Fig. 2.

Optimization of TAR monomer and dimer formation on native gels. (a) TARwt, A34U, and A34U:U37A variants dimerize to different extents under the same renaturation conditions. 3 µM RNA was renatured in 1×TE and 1×TEK₁₀₀ by incubating at 90 °C for 3 min, room temperature for 10 min, and 55 °C for 10 min. The sample was then either cooled at room temperature for 10 min (cooling condition 1) or on ice for 10 min (cooling condition 2). In the third set of salt conditions, MgCl₂ was added to 4 mM in the 1×TEK₁₀₀ samples, prior to the 55 °C step. Gel loading buffer contained 10 mM NaCl and 5% glycerol (b) Optimizing dimer formation in the A34U:U37A variant. 50 or 3 µM A34U:U37A was renatured in 1×TE, 1×TEK₁₀₀, or 1×TEK₅₀₀ by incubating at 90 °C for 3 min, followed by one of three cooling conditions: 1) ice for 10 min, 2) room temperature for 10 min, or 3) 55 °C for 10 min followed by room temperature for 10 min. The presence of salt and high RNA concentration drives dimer formation. All gels shown are 10% polyacrylamide native gels (0.5×TBE) fractionated for ~2 h at 16 °C. The percent of strands in dimer is provided below the gel.