QuShape: Rapid, accurate, and best-practices quantification of nucleic acid probing information, resolved by capillary electrophoresis

Abstract

Chemical probing of RNA and DNA structure is a widely used and highly informative approach for examining nucleic acid structure and for evaluating interactions with protein and small-molecule ligands. Use of capillary electrophoresis to analyze chemical probing experiments yields hundreds of nucleotides of information per experiment and can be performed on automated instruments. Extraction of the information from capillary electrophoresis electropherograms is a computationally intensive multistep analytical process, and no current software provides rapid, automated, and accurate data analysis. To overcome this bottleneck, we developed a platform-independent, user-friendly software package, QuShape, that yields quantitatively accurate nucleotide reactivity information with minimal user supervision. QuShape incorporates newly developed algorithms for signal decay correction, alignment of time-varying signals within and across capillaries and relative to the RNA nucleotide sequence, and signal scaling across channels or experiments. An analysis-by-reference option enables multiple, related experiments to be fully analyzed in minutes. We illustrate the usefulness and robustness of QuShape by analysis of RNA SHAPE (selective 29-hydroxyl acylation analyzed by primer extension) experiments.

High-throughput mutate-map-rescue evaluates SHAPE-directed RNA structure and uncovers excited states

“…We obtained results that were the same, within estimated experimental error, upon varying folding solution conditions (Wilkinson et al 2006;Deigan et al 2009;Kladwang et al 2011b), acylating reagents (1M7 and NMIA) (Merino et al 2005;Mortimer and Weeks 2007), reverse transcription conditions (Mills and Kramer 1979), quantitation software (Yoon et al 2011;Karabiber et al 2013), normalization schemes (Deigan et al 2009), and RNAstructure modeling software versions ( Fig. 1D; Supplemental Figs.…”

RNAIII of the Staphylococcus aureus agr system activates global regulator MgrA by stabilizing mRNA

“…The in vitro synthesized 303-nt mgrA UTR with adaptor sequences at both 5′ and 3′ ends was used as the template for the reaction and primer extension. The raw data were analyzed by QuShape software (28)(29)(30). This experimental design resulted in highly reproducible SHAPE reactivity data of the mgrA UTR (Fig.…”

The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA

“…These primers allowed analysis of the RNA structure from approximately nucleotide 560 to 100. The primer extension products were loaded onto an Applied Biosystems 3130xl genetic analyzer and the electropherograms were analyzed with the QuShape software [67]. The hSHAPE reactivities obtained with QuShape from three independent experiments were highly reproducible.…”