Specifying and verifying knowledge-based systems with KIV

Abstract

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 <i>acyclic</i> d-threoninol nucleic acid (d-<i>a</i>TNA), which is orthogonal to right-handed DNA and RNA. Because of its high thermal stability, use of an <i>a</i>TNA hairpin with a short 7 base-pair stem ensured clear ON-OFF control of the HCR circuit. The <i>a</i>TNA circuit was stable against nucleases. A circuit based on right-handed <i>acyclic</i> l-threoninol nucleic acid (l-<i>a</i>TNA) was also designed, and high orthogonality between d- and l-<i>a</i>TNA HCRs was confirmed by activation of each <i>a</i>TNA 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-<i>a</i>TNA circuits. The d-<i>a</i>TNA 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 <i>in vivo</i> and the FISH method.