Fluorolog spectrofluorimeter

All absorption data was taken on a Varian Cary 500 spectrometer. Rhodamine 6G emission measurements were taken on a Horiba Scientific Fluorolog spectrofluorimeter using a monochromated Xe lamp as the excitation source. CEP blend film and Stilbene-420 emission spectra were taken on a LaserStrobe spectrometer from Photon Technology International using a GL-3300 nitrogen laser and GL-302 dye laser attachment, also from Photon Technology International. Upconverted emission spectra were measured with the emission filtered by a 500 nm short-pass filter from Thorlabs, model FES0500, to prevent reflected excitation light from interfering with the measured emission signal. Laser power was measured with a 919P-003-10 thermopile sensor from Newport. Time-Resolved Single Photon Counting Data (in Extended Data section) was taken using excitation light generated by a Fianium SC400 supercontinuum fibre laser with wavelength selected by a Fianium AOTF system. Detection was measured via a photomultiplier tube connected to a Becker-Hickl SPC-130 system. All data was collected with signal count rate at <2% of excitation rep rate to ensure proper TCSPC statistics. All spectra were corrected for the spectral responsivities of the systems used for data collection.

AF488-labelled 120-nt, 30-nt and 20-nt long RNAs were used for estimating binding affinities of σNS to ssRNAs. All measurements were performed at 21°C using a Fluorolog spectrofluorimeter (Horiba Jobin-Yvon). σNS was titrated (10 nM–10 μM final concentrations) into 1 nM of each of the RNA in 10 mM HEPES–NaOH, pH 7.5, 150 mM NaCl, allowing equilibration for 30 min prior FA data collection. Normalized anisotropy was plotted as a function of protein concentration and fitted to a single-site binding model using OriginPro 9.0 software.

In order to form the binary complex EF-Tu–GTP, EF-Tu–GDP (40 µM) was incubated with GTP (2 mM), phosphoenol pyruvate (PEP) (6 mM), and pyruvate kinase (0.1 mg ml⁻¹) in buffer B at 37 °C for 15 min. MgCl₂ was added to compensate for GTP- and PEP-complexed Mg^{2+80 (link)}. EF-Tu–GTP was added to RCys-tRNA^Cys (10 nM) and incubated for 2 min at 37 °C and the fluorescence change monitored in the Fluorolog spectrofluorimeter (Horiba Jobin Yvon). NBD emission was monitored at 525 nm after excitation at 480 nm. BodipyFL emission was monitored at 510 nm after excitation at 470 nm. Atto520 emission was monitored at 545 nm after excitation at 500 nm. The fluorescence change $\mathrm{\Delta }\mathrm{F}$ was fitted to a hyperbolic binding model: $$\mathrm{\Delta }\mathrm{F}={\mathrm{F}}_0+{\mathrm{\Delta }\mathrm{F}}_{\mathit{max}}\frac{[EF-Tu]}{K_d+\left[E,F,-,T,u\right]},$$ where F₀ as initial fluorescence, ${\mathrm{\Delta }\mathrm{F}}_{\mathit{max}}$ the maximum fluorescence change and $K_d$ is the equilibrium dissociation constant. The respective values are reported in Fig. 1b.

The optical properties of all the annealed undoped Y₂O₃ and Y₂O₃:Bi thin films have been analyzed by photoluminescence (PL) and photoluminescence excitation (PLE) spectroscopy at room temperature (RT), performed by a Horiba Fluorolog spectrofluorimeter equipped with a 400 W Xe lamp. The excitation wavelength was varied between 300 nm and 550 nm with a scan step of 1 nm, while the luminescence was detected by a Hamamatsu Photomultiplier.
Cathodoluminescence (CL) properties have been investigated using a commercial Oxford CL system, fitted onto a Cambridge S360 standard tungsten gun scanning electron microscope. The CL system is equipped with a 1800 line/mm grating and a multialkali Photomultiplier sensitive in the range 350–830 nm. The spectroscopic CL analyses are carried out with an accelerating voltage of 3 kV at room temperature and at low temperature (liquid nitrogen, 77 K). The spectral resolution is 5 nm.

Dual-labeled stem-loop (Supplementary Table S1) was heat-annealed at 75°C for 5 min and cooled to 4°C. Folded RNA-alone and denatured RNA (in 50% v/v formamide) were initially measured in 100 μl volumes at a final RNA concentration of 10 nM. Serial 2-fold dilutions of NSP2 and σNS from 15 μM were incubated with 10 nM RNA at room temperature for 15 min prior to measurement. Measurements were performed using a Fluorolog spectrofluorimeter (Horiba Jobin-Yvon). Apparent FRET efficiencies were calculated using Equation (5).