A biosensor using semi-DNA walker and CHA -FRET loop for ultrasensitive detection of single nucleotide polymorphism

Single nucleotide polymorphisms (SNPs) are emerging as important biomarkers for disease diagnosis, prognostics and disease pathogenesis. However, it is still challenging to make highly specific and sensitive SNPs detection to distinguish target DNA with single-base difference, so that flap endonuclease 1 (FEN 1) manipulated semi-DNA walker reaction and catalytic hairpin assembly (CHA)-FRET loop for ultrasensitive SNP detection has been proposed. Herein, FEN 1 was employed to specifically recognize and cut the 5 ' flap of three-base overlapping structure formed by only hybridizing with mutant target (MT). Simultaneously, FEN 1 drove MT hybridization to autonomously walk along downstream probe (DP) and the cut sequence (CS) from semi-DNA walker reaction was collected by magnetic bead-based separation to avoid false-positive signal. Next, the cleaved CS triggered CHA and generated fluorescence resonance energy transfer (FRET) via Cy3 and Cy5. FEN 1 manipulated semi-DNA walker reaction to specifically recognize the target DNA and combined CHA-FRET loop to dually amplify signal. Thus, the SNP biosensor demonstrated superior performance for ultrasensitive detection of mutant KRAS gene with a low detection limit as low as 80 aM and was successfully applied to monitor the expression of mutant KRAS gene in human cancer cell lysates. This strategy provides an ultrasensitive way for the detection of bio-molecules and reveals an effective avenue for diseases diagnosis.

Automated summary

Single nucleotide polymorphisms (SNPs) are important biomarkers for disease diagnosis and prognosis. However, detecting SNPs with high specificity and sensitivity for DNA with single-base difference is challenging. This study proposes a strategy for ultrasensitive SNP detection using flap endonuclease 1 (FEN 1) manipulated semi-DNA walker reaction and catalytic hairpin assembly (CHA)-FRET loop. The strategy demonstrated superior performance for ultrasensitive detection of mutant KRAS gene and could be applied to monitor gene expression in cancer cells.