Rna oligonucleotide

The HPLC-purified RNA oligonucleotides for UV-absorbance-detected thermal denaturation experiments were purchased from Sigma. The ensemble thermal denaturation experiments for the pseudoknots and a control hairpin were conducted using a Shimadzu 2550 spectrometer. The control hairpin (residues 1 to 19) is a truncated form of the pseudoknot SF206 (residues 1 to 34) (see Fig. 1a and Supplementary Fig. S1). The RNA oligonucleotide sample concentrations were 3 μM. All pseudoknots and the control hairpin were heated at 85 °C then slowly cooled to 15 °C in more than 2 hours before the thermal melting experiments. The temperature ramping rate was 0.5 °C/min. Absorption values at 260 nm were recorded. The buffers contained 20 mM HEPES, 0.1 mM EDTA, pH 7.3 with varying NaCl concentrations (0 mM, 200 mM or 1 M NaCl). The measurement for each sample at each buffer condition was repeated three times. The melting temperatures were obtained from dA/dT versus temperature curves and presented as mean ± standard deviation.

Circular dichroism (CD) studies were performed on Jasco 815. RNA oligonucleotides (n = 6) were purchased from Merck for all biophysical experiments. RNA oligonucleotides were prepared in a final concentration of 10 μM with 10 mM sodium cacodylate buffer (pH −7.4) and 100 mM potassium chloride (KCl). The samples were slowly cooled to room temperature after heating at 90 °C for 5 min. Spectra at 220 to 320 nm wavelength range were recorded in a quartz cuvette of 1 mm path length, keeping a 1 nm step size and time of 1s per point. CD melt curves were obtained over 20 to 95 °C using the same oligonucleotide concentration (10 μM), either with or without BRACO19 as previously described by Kumar et al. (69 (link)). Melting temperatures (Tm) were calculated by fitting the curve into the Boltzmann function with Origin 9.7.5 (Origin Lab Corp).