Journal: Interface Focus

Article Title: Micro-compartmentalized strand displacement reactions with a random pool background

doi: 10.1098/rsfs.2023.0011

Figure Lengend Snippet: Toehold-mediated strand displacement with and without random pool strands. ( a ) Simplified depiction of a one-step TMSD process. The system consists of a single-stranded (ss) invader and a double-stranded (ds) reporter complex with a substrate strand which is labelled with a fluorophore and an incumbent labelled with a quencher. The short single-stranded overhang of the substrate, termed ‘toehold’ (depicted in red), is complementary to the toehold of the invader strand. Because of the sequence complementarity, the invader binds to the substrate and displaces the incumbent in a branch migration process. Eventually, the invader completely displaces the incumbent due to the higher thermodynamic stability of the invader–substrate complex. Separating the fluorophore from the quencher leads to an increase in fluorescence intensity, which can be used as a readout of the process. ( b ) TMSD process including a random pool strand that first forms a complex with the invader. The formation of a complex that occludes some of the toehold bases inhibits binding of the invader to the reporter, slowing down the overall displacement kinetics. ( c ) Schematic depiction of TMSD reactions in droplets. Invaders or invader–random pool complexes (green) are co-encapsulated with a reporter complex (orange) inside of a single droplet. After mixing of the droplet content, the TMSD reaction results in an increase in red fluorescence in the droplets. ( d ) Droplet production and monitoring of TMSD. TMSD reactants are encapsulated together in water-in-oil droplets in a microfluidic flow-focusing junction. Droplet sizes and mixing ratios can be controlled via the pressures applied to each inlet reservoir. In order to monitor TMSD reactions within droplets, the droplet flow is stopped instantly by applying a set of balanced pressures between inlets and outlets, directly followed by microscopy data acquisition downstream of the flow-focusing junction.

Article Snippet: All fluorescence microscopy time-lapse data were recorded with a 10X P-Apo air objective (NA 0.45) on a Nikon Ti-2E equipped with a SOLA SM II LED light source, a motorized stage and an Andor NEO 5.5 camera.

Techniques: Sequencing, Migration, Fluorescence, Binding Assay, Microscopy

Journal: Interface Focus

Article Title: Micro-compartmentalized strand displacement reactions with a random pool background

doi: 10.1098/rsfs.2023.0011

Figure Lengend Snippet: Monitoring TMSD reactions in emulsion droplets. ( a ) Workflow for the extraction of kinetic parameters. Droplets are individually tracked from time-lapse fluorescence microscopy images. Reference (green) and reporter (red) fluorescence intensity values are obtained for each droplet for each time frame. The initial slope of the reporter intensity time course is chosen as a measure for the reaction kinetics. ( b ) Swarm plots of individual slopes after dividing each by the mean value of the set of experiments. The initial slopes show a much larger variability in the presence of a random pool. The red lines indicate Gaussian fits to the data. ( c , d ) Kinetic curves of individual TMSD reactions in a set of droplets generated in one experiment. Also shown is the mean of all droplet kinetic curves, and the kinetics for the same TMSD process recorded in a bulk experiment. Experiments were performed without ( c ) and with random pool ( d ), respectively.

Article Snippet: All fluorescence microscopy time-lapse data were recorded with a 10X P-Apo air objective (NA 0.45) on a Nikon Ti-2E equipped with a SOLA SM II LED light source, a motorized stage and an Andor NEO 5.5 camera.

Techniques: Emulsion, Extraction, Fluorescence, Microscopy, Generated

Journal: Interface Focus

Article Title: Micro-compartmentalized strand displacement reactions with a random pool background

doi: 10.1098/rsfs.2023.0011

Figure Lengend Snippet: ( a ) Swarm plot of estimated invader concentration for the collection of droplets shown in ( c ). ( b ) Scatter plot of the initial slopes for each droplet versus the corresponding estimated invader concentration (blue dots). The grey parabola is the theoretically expected dependence of the slope on the invader concentration (see text). ( c ) Composite fluorescence microscopy image of halted droplets produced with different mixing ratios around 100 s after generation (green: reference dye; red: reporter dye). ( d ) Spatial map of the estimated invader concentration in the tracked droplets of the left panel. The concentrations follow the sinusoidal pattern imposed by the control pressure protocol. ( e ) Spatial map of the initial slope of the tracked droplets. The slope is highest in regions with approximate stoichiometry between invader and reporter.

Article Snippet: All fluorescence microscopy time-lapse data were recorded with a 10X P-Apo air objective (NA 0.45) on a Nikon Ti-2E equipped with a SOLA SM II LED light source, a motorized stage and an Andor NEO 5.5 camera.

Techniques: Concentration Assay, Fluorescence, Microscopy, Produced, Control