On Degradation-Induced Leak in Heterochiral DNA Strand Displacement Cascades

Heterochiral DNA strand displacement provides a powerful mechanism for converting nucleic acid input signals of one chirality to nucleic acid output signals of the opposite chirality. This is achieved by combining naturally occurring d-DNA with synthetic, chiral mirror-image l-DNA. Such systems have potential for practical applications, including autonomous biomedical diagnostics and therapeutic actuation, which could take advantage of the degradation resistance of l-nucleic acids to carry out diagnostic information processing using a robust l-DNA molecular circuit. One implementation of this approach uses a leakless molecular translator architecture to minimize off-target release of an l-nucleic acid output in the absence of a d-nucleic acid input. In nonpristine environments, however, degradation of the d-DNA components of a heterochiral system can lead to a slow leak that could cause off-target side effects in a biomedical application. Here, we report an investigation of degradation-induced leak in heterochiral DNA strand displacement systems that combines experimental measurements with the computational fitting of an abstract kinetic model. We explore both sequential and parallel leak hypotheses for the leak mechanism and conclude that initial degradation reactions provide otherwise leakless components with alternative means of activating the programmed reaction cascade in the absence of input. We validate this model by demonstrating that it predicts key behaviors of heterochiral translators when the d-DNA domains are hardened against degradation by using phosphorothioate backbone modifications. This work thus lays the groundwork for future exploration of degradation-induced leak that will enable the development and deployment of robust heterochiral DNA devices for biomedical applications.