Cary 3500 multicell uv vis spectrophotometer

Oligonucleotides (SI Appendix, Table S8) in 20 mM lithium cacodylate (pH 7.0) containing 10 or 100 mM KCl or LiCl (56 ) were annealed at 95 °C for 5 min followed by slow cooling to room temperature. UV spectral absorbances were measured between 200 and 330 nm using an Agilent Cary 3500 Multicell UV–Vis spectrophotometer. Measurements were made using a 1-cm path length quartz cuvette covered with a layer of mineral oil. Thermal differences were calculated by subtraction of the 20 and 90 °C spectra. Samples were heated from 20 to 90 °C and UV melting curves were recorded at a spectral bandwidth of 2 nm, with the temperature measured consecutively at a rate of 0.5 °C/min at 300 and 305 nm, with data collection in triplicate every 0.2 °C. T_m values were obtained from Van’t Hoff analysis of the melting profiles as the temperature at which the folded fraction (θ) equals 0.5 (57 (link)).

The reducing capacity (ability to reduce Fe³⁺ to Fe²⁺) of BR was investigated following the procedure for FRAP assay of Benzie and Strain [40 (link)]. Prior to the analysis, FRAP reagent, which contained 300 mmol/L of sodium acetate buffer (pH 3.60), 10 mmol/L of 2,4,6-tris(2-pyridyl)-(S)-triazine (TPTZ) in 40 mmol/L HCl, and 20 mmol/L FeCl₃∙6H₂O solution (10:1:1, v/v/v), was prepared. Then, 100 µL of the previously diluted BR was added to 3 mL of FRAP reagent. After 30 min incubation at 37 °C, the absorbance was measured at 593 nm. Methanol was used instead of extract to prepare blank, while the ascorbic acid was used as positive control. The results were expressed as mmol Fe²⁺/g of dry weight of extract, using the calibration curve ( $y=0.0209x+0.0015$ ; R² = 0.9976).
The spectrophotometric readings were conducted on Cary 3500 Multicell UV-Vis Spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). All measurements were performed in triplicates, and the results were expressed as mean value ± standard deviation.

UV thermal denaturation assays were conducted on a Cary 3500 Multicell UV-Vis Spectrophotometer (Agilent Technologies, Mulgrave, Australia) using quartz cuvettes (Starna Cells, Inc., Atascadero, CA, USA) with an optical path length of 1 cm. For each sample, the total RNA concentration (MALAT1/MENβ triple helix, MALAT1/MENβ SL with (1:1 stoichiometry) or without oligonucleotides) was maintained at 0.5 μM. Samples were prepared using a previously reported buffer containing 25 mM of sodium cacodylate (pH 7.0), 50 mM of KCl and 1 mM of MgCl₂ [16 (link)]. RNA folding was completed by heating (25 °C to 95 °C) and cooling (95 °C to 25 °C) the samples at a ramp rate of 5 °C/min. After the folding step, the oligonucleotides were added and incubated for 30 additional minutes at 25 °C. For the melting curves, the absorbance at 260 nm was recorded at 0.3 °C intervals as the temperature increased from 25 °C to 95 °C at a ramp rate of 0.8 °C/min. The buffer was subtracted from all the melting curves of the RNA. The melting temperatures were extrapolated from the peak maxima of the first derivatives of the melting curves (δA/δT) and normalized between 0 and 1, followed by Savitzky–Golay smoothing across 25 points using the OriginPro 2022 (64-bit) SR1 9.9.0.225 (Academic) graphing software (OriginLab Corporation, Northampton, MA, USA).