RNA cis-regulators are important for Streptococcus pneumoniae in vivo success.

Bacteria have evolved complex transcriptional regulatory networks, as well as many diverse regulatory strategies at the RNA level, to enable more efficient use of metabolic resources and a rapid response to changing conditions. However, most RNA-based regulatory mechanisms are not well conserved across different bacterial species despite controlling genes important for virulence or essential biosynthetic processes. Here, we characterize the activity of, and assess the fitness benefit conferred by, twelve cis-acting regulatory RNAs (including several riboswitches and a T-box), in the opportunistic pathogen Streptococcus pneumoniae TIGR4. By evaluating native locus mutants of each regulator that result in constitutively active or repressed expression, we establish that growth defects in planktonic culture are associated with constitutive repression of gene expression, while constitutive activation of gene expression is rarely deleterious. In contrast, in mouse nasal carriage and pneumonia models, strains with either constitutively active and repressed gene expression are significantly less fit than matched control strains. Furthermore, two RNA-regulated pathways, FMN synthesis/transport and pyrimidine synthesis/transport display exceptional sensitivity to mis-regulation or constitutive gene repression in both planktonic culture and in vivo environments. Thus, despite lack of obvious phenotypes associated with constitutive gene expression in vitro, the fitness benefit conferred on bacteria via fine-tuned metabolic regulation through cis-acting regulatory RNAs is substantial in vivo, and therefore easily sufficient to drive the evolution and maintenance of diverse RNA regulatory mechanisms.

A computational approach for the identification of distant homologs of bacterial riboswitches based on inverse RNA folding.

Riboswitches are conserved structural ribonucleic acid (RNA) sensors that are mainly found to regulate a large number of genes/operons in bacteria. Presently, >50 bacterial riboswitch classes have been discovered, but only the thiamine pyrophosphate riboswitch class is detected in a few eukaryotes like fungi, plants and algae. One of the most important challenges in riboswitch research is to discover existing riboswitch classes in eukaryotes and to understand the evolution of bacterial riboswitches. However, traditional search methods for riboswitch detection have failed to detect eukaryotic riboswitches besides just one class and any distant structural homologs of riboswitches. We developed a novel approach based on inverse RNA folding that attempts to find sequences that match the shape of the target structure with minimal sequence conservation based on key nucleotides that interact directly with the ligand. Then, to support our matched candidates, we expanded the results into a covariance model representing similar sequences preserving the structure. Our method transforms a structure-based search into a sequence-based search that considers the conservation of secondary structure shape and ligand-binding residues. This method enables us to identify a potential structural candidate in fungi that could be the distant homolog of bacterial purine riboswitches. Further, phylogenomic analysis and evolutionary distribution of this structural candidate indicate that the most likely point of origin of this structural candidate in these organisms is associated with the loss of traditional purine riboswitches. The computational approach could be applicable to other domains and problems in RNA research.

Revisiting the Relationships Between Genomic G + C Content, RNA Secondary Structures, and Optimal Growth Temperature.

Over twenty years ago Galtier and Lobry published a manuscript entitled "Relationships between Genomic G + C Content, RNA Secondary Structure, and Optimal Growth Temperature" in the Journal of Molecular Evolution that showcased the lack of a relationship between genomic G + C content and optimal growth temperature (OGT) in a set of about 200 prokaryotes. Galtier and Lobry also assessed the relationship between RNA secondary structures (rRNA stems, tRNAs) and OGT, and in this case a clear relationship emerged. Increasing structured RNA G + C content (particularly in regions that are double-stranded) correlates with increased OGT. Both of these fundamental relationships have withstood test of many additional sequences and spawned a variety of different applications that include prediction of OGT from rRNA sequence and computational ncRNA identification approaches. In this work, I present the motivation behind Galtier and Lobry's original paper and the larger questions addressed by the work, how these questions have evolved over the last two decades, and the impact of Galtier and Lobry's manuscript in fields beyond these questions.

Siblings or doppelgängers? Deciphering the evolution of structured cis-regulatory RNAs beyond homology.

Structured cis-regulatory RNAs have evolved across all domains of life, highlighting the utility and plasticity of RNA as a regulatory molecule. Homologous RNA sequences and structures often have similar functions, but homology may also be deceiving. The challenges that derive from trying to assign function to structure and vice versa are not trivial. Bacterial riboswitches, viral and eukaryotic IRESes, CITEs, and 3' UTR elements employ an array of mechanisms to exert their effects. Bioinformatic searches coupled with biochemical and functional validation have elucidated some shared and many unique ways cis-regulators are employed in mRNA transcripts. As cis-regulatory RNAs are resolved in greater detail, it is increasingly apparent that shared homology can mask the full spectrum of mRNA cis-regulator functional diversity. Furthermore, similar functions may be obscured by lack of obvious sequence similarity. Thus looking beyond homology is crucial for furthering our understanding of RNA-based regulation.

Comparative Metatranscriptomics of Periodontitis Supports a Common Polymicrobial Shift in Metabolic Function and Identifies Novel Putative Disease-Associated ncRNAs.

Periodontitis is an inflammatory disease that deteriorates bone supporting teeth afflicting ∼743 million people worldwide. Bacterial communities associated with disease have been classified into red, orange, purple, blue, green, and yellow complexes based on their roles in the periodontal pocket. Previous metagenomic and metatranscriptomics analyses suggest a common shift in metabolic signatures in disease vs. healthy communities with up-regulated processes including pyruvate fermentation, histidine degradation, amino acid metabolism, TonB-dependent receptors. In this work, we examine existing metatranscriptome datasets to identify the commonly differentially expressed transcripts and potential underlying RNA regulatory mechanisms behind the metabolic shifts. Raw RNA-seq reads from three studies (including 49 healthy and 48 periodontitis samples) were assembled into transcripts de novo. Analyses revealed 859 differentially expressed (DE) transcripts, 675 more- and 174 less-expressed. Only ∼20% of the DE transcripts originate from the pathogenic red/orange complexes, and ∼50% originate from organisms unaffiliated with a complex. Comparison of expression profiles revealed variations among disease samples; while specific metabolic processes are commonly up-regulated, the underlying organisms are diverse both within and across disease associated communities. Surveying DE transcripts for known ncRNAs from the Rfam database identified a large number of tRNAs and tmRNAs as well as riboswitches (FMN, glycine, lysine, and SAM) in more prevalent transcripts and the cobalamin riboswitch in both more and less prevalent transcripts. In silico discovery identified many putative ncRNAs in DE transcripts. We report 15 such putative ncRNAs having promising covariation in the predicted secondary structure and interesting genomic context. Seven of these are antisense of ribosomal proteins that are novel and may involve maintaining ribosomal protein stoichiometry during the disease associated metabolic shift. Our findings describe the role of organisms previously unaffiliated with disease and identify the commonality in progression of disease across three metatranscriptomic studies. We find that although the communities are diverse between individuals, the switch in metabolic signatures characteristic of disease is typically achieved through the contributions of several community members. Furthermore, we identify many ncRNAs (both known and putative) which may facilitate the metabolic shifts associated with periodontitis.

Debating tRNA Origins.

The tRNA is a critical component in all modern translation systems as well as an important intermediate in models of early protein coding systems. In the following works, proponents for each of the major hypotheses for tRNA origin and evolution engage in discussion of the merits for each model.

Regulatory context drives conservation of glycine riboswitch aptamers.

In comparison to protein coding sequences, the impact of mutation and natural selection on the sequence and function of non-coding (ncRNA) genes is not well understood. Many ncRNA genes are narrowly distributed to only a few organisms, and appear to be rapidly evolving. Compared to protein coding sequences, there are many challenges associated with assessment of ncRNAs that are not well addressed by conventional phylogenetic approaches, including: short sequence length, lack of primary sequence conservation, and the importance of secondary structure for biological function. Riboswitches are structured ncRNAs that directly interact with small molecules to regulate gene expression in bacteria. They typically consist of a ligand-binding domain (aptamer) whose folding changes drive changes in gene expression. The glycine riboswitch is among the most well-studied due to the widespread occurrence of a tandem aptamer arrangement (tandem), wherein two homologous aptamers interact with glycine and each other to regulate gene expression. However, a significant proportion of glycine riboswitches are comprised of single aptamers (singleton). Here we use graph clustering to circumvent the limitations of traditional phylogenetic analysis when studying the relationship between the tandem and singleton glycine aptamers. Graph clustering enables a broader range of pairwise comparison measures to be used to assess aptamer similarity. Using this approach, we show that one aptamer of the tandem glycine riboswitch pair is typically much more highly conserved, and that which aptamer is conserved depends on the regulated gene. Furthermore, our analysis also reveals that singleton aptamers are more similar to either the first or second tandem aptamer, again based on the regulated gene. Taken together, our findings suggest that tandem glycine riboswitches degrade into functional singletons, with the regulated gene(s) dictating which glycine-binding aptamer is conserved.

