Analysis of RNA folding by native polyacrylamide gel electrophoresis

Abstract

Polyacrylamide gel electrophoresis under native conditions (native PAGE) is a well-established and versatile method for probing nucleic acid conformation and nucleic acid-protein interactions. Native PAGE has been used to measure RNA folding equilibria and kinetics under a wide variety of conditions. Advantages of this method are its adaptability, absolute determination of reaction endpoints, and direct analysis of conformational hetereogeneity within a sample. Native PAGE is also useful for resolving ligand-induced structural changes.

Figure 1.

Models for macromolecular electrophoresis. (a) Reptation of long DNA fragments in an electric field. Redrawn from (Bloomfield et al., 2000). (b) Ogston sieve model, which applies when R_g of the macromolecule is smaller than the diameter of the pore. Counterions move in the opposite direction of the RNA. (c) Scanning electron micrograph of 7.5% polyacrylamide gel. Reprinted from (Yuan et al., 2006).

Figure 2.

Native gel electrophoresis of the Tetrahymena P4-P6 RNA. (A) The folded and extended forms equilibrate rapidly, producing a single band whose mobility reflects the average structure of the RNA. U1, U2, BP5/5a refer to mutations in a hinge region. RNAs were run on native 6% (19:1) polyacrylamide gel in TBE + 10 mM MgCl₂ at 25 ˚C. (B) Ferguson plot shows that the relative mobility depends linearly on gel concentration, as predicted by the Ogston model. Reprinted from (Szewczak and Cech, 1997).

Figure 3.

Native gel electrophoresis of the Tetrahymena ribozyme. (A) Folded (N) and misfolded (I) forms of the RNA are in slow exchange and are trapped within the matrix of the gel, migrating at different rates. Folded RNA is stabilized by Mg²⁺ in the running buffer. (B) Folding kinetics. Ribozyme was incubated in folding buffer containing MgCl₂ for 0–10 min before samples were loaded on a native 8% (29:1) polyacrylamide gel in THEM₃ at 4 ˚C. wt, wild type; L2P5cP3 is a mutant that increases the folding rate. The fraction of native RNA is determined from the volume of counts in the native band relative to the total counts in the lane. (C) Folding equilibrum of the ribozyme in different ions measured by native PAGE. The fraction of N was fit to the Hill equation. Redrawn from (Pan et al., 2000) and (Heilman-Miller et al., 2001).

Figure 4.

Mobility of the Tetrahymena ribozyme in different divalent metal ions. (A) The unfolded (U) and folded (F) ribozyme was run next to ΦX DNA size markers on native 8% PAGE in THE buffer with 3 mM MgCl₂, CaCl₂ or BaCl₂. (B) The relative RNA mobility decreased with the size and charge density of the metal ion. Reprinted from (Koculi et al., 2007).