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. 2023 Dec 1;25(1):bbad421. doi: 10.1093/bib/bbad421

Table 2.

Accessibility-based RIP tools based on MFE algorithms

Characteristic Accessibility-based RIP Tool Description Input Output Applicable
Species		 Active (T)/
Inactive (F)
Conservation Suboptimal Prediction Local Interaction Length LncRRIsearch (lncRNA–lncRNA and lncRNA–mRNA) [258]

  • A web server for pairwise alignment and comprehensive prediction of human and mouse lncRNA–lncRNA and lncRNA–mRNA interaction, including tissue-specific or subcellular localised lncRNA interactions

 Longest mRNA and lncRNA transcript sequences excluding excluded transcripts in the pseudoautosomal region on the Y-chromosome

  • Prediction of the RRIs (seed-and-extension approach), accessible energy and hybridization energy

  • Expression analysis for tissue-specific RRIs

  • Prediction of the subcellular localised RRIs

  • RIblast program for comprehensive RIP

 Human,
animal		 T
RIblast (sRNA and lncRNA TINCR) [141]

  • The fastest software for pairwise alignment and large-scale lncRNA datasets by parallelization method

 A query RNA and a target RNA

  • Prediction of the intermolecular base pair only

  • Prediction of the sRNA target

  • Prediction of the lncRNA TINCR target

 All species T
TargetRNA2 (sRNA-mRNA) [142]

  • A tool that identifies targets of srRNAs in bacteria, allows incorporation of RNA-Seq data, and comparative RIP with MSA

 Sequence of an sRNA in FASTA format and the name of a sequenced bacterial replicon

  • Identification of message targets of sRNA regulation, including sRNA conservation regions, structural accessibility of regions of sRNA and mRNA, and hybridization energy between the two RNAs

 Bacteria T
RNAstructure (including ProbKnot, OligoWalk, bipartition, bifold, DuplexFold, Dynalign, Multilign, PARTS, TurboFold, etc.) (RNA–RNA) [259]

  • A software package for RNA secondary structure prediction and analysis, using both MFE and comparative approaches (more accurate than single sequence secondary structure prediction)

 Accept MSA as input, the name of a sequence file (SEQ, FASTA) or structure file (CT, DBN) containing the input sequence

  • Constrained/Restrained structure prediction based on chemical mapping, enzymatic mapping, NMR, and SHAPE data

  • Prediction of the accessible regions in an RNA target to oligonucleotide hybridization

  • Calculation of thermodynamic features of sense–antisense hybridization

  • Summary of tools:

 
  • a)

    ProbKnot: Prediction of base-pairing probabilities, bimolecular structures with and without intramolecular structure

  • b)

    OligoWalk: Equilibrium binding affinity of an oligonucleotide to a structured RNA target

  • c)

    Bipartition: Partition function calculation for two interacting NA sequences without intramolecular pairs

  • d)

    Bifold: Prediction of lowest free energy structure for two interacting sequences with or without intramolecular base pairs (DuplexFold)

  • e)

    TurboFold: Calculation of conserved structures of more than 3 unaligned sequences using iteratively refined partition functions

  • f)

    Dynalign, Multilign: Prediction of secondary structures common to two, unaligned sequences

  • g)

    PARTS: Prediction of the common secondary structure, including base pair probabilities, for two unaligned sequences

 All species T
Conservation Suboptimal Prediction Local Interaction Length OligoWalk (siRNA-mRNA) [137]

  • An online siRNA design tool using hybridization thermodynamics to predict efficient siRNA candidates for an mRNA sequence based on the statistical mechanics of the siRNA-target interaction with 78.6% efficient silencing

 Only RNA oligomer is allowed, and 19 bases are recommended for siRNA design

  • Generation of a siRNA candidate table ranked by the probability of being efficient at knock-down.

