Sensitive detection of uracil-DNA glycosylase (UDG) activity is very important in the study of many fundamental biochemical processes and clinical applications. Here, we develop a novel assay for the detection of UDG activity by using the self-initiating multiple rolling circle amplification (SM-RCA) strategy. We first design a trigger probe modified with NH2 at its 3'-terminal and uracil base in the middle of sequence, which is complementary to a cyclized padlock probe. In the presence of UDG, a uracil base can be excised by UDG to generate an apurinic/apyrimidinic (AP) site. The AP site is recognized and cleaved by endonuclease IV (Endo IV), releasing the primer with 3'-OH. The primer can trigger the rolling circle amplification (RCA) reaction, producing a long and repeated DNA strand embedded some uracil bases. These uracil bases can be cleaved by UDG and Endo IV again, and then, more primers are generated to initiate SM-RCA reaction, producing large amounts of DNA product. Afterward, the DNA product is measured by a specific DNA fluorescence dye for quantitative detection of UDG activity. The linear range of the method is 5 × 10-5 to 1.25 × 10-3 U/mL, and the detection limit is 1.7 × 10-5 U/mL. This method not only utilizes the target UDG itself to trigger RCA but also further induces SM-RCA reaction, providing a simple, sensitive, and cost-effective strategy for the detection of glycosylase and clinical diagnosis.

A novel and highly sensitive method for detection of microRNA (miRNA) was developed by integration of 17 exonuclease-triggered amplification and cationic conjugated polymer (CCP) biosensing. First, a fluorescein-labeled probe was designed with the complementary sequence to the target miRNA. When target miRNA was absent in the solution, the fluorescence probe interacted with CCP through the strong electrostatic interactions, leading to the highly efficient fluorescence resonance energy transfer (FRET) from CCP to fluorescein. In the presence of target miRNA, the probe hybridized with the miRNA to form DNA/miRNA duplex hybrids. Then, 17 exonuclease digested cyclically the fluorescence probes in hybrids and triggered the enzyme amplification reaction, generating a large number of single nucleotides. Owing to the weak electrostatic interaction between CCP and the single nucleotide, the FRET from CCP to fluorescein would not take place, which effectively reduced the background and significantly enhanced the sensitivity and the dynamic range of miRNA detection. The linear range of the assay was 0.2-100 pM and the detection limit 0.08 pM was 58 times lower than that of the endonuclease-based assay. The method is simple, cost-effective, and with no need for the sophisticated instrument, and has broad application prospects for miRNA detection and early diagnosis.