Monovalent cations use multiple mechanisms to resolve ribozyme misfolding

(A) Experimental schemes. (Scheme I) Analyzing the structure of different folding starting states renatured at 0–20 mM monovalent cations. The Candida ribozyme Ca.L-11 was denatured at 95°C for 1.5 min and renatured by a rapid annealing (on ice). Tris-HCl (pH 7.5) and NaCl were added on ice to reach the final concentration of 100 mM for each cation. Ribonuclease T1 at a final concentration of 0.4 U/μL was then added to initiate the cleavage reaction on ice for 10 min. (Scheme II) Analyzing how the monovalent cation-promoted folding starting states impact the Mg²⁺-induced ribozyme folding by exploring the altered ribozyme structure (T1 footprinting) and ribozyme activity (k_obs). The ribozyme was denatured and renatured as in Scheme I. However, in addition to the same concentrations of Tris-HCl and NaCl, a final concentration of 10 mM MgCl₂ was added to initiate tertiary folding. In order to probe the rapidly compacted ribozyme structure, a final concentration of 0.1 U/μL T1 ribonuclease was added, and the reactions proceeded at 37°C for 1 min. In order to probe the ribozyme activity, GTP and the 5′-end-labeled substrate RNA at a final concentration of 0.1 mM and 20 nM, respectively, were added, and the reactions proceeded at 37°C from 0 to 60 min to obtain the k_obs for each ribozyme sample (10 nM) renatured at a distinct concentration of Tris-HCl or NaCl. (Scheme III) Analyzing if monovalent cations present during the Mg²⁺-induced RNA folding could relieve ribozyme misfolding. The ribozyme RNA was denatured and renatured in water following the same procedure as in Scheme I. Then 0–400 mM NaCl was added together with 10 or 15 mM MgCl₂ to the ribozyme RNA on ice. T1 footprinting analysis and activity analysis were then performed as in Scheme II. (B) The observed catalytic activity (k_obs) of the Candida ribozyme following Scheme II. The k_obs of each reaction was obtained and plotted against the concentrations of Na⁺ and Tris⁺. The half-maximal activity for Na⁺ and Tris⁺ was 3.81 and 2.82 mM, respectively. The Hill coefficients were 2.69 ± 0.262 and 3.50 ± 0.613 for Na⁺ and Tris⁺, respectively. (C) The observed catalytic activity (k_obs) of the Candida ribozyme following Scheme III. The half-maximal activity was achieved at 99.25 mM and 199.3 mM Na⁺ in the presence of 10 mM and 15 mM Mg²⁺, respectively. The corresponding Hill coefficients were 3.73 ± 1.705 and 3.516 ± 0.9829.