  • Prefilter score: The score calculated using Reynold et al. method [260]

  • Generation of a thermodynamic table which includes:

 
  • a)

    Net free energy change

  • b)

    Free energy change of hybridised duplex between oligomer and target

  • c)

    Melting temperature

  • d)

    Free energy cost for opening base pairs in the region of complementarity to the target

  • e)

    Free energy change of the self-structure of unimolecular oligo

  • f)

    Free energy change of oligo-oligo dimer

  • g)

    The number of suboptimal structures of the target used before and after the binding of oligomer

  • h)

    Free energy difference between the 5′ and 3′ end of the antisense strand of siRNA, with windows of 2

 All species T
RNAplex-aA and RNAplex-cA (RNA–RNA) [128]

  • A general RIP tool designed to quickly search possible hybridisation sites for a query RNA in large RNA databases as well as short interactions between two long RNAs

 At least 1 FASTA file containing target and query RNA sequences or 2 CLUSTAL files as input

  • Computation of optimal and suboptimal structure (one structure per line)

  • Conservation profile, consensus structure, and interactions with one, two and three types of base pairs

  • Types of RNAplex:

 
  • a)

    RNAplex-aA (accessibility and MSA as input)

  • b)

    RNAplex-cA (interaction-only and MSA as input)

 Virus, animal T
No Conservation RNAplex-a and RNAplex-c (RNA–RNA) [128]
  • c)

    RNAplex-a (accessibility)

  • d)

    RNAplex-c (interaction-only)
RIsearch2 (RIsearch and GUUGle) (RNA–RNA) [140]

  • The first large-scale RIP tool using a seed-and-extend framework based on suffix arrays with a focus on perfect-complementary seed regions and extensions on both ends, applicable to all kinds of interaction predictions, and can be accessed via the conda package manager

 RNA sequences in FASTA format

  • Quick localization of potential near-complementary interactions between given query and target sequences

  • A modified Smith–Waterman-Gotoh algorithm based on di-nucleotides to approximate nearest-neighbour energy parameters

  • Discovery of RRIs on genome/transcriptome-wide scale

  • Parallel suffix array matching and seed extension

  • Prediction of siRNA off-targets, including:

 
  • a)

    Putative siRNA–RNA interactions

  • b)

    Intersection with transcriptomic data

  • c)

    Partition function

  • d)

    Accessibility of binding sites

  • e)

    Evaluation of siRNA off-target predictions and potential measurements

  • f)

    Relationship between inhibition efficiency and off-targeting potential of siRNAs

  • g)

    Validation of off-targeting potential measures

 Multiple species T
No Conservation Suboptimal Prediction Local Interaction Length IntaRNA 2.0 (ncRNA-ncRNA) [134]

  • An upgraded version of IntaRNA, offers enhanced parameterization, flexible prediction modes and output formats, can be accessed via the conda package manager, integrated into the Galaxy workflow framework or ad hoc usage in the web interface

 At least 1 FASTA file containing target and query ncRNA sequences

  • Visualization of new minimal energy profiles of RRIs

  • Detailed investigation of interaction alternatives and detection of potential interaction site multiplicity

  • Seed stability constraints

  • Dangling end contributions

 Bacteria T
      IntaRNA (ncRNA-ncRNA) [135]

  • A program for fast and accurate RIP by incorporating seed constraints and interaction site accessibility, offers accurate sRNA binding site identification via optimal solution, prediction of optimal and suboptimal hybridisations (similar performance with RNAplex)

 At least 1 set of noncoding RNA sequences in FASTA format with more than 1 but at most 100 sequences, each with a length ranging from 7 to 2000 nt

  • Prediction of the interactions in single organisms

  • Summary of the best 100 predicted interactions

  • Functional enrichment with region plots of top-25 predicted targets (an overview of the regions in the target and query sequences that play predominant roles)

  • RRI output with ASCII chart

  • Types of IntaRNA:

 
  • a)

    IntaRNA- fast, heuristic RIP

  • b)

    IntaRNAhelix- helix-based predictions

  • c)

    IntaRNAexact- exact predictions like RNAup

  • d)

    IntaRNAduplex- hybrid-only optimisation like RNAduplex

  • e)

    IntaRNAsTar- optimised for sRNA-target prediction

  • f)

    IntaRNAseed- identification of seed interactions only

  • g)

