Addition of mirror-image L-DNA elements to DNA amplification circuits to distinguish leakage from target signal

DNA amplification circuits that rely on thermodynamically-driven hybridization events triggered by a target nucleic acid are becoming increasingly utilized due to their relative simplicity. A drawback of these circuits is that non-specific amplification, or circuit leakage, must be estimated using a separate “no-target” control reaction to eliminate false positives. Aside from requiring an additional reaction, the problem with this approach is the difficulty of creating a no-target control for biological specimens. To overcome this limitation, we propose a strategy that combines both reactions into the same tube using naturally-occurring right-handed D-DNA circuit elements for the target detection reaction and identical synthetic mirror-image left-handed L-DNA circuit elements for the no-target control reaction. We illustrate this approach using catalyzed hairpin assembly (CHA), one of the most studied DNA amplification circuits. In a dual-chirality CHA design, the right-handed circuit signal is produced by target-specific amplification and circuit leakage, whereas the left-handed circuit signal is produced only by circuit leakage. The target-specific amplification is calculated as the difference between the two signals. The limit of detection of this dual-chirality CHA reaction was found to be similar to that of traditional CHA (81 vs 92 pM, respectively). Furthermore, the left-handed no-target signal matched the right-handed leakage across a wide range of sample conditions including background DNA, increased salt concentration, increased temperature, and urine. These results demonstrate the robustness of a dual-chirality design and the potential utility of left-handed DNA in the development of new DNA amplification circuits better-suited for target detection applications in biological samples. •Identical D-DNA and L-DNA circuits operate independently in one reaction.•Left-handed leakage matches right-handed across a wide range of sample types.•Promising approach to simultaneously differentiate leakage from amplification.