Not very nice logo, but the first author insisted on it. The last author thinks it could be much better. Or the pure text instead.

RNAProbe - a web server for normalization and analysis of RNA structure probing data. You can take a (quick) NAP, while we analyze your data :)

About SHAPE probing

Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) is a technique regarded as a gold standard chemical strategy, widely applied to study RNA secondary structure at a single-nucleotide resolution [1]. SHAPE probing allows for studying the flexibility of RNA backbone, based on the susceptibility of nucleotides to chemical modification. RNA-modifying agents, such as NMIA most commonly used in in vitro experiments or 1M7 applied in probing RNA in vivo in the cellular context [2] [3], are sequence-unbiased reagents. They form an adduct with the sugar 2′-hydroxyl group of flexible or disordered nucleotides and discriminate them from residues that are rigid, e.g., involved in base pairing in double-stranded, helical regions.

Briefly, RNA is first subjected to the modification and then serves as a template for the reverse transcription reaction. The synthesis of the cDNA strands halts at the sites of modification at highly reactive nucleotides. Samples are then submitted to capillary electrophoresis, and resulting chromatograms can subsequently be analyzed in dedicated QuShape software [4]. Raw data coming from the analysis of modified vs. non-modified RNA requires normalization and then can be used for the secondary structure prediction; however, those steps, including data visualization, so far have to be executed manually by using multiple tools available from different sources.

About DMS and CMCT probing

DMS (dimethyl sulfate) and CMCT (N-cyclohexyl-N-(2-morpholinoethyl)carbodiimide methyl-p-toluenesulfonate) are commonly used chemicals that react with the nitrogen base moiety of RNA in a sequence-specific manner. Both compounds modify the Watson-Crick edge of nucleotides that are free from the Watson-Crick base pairing [5]. DMS methylates adenine at the N1 position and cytosine at the N3 position [6], while CMCT is used mostly for modification of uracil in position N3, and can also react with the nitrogen in the N3 position of guanine [7]. These modifications create blocks to reverse transcriptase and they can be detected as stops in the primer extension reaction. Because DMS and CMCT modify only single-stranded RNA, double-stranded regions are inferred by the lack of modification. In addition, DMS modifies guanine at the N7 position of the Hoogsten edge, which does not create reverse transcriptase stops, but can be detected by cleavage of the modified RNA after borohydride reduction and aniline cleavage [5].

Related resources

RNAex [8], Structure Surfer [9], Fold Atlas [10], and RSVdb [11] databases serve as repositories for high-throughput probing data from various types of experiments (see the table below). They allow for browsing and visualizing transcriptome structural data. None of these four databases contain data or secondary structures based on CMCT chemical probing. The key difference compared to RNAProbe is that these databases do not allow the user to analyze their own experimental data on the fly. Instead, they provide results of previous experiments, mostly done in a high-throughput fashion for entire transcriptomes of model organisms. Hence, there is no overlap of RNAProbe with these databases.
Database Species Probing methods Secondary structure
RNAex A. thaliana, S. cerevisiae, H. sapiens,M. musculus PARS, DMS-seq, Structure-seq, icSHAPE, Frag-seq, CIRS-seq restrained MaxExpect, SeqFold, RNAstructure (Fold), RNAfold
Structure Surfer Mammalian transcriptomes PARS, DMS-seq, icSHAPE, ds/ssRNA-Seq SAVoR
Fold Atlas A. thaliana High-depth Structure-seq DMS analysis RNAstructure (Fold)
RSVdb A. thaliana, D. melanogaster, E. coli, H. sapiens, M. musculus, O. sativa, S. cerevisiae,D. rerio DMS-seq, structure-seq, structure-seq2, CIRS-seq, and DMS-MaPseq RNAstructure (Fold)
RNAFramework [12] and Galaxy StructureFold [13] are multifunctional tools, designed for analyzing high-throughput data. RNAFramework requires good bioinformatic skills, which excludes less advanced users. StructureFold has a browser interface and it is possible to harness it to process the QuSHAPE output data, though the user has to modify the format of the input  data (which again can exclude less advanced users). StructureFold output files provide similar datasets as RNAProbe (bar plots, the secondary structure provided in dot-bracket notation, and graphical visualization), but in different file formats. During the revision of the RNAProbe manuscript, RNAex server has been inaccessible due to ongoing maintenance and we could not test it or compare it to our tool. According to the publication, RNAex allows the user to analyze the data obtained from different high-throughput probing methods (Structure-seq, DMS-seq, PARS, Frag-seq, CIRS-seq, icSHAPE) and predicts and visualizes secondary structures with mapped reactivities, but it is limited to a number of species from which the sequencing result can be analyzsed. The main purpose of RNAex is to analyze data from NGS- based probing methods, while RNAProbe was created to facilitate the analysis of low-pass probing experiments analyzed with capillary electrophoresis. When RNAex is available again, we will add the relevant comparison to the table on the RNAProbe website.
Tool: RNA Framework StructureFold RNAProbe
Browser interface NO YES YES
Local installation required YES NO NO
Programming skills required YES NO NO
Tool content Set of programs Set of programs Fully integrated pipeline
Supported methods CIRS-seq, SHAPE-seq, Structure-seq, PARS,SHAPE-MaP, DMS-MaPseq Structure-Seq, Mod-seq, DMS-seq SHAPE, DMS and CMCT probing
Processing of high-throughput sequencing data YES YES NO
Processing of data from single experiments analyzed with capillary electrophoresis NO YES (but requires preprocessing) YES
Secondary structure prediction RNAstructure, ViennaRNA, SeqFold RNAstructure, ViennaRNA RNAstructure (ShapeKnots for SHAPE, Fold for DMS and CMCT), ViennaRNA
Input RNA structure profiling data, reference genome or transcriptome RNA structure profiling data, reference genome or transcriptome Nucleotide reactivity table obtained from QuShape analysis
Output Bar plot with the nucleotide reactivity heatmap, predicted secondary structure in a dot bracket notation and graphical visualization (*.ps or *.svg file) Bar plot with nucleotide reactivity, predicted secondary structure in a dot bracket notation and graphical visualization (*.ps file) Bar plot and table with the nucleotide reactivity heatmap, predicted secondary structure in a dot bracket notation, mapping reactivity to 2D and 3D RNA structure, and graphical visualization (*.png file)