Typhoon 9210 imager

We have previously described the isolation [38 (link),42 (link)] and quantification [40 (link)] of active recombinant G4R1 from Rosetta 2 cells. The apparent K_d was estimated using gel mobility shift assays (GMSA) as previously described [40 (link)]. Briefly, recombinant G4R1 at concentrations of 10–500 pM was incubated with 1 pM 5’-[32P]-end-labeled G4-DNA in Res buffer (50 mM KCl, 10 mM NaCl, 3 mM MgCl₂, 50 mM Tris acetate, pH 7.8, 70 mM glycine, 0.012% bovine lactalbumin, 10% glycerol) with 10 mM EDTA at 37°C for 0.5 or 24 h. Binding reactions were loaded onto 10% non-denaturing polyacrylamide gels and electrophoresis was performed at 70 V for 15 h in a cold room (7°C). Gels were imaged on a Typhoon 9210 Imager (GE Healthcare), and band densities were analyzed using Multi Gauge software (Fuji). The apparent K_d was derived from the Scatchard equation using SigmaPlot 11.0 software to perform direct curve- fitting analysis. For binding experiments in which the monovalent cation composition was varied, recombinant G4R1 at concentrations of 50–500 pM was incubated with 1 pM 5’-[32P]-end labeled G4-DNA and Res buffer with either 50 mM KCl/10 mM NaCl, 50 mM KCl or 50 mM NaCl. Analysis was performed as described above.

The procedure of transformation and purification of RHAU was the same as the previously described BG4. Gel shift assay was performed as previously described.^{66 (link)} Recombinant RHAU purified at concentration of 1 μM was incubated with 1 pM of 5′-³²P-labeled G4 nucleic acid in the RES-EDTA buffer (100 mM KCl, 10 mM NaCl, 3 mM MgCl₂, 50 mM Tris-acetate, pH 7.8, 70 mM glycine, 10% glycerol, 10 mM EDTA) at 37 °C for 30 min. Bound mixtures were then analyzed by 10% non-denaturing polyacrylamide gel. Electrophoresis was performed at 100 V for 2 h in a cold room. Gels were imaged on a Typhoon 9210 Imager (GE Healthcare).
To demonstrate the effect of ATP on RHAU/Kit association, we referred the previously described method.^{56 (link)} 1 μM RHAU was incubated with 1 pM 5′-³²P-labeled self-annealed kit in the presence or absence of 5 mM ATP at 37 °C for 30 min, respectively. All the samples were analyzed on 10% non-denaturing polyacrylamide gel at 150 V for 2 h. The following steps were the same as the procedure described above.

To test whether MazE-Da neutralized MazF-Da toxicity, 1 or 10pmol MazF-Da was pre-incubated with 20 or 40pmol of MazE-Da in a reaction buffer at 25°C for 10min. Next, RNA 1500-1 (200ng) was added to this mixture followed by incubation at 60°C for 90min. These RNAs were then purified using RNA Clean and Concentrator^™-5 (Zymo Research). Gel loading buffer II (Ambion) was added to each sample followed by incubation at 95°C for 5min. Samples were separated on a 7.5% polyacrylamide gel containing 7M urea, stained with SYBR Gold (Life Technologies), and detected using a Typhoon 9210 imager (GE Healthcare).

To test the enzymatic activity of MazF-nd1, 0.373, 3.73, or 18.6 pmol of MazF-nd1 or 0.250, 1.25, or 6.25 U of E. coli MazF (Takara, Shiga, Japan) was added to 200 ng of RNA 500-2. Five pmol of MazF-nd1 was first pre-incubated with 1, 5, or 25 pmol of MazE-nd1 at 25 °C for 15 min. As a control reaction, 5 pmol of MazF-nd1 and 25 pmol of MazE-nd1 was pre-incubated solely at 25 °C for 15 min. Next, 50 ng of RNA 500-2 was added to each sample. All samples were incubated at 37 °C for 1 h in a total of 30 µL of MazF reaction buffer (20 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, 0.01% Triton X-100, and 4 U of recombinant RNase inhibitor (Takara)). RNAs were purified with RNA Clean and Concentrator™-5 (Zymo Research, Irvine, CA, USA) and then incubated with gel loading buffer II (Ambion, Austin, TX, USA) at 95 °C for 5 min. Samples were separated on 10% polyacrylamide gel containing 7 M urea. RNA was stained using SYBR Gold (Life Technologies, Carlsbad, CA, USA) and detected using a Typhoon 9210 imager (GE Healthcare).

Synthetic RNA constructs were prepared as described in our previous study (Miyamoto et al. 2016). One hundred nanograms of RNA 500‐2 was incubated with 1.2, 6, or 30 pmol of MazF_DR0417 at 37°C for 2 h in MazF reaction buffer (20 mmol/L Tris‐HCl (pH 8.0), 1 mmol/L dithiothreitol, 0.01% Triton X‐100, and 4 U of recombinant RNase inhibitor (Takara)) in a 50‐μL reaction volume. As a control reaction, 30 pmol of MazF_DR0417 was preincubated with 300 pmol of MazE_DR0416 at room temperature for 10 min, and this mixture was incubated with 100 ng of RNA 500‐2 at 37°C for 2 h in MazF reaction buffer in a final volume of 50 μL. These RNAs were purified with RNA Clean and Concentrator^™‐5 (Zymo Research, Orange, CA, USA). Next, gel loading buffer II (Ambion, Austin, TX, USA) was added to each sample. The samples were incubated at 95°C for 5 min and then separated on a 10% polyacrylamide gel containing 7 mol/L urea. The RNA was stained using SYBR Gold (Life Technologies) and then detected using a Typhoon 9210 imager (GE Healthcare).

One unit of MazF-Ec (TaKaRa) or 7.1pmol of MazF-Da, and MazF reaction buffer (20mM Tris–HCl [pH 8.0], 1mM dithiothreitol, 0.01% Triton X-100, and 4U of recombinant RNase inhibitor [TaKaRa]) were mixed, adjusted to a total volume of 30μl, and incubated at 4, 37, 50, 60, 70, 80, and 90°C for 10min. The reaction mixture was placed on ice, followed by the addition of substrate RNA 1500-1 (300ng) and incubation at each temperature for 60min. The resultant digested RNA fragments were purified using RNA Clean and Concentrator^™-5 (Zymo Research, Irvine, CA, United States). Gel loading buffer II (Ambion, Austin, TX, United States) was added to each sample followed by incubation at 95°C for 5min. The purified RNA was separated on a 7.5% polyacrylamide gel containing 7M urea, stained with SYBR Gold (Life Technologies, Carlsbad, CA, United States), and detected using a Typhoon 9210 imager (GE Healthcare).