Tuning the specificity of DNA probes using bulge-loops for low-abundance SNV detection

Tuning the free energy difference between a molecular probe and the target has been regarded as a feasible way to realize selective mutant recognition. But due to limited extent of variation on the probing sequences, it remains a challenge to moderately leverage the thermodynamic kinetics simply by changing the base composition of probes. Herein we propose the modulation of discrimination capability for single nucleotide variations (SNVs) detection by insertion of bulge-loop into duplex DNA probes. Based on controllable tuning of free energy change (ΔG) before and after strand exchange with either mutated or wild-type DNAs, much higher specificity than conventional linear probes is obtained. As-proposed bulge-loop probes allows excellent discrimination of SNVs in high guanine and cytosine (GC) rich regions, and reaches a detection limit of 0.02% abundance with down to 2 femtomolar target gene. The probes also demonstrate excellent consistence with droplet digital PCR (ddPCR) in identifying low abundant L858R mutant in lung tissue samples that are not resolved by either a commercial PCR kit or Sanger sequencing. Our work not only provides insight into the rational design of strand exchange probes for point-of-care diagnosis but also advance the construction of customizable cascade reactions in dynamic DNA nanotechnology more broadly.