Thermodynamic analysis of an RNA combinatorial library contained in a short hairpin.
Prediction of nucleic acid structure from sequence requires thermodynamic parameters for a variety of motifs, many of which are complex and consist of a large number of possible sequence combinations. Here we report an experimental approach for identifying the stable and unstable members of an RNA combinatorial library. Short model RNA hairpins consisting of 13 base pairs (bp) flanked by primer binding sites are constructed and separated according to their relative thermodynamic stabilities using temperature gradient gel electrophoresis (TGGE). Partially denaturing TGGE is carried out with potassium chloride, sodium chloride, or magnesium chloride salts in the gel. The TMs of model hairpins can be tuned by adjusting the concentration of urea in the gel while maintaining the correct order of stabilities for the hairpins. Mixtures of RNAs differing by a single Watson-Crick base pair are resolved according to their relative thermodynamic stabilities, as are mixtures of GC or AU base pair transversions differing in DeltaG degrees37 by only 0.3-0.5 kcal/mol. In addition, a simple combinatorial library with one position of randomization opposite a guanosine is prepared and separated into its four members by parallel and perpendicular TGGE. The order of thermodynamic stabilities for the library determined by TGGE is shown to be the same when assayed by UV-melting experiments. Analysis of the thermodynamics of folding of combinatorial libraries is general and may be applied to a wide variety of complex nucleic acid secondary and tertiary motifs in order to identify the stable and unstable members.
Bevilacqua
J M
JM
Department of Chemistry, The Pennsylvania State University, University Park 16802, USA.
Bevilacqua
P C
PC
D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics; D004587:Electrophoresis, Agar Gel / Q000379:methods; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D012367:RNA, Viral / Q000737:chemistry*; D013696:Temperature; D013698:Templates, Genetic; D013816:Thermodynamics; D014764:Viral Proteins
D009838:Oligodeoxyribonucleotides; D012367:RNA, Viral; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases