Pyrophosphatase

PCR droplets were reinjected and spaced into a fusion device at a rate of ~1500 droplets/s. Each PCR droplet was then synchronized with a 16 pL in vitro transcription (IVT) droplet containing 2 mM each NTP (Larova), 25 mM MgCl₂, 44 mM Tris-HCl pH 8.0 (at 25°C), 5 mM DTT, 1 mM Spermidine, 0.1% of Pluronic F68 (Sigma), 1 μg of pyrophosphatase (Roche), 500 nM Gemini-561, 1 μM coumarin acetate (Sigma) and 17.5 μg/mL T7 RNA polymerase (prepared in the laboratory). IVT mixture was loaded in a length of PTFE tubing and kept on ice during all experiment. PCR droplets were spaced and IVT droplets produced using a single stream of Novec 7500 fluorinated oil (3M) supplemented with 2% (w/w) of fluorinated. Flow-rates (MFCS, Fluigent) were adjusted to generate 16 pL IVT droplets and maximize synchronization of 1 PCR droplet with 1 IVT droplet. Pairs of droplets were then fused with an AC field (400 V at 30 kHz) and the resulting emulsion was collected off-chip and incubated for 2 h at 37°C.

The assay was modified from the original method [13 (link)]. A 20 µl reaction contained the following components: 50 mM HEPES-KOH (pH 7.2), 25 mM KCl, 10 mM MgCl₂, 5 mM DTT, 10 mM ATP, 25 µg/ml pyrophosphatase (Roche Applied Science), 1 µM tRNA. All tRNA aminoacylation levels were determined at 37°C with synthetase, 10 nM ³²P-labeled tRNA. Time points were taken at 5 min, 10 min and 30 min by removing 2 µl aliquots from the reaction and immediately quenching the reaction into an ice-cold 3 µl quench solution (0.66 µg/µl nuclease P1 (Sigma) in 100 mM sodium citrate (pH 5.0)). For each reaction, 2 µl of blank reaction mixture (containing no enzyme) was added to the quench solution as the start time point. The nuclease P1 mixture was then incubated a room temperature for 30 min and 1 µl aliquots were spotted on PEI-cellulose plates (Merck) and developed in running buffer containing 5% acetic acid and 100 mM ammonium acetate. Radioactive spots of AMP and AA-AMP (representing free tRNA and aminoacyl-tRNA, respectively) were separated and then visualized and quantified by phosphorimaging in a Molecular Dynamics Storm 860 phosphorimager (Amersham Biosciences). The ratio of amino-acyl-tRNA to total tRNA was determined to monitor reaction progress.

A 20 μl aminoacylation reaction contained the following components: 50 mM HEPES-KOH (pH 7.2), 25 mM KCl, 10 mM MgCl2, 5 mM DTT, 10 mM ATP, 25 μg/ml pyrophosphatase (Roche Applied Science), 2 mM amino acids. All plateau tRNA aminoacylation levels were determined at 37 °C according to the reaction conditions described above with 500 nM enzyme, 5 μM unlabeled tRNA plus 100 nM ³²P-labeled tRNA. Time points were taken at 5 min, 20 min and 60 min by removing 2 μl aliquots from the reaction and immediately quenching the reaction into an ice-cold 3 μl quench solution (0.66 μg/μl nuclease P1 (Sigma) in 100 mM sodium citrate (pH 5.0)). For each reaction, 2 μl of blank reaction mixture containing no enzymes was added to the quench solution as the start time point. The nuclease P1 mixture was then incubated at room temperature for 30 min and 1 μl aliquots were spotted on PEI-cellulose plates (Merck) and developed in running buffer containing 5% acetic acid and 100 mM ammonium acetate. Radioactive spots for AMP and AA-AMP (representing free tRNA and aminoacyl-tRNA, respectively) were separated and then visualized and quantified by phosphorimaging by a Molecular Dynamics Storm 860 phosphorimager (Amersham Biosciences). The ratio of aminoacylated tRNA to total tRNA was determined to monitor reaction progress.