    IntaRNAens- ensemble-based prediction and partition function computation

 Bacteria T
  No Suboptimal   InRNAs (RNA–RNA) [139]

  • A fast heuristic method to predict the specific (multiple) binding sites of two interacting RNAs, determine pairwise alignment, and handle complex joint structures

 RNA pairs ranging from 20 to 60 nt

  • Prediction of the competitive RNA–RNA binding sites and RRIs

  • Computation of the MFE joint secondary structure without pseudoknots, crossing interactions, and zigzags

 All species F
      BistaRNA (mRNA of ncRNA) [138]

  • A method for predicting multiple binding sites of target RNAs with reduced computational cost by binding profiles representing scores for hybridised structures

 mRNA sequences of specific ncRNA

  • Prediction binding sites of target RNAs that are expected to interact with regulatory antisense RNAs

  • Prediction of multiple binding sites of target RNAs

  • Prediction of binding profiles that represent scores for hybridised structures

  • Computation of accessible regions of the antisense RNA sequence

 All species T
      RNAup (RNA–RNA) [133]

  • A program that calculates the thermodynamics of RRIs by assessing the probability of a potential unpaired binding site, combining it with interaction energy to obtain the total binding energy, making it ideal for in-depth RIP especially when the interaction partners are known or when a candidate set has already been obtained by faster, less accurate methods

 One (accessibility) or 2 (interaction) RNA sequences in FASTA format with a limit of 5000 nt per sequence

  • Two modes:

 
  • a)

    Accessibility: identification of the region with the highest accessibility and its opening energy

  • b)

    Interaction: Calculation of RRI between 2 RNA sequences, the best free energy of binding, its location, the optimal region of interaction, and its optimal structure (RNAduplex)

 All species T
No Conservation No Suboptimal Global Interaction Length Sfold (siRNA and miRNA) [132, 261]

  • A software program developed to predict RNA secondary structures, assess mRNA and viral RNA target accessibility

 RNA sequences in raw format, in FASTA format, or GenBank format (200 bases for an interactive job and 5000 bases for a batch job)

  • Two application modules:

    • – STarMirDB: a database of precomputed transcriptome scale predictions

    • – STarMir: miRNA binding site predictions for mRNA and target sequences)

  • Outputs:

    • – Prediction of target accessibility and rational design of antisense oligonucleotides or trans-cleaving ribozymes

    • – Prediction of target accessibility and RNA duplex thermodynamics for rational siRNA design

    • – Energetic characteristics of hybridisation between a structured target and a miRNA

    • – Visualisation of comprehensive sequence, thermodynamic, and target structure features, a logistic probability as a measure of confidence for each predicted site, and a publication-quality diagram of the predicted miRNA–target hybrid

 All species T
  Suboptimal Prediction   RNApredator (RNA–RNA) [136]

  • A fast accessibility-based prediction of single sRNA targets via RNAplex with improved prediction specificity via the inclusion of accessibility and a database of over 2155 genomes and plasmids from 1183 bacterial species

 A single small RNA sequence consisting of lower or uppercase letters (A, T, C, G, U), where T is automatically converted into U (with confirmed genome)

  • Computation of the full nonpseudoknot partition function of interacting strands in dilute solution

  • Calculation of the concentrations, MFEs, and base-pairing probabilities of the ordered complexes below a certain complexity

  • Computation of the partition function and base pairing of single strands including a class of pseudoknotted structures

  • Prediction of the suboptimal interactions

  • Design of ordered complexes

  • Computation of putative target via RNAplex

 Bacteria T

A: Adenine; C: cytosine; G: guanine; lncRNA: long noncoding RNA; MFE: minimum free energy; miRNA: microRNA; mRNA: messenger RNA; MSA: multiple sequence alignment; NA: nucleic acid; ncRNA: noncoding RNA; NMR: nuclear magnetic resonance; nt: number of nucleotides; RIP: RNA–RNA interaction prediction; RRI: RNA–RNA interaction; RNA: ribonucleic acid; siRNA: short interfering RNA; srRNA: small regulatory RNA; sRNA: small RNA; T: thymine; TINCR: terminal differentiation-induced noncoding RNA; U: uracil