Table 3.

Concatenation-based RIP tools based on MFE algorithms

Characteristic			Concatenation-based RIP Tool	Description	Input	Output	Applicable Species	Active (T)/ Inactive (F)
Conservation	Suboptimal Prediction	Local Interaction Length	Nucleic Acid Package 4.0 (NUPACK 4.0) [148, 149]	• A growing software suite for the analysis and design of one or more species of interacting RNA strands. It enables analysis of nucleic acid sequences over complex and test tube ensembles containing arbitrary numbers of interacting strand species	At least 2 alignments of RNA sequences and allow specifications for the components, conditions of the RNA solution of interest, temperature, number of strand species, maximum complex size, strand sequences and strand concentrations	• Calculation of partition function, equilibrium base-pairing probabilities, MFE energy, proxy structure, suboptimal proxy structures, and Boltzmann sampled structures • Calculation of the partition function and MFE secondary structure for nonpseudoknot complexes of arbitrary numbers of interacting RNA strands • Calculation of the equilibrium concentrations for arbitrary species of complexes in a dilute solution • Calculation of equilibrium base-pairing observables for dilute solutions of interacting strand species via partition function and concentration information • Sequence design for >1 strand intended to adopt a nonpseudoknot target secondary structure at equilibrium	All species	T
No Conservation	No Suboptimal		UNAFold (Unified Nucleic Acid Folding and hybridization package) (DNAmelt & mFold) [146, 152]	• A tool with several closely related software applications available on the Worldwide Web for the prediction of the secondary structure of single-stranded nucleic acids; mFold has been replaced by UNAFold	One or 2 single-stranded RNA sequences in FASTA format with sequence name	• Prediction of RNA secondary structure (excluding pseudoknots) • Simulation of folding, hybridization, and melting pathways for one or two single-stranded NA sequences • Folding (secondary structure) prediction for single-stranded RNA via free energy minimisation, partition function calculations and stochastic sampling • Computation of entire melting profiles (plots), including melting temperatures (UV absorbance at 260 nm, heat capacity change (C(p)), and mole fractions of different molecular species	All species	T
	Suboptimal Prediction	Global Interaction Length	AccessFold (RNA–RNA, miRNA–mRNA, sRNA-mRNA) [262]	• A program for RIP with consideration for competing self-structure and allowing accessibility-based prediction as well as pairwise alignment	Two sequence files with sequence names for the first and second sequence	• Two approaches to evaluate accessibility: • Free energy density minimization • Pseudoenergy minimization • Minimization of the sum of free energy change and a pseudofree energy penalty for bimolecular pairing of nucleotides that are unlikely to be accessible for bimolecular structure • Prediction of binding sites that are split by unimolecular structures • Output is written to a CT file where the sequences are concatenated, with an intermolecular linker between them	All species	T
			PairFold [144]	• The first tool to predict suboptimal secondary structures of two interacting RNA strands and can handle complex joint structures	At least 2 sets of RNA sequences	• Prediction of the MFE pseudoknot-free secondary structure of two or more nucleic acid molecules via an extension of the Zuker and Stiegler algorithm [123] • Prediction of alternative low-energy suboptimal secondary structures for two NA molecules via suboptimal folding algorithm by Wuchty et al. [263] • Prediction of interactions between a probe and target RNA molecule or between pairs of strands in biomolecular nanostructures	All species
No Conservation	Suboptimal Prediction	Global Interaction Length	MultiFold [144]	• The first program to handle multiple RNA strands	At least 2 RNA sequences and accept MSA as input	• Standard thermodynamic parameters of the Turner group prediction of the MFE pseudoknot-free secondary structure of two or more nucleic acid molecules • Prediction of alternative low-energy (suboptimal) secondary structures for two nucleic acid molecules	All species
			RNAsoft (PairFold, CombFold, RNA designer, AveRNA, & HotKnots 2.0) (RNA–RNA) [143]	• A suite of RNA secondary structure prediction and design software tools, applicable for DNA sequences, and handles complex joint structures	Two RNA sequences with a description of a combinatorial set of RNA strands	• Summary of tools: • PairFold: Prediction of the MFE secondary structure formed by two input RNA molecules and interactions between a probe and target RNA molecule or between pairs of strands in biomolecular nanostructures • ComdFold: Prediction of the origin of a strand from a combinatorial set formed from RNA input strands and folding to a secondary structure with the lowest MFE • RNA designer: Designing an RNA sequence that folds to a given input secondary structure • AveRNA: Combination of the RNA secondary structures predicted by different algorithms to increase the overall accuracy • HotKnots 2.0: Prediction of short RNA secondary structures that are expected to form pseudoknots	All species
	No Suboptimal		RNANUE (RNA–RNA) [147]	• A comprehensive and efficient analysis to detect RRIs from DDD (direct-duplex-detection) data	RNA sequencing files in a specific folder structure (the root folders must be specified for both treatment and control groups, and subfolders should represent arbitrary conditions that contain the read files)	• Split reads generation in SAM format • Clusters identification, including the IDs of the clusters, its length, size and genomic coordinates • Detection of RRIs, complementarity scores, and hybridization energies identification • MFE hybrid structure prediction and the probability in the ensemble of all possible interactions via RNAlib	All species	T
			RNAfold (RNA–RNA) [125]	• A web server which predicts secondary structures of single-stranded RNA sequences	RNA or DNA sequence in FASTA format with a limit of 7500 nt for partition function calculations and 10,000 nt for MFE-only predictions	• Interactive RNA secondary structure plot • RNA secondary structure plots with reliability annotation (partition function folding only) • Mountain plot (to predict and plot secondary structures)	All species	T
			RNAcofold (RNA–RNA) [125, 145]	• A program like RNAfold, but allows users to specify two RNA sequences that can form a dimer structure, capable of interaction-only MFE-based method, and can handle complex joint structures	RNA sequences are read from stdin in the usual format	• Calculation of secondary structures of two RNAs with dimerization • Computation of the hybrid structure of two molecules • Computation of MFE structures, partition function (pf) and base pairing probability matrix (using the −p switch) • Computation of equilibrium concentrations for all five monomers and (homo/hetero)-dimer species, given input concentrations for the monomers (since dimer formation is concentration dependent) • Generation of PostScript structure plots and "dot plot" files containing the pair probabilities	All species	T
			RNA–RNA interACTion prediction using Integer Programming (RactIP) (RNA–RNA) [163]	• A fast and accurate ML and probabilistic approach to predict RRI using integer programming, and handling complex joint structures	Two RNA sequences in FASTA format	• Integration of approximate information on an ensemble of equilibrium joint structures into the objective function of integer programming using posterior internal and external base-pairing probabilities • Prediction of RNA joint secondary structures under the general type of interaction including kissing hairpins • Prediction of the maximum expected accuracy (MEA) structure using integer programming (IP) with threshold cut via GNU Linear Programming Kit (GLPK)	All species	T

CT: continuous tone; DNA: deoxyribonucleic acid; GNU: Gnu's Not Unix; MFE: minimum free energy; MSA: multiple sequence alignment; NA: nucleic acid; nt: number of nucleotides; RIP: RNA–RNA interaction prediction; RRI: RNA–RNA interaction; RNA: ribonucleic acid; UV: ultraviolet