Cary 100 spectrophotometer

All reagents and solvents were purchased from commercial sources and were of analytical grade. ¹H and ¹³C NMR spectra in CDCl₃ or DMSO-d₆ were recorded on a Bruker AV 300 MHz NMR instrument with tetramethylsilane (TMS) as the internal standard. Data for ¹H NMR spectra is reported as follows: chemical shift (ppm) and multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet). Data for ¹³C NMR spectra is reported in ppm. High Resolution Mass Spectroscopy (HRMS) spectra were recorded on a Waters Q-Tof Premier™ Mass Spectrometer. UV/Vis spectra were measured with a Varian Cary 100 spectrophotometer (1 cm quartz cell). Emission spectra were measured with a Varian Cary Eclipse (1 cm quartz cell). The time dependent fluorescence study was conducted by fluorescence induction in situ and measured with a Varian Cary Eclipse (1 cm quartz cell) at 37 °C. Purification by flash column chromatography was carried out using silica gel 60 (0.010–0.063 mm) with eluents as noted in the experimental data sections for the respective compounds. Deionized water was used in the preparation of all samples.

UV-visible spectra were recorded in a Cary 100 spectrophotometer (Varian) and SpnFADS was quantified using the theoretical ε_279nm = 28.88 mM⁻¹·cm⁻¹ and a molecular weight of 34,521 Da (ProtParam). Circular dichroism (CD) spectra were recorded with a Chirascan spectropolarimeter (Applied Photophysics Ltd.) at 25 °C. Samples containing ∼5 and ∼20 µM SpnFADS in 25 mM Tris/HCl, pH 7.5 were used in the far-UV (cuvette path length, 0.1 cm) and near-UV CD (cuvette path length, 0.4 cm), respectively.

Commercially available reagents were purchased from Fluka (Sydney, NSW, Australia), Aldrich (Sydney, NSW, Australia), Acros Organics (Morris Plains, NJ, USA), Alfa Aesar (Lancashire, UK) and Lancaster (Lancashire, UK) and purified if necessary. The synthetic procedures have been reported for all compounds as general methods and appropriate references have been given for known compounds. ¹H (300 MHz) and ¹³C-NMR (75 MHz) spectra were obtained in the designated solvents on a DPX 300 spectrometer (Bruker, Sydney, NSW, Australia). Melting points were measured using a Mel-Temp melting point apparatus and are uncorrected. Infrared spectra were recorded on Avatar Series FT-IR spectrophotometer as KBr disks (Thermo Nicolet, Waltham, MA, USA). Ultraviolet spectra were measured using a Cary 100 spectrophotometer (Varian, Santa Clara, CA, USA) in the designated solvents and data reported as wavelength (λ) in nm and adsorption coefficient (ε) in cm⁻¹M⁻¹. High-resolution [ESI] mass spectra were recorded by the UNSW Bioanalytical Mass Spectrometry Facility, on an Orbitrap LTQ XL (Thermo Scientific, Waltham, MA, USA) ion trap mass spectrometer using a nanospray (nano-electrospray) ionization source.

TEM was used to observe the particle size, structure, and morphology of AuÅPs or AuÅPs-AL. In short, gold particles at a concentration of 500 ng/µL were permitted to adsorb onto carbon-coated copper grids for approximately 30 min, after which the grids were rinsed in water. TEM imaging was then performed (H-7000FA, Hitachi, Tokyo, Japan). The size distribution of gold particles in aqueous solution was measured by DLS (Nano-ZS90, Malvern Instruments, Malvern, UK). NMR spectra were obtained using a Bruker NMR spectrometer (600 MHz). To measure the stability of AuÅPs-AL in water over 48 h at room temperature, UV and visible light absorption spectra were recorded using a Cary 100 spectrophotometer (Varian, Palo Alto, USA), and fluorescence spectra were recorded using a Fluorolog-3 Spectrofluorometer (HORIBA Jobin Yvon, Edison, USA).

To observe the spectra of the different redox states of SpFld, a sodium dithionite reduction was performed at 10°C using 20 μM protein in 150 mM NaCl, 100mM MES buffer, pH 6.5 under pseudo-anaerobic conditions. A concentrated anaerobic sodium dithionite solution (50 mM) was prepared in the same buffer by sequential evacuation and re-equilibration with oxygen-free argon. Pseudo-anaerobic conditions were achieved in the spectrophotometer cell by argon flux. Stepwise SpFld reduction was achieved by addition of 2.5–10 ml aliquots of sodium dithionite to the protein sample. After each addition, the UV-Vis spectrum was recorded in a Cary 100 spectrophotometer (Varian).

RNA samples were lyophilized as triethylammonium or sodium salts, dissolved in 800 or 330 μl buffer (10 mM Na₂HPO₄ (pH 7.0) containing 150 mM NaCl) and transferred into UV permeable high precision cells made of quartz SUPRASIL^® with a light path of 10 mm or 1 mm. UV melting profiles were recorded at 250 and 260 nm on a Varian Cary-100 spectrophotometer equipped with a multiple cell holder and a peltier temperature control device. Each RNA was measured at five different concentrations (between ∼1 and ∼100 μM) and with at least four ramps (heating-cooling-heating-cooling; 1°C min^–1 heating/cooling rate). T_m values were determined by calculating the first derivative, usually from data of the fourth ramp (cooling). Thermodynamic parameters were determined according to reference (18 (link),19 (link)). The error limits for ΔG, ΔH and ΔS reflect the standard deviation of at least three independent measurements for a confidence interval of 95%.
For the pH dependent UV-melting curve experiments the following buffers were used: pH 5.0: 10 mM Na₂HPO₄ containing 150 mM NaCl; pH 5.99: 10 mM Na₂HPO₄ containing 150 mM NaCl.