The abbreviated name, 'mfold web server', describes a number of closely related software applications available on the World Wide Web (WWW) for the prediction of the secondary structure of single stranded nucleic acids. The objective of this web server is to provide easy access to RNA and DNA folding and hybridization software to the scientific community at large. By making use of universally available web GUIs (Graphical User Interfaces), the server circumvents the problem of portability of this software. Detailed output, in the form of structure plots with or without reliability information, single strand frequency plots and 'energy dot plots', are available for the folding of single sequences. A variety of 'bulk' servers give less information, but in a shorter time and for up to hundreds of sequences at once. The portal for the mfold web server is http://www.bioinfo.rpi.edu/applications/mfold. This URL will be referred to as 'MFOLDROOT'.

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This paper presents a new computer method for folding an RNA molecule that finds a conformation of minimum free energy using published values of stacking and destabilizing energies. It is based on a dynamic programming algorithm from applied mathematics, and is much more efficient, faster, and can fold larger molecules than procedures which have appeared up to now in the biological literature. Its power is demonstrated in the folding of a 459 nucleotide immunoglobulin gamma 1 heavy chain messenger RNA fragment. We go beyond the basic method to show how to incorporate additional information into the algorithm. This includes data on chemical reactivity and enzyme susceptibility. We illustrate this with the folding of two large fragments from the 16S ribosomal RNA of Escherichia coli.

A dynamic programming algorithm for prediction of RNA secondary structure has been revised to accommodate folding constraints determined by chemical modification and to include free energy increments for coaxial stacking of helices when they are either adjacent or separated by a single mismatch. Furthermore, free energy parameters are revised to account for recent experimental results for terminal mismatches and hairpin, bulge, internal, and multibranch loops. To demonstrate the applicability of this method, in vivo modification was performed on 5S rRNA in both Escherichia coli and Candida albicans with 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate, dimethyl sulfate, and kethoxal. The percentage of known base pairs in the predicted structure increased from 26.3% to 86.8% for the E. coli sequence by using modification constraints. For C. albicans, the accuracy remained 87.5% both with and without modification data. On average, for these sequences and a set of 14 sequences with known secondary structure and chemical modification data taken from the literature, accuracy improves from 67% to 76%. This enhancement primarily reflects improvement for three sequences that are predicted with <40% accuracy on the basis of energetics alone. For these sequences, inclusion of chemical modification constraints improves the average accuracy from 28% to 78%. For the 11 sequences with <6% pseudoknotted base pairs, structures predicted with constraints from chemical modification contain on average 84% of known canonical base pairs. R ecent discoveries have shown that RNA plays a larger role in biology than previously realized, e.g., in posttranscriptional regulation (1), development (2, 3), immunity (4, 5), and peptide bond formation (6, 7). It is necessary to determine the native structures of RNAs to understand their mechanisms of action, and determining secondary structure is a crucial step in this process.RNA secondary structure can be predicted by free energy minimization with nearest neighbor parameters to evaluate stability (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). Previous studies demonstrated that nuclease cleavage data can be used to refine structure prediction and improve accuracy (8, 11). A predicted secondary structure can guide further experiments or comparative sequence analysis (19) and also aid in the design of RNA molecules (20,21).Chemical modification is a technique that reveals solvent accessible nucleotides (22). The nucleotides accessible to 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-ptoluene sulfonate, dimethyl sulfate, and kethoxal are unpaired, in A-U or G-C pairs at helix ends, in G-U pairs anywhere, or adjacent to G-U pairs. This limited specificity differs from that observed with nucleases, and an algorithm allowing constraints from such chemical modification has not been reported. Chemical modification is used extensively to test hypothesized RNA secondary structures (19,(23)(24)(25)(26)(27)(28). Chemical modification can also be used to deduce possible tertia...