The Transcriptional landscape of Streptococcus pneumoniae TIGR4 reveals a complex operon architecture and abundant riboregulation critical for growth and virulence.

Efficient and highly organized regulation of transcription is fundamental to an organism's ability to survive, proliferate, and quickly respond to its environment. Therefore, precise mapping of transcriptional units and understanding their regulation is crucial to determining how pathogenic bacteria cause disease and how they may be inhibited. In this study, we map the transcriptional landscape of the bacterial pathogen Streptococcus pneumoniae TIGR4 by applying a combination of high-throughput RNA-sequencing techniques. We successfully map 1864 high confidence transcription termination sites (TTSs), 790 high confidence transcription start sites (TSSs) (742 primary, and 48 secondary), and 1360 low confidence TSSs (74 secondary and 1286 primary) to yield a total of 2150 TSSs. Furthermore, our study reveals a complex transcriptome wherein environment-respondent alternate transcriptional units are observed within operons stemming from internal TSSs and TTSs. Additionally, we identify many putative cis-regulatory RNA elements and riboswitches within 5'-untranslated regions (5'-UTR). By integrating TSSs and TTSs with independently collected RNA-Seq datasets from a variety of conditions, we establish the response of these regulators to changes in growth conditions and validate several of them. Furthermore, to demonstrate the importance of ribo-regulation by 5'-UTR elements for in vivo virulence, we show that the pyrR regulatory element is essential for survival, successful colonization and infection in mice suggesting that such RNA elements are potential drug targets. Importantly, we show that our approach of combining high-throughput sequencing with in vivo experiments can reconstruct a global understanding of regulation, but also pave the way for discovery of compounds that target (ribo-)regulators to mitigate virulence and antibiotic resistance.

Fitness advantages conferred by the L20-interacting RNA cis-regulator of ribosomal protein synthesis in Bacillus subtilis.

In many bacteria, ribosomal proteins autogenously repress their own expression by interacting with RNA structures typically located in the 5'-UTRs of their mRNA transcripts. This regulation is necessary to maintain a balance between ribosomal proteins and rRNA to ensure proper ribosome production. Despite advances in noncoding RNA discovery and validation of RNA-protein regulatory interactions, the selective pressures that govern the formation and maintenance of such RNA cis-regulators in the context of an organism remain largely undetermined. To examine the impact disruptions to this regulation have on bacterial fitness, we introduced point mutations that abolish ribosomal protein binding and regulation into the RNA structure that controls expression of ribosomal proteins L20 and L35 within the Bacillus subtilis genome. Our studies indicate that removing this regulation results in reduced log phase growth, improper rRNA maturation, and the accumulation of a kinetically trapped or misassembled ribosomal particle at low temperatures, suggesting defects in ribosome synthesis. Such work emphasizes the important role regulatory RNAs play in the stoichiometric production of ribosomal components for proper ribosome composition and overall organism viability and reinforces the potential of targeting ribosomal protein production and ribosome assembly with novel antimicrobials.

rRNA Mimicry in RNA Regulation of Gene Expression.

The rRNA is the largest and most abundant RNA in bacterial and archaeal cells. It is also one of the best-characterized RNAs in terms of its structural motifs and sequence variation. Production of ribosome components including >50 ribosomal proteins (r-proteins) consumes significant cellular resources. Thus, RNA cis-regulatory structures that interact with r-proteins to repress further r-protein synthesis play an important role in maintaining appropriate stoichiometry between r-proteins and rRNA. Classically, such mRNA structures were thought to directly mimic the rRNA. However, more than 30 years of research has demonstrated that a variety of different recognition and regulatory paradigms are present. This review will demonstrate how structural mimicry between the rRNA and mRNA cis-regulatory structures may take many different forms. The collection of mRNA structures that interact with r-proteins to regulate r-protein operons are best characterized in Escherichia coli, but are increasingly found within species from nearly all phyla of bacteria and several archaea. Furthermore, they represent a unique opportunity to assess the plasticity of RNA structure in the context of RNA-protein interactions. The binding determinants imposed by r-proteins to allow regulation can be fulfilled in many ways. Some r-protein-interacting mRNAs are immediately obvious as rRNA mimics from primary sequence similarity, others are identifiable only after secondary or tertiary structure determination, and some show no obvious similarity. In addition, across different bacterial species a host of different mechanisms of action have been characterized, showing that there is no simple one-size-fits-all solution.

