Table 9.

RSP and RIP tools involving pseudoknots

Strategy	RSP and RSP tools involving pseudoknots	Description	Input	Output	Applicable Species	Active (T)/Inactive (F)
Thermodynamic-based approach	PknotsRG [275]	A web tool for folding and single sequence RNA secondary structure prediction including pseudoknots	A file containing one single RNA sequence in FASTA format	Folding and RSP, including pseudoknots, near-optimal structures and sliding windows Enumeration of suboptimal folding Visualization of RNA structure Alignment of RNA secondary structure Analysis of RNA secondary structure	Human, virus, bacteria	T
Thermodynamic-based approach	Kinefold (RNA–RNA) [223]	A web interface for RNA/DNA folding path and structure prediction including pseudoknots and knots	A string of unmodified RNA/DNA bases (limit of 400 bases for renaturation fold and cotranscriptional fold)	Folding kinetics of RNA/DNA sequences including pseudoknots and entangled helices Generation of a series of low free energy structures Generation of an online animated folding path Generation of a programmable trajectory plot focusing on a few helices of interest to each user	Virus, eukaryote	T
Thermodynamic-based approach	RNAMotif [276]	An RNA secondary structure definition and search algorithm including single strands, duplexes (antiparallel and parallel), pseudoknots, triplexes, and quadruplexes	A formal description of the permissible forms of the structure and the sequences contained within it	Description of RNA structural element, followed by the results of search in sequence databases, including the complete prokaryotic and eukaryotic genomes	Bacteria, virus	T
Thermodynamic-based approach	RCPred (RNA–RNA) [277]	A tool for secondary structure prediction of RNA complexes	Multiple RNA secondary structures in the complex with possible interactions in each RNA pairs	Prediction of internal and external pseudoknots, crossing interactions, and zigzags Generation of several suboptimal secondary structures	Bacteria, virus	T
Thermodynamic-based approach	Hyperfold (RNA–RNA) [278]	A web server for predicting NA complexes by interacting RNA strands with nonnested base pairings needed in silico secondary structure prediction	RNA and DNA strand sequences (including temperature and concentration)	Prediction of RNA multistrand structures, including RNA assemblies Prediction of structural information such as complex concentrations and base pairing Prediction of folding properties of RNA switches, RNA–DNA hybrid duplexes and RNA nanostructures resembling cubes and hexagons	Human	T
Thermodynamic-based approach	VfoldCPX (RNA–RNA) [225]	A web server for predicting RNA–RNA complex structure and stability	Two RNA sequences including temperature (recommendation: 300 nt for RNA secondary structures without crossing base pairs, ≤150 nt for structures with H-type pseudoknots, and ≤ 120 nt for RNA secondary structures with pseudoknots and hairpin-hairpin kissed structures)	Prediction of 2D RNA–RNA complex structures with at most one intermolecular crossing base pairing helix Prediction of RNA folding thermodynamics Prediction of RNA structure stability Prediction of kissing interactions in miRNA–target complex and assessment of miRNA activity	Eukaryote	T
Thermodynamic-based approach (statistical mechanics)	Vfold (ncRNA-RNA) [279]	A web server to predict RNA 2D, 3D structures and folding thermodynamics	RNA sequence in plain text form	Prediction of 2D structure (base pairs), via generation of RNA ensemble structures, including loop structure with different intraloop mismatches Prediction of 3D structure via motif scaffold assembly using structure templates from known PDB structures and refinement of structures through all-atom energy minimization Prediction of folding thermodynamics (heat capacity melting curve) and evaluation of free energies via experimental parameters for base stacks and loop entropy parameters	Human, virus	T
Thermodynamic-based approach (DDP heuristic algorithm)	HotKnots (RNA–RNA) [216]	A heuristic prediction of RNA secondary structures with or without pseudoknots	RNA sequences or sequence fragments	Identification of the lowest free energy structures at tree nodes via a standard free energy model Determination of tree pruning to explore alternatives from the most promising partial structures	Virus	T
Thermodynamic-based approach (DDP algorithm)	Pknots (RNA–RNA) [215]	An experimental code demonstrating a dynamic programming algorithm for RNA pseudoknot prediction	A single RNA sequence	Prediction of RNA structure with pseudoknots Prediction of the optimal minimum energy structure for a single RNA sequence Folding of optimal pseudoknotted RNAs ranging from 100 to 200 nt	Bacteria, virus	T
Thermodynamic-based approach (heuristic algorithm)	FlexStem (RNA–RNA) [222]	An algorithm improving predictions of RNA secondary structures with pseudoknots by reducing the search space	A ≥ 2 bp RNA secondary structure with a helical region or stem defined as an anti-parallel complementary strand	Simulation of the RNA folding process by successive addition of maximal stems Prediction of RNA structure with pseudoknots	Virus	T
Thermodynamic-based approach (empirical scoring function)	NanoFolder (RNA–RNA) [224]	A method for the prediction of the base pairing of potentially pseudoknotted multistrand RNA nanostructures	A set of RNA sequences combined with a descriptor for the desired target secondary structure	Prediction of the base pairing of potentially pseudoknotted multistrand RNA nanostructures Prediction of RNA complexes with nonnested base pairings; better performance than NUPACK, RNAcofold and PairFold Design of RNA sequence	Bacteria, human	T
Thermodynamic- or comparative-based approach (heuristic algorithm)	Iterated loop matching algorithm (RNA–RNA) [220]	An iterated loop matching approach to predict RNA secondary structures with pseudoknots	RNA homologous sequences	Identification of base-pairs for short sequences Prediction of pseudoknots with high accuracy on individual sequences Higher sensitivity and specificity than the maximum weighted matching method [219]	Eukaryote	T
Thermodynamic- or comparative-based approach	ProbKnot (part of RNAstructure) (RNA–RNA) [280]	A fast prediction of RNA secondary structure including pseudoknots	A sequence file of DNA or RNA	Prediction of the presence of pseudoknots in its folded configuration Visualization of pseudoknot in a circular structure Better performance than ILM, pknotsRG and HotKnots	Human, virus	T
Comparative-based approach	IPknot (RNA–RNA) [226]	A fast and accurate prediction of RNA secondary structures with pseudoknots using integer programming	A single sequence of RNA or MSA	Prediction of the MEA structure using IP with threshold cut and the consensus secondary structure with pseudoknots given an MSA input Decomposition of a pseudoknotted structure into a set of pseudoknot-free substructures Prediction of a base-pairing probability distribution that considers pseudoknots via a heuristic algorithm for refinement	Virus, eukaryote	T

2D: two-dimensional; 3D three-dimensional; bp: base pair; DNA: deoxyribonucleic acid; IP: integer programming; ILM: iterated loop matching; MEA: maximum expected accuracy; miRNA: microRNA; MSA: multiple sequence alignment; NA: nucleic acid; ncRNA: noncoding RNA; nt: number of nucleotides; RNA: ribonucleic acid