In order to check for structure homogeneity of the RNA molecules, all labeled transcripts were analyzed on 5–10% polyacrylamide gels (400/300/0.8 mm, acrylamide/bisakrylamide–29/1) in non-denaturing conditions. The electrophoresis was conducted at a constant power of 10 or 20 W for 4–6 h in 0.5 × TB buffer (45 mM Tris–borate) at 20°C (identical to the temperature of chemical and enzymatic structure probing reactions). Prior to gel electrophoresis, the 32P-labeled transcripts were subjected to a denaturation/renaturation procedure in a solution containing 10 mM Tris–HCl (pH 7.2), 40 mM NaCl and 10 mM MgCl2, by heating the sample at 90°C for 1 min. and slowly cooling it to 20°C (∼1°C/min), and mixed with an equal volume of 7% sucrose with dyes. Electrophoresis performed in the presence of 1–10 mM Mg2+ and constant buffer circulation did not reveal any significant differences in the formation of stable conformers. The specific conditions for the temperature and pH dependence experiments are described in the legend to the Supplementary Figure 2.
Two different electrophoretic migration standards were used: ds69 and ds107. The ds69 represents dsRNA molecule, 69 bp long, obtained by hybridization of two complementary RNA oligomers: 5′ GGG(CUG)21CCC and 5′ GGG(CAG)21 CCC. The second marker, ds107, was obtained by annealing of the fx4 transcript with its complementary molecule containing 23 CCG repeats.
A number of transcripts analyzed in this study migrated on the native gels as two distinct conformers. In all cases, the contribution of the less prevalent conformer was too high to be neglected in the structure studies. Two assays were used to obtain conformer-specific structural data. First, the preparative amount of intact conformers was separated on a native 8% polyacrylamide gel, exposed to the X ray film and then separately excised and eluted from the gel with 20 mM Tris–HCl, pH 7.2. The conformer-specific structure probing was performed without the initial denaturation/renaturation step as described below. Alternatively, structure probing reactions were performed on the mixture of coexisting stable conformers and partially nicked RNA molecules that were resolved on native polyacrylamide gels. Nicked transcripts (due to the nuclease or lead ion hydrolysis), which migrate on native gels at the same rate as intact conformers, were eluted from the gel (with 0.3 M potassium acetate, pH 5.1, 1 mM EDTA and 0.1% SDS), precipitated and analyzed on denaturing polyacrylamide gels. Although, both methods led to identical results, the first, more straightforward approach was used more frequently. In order to rule out the possibility of sequence heterogeneity between the stable coexisting conformers, RNA sequencing analysis of each conformer was conducted using RNA Sequencing Kit (Pharmacia Biotech Inc.) according to the manufacturer's recommendations.
Two different electrophoretic migration standards were used: ds69 and ds107. The ds69 represents dsRNA molecule, 69 bp long, obtained by hybridization of two complementary RNA oligomers: 5′ GGG(CUG)21CCC and 5′ GGG(CAG)21 CCC. The second marker, ds107, was obtained by annealing of the fx4 transcript with its complementary molecule containing 23 CCG repeats.
A number of transcripts analyzed in this study migrated on the native gels as two distinct conformers. In all cases, the contribution of the less prevalent conformer was too high to be neglected in the structure studies. Two assays were used to obtain conformer-specific structural data. First, the preparative amount of intact conformers was separated on a native 8% polyacrylamide gel, exposed to the X ray film and then separately excised and eluted from the gel with 20 mM Tris–HCl, pH 7.2. The conformer-specific structure probing was performed without the initial denaturation/renaturation step as described below. Alternatively, structure probing reactions were performed on the mixture of coexisting stable conformers and partially nicked RNA molecules that were resolved on native polyacrylamide gels. Nicked transcripts (due to the nuclease or lead ion hydrolysis), which migrate on native gels at the same rate as intact conformers, were eluted from the gel (with 0.3 M potassium acetate, pH 5.1, 1 mM EDTA and 0.1% SDS), precipitated and analyzed on denaturing polyacrylamide gels. Although, both methods led to identical results, the first, more straightforward approach was used more frequently. In order to rule out the possibility of sequence heterogeneity between the stable coexisting conformers, RNA sequencing analysis of each conformer was conducted using RNA Sequencing Kit (Pharmacia Biotech Inc.) according to the manufacturer's recommendations.
