Table 4.

Align-then-fold RSP tools based on comparative sequence analysis

Characteristic			Align-then-fold RSP Tool	Description	Input	Output	Applicable Species	Active (T)/Inactive (F)
Conservation	Suboptimal	Local Interaction Length	TurboFold II (part of RNAstructure) (RNA–RNA) [264]	An RNA structural alignment and secondary structure prediction informed by multiple RNA homologues, involving MFE	Homologous RNA sequences	Folding of a collection of RNA homologues via an iterative process instead of solving the joint problem of aligning and folding multiple RNA sequences Estimation of base pairing probabilities for each sequence and alignment posterior probabilities for each pair of sequences	All species	T
	No Suboptimal		RNAalifold (RNA–RNA) [172]	One of the oldest and most widely used tools for consensus structure prediction from RNA alignments, involving MFE	Multiple RNA alignments in CLUSTAL W and FASTA format	Computation of the MFE structure that is simultaneously formed by a set of aligned sequences Improved consensus RSP for RNA alignments Interactive RNA secondary structure plot RNA secondary structure plots with reliability annotation (partition function folding only) Mountain plot formation	Virus, bacteria, human	T
			Pfold [173]	An improved RNA secondary structure prediction software using the SCFG model, instead of using an explicit evolutionary model and a probabilistic model of structures	An alignment of up to 40 sequences and 500 positions in FASTA format with a phylogenetic tree relating the sequences	Calculation of structure posterior probability based on individual probabilities for alignment columns or pairs of columns in the case of a base-pair Estimation of the tree using a maximum likelihood approach in the SCFG model [265] Prediction of structure given as a bracket notation via CYK algorithm [266] Evaluation of the reliability of the prediction for each position A dot plot representing the overview of the prediction	Virus, bacteria, human	T
		Global interaction length	PETfold (RNA–RNA) [174]	A web server for intra- and intermolecular structures of multiple RNA sequences, involving concatenation MFE-based method	One MSA in FASTA format	Integration of both the thermodynamic and evolutionary paradigms into one model to predict:   (a)Intra- and intermolecular RNA structures (b)Pairing reliability of base pairs	Bacteria, virus	T

CYK: Cocke–Younger–Kasami; MFE: minimum free energy; MSA: multiple sequence alignment; RNA: ribonucleic acid; RSP: RNA structure prediction; SCFG: stochastic context-free grammars