivity.