Abstract
Despite recent developments in analyzing RNA secondary structures, relatively few RNA structures have been determined. To date, many investigators have relied on the traditional method of using structure-specific RNAse enzymes to probe RNA secondary structures. However, if these data were combined with novel computational approaches, investigators would have an informative and valuable tool for RNA structural analysis. To this end, we created the web server “RNAdigest.” RNAdigest uses mfold RNA structural models in order to predict the results of RNAse digestion experiments. Furthermore, RNAdigest also utilizes both RNA sequence and the experimental digestion patterns to formulate the constraints for predicting secondary structures of the RNA. Thus, RNAdigest allows for the structural interpretation of RNAse digestion experiments. Overall, RNAdigest simplifies RNAse digestion result analyses while allowing for the identification of unique fragments. These unique fragments can then be used for testing predicted mfold structures and for designing structural-specific DNA/RNA probes.
Highlights
Recent advances in experimental and computational approaches for predicting and analyzing RNA secondary structure, along with the use of deep sequencing technology, have contributed to the prediction and establishment of accurate structures for many RNAs [1,2]
The results of RNAse digestion are visualized on sequencing gels, and the presence of unique structural motifs related to specific digestion products can be confirmed with PCR and northern blots using well-designed primers or probes
There is a need for a freely-available, simple and user-friendly software that facilitates the biochemical validation of theoretical structural models and allows the user to incorporate the derived biochemical data into predictive models
Summary
Recent advances in experimental and computational approaches for predicting and analyzing RNA secondary structure, along with the use of deep sequencing technology, have contributed to the prediction and establishment of accurate structures for many RNAs [1,2]. Despite these in silico improvements, deep sequencing has limited availability, and it is still necessary to validate those predicted structures experimentally. RNAdigest is a user-friendly web tool for (1) identifying structural-specific RNAse restriction sites based on the mfold RNA secondary structure program; (2) identifying sequences of unique RNAse digestion products; and most importantly, (3) computing the mRNA secondary structure constraints based on experimentally obtained sequences of the digestion products
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