title: A neutral pH thermal hydrolysis method for quantification of structured RNAs.
abstract: Riboswitch aptamers adopt diverse and complex tertiary structural folds that contain both single-stranded and double-stranded regions. We observe that this high degree of secondary structure leads to an appreciable hypochromicity that is not accounted for in the standard method to calculate extinction coefficients using nearest-neighbor effects, which results in a systematic underestimation of RNA concentrations. Here we present a practical method for quantifying riboswitch RNAs using thermal hydrolysis to generate the corresponding pool of mononucleotides, for which precise extinction coefficients have been measured. Thermal hydrolysis can be performed at neutral pH without reaction quenching, avoids the use of nucleases or expensive fluorescent dyes, and does not require generation of calibration curves. The accuracy of this method for determining RNA concentrations has been validated using quantitative (31)P-NMR calibrated to an external standard. We expect that this simple procedure will be generally useful for the accurate quantification of any sequence-defined RNA sample, which is often a critical parameter for in vitro binding and kinetic assays.
authors.affiliation: Department of Chemistry, University of California, Berkeley, California 94720, USA.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Wilson
authors.affiliation: Department of Chemistry, University of California, Berkeley, California 94720, USA.
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Cohen
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
authors.forename: Xin C
authors.initials: XC
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of California, Berkeley, California 94720, USA Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
authors.forename: Ming C
authors.initials: MC
authors.lastname: Hammond
mesh_terms: D052157:Aptamers, Nucleotide / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D012313:RNA / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D058928:Riboswitch; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
chemical_list: D052157:Aptamers, Nucleotide; D058928:Riboswitch; D012313:RNA
keywords: concentration; extinction coefficient; quantitation; riboswitch; thermal denaturation; ultraviolet spectroscopy