A Methodology for Ultrasensitive Detection of Sequence-Specific DNA or Uracil-DNA Glycosylase Activity

Abstract Ultrasensitive detection of sequence-specific DNA and uracil-DNA glycosylase (UDG) activity shows great practical significance in clinical diagnostic and biomedical studies. Here, a methodology based on a CRISPR/Cas12a system coupled with enhanced strand displacement amplification (E-SDA) was innovatively established for sequence-specific DNA or UDG activity detection. Sequence-specific DNA or DNA primers processed by UDG and Endonuclease IV can initiate E-SDA, generating auxiliary DNA chains, which act as activators to unlock the indiscriminate collateral cleavage activities (trans-cleavage) of the CRISPR/Cas12a. Then, the activated CRISPR/Cas12a, which intrinsically possesses the ability of significant signal amplification, can indiscriminately cleave the added cleavage reporters in the system. Thus, the multistep amplification of the method was obtained. Under the selected experimental conditions, the established method can achieve an actual sensitivity of sequence-specific DNA up to 100 aM within 2.5 h or ultralow UDG activity (3.1×10–5 U/mL) detection within 3.5 h. We believe that the proposed method will have great potential for practical application in ultrasensitive detection of sequence-specific DNA or UDG activity.