Ago2 directly binds preannealed miRNA–mRNA in live cells. HeLa cells transiently expressing a chimeric human Ago2 with an N-terminal monomeric eGFP protein were transfected with Alexa568-labeled CXCR4 preannealed to the target FL6X and incubated for an additional 16 h before imaging. (A) Confocal image showing colocalization of eGFP-Ago2 and P-body–targeted miRNA–mRNA duplexes (white squares). (B) Color-coded FLIM image of HeLa cells expressing eGFP-Ago2 transfect with nonlabeled (CXCR4) or Alexa568-labeled (568CXCR4) miRNA preannealed to target mRNA. Higher magnification FLIM acquisition shown in inset (center panels) features a red ROI, which was used to generate color-coded FLIM histogram (right panels). (C) Fluorescence lifetimes were measured for 30 cells in each condition, and mean lifetime was displayed as bar graph. A decrease of 11% of fluorescence lifetime was measured only in the presence of labeled miRNA, which indicates the presence of FRET. Differences were statistically significant, with a P-value of less than 0.0001. (D) A single pixel (blue cross) was chosen from the ROI in B in order to generate a fluorescence decay curve to which was fitted the FLIM values for the remainder of the pixel within the image. Displayed are relative photon counts per nanosecond, where the highest density of photons measured was set at the value of 1. Curve fitting was optimized to a χ2 nearest to one (1.00 for CXCR4 and 1.02 for 568CXCR4) using single or double exponential curve fitting algorithms.