In Vivo Behavior of the Tandem Glycine Riboswitch in Bacillus subtilis.

In many bacterial species, the glycine riboswitch is composed of two homologous ligand-binding domains (aptamers) that each bind glycine and act together to regulate the expression of glycine metabolic and transport genes. While the structure and molecular dynamics of the tandem glycine riboswitch have been the subject of numerous in vitro studies, the in vivo behavior of the riboswitch remains largely uncharacterized. To examine the proposed models of tandem glycine riboswitch function in a biologically relevant context, we characterized the regulatory activity of mutations to the riboswitch structure in Bacillus subtilis using ß-galactosidase assays. To assess the impact disruptions to riboswitch function have on cell fitness, we introduced these mutations into the native locus of the tandem glycine riboswitch within the B. subtilis genome. Our results indicate that glycine does not need to bind both aptamers for regulation in vivo and mutations perturbing riboswitch tertiary structure have the most severe effect on riboswitch function and gene expression. We also find that in B. subtilis, the glycine riboswitch-regulated gcvT operon is important for glycine detoxification.IMPORTANCE The glycine riboswitch is a unique cis-acting mRNA element that contains two tandem homologous glycine-binding domains that act on a single expression platform to regulate gene expression in response to glycine. While many in vitro experiments have characterized the tandem architecture of the glycine riboswitch, little work has investigated the behavior of this riboswitch in vivo In this study, we analyzed the proposed models of tandem glycine riboswitch regulation in the context of its native locus within the Bacillus subtilis genome and examined how disruptions to glycine riboswitch function impact organismal fitness. Our work offers new insights into riboswitch function in vivo and reinforces the potential of riboswitches as novel antimicrobial targets.

Recognizing RNA structural motifs in HT-SELEX data for ribosomal protein S15.

BACKGROUND: Proteins recognize many different aspects of RNA ranging from single stranded regions to discrete secondary or tertiary structures. High-throughput sequencing (HTS) of in vitro selected populations offers a large scale method to study RNA-proteins interactions. However, most existing analysis methods require that the binding motifs are enriched in the population relative to earlier rounds, and that motifs are found in a loop or single stranded region of the potential RNA secondary structure. Such methods do not generalize to all RNA-protein interaction as some RNA binding proteins specifically recognize more complex structures such as double stranded RNA. RESULTS: In this study, we use HT-SELEX derived populations to study the landscape of RNAs that interact with Geobacillus kaustophilus ribosomal protein S15. Our data show high sequence and structure diversity and proved intractable to existing methods. Conventional programs identified some sequence motifs, but these are found in less than 5-10% of the total sequence pool. Therefore, we developed a novel framework to analyze HT-SELEX data. Our process accounts for both sequence and structure components by abstracting the overall secondary structure into smaller substructures composed of a single base-pair stack, which allows us to leverage existing approaches already used in k-mer analysis to identify enriched motifs. By focusing on secondary structure motifs composed of specific two base-pair stacks, we identified significantly enriched or depleted structure motifs relative to earlier rounds. CONCLUSIONS: Discrete substructures are likely to be important to RNA-protein interactions, but they are difficult to elucidate. Substructures can help make highly diverse sequence data more tractable. The structure motifs provide limited accuracy in predicting enrichment suggesting that G. kaustophilus S15 can either recognize many different secondary structure motifs or some aspects of the interaction are not captured by the analysis. This highlights the importance of considering secondary and tertiary structure elements and their role in RNA-protein interactions.

The role of mRNA structure in bacterial translational regulation.

The characteristics of bacterial messenger RNAs (mRNAs) that influence translation efficiency provide many convenient handles for regulation of gene expression, especially when coupled with the processes of transcription termination and mRNA degradation. An mRNA's structure, especially near the site of initiation, has profound consequences for how readily it is translated. This property allows bacterial gene expression to be altered by changes to mRNA structure induced by temperature, or interactions with a wide variety of cellular components including small molecules, other RNAs (such as sRNAs and tRNAs), and RNA-binding proteins. This review discusses the links between mRNA structure and translation efficiency, and how mRNA structure is manipulated by conditions and signals within the cell to regulate gene expression. The range of RNA regulators discussed follows a continuum from very complex tertiary structures such as riboswitch aptamers and ribosomal protein-binding sites to thermosensors and mRNA:sRNA interactions that involve only base-pairing interactions. Furthermore, the high degrees of diversity observed for both mRNA structures and the mechanisms by which inhibition of translation occur have significant consequences for understanding the evolution of bacterial translational regulation. WIREs RNA 2017, 8:e1370. doi: 10.1002/wrna.1370 For further resources related to this article, please visit the WIREs website.

