Acrylamide and bis acrylamide solution

Copper (II) sulfate pentahydrate, CuSO₄∙5H₂O (>98.0%); ammonium sulfate, (NH₄)₂SO₄ (99%); calcium chloride, CaCl₂ (99.9%); magnesium sulfate heptahydrate, MgSO₄∙7H₂O (99%); potassium chloride, KCl (99%); potassium phosphate monobasic, K₂HPO₄ (99%); thiamine hydrochloride (99%) sodium citrate dihydrate HOC(COONa)(CH₂COONa)₂∙2H₂O (>99%), and sodium hydroxide anhydrous pellets, NaOH (>98%) were purchased from Merck (Darmstadt, Germany). The citric acid (99.5%), malt extract agar, and yeast extract were supplied by Scharlau (Barcelona, Spain). 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonate), ABTS (>98.0%), 3,5-dinitrosalicylic acid, and DNS (>98.0%) were manufactured from Sigma–Aldrich, St. Louis, MO, USA. D(+)-Glucose anhydrous PA-ACS and sodium acetate anhydrous (99.9%) were obtained from PanReac AppliChem (Barcelona, Spain). Acrylamide and bis-acrylamide solution (30%, 29:1), 0.5 M Tris-HCl, pH 6.8, 1.5 M Tris-HCl, pH 8.8 solutions were provided for Bio-Rad, Alcobendas, Spain.

Gels of different concentrations were prepared using 30% acrylamide and bis-acrylamide solution (Bio-Rad, 29:1) with 7 M urea in 0.5 × TBE buffer (Bio-Rad) and run on a PROTEAN II xi cell (Bio-Rad) or a Mini-PROTEAN Tetra cell (Bio-Rad). Samples were mixed 1:1 with TBE-urea sample buffer (Bio-Rad) and heated at 70 °C for 5 min before they were loaded into the wells. Gel concentrations were carefully chosen to ensure the proper separations between different topologies as well as the references. For imaging, gels were stained with GelRed (Biotium), and images were taken by Gel Doc XR+ (Bio-Rad) imaging system and processed by software Image Lab (v.4.0.1, Bio-Rad). For purification, gels (without staining) were visualized by UV shadowing against a fluorescent thin-layer chromatography plate and bands of interest were cut. The bands were then eluted using the crush-and-soak method and the eluent was purified and concentrated on 3 K Nanosep filters (Pall). The concentration of product was determined by measuring the OD₂₆₀. Optionally, the ssRNA knots and circles can be digested by RNase R (Epicentre) after the gel extraction to remove the unavoidable cleaved linear RNA during the purification. However, RNase R digestion is not useful for the hybrid BR.