Metastable Dumbbell Probe-Based Hybridization Chain Reaction for Sensitive and Accurate Imaging of Intracellular-Specific MicroRNAs In Situ in Living Cells.
Jun ChenHui-Hui YangWen YinYanfei ZhangYingjun MaDanping ChenYuzhi XuSi-Yang LiuLi ZhangZong DaiXiaoyong ZouPublished in: Analytical chemistry (2019)
Sensitive and accurate imaging of intracellular-specific microRNAs (miRNAs) in situ in living cells is seriously challenged by the susceptibility of nucleic acid probes and the low dynamics of the hybridization reaction in cellular environments. Herein, we engineer a set of new metastable dumbbell probes (M xDPs) to overcome these key limitations by concurrently boosting transfection, antidigestibility, assembly dynamics, and nanostructural uniformity. The M xDPs can maintain their stability up to 16 h in living cells and produce uniform and dense DNA nanostructures rapidly (<2 h) and specifically from a hybridization chain reaction (HCR). A sharp signal from the cascade accumulation of fluorescence resonance energy transfer (FRET) further minimizes the effect of system fluctuations. The M xDPs-based HCR (M xDPHCR) method showed identical performance in the analysis of miR-27a in cell lysate and buffer condition and obtained a limit of detection down to 3.2 pM (corresponding to 160 amol per 50 μL), which is 44-fold lower than on conventional hairpin probes. The M xDPHCR method clearly distinguished normal cells from tumor cells and provided more accurate quantitative information on the intracellular-specific miRNAs. The strategy would offer a powerful tool for visualizing and localizing desired nucleic acids in living cells.
Keyphrases
- living cells
- single molecule
- nucleic acid
- energy transfer
- high resolution
- fluorescent probe
- reactive oxygen species
- quantum dots
- cell proliferation
- long non coding rna
- healthcare
- stem cells
- label free
- air pollution
- single cell
- cell therapy
- particulate matter
- mesenchymal stem cells
- bone marrow
- loop mediated isothermal amplification
- electron transfer