RNA regulators responding to ribosomal protein S15 are frequent in sequence space.

There are several natural examples of distinct RNA structures that interact with the same ligand to regulate the expression of homologous genes in different organisms. One essential question regarding this phenomenon is whether such RNA regulators are the result of convergent or divergent evolution. Are the RNAs derived from some common ancestor and diverged to the point where we cannot identify the similarity, or have multiple solutions to the same biological problem arisen independently? A key variable in assessing these alternatives is how frequently such regulators arise within sequence space. Ribosomal protein S15 is autogenously regulated via an RNA regulator in many bacterial species; four apparently distinct regulators have been functionally validated in different bacterial phyla. Here, we explore how frequently such regulators arise within a partially randomized sequence population. We find many RNAs that interact specifically with ribosomal protein S15 from Geobacillus kaustophilus with biologically relevant dissociation constants. Furthermore, of the six sequences we characterize, four show regulatory activity in an Escherichia coli reporter assay. Subsequent footprinting and mutagenesis analysis indicates that protein binding proximal to regulatory features such as the Shine-Dalgarno sequence is sufficient to enable regulation, suggesting that regulation in response to S15 is relatively easily acquired.

Co-evolution of Bacterial Ribosomal Protein S15 with Diverse mRNA Regulatory Structures.

RNA-protein interactions are critical in many biological processes, yet how such interactions affect the evolution of both partners is still unknown. RNA and protein structures are impacted very differently by mechanisms of genomic change. While most protein families are identifiable at the nucleotide level across large phylogenetic distances, RNA families display far less nucleotide similarity and are often only shared by closely related bacterial species. Ribosomal protein S15 has two RNA binding functions. First, it is a ribosomal protein responsible for organizing the rRNA during ribosome assembly. Second, in many bacterial species S15 also interacts with a structured portion of its own transcript to negatively regulate gene expression. While the first interaction is conserved in most bacteria, the second is not. Four distinct mRNA structures interact with S15 to enable regulation, each of which appears to be independently derived in different groups of bacteria. With the goal of understanding how protein-binding specificity may influence the evolution of such RNA regulatory structures, we examine whether examples of these mRNA structures are able to interact with, and regulate in response to, S15 homologs from organisms containing distinct mRNA structures. We find that despite their shared RNA binding function in the rRNA, S15 homologs have distinct RNA recognition profiles. We present a model to explain the specificity patterns observed, and support this model by with further mutagenesis. After analyzing the patterns of conservation for the S15 protein coding sequences, we also identified amino acid changes that alter the binding specificity of an S15 homolog. In this work we demonstrate that homologous RNA-binding proteins have different specificity profiles, and minor changes to amino acid sequences, or to RNA structural motifs, can have large impacts on RNA-protein recognition.

An S6:S18 complex inhibits translation of E. coli rpsF.

More than half of the ribosomal protein operons in Escherichia coli are regulated by structures within the mRNA transcripts that interact with specific ribosomal proteins to inhibit further protein expression. This regulation is accomplished using a variety of mechanisms and the RNA structures responsible for regulation are often not conserved across bacterial phyla. A widely conserved mRNA structure preceding the ribosomal protein operon containing rpsF and rpsR (encoding S6 and S18) was recently identified through comparative genomics. Examples of this RNA from both E. coli and Bacillus subtilis were shown to interact in vitro with an S6:S18 complex. In this work, we demonstrate that in E. coli, this RNA structure regulates gene expression in response to the S6:S18 complex. ß-galactosidase activity from a lacZ reporter translationally fused to the 5' UTR and first nine codons of E. coli rpsF is reduced fourfold by overexpression of a genomic fragment encoding both S6 and S18 but not by overexpression of either protein individually. Mutations to the mRNA structure, as well as to the RNA-binding site of S18 and the S6-S18 interaction surfaces of S6 and S18, are sufficient to derepress ß-galactosidase activity, indicating that the S6:S18 complex is the biologically active effector. Measurement of transcript levels shows that although reporter levels do not change upon protein overexpression, levels of the native transcript are reduced fourfold, suggesting that the mRNA regulator prevents translation and this effect is amplified on the native transcript by other mechanisms.

