Journal: ACS synthetic biology

Article Title: Nucleic Acid Strand Displacement with Synthetic mRNA Inputs in Living Mammalian Cells

doi: 10.1021/acssynbio.8b00288

Figure Lengend Snippet: a) Domain-level schematic of SDprobe_v1: Individual functional domains are annotated with their respective primary features in parentheses. Half-arrows indicate 3’-ends of the corresponding strands. The fluorophore and quencher are denoted by red and black circles respectively. b) Schematic of probe interaction with the 3’-UTR target sites (blue domains). Probes bind to the target sites via the short toehold domain, which initiates a 3-way branch migration step, ultimately displacing the quencher-labeled strand and thereby unquenching the fluorophore. Co-localization of multiple fluorophores on a single mRNA significantly increases the signal. c) Experimental workflow for detection of mRNAs using strand displacement probes: Probes are delivered via electroporation to cultured cells. Cells are incubated for 1 hour at 37°C allowing the probes to interact with mRNA target sites. Co-localization of up to 96 probes per mRNA generates a bright fluorescent spot detectable with fluorescence microscopy. After incubation, sequential image frames are collected at every 0.3 um z-distance across the cell height and the acquired frames are combined to generate maximum intensity projections representing the entire cell volume. These images are processed using the FISH-Quant33 MATLAB tool; a filtering step performs background signal correction and spots are pre-detected based on a threshold signal cut-off. The pre-detected spots are then fitted and examined for accuracy followed by spot quantification, where the spots are either selected or rejected based on signal intensity and elimination of false-positives. The selected spots represent the number of detected mRNAs per cell.

Article Snippet: These images are processed using the FISH-Quant 33 MATLAB tool; a filtering step performs background signal correction and spots are pre-detected based on a threshold signal cut-off.

Techniques: Functional Assay, Migration, Labeling, Electroporation, Cell Culture, Incubation, Fluorescence, Microscopy