Table 5.

Sankoff-type (sequence-based heuristics) RSP tools based on comparative sequence analysis

Characteristic			Sankoff-type (Sequence-based Heuristics) RSP Tool	Description	Input	Output	Applicable Species	Active (T)/Inactive (F)
Conservation	Suboptimal Prediction	Local Interaction Length	Dynalign (part of RNAstructure) [267]	A web server for predicting common secondary structure in RNA homologues with domain insertions, including structural alignment and offering improved RSP accuracy by combining MFE and comparative RSP	Two RNA sequences (homologues)	Output a sequence alignment and a common structure for the two sequences. Prediction of the conserved pseudoknot-free secondary structure and the structural alignment of the sequences	Bacteria, virus, eukaryote	T
			Multilign [268]	An algorithm to predict secondary structures conserved in multiple RNA sequences, similar to Dynalign but with computational complexity that scales linearly in the number of sequences	At least two RNA sequence alignments	Prediction of the lowest free energy RNA secondary structure common to multiple sequences Improved prediction accuracy by keeping genuine base pairs and excluding competing false base pairs	Bacteria, virus, eukaryote	T
			MXSCARNA (multiplex stem candidate aligner for RNAs) [185]	A multiple alignment tool for RNA sequences using a progressive alignment approach based on the pairwise structural alignment algorithm of SCARNA, consuming less computational time and memory for large-scale analyses with better alignment accuracies	Two RNA sequences and accept MSA as input	Output a sequence alignment from a pair of RNA sequences based on the predicted common secondary structure Output from pairwise alignments to progressive multiple alignments with improved score functions, and simultaneously construct multiple alignments and the associated common secondary structures	Bacteria, virus, eukaryote	T
		Global interaction length	Stemloc [184]	A ML and probabilistic-based program for multiple alignment of RNA using SCFG, including structural alignment	Two RNA sequences (homologues), capable of pairwise alignment of multiple sequences	Output in Stockholm format, including the sequence names, the coordinates of matches, the alignment, the consensus primary sequence, the secondary structure of each sequence, the consensus secondary structure, and the log-odds score of the alignment in bits	Bacteria	T
	No Suboptimal	Local interaction length	CARNA (constraint-based alignment of RNA ensembles) [269]	A tool for multiple alignment of RNA molecules, involving MFE and predicting base pair probability for each RNA sequence with options for handling pseudoknots using RNAfold or without pseudoknots via NUPACK	A set of RNA sequences in FASTA format and one dot plot per sequence in PostScript format	Computation of optimal alignment of the sequences with respect to a sequence and structure similarity-based scoring Generation of conservation dot plots with the most likely base pairs of the consensus Representation diagram of sequence conservation and compatibility of base pair	Bacteria, eukaryote, virus	T
		Global interaction length	Foldalign version 2.5 (ncRNA) [182]	A new multithreaded version of Foldalign for pairwise structural RNA alignment, including structural alignment	Two RNA sequences or entire sequences with lengths up to 10,000 nt and a maximum alignment length of 1000 nt	Scanning for more than the best-scoring RNA structures Capable of discovery of ncRNAs Effective in local structural alignments of sequences with low similarity	Bacteria	T

MFE: minimum free energy; ncRNA: noncoding RNA; nt: number of nucleotides; RNA: ribonucleic acid; RSP: RNA structure prediction; SCFG: stochastic context-free grammars