DNAzyme-assisted the detection of rps27l mRNA in protein nanopores

Quantifying mRNA is crucial for elucidating the physiological condition of organisms in life science research and clinical diagnostics. Recently, nanopore technology has been demonstrated as a versatile tool with a myriad of applications and successful implementations for genomic analyte identification. However, its application in mRNA quantification encounters challenges such as the presence of numerous background species, low expression levels, and complex mRNA structures. Herein, we propose the implementation of a DNAzyme-assisted approach for rp27l mRNA quantification in MspA protein nanopores. The strategy involves converting the quantification of mRNA through a DNAzyme reaction, where the sequence of GR-5 DNAzyme is integrated into Probe 2 (P2). The sandwich structure (P1-T-P2) is constructed between the target molecules and two types of probes, enabling the identification of target molecules and the retention of the enzyme strand in magnetic fields. As a result, GR-5 DNAzyme not only effectively hydrolyzes the corresponding substrates to yield external probes for subsequent nanopore analysis but also functions as an ingenious molecular amplification method by persistently digesting excess substrates to produce abundant external probes. This sensor acquires the amplification capability and attains a high sensitivity with a detection limit of 40 pM within 15-min measurements, improving the sensitivity of protein nanopores for nucleic acid detection. Thus, the proposed DNAzyme-based protein nanopore sensor for rp27l mRNA quantification demonstrates remarkable advantages in its label-free, rapid, and sensitive nature, which promotes the performance of protein nanopores in genomic analyte detection. Moreover, this sensor may open a novel approach for mRNA quantification, significantly impacting disease diagnosis, customized treatment, and metabolic studies.