Sampled ensemble neutrality as a feature to classify potential structured RNAs.

BACKGROUND: Structured RNAs have many biological functions ranging from catalysis of chemical reactions to gene regulation. Yet, many homologous structured RNAs display most of their conservation at the secondary or tertiary structure level. As a result, strategies for structured RNA discovery rely heavily on identification of sequences sharing a common stable secondary structure. However, correctly distinguishing structured RNAs from surrounding genomic sequence remains challenging, especially during de novo discovery. RNA also has a long history as a computational model for evolution due to the direct link between genotype (sequence) and phenotype (structure). From these studies it is clear that evolved RNA structures, like protein structures, can be considered robust to point mutations. In this context, an RNA sequence is considered robust if its neutrality (extent to which single mutant neighbors maintain the same secondary structure) is greater than that expected for an artificial sequence with the same minimum free energy structure. RESULTS: In this work, we bring concepts from evolutionary biology to bear on the structured RNA de novo discovery process. We hypothesize that alignments corresponding to structured RNAs should consist of neutral sequences. We evaluate several measures of neutrality for their ability to distinguish between alignments of structured RNA sequences drawn from Rfam and various decoy alignments. We also introduce a new measure of RNA structural neutrality, the structure ensemble neutrality (SEN). SEN seeks to increase the biological relevance of existing neutrality measures in two ways. First, it uses information from an alignment of homologous sequences to identify a conserved biologically relevant structure for comparison. Second, it only counts base-pairs of the original structure that are absent in the comparison structure and does not penalize the formation of additional base-pairs. CONCLUSION: We find that several measures of neutrality are effective at separating structured RNAs from decoy sequences, including both shuffled alignments and flanking genomic sequence. Furthermore, as an independent feature classifier to identify structured RNAs, SEN yields comparable performance to current approaches that consider a variety of features including stability and sequence identity. Finally, SEN outperforms other measures of neutrality at detecting mutational robustness in bacterial regulatory RNA structures.

Heterochromatin assembly and transcriptome repression by Set1 in coordination with a class II histone deacetylase.

Histone modifiers play essential roles in controlling transcription and organizing eukaryotic genomes into functional domains. Here, we show that Set1, the catalytic subunit of the highly conserved Set1C/COMPASS complex responsible for histone H3K4 methylation (H3K4me), behaves as a repressor of the transcriptome largely independent of Set1C and H3K4me in the fission yeast Schizosaccharomyces pombe. Intriguingly, while Set1 is enriched at highly expressed and repressed loci, Set1 binding levels do not generally correlate with the levels of transcription. We show that Set1 is recruited by the ATF/CREB homolog Atf1 to heterochromatic loci and promoters of stress-response genes. Moreover, we demonstrate that Set1 coordinates with the class II histone deacetylase Clr3 in heterochromatin assembly at prominent chromosomal landmarks and repression of the transcriptome that includes Tf2 retrotransposons, noncoding RNAs, and regulators of development and stress-responses. Our study delineates a molecular framework for elucidating the functional links between transcriptome control and chromatin organization.

Complete RNA inverse folding: computational design of functional hammerhead ribozymes.

Nanotechnology and synthetic biology currently constitute one of the most innovative, interdisciplinary fields of research, poised to radically transform society in the 21st century. This paper concerns the synthetic design of ribonucleic acid molecules, using our recent algorithm, RNAiFold, which can determine all RNA sequences whose minimum free energy secondary structure is a user-specified target structure. Using RNAiFold, we design ten cis-cleaving hammerhead ribozymes, all of which are shown to be functional by a cleavage assay. We additionally use RNAiFold to design a functional cis-cleaving hammerhead as a modular unit of a synthetic larger RNA. Analysis of kinetics on this small set of hammerheads suggests that cleavage rate of computationally designed ribozymes may be correlated with positional entropy, ensemble defect, structural flexibility/rigidity and related measures. Artificial ribozymes have been designed in the past either manually or by SELEX (Systematic Evolution of Ligands by Exponential Enrichment); however, this appears to be the first purely computational design and experimental validation of novel functional ribozymes. RNAiFold is available at http://bioinformatics.bc.edu/clotelab/RNAiFold/.