Quantitative Analysis of Mitochondrial RNA in Living Cells with a Dual‐Color Imaging System

Abstract Accurate quantification and dynamic expression profiling of mitochondrial RNA (mtRNA for short) are critical for illustrating their cellular functions. However, there lack methods for precise detection of mtRNA in situ due to the delivery restrictions and complicated cellular interferences. Herein, a dual‐color imaging system featured with signal amplification and normalization capability for quantitative analysis of specific mtRNA is established. As a proof‐of‐concept example, an enzyme‐free hairpin DNA cascade amplifier fine‐tailored to specifically recognize mtRNA encoding NADH dehydrogenase subunit 6 (ND6) is employed as the signal output module and integrated into the biodegradable mitochondria‐targeting black phosphorus nanosheet ( BP‐PEI‐TPP ) to monitor spatial‐temporal dynamics of ND6 mtRNA. An internal reference module targeting β‐actin mRNA is sent to the cytoplasm via BP‐PEI for signal normalization, facilitating mtRNA quantification inside living cells with a degree of specificity and sensitivity as high as reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR). With negligible cytotoxicity, this noninvasive “RT‐qPCR mimic” can accurately indicate target mtRNA levels across different cells, providing a new strategy for precise analysis of subcellular RNAs in living systems.