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 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 into 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 PC-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 selfcalibration performance, effectively shielding the influences of operating conditions and cell-to-cell variations,

Synergistic Incorporation of Two ssDNA Activators Enhances the Trans-Cleavage of CRISPR/Cas12a

“…In another study, modification of 2′-O-methyl on gRNA enables one to promote Cas12a’s discrimination ability against single-base mutations, with a specificity twice that of the original gRNA . Despite these major advances in crRNA reconstruction, systematic optimization of DNA − may be more attainable than screening RNA to improve the performance of the Cas12a system, from a stability and cost perspective.…”

Compute-and-Release Logic-Gated DNA Cascade Circuit for Accurate Cancer Cell Imaging

“…Next, we verified the cell recognition ability of the CAR-CHA-HCR circuit with MCF-7 (human breast cancer breast cancer cell line) as the targeted cells, which expressed high levels of miR-21 and low levels of miR-892b. ,,,− Figure S17 demonstrates that the red fluorescence in MCF-7 cells was gradually brightened after incubation with CAR-CHA-HCR circuits for 120 min, suggesting an optimized incubation time (120 min) for the following cell testing. In Figure A, a bright red fluorescence was shown in MCF-7 cells, indicating that “ON” fluorescence signals of the CAR-CHA-HCR circuit could be activated in targeted cells as expected.…”

“…To further evaluate the accurate cancer identification of the CAR-CHA-HCR circuit, we selected MCF-7 as positive cells and HEPG2 (human liver cancer cell line) and MCF-10A (normal human renal epithelial cells) as negative cells. MCF-7 cells express high levels of miR-21 while low levels of miR-892b. ,,,− Both miR-21 and miR-892b are overexpressed in HEPG2 cells, but both are expressed lowly in MCF-10A cells. ,,,, For comparison, the conventional fluorescence system based on a single-biomarker-controlled strategy (YES logic gate), miR-21-triggered CHA-HCR circuit, was also tested. Figures S18 and A outline the signal changes of the YES logic gate composed of the CHA-HCR circuit in the three cell lines.…”

“…17,18,20,42−44 Both miR-21 and miR-892b are overexpressed in HEPG2 cells, but both are expressed lowly in MCF-10A cells. 20,35,38,42,43 For comparison, the conventional fluorescence system based on a single-biomarker-controlled strategy (YES logic gate), miR-21-triggered CHA-HCR circuit, was also tested. Figures S18 and 4A outline the signal changes of the YES logic gate composed of the CHA-HCR circuit in the three cell lines.…”

“…Considerable effort has been invested in improving the sensitivity of sensing systems, particularly the introduction of various DNA cascade circuit-based isothermal amplification techniques, such as catalytic hairpin assembly (CHA), entropy-driven catalysis (EDC), and hybridization chain reaction (HCR). − These amplification approaches, performing one-to-many signal output, allow a single target molecule to activate many signal molecules by programmed DNA assembly or a DNA chain-growth polymerization reaction, paving a facial path for nonenzymatic signal amplification using only several artificially programmable DNA sequences. Accordingly, extensive research endeavor has been directed toward utilizing DNA cascade circuits to identify cancer-related biomarkers, such as microRNAs, adenosine triphosphate (ATP), and telomerase. − However, the DNA cascade circuits triggered by a single biomarker are prone to unreliable cellular imaging output due to the lack of gating for specific cancer cells.…”