Combination of RCA and DNAzyme for Dual-Signal Isothermal Amplification of Exosome RNA.

The RNA contained in exosomes plays a crucial role in information transfer between cells in various life activities. The accurate detection of low-abundance exosome RNA (exRNA) is of great significance for cell function studies and the early diagnosis of diseases. However, their intrinsic properties, such as their short length and high sequence homology, represent great challenges for exRNA detection. In this paper, we developed a dual-signal isothermal amplification method based on rolling circle amplification (RCA) coupled with DNAzyme (RCA-DNAzyme). The sensitive detection of low-abundance exRNA, the specific recognition of their targets and the amplification of the detection signal were studied and explored. By designing padlock probes to specifically bind to the target exRNA, while relying on the ligation reaction to enhance recognition, the precise targeting of exosome RNA was realized. The combination of RCA and DNAzyme could achieve a twice-as-large isothermal amplification of the signal compared to RCA alone. This RCA-DNAzyme assay could sensitively detect a target exRNA at a concentration as low as 527 fM and could effectively distinguish the target from other miRNA sequences. In addition, this technology was successfully proven to be effective for the quantitative detection of miR-21 by spike recovery, providing a new research approach for the accurate detection of low-abundance exRNA and the exploration of unknown exRNA functions.