RiboCAT: a new capillary electrophoresis data analysis tool for nucleic acid probing

Abstract

Chemical and enzymatic probing of RNA secondary structure and RNA/protein interactions provides the basis for understanding the functions of structured RNAs. However, the ability to rapidly perform such experiments using capillary electrophoresis has been hampered by relatively labor-intensive data analysis software. While these computationally robust programs have been shown to calculate residue-specific reactivities to a high degree of accuracy, they often require time-consuming manual intervention and lack the ability to be easily modified by users. To alleviate these issues, RiboCAT (Ribonucleic acid capillaryelectrophoresis analysis tool) was developed as a user-friendly, Microsoft Excel-based tool that reduces the need for manual intervention, thereby significantly shortening the time required for data analysis. Features of this tool include (i) the use of an Excel platform, (ii) a method of intercapillary signal alignment using internal size standards, (iii) a peak-sharpening algorithm to more accurately identify peaks, and (iv) an open architecture allowing for simple user intervention. Furthermore, a complementary tool, RiboDOG (RiboCAT data output generator) was designed to facilitate the comparison of multiple data sets, highlighting potential inconsistencies and inaccuracies that may have occurred during analysis. Using these new tools, the secondary structure of the HIV-1 5 ′ untranslated region (5 ′ UTR) was determined using selective 2 ′ -hydroxyl acylation analyzed by primer extension (SHAPE), matching the results of previous work.

Argonaute-based programmable RNase as a tool for cleavage of highly-structured RNA

“…SHAPE probing experiments of HIV-1 ΔDIS 5′UTR were performed as described previously ( 15 ). Briefly, in vitro transcribed and gel-purified RNA was refolded as described above.…”

“…SHAPE reactivities were determined via primer extension and capillary electrophoresis using a NED™ (Thermo-Fisher)-labeled DNA primer and SuperScriptIII (Invitrogen). SHAPE reactivities were determined using the RiboCAT data analysis tool ( 15 ). Results are the average of four replicates.…”

The evolution of RNA structural probing methods: From gels to next‐generation sequencing

“…Several analytical pipelines have been developed to semi‐automatically convert CE electropherograms into reactivities. These include capillary automated footprinting analysis (CAFA) (Mitra, Shcherbakova, Altman, Brenowitz, & Laederach, ), ShapeFinder (Vasa, Guex, Wilkinson, Weeks, & Giddings, ), high‐throughput robust analysis for capillary electrophoresis (HiTRACE) (Yoon et al, ), Fast Analysis of SHAPE traces (FAST) (Pang, Elazar, Pham, & Glenn, ), SHAPE‐CE (Aviran et al, ), QuShape (Karabiber, McGinnis, Favorov, & Weeks, ), and RiboCAT (Cantara, Hatterschide, Wu, & Musier‐Forsyth, ). All these programs require electropherograms for the chemically modified RNA, the unmodified reference and one or two sequencing reactions to allow mapping of electropherogram peaks to the RNA sequence.…”

Human T-cell leukemia virus type 1 Gag domains have distinct RNA-binding specificities with implications for RNA packaging and dimerization

“…Raw electropherograms were converted to normalized SHAPE reactivity using the recently described RNA capillaryelectrophoresis analysis tool (RiboCAT) (97). Briefly, RiboCAT uses internal size standards to align signals from different sample-containing capillaries as well as a peak-sharpening algorithm to enhance peak identification.…”

“…Briefly, RiboCAT uses internal size standards to align signals from different sample-containing capillaries as well as a peak-sharpening algorithm to enhance peak identification. For SHAPE probing experiments of RNA alone, (Ϫ)-reaction peak areas were scaled to the (ϩ)-reaction based on the average of the lowest 20% of peak areas in each trace prior to normalization as described (97). At least three independent experiments were performed for each of the three primers used.…”