Specific Recognition Of Double-Stranded Nucleic Acids By Nucleases Driven Strand Displacement

The recognition and detection of nucleic acid molecules are very important for the prevention,control,diagnosis and treatment of tumor,virus infection and other diseases.At present,most of the existing nucleic acid detection technologies are based on PCR amplification.Due to the strict temperature gradient control,there are certain restrictions in the actual detection.The genomes of biological samples are mostly double stranded nucleic acids.The detection of double stranded nucleic acids in nucleic acid molecular diagnosis needs special primer design and enzyme digestion and other pretreatment steps.The genomic nucleic acid fragments are digested into single stranded oligonucleotide fragments before they can enter the detection system for effective detection,which greatly limits the efficiency of nucleic acid molecular detection.Combined with the above factors,it is of great significance to detect dsna at room temperature.Currently,the existing technologies include multi enzyme linked nucleic acid detection and gene editing(CRISPR).The multi enzyme linked method mainly uses the functions of recombinase,helicase,etc.to spin the double stranded nucleic acids at room temperature,and the substrate recognition and degradation functions of some nucleases(endonuclease IV,APE1,Exo III,etc.).Due to the complexity and instability of these methods,it is of great significance to construct a new method for detection of dsna at room temperature.In this study,a fluorescent probe with universal structure was designed based on the selective specificity of λ exonuclease to construct substrate suitable for enzyme digestion by driving chain replacement reaction.The probe can directly detect the double stranded nucleic acid target at room temperature,and the enzyme can recognize and digest the 5’ phosphate end of nucleic acid,trigger the digestion reaction of the enzyme,and produce a fluorescent signal,which is strictly corresponding to the target nucleic acid sequence,and the detection sensitivity can reach the level of single base pair mutation.The mechanism of this detection reaction was preliminarily explained by exploring the effect of enzyme on strand displecement and analyzing the kinetics of toehold-mediated strand displecement.The detection of positive samples of COVID-2019 using the designed fluorescent probe proved the potential of this detection system.