Sensitive detection of cancer gene based on a nicking-mediated RCA of circular DNA nanomachine

- A DNA nanomachine was developed by inserting molecular beacon into a padlock probe.
- RCA and SDA were integrated into a circular padlock-based DNA nanomachine.
- Two types of nicked fragments flow out from CMB-PP nanomachine and amplify signal.
- The newly-proposed strategy can detect cancer genes with high sensitivity and selectivity.

We propose a DNA nanomachine (simplified as MB-PP) by inserting molecular beacon into a padlock probe for the sensitive and specific detection of K-ras gene mutation. In the presence of target DNA, MB-PP is able to be cyclized by ligase because its two ends are pulled together by target species. In this case, rolling circle amplification (RCA) occurs via the extension of primer by polymerase on cyclized MB-PP (CMB-PP), and thus two complete binding sites of nicking endonuclease are obtained due to the formation of RCA product/CMB-PP duplex. Naturally, a steady stream of two types of nicked fragments flow out from continuous rolling CMB-PP nanomachine after introduction of nickase, one of which can in turn triggers strand-displacement amplification (SDA), leading to the dramatic accumulation of nicked fragments. As a result, even in the presence of a trace amount of wild target DNA, the fluorescence intensity will substantially increase. Utilizing this DNA machine, the K-ras gene can be detected down to 50 pM with the linear response range from 50 pM to 10 nM. Moreover, the point mutation existing in the codon 12 of K-ras can be easily distinguished from the wild type.