Abstract
The accurate detection of nucleic acids from certain biological pathogens is critical for the diagnosis of human diseases. However, amplified detection of RNA molecules from a complex sample by direct detection of RNA/DNA hybrids remains a challenge. Here, we show that type IIS endonuclease FokI is able to digest DNA duplexes and DNA/RNA hybrids when assisted by a dumbbell-like fluorescent sensing oligonucleotide. As proof of concept, we designed a battery of sensing oligonucleotides against specific regions of the SARS-CoV-2 genome and interrogated the role of FokI relaxation as a potential nicking enzyme for fluorescence signal amplification. FokI-assisted digestion of SARS-CoV-2 probes increases the detection signal of ssDNA and RNA molecules and decreases the limit of detection more than 3.5-fold as compared to conventional molecular beacon approaches. This cleavage reaction is highly specific to its target molecules, and no detection of other highly related B-coronaviruses was observed in the presence of complex RNA mixtures. In addition, the FokI-assisted reaction has a high multiplexing potential, as the combined detection of different viral RNAs, including different SARS-CoV-2 variants, was achieved in the presence of multiple combinations of fluorophores and sensing oligonucleotides. When combined with isothermal rolling circle amplification technologies, FokI-assisted digestion reduced the detection time of SARS-CoV-2 in COVID-19-positive human samples with adequate sensitivity and specificity compared to conventional reverse transcription polymerase chain reaction approaches, highlighting the potential of FokI-assisted signal amplification as a valuable sensing mechanism for the detection of human pathogens.
【초록키워드】 SARS-CoV-2, Diagnosis, RNA, polymerase chain reaction, amplification, nucleic acid, DNA, Region, pathogen, Sensitivity and specificity, SARS-CoV-2 variants, nucleic acids, SARS-CoV-2 genome, limit of detection, cleavage, Pathogens, reverse transcription, molecular, Critical, mechanism, Combination, Rolling circle amplification, isothermal, digestion, Chain Reaction, proof of concept, viral RNAs, human diseases, approaches, enzyme, battery, complex, proof, oligonucleotides, fluorescent, oligonucleotide, RNA molecules, ssDNA, probe, decrease, FokI, polymerase chain, amplified, addition, reduced, increase, RNA molecule, highlighting, the SARS-CoV-2 genome, 【제목키워드】 detection, RNA, DNA, hybrid, enhanced, Specy,