A multivalent dual lock-and-key system and its toehold-mediated orthogonal activation for highly specific strand displacement amplification

DNA strand displacement reaction plays an essential role in dynamic DNA nanotechnology and has proven powerful in DNA nano-computers and biosensors. However, the poor specificity and/or kinetics hamper its practical applications. Here we report a multivalent dual lock-and-key (Multi-DLK) system that can be accurately activated to perform a toehold-mediated orthogonal DNA circuitry (ODC) for highly specific and rapid nucleotide polymorphism assay. The “dual lock” can be opened only when target as the unique “key” is introduced and perform matching operation twice in an exact order separately by using the two fragments of target as inputs. The two operations are designed based on toehold-mediated strand displacement reaction, separately named TSDR1 and TSDR2, offering an orthogonal activation strategy for accurate identification. After that, the palindromic end of linker strand is released and taken as self-primer by DNA polymerase to perform a highly target-specific strand displacement amplification (TSSDA). We demonstrate that Multi-DLK system comes with the benefits of orthogonal activation strategy and TSSDA-based amplifier, which make the system ideally suitable for gene typing with a high specificity and efficiency. Comparative analysis and the final application of Multi-DLK system for real samples have shown the great potential for accurate and timely diagnosis.