Simulation-guided engineering of an enzyme-powered three dimensional DNA nanomachine for discriminating single nucleotide variants

Single nucleotide variants (SNVs) are important both clinically and biologically because of their profound biological consequences. Herein, we engineered a nicking endonuclease-powered three dimensional (3D) DNA nanomachine for discriminating SNVs with high sensitivity and specificity. Particularly, we performed a simulation-guided tuning of sequence designs to achieve the optimal trade-off between device efficiency and specificity. We also introduced an auxiliary probe, a molecular fuel capable of tuning the device in solution via noncovalent catalysis. Collectively, our device produced discrimination factors comparable with commonly used molecular probes but improved the assay sensitivity by similar to 100 times. Our results also demonstrate that rationally designed DNA probes through computer simulation can be used to quantitatively improve the design and operation of complexed molecular devices and sensors.