A Cell-Anchored and Self-Calibrated DNA Nanoplatform for in Situ Imaging and Quantification of Endogenous MicroRNA in Live Cells: Introducing Two Controls to Normalize the Sensing Signals

Abstract

Quantifying the microRNAs (miRNAs) levels in living cells, while essential for the study of fundamental biology and medical diagnostics, has barely been achieved due to insufficient probe delivery and unquantifiable signals. We report a cell-anchored and self-calibrated DNA nanoplatform, a cholesterol-headed DNA nanowire that is capable of efficiently delivering to various cells and simultaneously detecting two target miRNAs. One miRNA target can be utilized as an endogenous control against cell-to-cell variations. Moreover, the photocleavable linkers inserted in the nanostructures allow us to precisely regulate the probe structure and fluorescence signaling at the desired time and location in vivo. As a second control, the maximum fluorescence can be elicited by UV light, which further facilitates the normalization of the absolute fluorescence signal. With two introduced internal controls, the maximum fluorescence and endogenous control gene, this approach displays excellent stability and self-calibration performance, effectively avoiding the interference from operating conditions and cell-to-cell variations, such as the laser powers and intracellular probe concentrations. Importantly, this design is capable of unifying the output signal intensity between in vitro test and cell imaging, making the in vitro linear calibration curve appropriate for the quantification of miRNA expression in living cells.