For a signal amplification system that is orthogonal to DNA, we designed a simplified seesaw gate composed of only D‐aTNA. This new system performed signal amplification by toehold exchange reaction just as the DNA circuit did. Moreover, the D‐aTNA circuit was not affected by natural nucleic acids carrying sequences complementary to the D‐aTNA. In the presence of an SNA interface, however, an RNA signal was converted to D‐aTNA signal, resulting in successful activation of D‐aTNA circuit. This system can be used to design signal‐amplification circuits that are not influenced by contaminating DNA and RNA.

Construction of complex DNA circuits is difficult due to unintended hybridization and degradation by enzymes under biological conditions. We herein report a hybridization chain reaction (HCR) circuit composed of left-handed acyclic d-threoninol nucleic acid (d-aTNA), which is orthogonal to right-handed DNA and RNA. Because of its high thermal stability, use of an aTNA hairpin with a short 7 base-pair stem ensured clear ON–OFF control of the HCR circuit. The aTNA circuit was stable against nucleases. A circuit based on right-handed acyclic l-threoninol nucleic acid (l-aTNA) was also designed, and high orthogonality between d- and l-aTNA HCRs was confirmed by activation of each aTNA HCR via a corresponding input strand. A dual OR logic gate was successfully established using serinol nucleic acid (SNA), which could initiate both d- and l-aTNA circuits. The d-aTNA HCR was used for an RNA-dependent signal amplification system via the SNA interface. The design resulted in 80% yield of the cascade reaction in 3000 s without a significant leak. This work represents the first example of use of heterochiral HCR circuits for detection of RNA molecules. The method has potential for direct visualization of RNA in vivo and the FISH method.