Catalase

Single molecule fluorescence recordings were carried out at 23°C. For the dsDNA oligos, nuclease-free duplex buffer (IDT) was used, while for ribosomes, TAM₁₅ buffer (50 mM Tris- HCl [pH 7.5], 15 mM Mg(OAc)₂, 30 mM NH₄Cl, 70 mM KCl, 1 mM DTT) [49 (link)] was used. An enzymatic deoxygenation system of 0.3% (w/v) glucose, 300 μg/ml glucose oxidase (Sigma-Aldrich), 120 μg/ml catalase (Roche), and aged 1.5 mM 6-hydroxy-2,5,7,8-tetramethyl-chromane-2-carboxylic acid (Trolox, Sigma-Aldrich) was added to the final single-molecule imaging solutions to reduce fluorophore photobleaching and blinking [49 (link)].

All STORM imaging was performed in a closed chamber with buffer containing reducing and oxygen-scavenging compounds. The stained cells were imaged in PBS with the addition of 50 mM mercaptoethylamine, 5% glucose (wt/vol) and oxygen-scavenging enzymes (0.5 mg ml^–1 glucose oxidase (Sigma-Aldrich), and 40 mg ml^–1 catalase (Roche Applied Science)^{33 (link)}. The ensemble fluorescence was converted into the desired density of single molecules by using the 640 nm laser at high power. For TIRF imaging, reduction was performed by briefly placing the setup into HILO and performing a z-stack with high laser power. For soSPIM imaging, we increased the power delivered via the light sheet and performed a z-stack prior to refocusing on the plane of interest using the printed fiducials as references. Prior to reducing dyes into a dark state, a standard TIRF or soSPIM light-sheet image was acquired. For image acquisition the laser power was reduced, and a UV laser (405 nm) turned on to aid switching. Approximately 10,000–20,000 images were recorded and analyzed.

The silver nanowires were first resuspended in 0.5 ml ethanol solution of biotin-terminated poly(ethylene glycol) (~2.5 mM, Nanoscience Instruments). Then the mixture was bubbled with argon gas for two minutes and kept shaking overnight. The suspension was centrifuged at 4,000 r.p.m. and the supernatant exchanged with pure ethanol. This process was repeated twice. The biotinated nanowires were then kept in 0.5 ml of PBS solution of avidin (~0.6 mg ml⁻¹) at 4 °C for 2 h, and purified twice using centrifugation at 4,000 r.p.m. Those samples were immersed in PBS buffer of biotinated HIS-tag antibody (IgG1, SM1693B, Acris Antibodies; ~3 μg ml⁻¹) at 4 °C for 2 h and the excess antibody was removed. Finally, the nanowires with antibody were further functionalized with Alexa 647 Goat Anti-Mouse IgG (Life Technologies) in PBS buffer at 4 °C for 2 h and the excess antibody removed. The silver nanowires were then drop-cast onto a cover slip which had been silanized by (3-aminopropyl)trimethoxysilane (Sigma-Aldrich). After that, the sample was kept in a sealed chamber filled with switching buffer, PBS (pH 7.4), containing oxygen scavenger (0.5 mg ml⁻¹ glucose oxidase (Sigma-Aldrich), 40 mg ml⁻¹ catalase (Roche Applied Science), 10% w/v glucose) and 50 mM β-mercaptoethylamine20 .

smFRET studies were carried out at 21°C. 70S initiation complexes formed using a 5′-biotinylated mRNA (Dharmacon RNAi Tech.) were immobilized on a biotin/PEG-streptavidin coated glass surface (20 (link)). After washing away unbound complexes, collection of real-time fluorescence traces began 5 s prior to injecting 5 or 10 nM ternary complexes, which were preformed from EF-Tu, GTP and charged tRNAs. Recording continued for 60 s without further washing. An enzymatic deoxygenation system of 3 mg/ml glucose, 100 μg/ml glucose oxidase (Sigma-Aldrich), 40 μg/ml catalase (Roche) and 1.5 mM 6-hydroxy-2,5,7,8-tetramethyl-chromane-2-carboxylic acid (Trolox, Sigma-Aldrich—by dilution from a DMSO solution) was present in the final single-molecule imaging solutions to diminish fluorophore photobleaching and blinking. A custom-built objective-type total internal reflection fluorescence (TIRF) microscope was used to collect Cy3 and Cy5 (due to FRET from Cy3) fluorescence intensities on excitation by a 532 nm laser (16 (link)). The 11 ms integration time per frame was achieved by cutting the exposure area down to 128 pixels × 512 pixels without further binning. Other details of materials preparation, experimental setup, and data analysis were as previously described (16 (link)).

Animals were imaged in PBS buffer containing 100 mM cysteamine (Sigma), 5% glucose (Sigma), glucose oxidase (0.8 mg/ml) (Sigma), and catalase (40 μg/ml) (Roche Applied Science).

DNA fragments for cyclization measurement were immobilized on a PEG-coated microscope slide via biotin-neutravidin linkage. The fragments had complementary 10 nt 5’ overhang at either end, which permit looping via annealing. Cy3 and Cy5 were also present at the two 5’ ends, resulting in high FRET in the looped state. Measuring FRET allowed us to quantify the fraction of looped molecules as a function of time since introduction of a high salt buffered solution 10 mM Tris-HCl pH 8.0, 1 M NaCl, 0.5% w/v D-Glucose (Sigma), 165 U/ml glucose oxidase (Sigma), 2170 U/ml catalase (Roche) and 3 mM Trolox (Sigma). Approximately 2500–3500 molecules were quantified at each timestamp during the experiment, and three independent experiments were performed for each sequence (Figure 5—figure supplement 1). The rate of loop formation was used as an operational measurement of DNA flexibility.

A microscope quartz slide was coated with polyethyleneglycol (PEG) (mixture of mPEG-SVA and Biotin-PEG-SVA, Laysan Bio) according to (25 (link)). The nucleosome sample was immobilized on the PEG-coated slide at 50 pM in nucleosome dilution buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM MgCl₂) through a biotin/neutravidin linker. Single-molecule FRET data were taken in imaging buffer (50 mM Tris-HCl pH 8, 1 mM MgCl₂, 0.5% w/v D-Glucose (Sigma), 165 U/ml glucose oxidase (Sigma), 2170 U/ml catalase (Roche), 3 mM Trolox (Sigma)) and a desired amount of NaCl) using a home-build prism-type total internal reflection fluorescence microscope (25 (link)). Image integration times were 500 ms for obtaining long single-molecule FRET traces and 50 ms for building FRET histogram.