(A) Constructing a Three-Dimensional DNA Nanomachine to Achieve Rapid Isothermal Signal Amplification for Nucleic Acid Detection (B) Regulation of DNA Strand Displacement Using an Allosteric DNA Toehold

Abstract

(A) We developed a new strategy to achieve rapid isothermal signal amplification through the construction of DNA nanomachine. DNA nanomachine built from a DNA functionalized gold nanoparticle (DNA−AuNP), which moves a DNA walker along a three-dimensional (3-D) DNA−AuNP track and executes the task of releasing signal reporters (SRs) to generate fluorescence. The movement of the DNA walker is powered by a nicking endonuclease that cleaves specific DNA substrates on the track. During the movement, each DNA walker cleaves multiple substrates, resulting in the rapid release of SRs to achieve signal amplification at a constant temperature. The 3-D DNA nanomachine is highly efficient due to the high local effective concentrations of all DNA components that have been co-conjugated on the same AuNP. Moreover, the activity of the 3-D DNA nanomachine can be controlled by introducing a protecting DNA probe that can hybridize to or dehybridize from the DNA walker in a target-specific manner. This property allows us to tailor the DNA nanomachine into a DNA nanosensor that is able to achieve rapid, isothermal, and homogeneous signal amplification for detection of nucleic acids in both buffer and a complicated biomatrix. (B) Toehold-mediated DNA strand displacement has proven extremely powerful in the construction and operation of DNA devices, including reconfigurable structures, DNA circuits, and amplifications. To achieve the construction of such DNA devices, toeholds are required for controllable activation and regulation. Usually, the complicated strand displacement behaviors and functions are achieved by combining conventional toehold-mediated strand displacement, associative toehold-mediated strand displacement, and remote toehold strand displacement toehold activation mechanisms. We still need to enrich the toolbox of strand displacement techniques with alternative approaches for toehold activation to construct devices of higher complexity. Here we introduce an allosteric DNA toehold (A-toehold) design that allows flexible activation or regulation of DNA strand displacement.