Tris-borate is a poor counterion for RNA: a cautionary tale for RNA folding studies.
Native polyacrylamide gel electrophoresis is a powerful approach for visualizing RNA folding states and folding intermediates. Tris-borate has a high-buffering capacity and is therefore widely used in electrophoresis-based investigations of RNA structure and folding. However, the effectiveness of Tris-borate as a counterion for RNA has not been systematically investigated. In a recirculated Hepes/KCl buffer, the catalytic core of the bI5 group I intron RNA undergoes a conformational collapse characterized by a bulk transition midpoint, or Mg1/2, of approximately 3 mM, consistent with extensive independent biochemical experiments. In contrast, in Tris-borate, RNA collapse has a much smaller apparent Mg1/2, equal to 0.1 mM, because in this buffer the RNA undergoes a different, large amplitude, folding transition at low Mg2+ concentrations. Analysis of structural neighbors using a short-lived, RNA-tethered, photocrosslinker indicates that the global RNA structure eventually converges in the two buffer systems, as the divalent ion concentration approaches approximately 1 mM Mg2+. The weak capacity of Tris-borate to stabilize RNA folding may reflect relatively unfavorable interactions between the bulky Tris-borate ion and RNA or partial coordination of RNA functional groups by borate. Under some conditions, Tris-borate is a poor counterion for RNA and its use merits careful evaluation in RNA folding studies.
Buchmueller
Karen L
KL
Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599-3290, USA.
Weeks
Kevin M
KM
D001483:Base Sequence; D001888:Boric Acids / Q000737:chemistry*; D002021:Buffers; D004591:Electrophoresis, Polyacrylamide Gel; D006531:HEPES / Q000737:chemistry; D007438:Introns; D007477:Ions / Q000737:chemistry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry*; D014325:Tromethamine / Q000737:chemistry*
D001888:Boric Acids; D002021:Buffers; D007477:Ions; D014325:Tromethamine; D012313:RNA; D006531:HEPES