Using the RNAstructure Software Package to Predict Conserved RNA Structures

The structures of many non-coding RNAs (ncRNA) are conserved by evolution to a greater extent than their sequences. By predicting the conserved structure of two or more homologous sequences, the accuracy of secondary structure prediction can be improved as compared to structure prediction for a single sequence. Here, we provide protocols for the use of four programs in the RNAstructure suite to predict conserved structures: Multilign, TurboFold, Dynalign, and PARTS. TurboFold iteratively aligns multiple homologous sequences and estimates the pairing probabilities for the conserved structure. Dynalign, PARTS, and Multilign are dynamic programming algorithms that simultaneously align sequences and identify the common secondary structure. Dynalign uses a pair of homologs and finds the lowest free energy common structure. PARTS uses a pair of homologs and estimates pairing probabilities from the base pairing probabilities estimated for each sequence. Multilign uses two or more homologs and finds the lowest free energy common structure using multiple pairwise calculations with Dynalign. It scales linearly with the number of sequences. We outline the strengths of each program. These programs can be run through web servers, on the command line, or with graphical user interfaces. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Predicting a structure conserved in three or more sequences with the RNAstructure web server Basic Protocol 2: Predicting a structure conserved in two sequences with the RNAstructure web server Alternative Protocol 1: Predicting a structure conserved in multiple sequences in the RNAstructure graphical user interface Alternative Protocol 2: Predicting a structure conserved in two sequences with Dynalign in the RNAstructure graphical user interface Alternative Protocol 3: Running TurboFold on the command line.