Computational programs for predicting RNA sequences with desired folding properties have been extensively developed and expanded in the past several years. Given a secondary structure, these programs aim to predict sequences that fold into a target minimum free energy secondary structure, while considering various constraints. This procedure is called inverse RNA folding. Inverse RNA folding has been traditionally used to design optimized RNAs with favorable properties, an application that is expected to grow considerably in the future in light of advances in the expanding new fields of synthetic biology and RNA nanostructures. Moreover, it was recently demonstrated that inverse RNA folding can successfully be used as a valuable preprocessing step in computational detection of novel noncoding RNAs. This review describes the most popular freeware programs that have been developed for such purposes, starting from RNAinverse that was devised when formulating the inverse RNA folding problem. The most recently published ones that consider RNA secondary structure as input are antaRNA, RNAiFold and incaRNAfbinv, each having different features that could be beneficial to specific biological problems in practice. The various programs also use distinct approaches, ranging from ant colony optimization to constraint programming, in addition to adaptive walk, simulated annealing and Boltzmann sampling. This review compares between the various programs and provides a simple description of the various possibilities that would benefit practitioners in selecting the most suitable program. It is geared for specific tasks requiring RNA design based on input secondary structure, with an outlook toward the future of RNA design programs.

Riboswitches are metabolite or ion sensing cis-regulatory elements that regulate the expression of the associated genes involved in biosynthesis or transport of the corresponding metabolite. Among the nearly 40 different classes of riboswitches discovered in bacteria so far, only the TPP riboswitch has also been found in algae, plants, and in fungi where their presence has been experimentally validated in a few instances. We analyzed all the available complete fungal and related genomes and identified TPP riboswitch-based regulation systems in 138 fungi and 15 oomycetes. We find that TPP riboswitches are most abundant in Ascomycota and Basidiomycota where they regulate TPP biosynthesis and/or transporter genes. Many of these transporter genes were found to contain conserved domains consistent with nucleoside, urea and amino acid transporter gene families. The genomic location of TPP riboswitches when correlated with the intron structure of the regulated genes enabled prediction of the precise regulation mechanism employed by each riboswitch. Our comprehensive analysis of TPP riboswitches in fungi provides insights about the phylogenomic distribution, regulatory patterns and functioning mechanisms of TPP riboswitches across diverse fungal species and provides a useful resource that will enhance the understanding of RNA-based gene regulation in eukaryotes.

Riboswitches are RNA genetic control elements that were originally discovered in bacteria and provide a unique mechanism of gene regulation. They work without the participation of proteins and are believed to represent ancient regulatory systems in the evolutionary timescale. One of the biggest challenges in riboswitch research is to find additional eukaryotic riboswitches since more than 20 riboswitch classes have been found in prokaryotes but only one class has been found in eukaryotes. Moreover, this single known class of eukaryotic riboswitch, namely the TPP riboswitch class, has been found in bacteria, archaea, fungi and plants but not in animals. The few examples of eukaryotic riboswitches were identified using sequence-based bioinformatics search methods such as a combination of BLAST and pattern matching techniques that incorporate base-pairing considerations. None of these approaches perform energy minimization structure predictions. There is a clear motivation to develop new bioinformatics methods, aside of the ongoing advances in covariance models, that will sample the sequence search space more flexibly using structural guidance while retaining the computational efficiency of sequence-based methods. We present a new energy minimization approach that transforms structure-based search into a sequence-based search, thereby enabling the utilization of well established sequence-based search utilities such as BLAST and FASTA. The transformation to sequence space is obtained by using an extended inverse RNA folding problem solver with sequence and structure constraints, available within RNAfbinv. Examples in applying the new method are presented for the purine and preQ1 riboswitches. The method is described in detail along with its findings in prokaryotes. Potential uses in finding novel eukaryotic riboswitches and optimizing pre-designed synthetic riboswitches based on ligand simulations are discussed. The method components are freely available for use.

In recent years, new methods for computational RNA design have been developed and applied to various problems in synthetic biology and nanotechnology. Lately, there is considerable interest in incorporating essential biological information when solving the inverse RNA folding problem. Correspondingly, aims at including biologically meaningful constraints and is the only program to-date that performs a fragment-based design of RNA sequences. In doing so it allows the design of sequences that do not necessarily exactly fold into the target, as long as the overall coarse-grained tree graph shape is preserved. Augmented by the weighted sampling algorithm of , our web server called implements the method devised in and offers an interactive environment for the inverse folding of RNA using a fragment-based design approach. It takes as input: a target RNA secondary structure; optional sequence and motif constraints; optional target minimum free energy, neutrality and GC content. In addition to the design of synthetic regulatory sequences, it can be used as a pre-processing step for the detection of novel natural occurring RNAs. The two complementary methodologies and are merged together and fully implemented in our web server , available at http://www.cs.bgu.ac.il/incaRNAfbinv.

Searching for RNA sequence-structure patterns is becoming an essential tool for RNA practitioners. Novel discoveries of regulatory non-coding RNAs in targeted organisms and the motivation to find them across a wide range of organisms have prompted the use of computational RNA pattern matching as an enhancement to sequence similarity. State-of-the-art programs differ by the flexibility of patterns allowed as queries and by their simplicity of use. In particular—no existing method is available as a user-friendly web server. A general program that searches for RNA sequence-structure patterns is RNA Structator. However, it is not available as a web server and does not provide the option to allow flexible gap pattern representation with an upper bound of the gap length being specified at any position in the sequence. Here, we introduce RNAPattMatch, a web-based application that is user friendly and makes sequence/structure RNA queries accessible to practitioners of various background and proficiency. It also extends RNA Structator and allows a more flexible variable gaps representation, in addition to analysis of results using energy minimization methods. RNAPattMatch service is available at http://www.cs.bgu.ac.il/rnapattmatch. A standalone version of the search tool is also available to download at the site.