Thermodynamic Analysis of an RNA Combinatorial Library Contained in a Short Hairpin

Abstract

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.

Molecular crowders and cosolutes promote folding cooperativity of RNA under physiological ionic conditions

“…1A). The folding behavior of MT tRNA Phe was examined through perpendicular temperature-gradient gel electrophoresis (TGGE) (Bevilacqua and Bevilacqua 1998;Chadalavada and Bevilacqua 2009) displayed in Figure 5. The MT tRNA Phe migrates slower than WT at low temperatures, has a single lower-melting unfolding transition associated with loss of secondary structure, and then migrates identically to WT at higher temperatures.…”

“…Electrophoresis was performed in 13% polyacrylamide, 1× THEN 10 M 0.9 (pH 7.5) (33 mM Tris, 66 M HEPES, 0.1 mM EDTA, 10 mM NaCl, 0.9 mM MgCl 2 ), and a semi-denaturing background of 4 M urea in order to facilitate melting in the TGGE temperature range (Bevilacqua and Bevilacqua 1998;Chadalavada and Bevilacqua 2009). Briefly, the experiment was conducted in perpendicular format in which samples were layered across a single wide well to which a temperature gradient of ∼10°C-60°C was applied.…”

A simple ligation-based method to increase the information density in sequencing reactions used to deconvolute nucleic acid selections

“…1; Bevilacqua and Bevilacqua 1998;Childs-Disney et al 2007). The library was amplified by RT-PCR with 59-biotinylated primers designed to incorporate BamHI restriction sites flanking both sides of the random region (Fig.…”

“…SELEX experiments have been used to study the origins of life (Szostak et al 2001), as analytical tools (Seetharaman et al 2001;Hartig et al 2002), in the development of therapeutics (Gold 2002), and in reprogramming the genetic code (Bessho et al 2002). Single rounds of nucleic acid selection have also been used to identify members of RNA structure libraries that have interesting thermodynamic properties from temperature gradient gel electrophoresis experiments (Bevilacqua and Bevilacqua 1998;Shu and Bevilacqua 1999;Nakano et al 2002;Proctor et al 2002). In each type of selection experiment, an ensemble of active sequences and/or structures is isolated.…”

Phylogenetic conservation of RNA secondary and tertiary structure in the trpEDCFBA operon leader transcript in Bacillus

“…Glycerol was added to 10% final concentration, and the samples were fractionated on a 6% (29:1 acrylamide/ bis)/0.5X TBE native gel at 330 V for 4-5 h. The temperature of the apparatus was controlled by an external bath that flowed into a heat exchanger located behind the gel plates. The external bath temperature was 12°C, and the actual gel temperature was measured at 16°C by insertion of a thermocouple probe into the gel (Bevilacqua and Bevilacqua 1998). Gels were dried, visualized using a PhosphorImager (Molecular Dynamics), and quantified using ImageQuant software (Molecular Dynamics).…”