Computational design and experimental verification of pseudoknotted ribozymes.

The design of new RNA sequences that retain the function of a model RNA structure is a challenge in bioinformatics because of the structural complexity of these molecules. RNA can fold into its secondary and tertiary structures by forming stem-loops and pseudoknots. A pseudoknot is a set of base pairs between a region within a stem-loop and nucleotides outside of this stem-loop; this motif is very important for numerous functional structures. It is important for any computational design algorithm to take into account these interactions to give a reliable result for any structures that include pseudoknots. In our study, we experimentally validated synthetic ribozymes designed by Enzymer, which implements algorithms allowing for the design of pseudoknots. Enzymer is a program that uses an inverse folding approach to design pseudoknotted RNAs; we used it in this study to design two types of ribozymes. The ribozymes tested were the hammerhead and the glmS, which have a self-cleaving activity that allows them to liberate the new RNA genome copy during rolling-circle replication or to control the expression of the downstream genes, respectively. We demonstrated the efficiency of Enzymer by showing that the pseudoknotted hammerhead and glmS ribozymes sequences it designed were extensively modified compared to wild-type sequences and were still active.

Toehold-mediated strand displacement to measure released product from self-cleaving ribozymes.

This paper presents a probe comprising a fluorophore and a quencher, enabling measurement of released product from self-cleaving hammerhead ribozyme, without labeled RNA molecules, regular sampling or use of polyacrylamide gels. The probe is made of two DNA strands; one strand is labeled with a fluorophore at its 5'-end, while the other strand is labeled with a quencher at its 3'-end. These two DNA strands are perfectly complementary, but with a 3'-overhang of the fluorophore strand. These unpaired nucleotides act as a toehold, which is utilized by a detached cleaved fragment (coming from a self-cleaving hammerhead ribozyme) as the starting point for a strand displacement reaction. This reaction causes the separation of the fluorophore strand from the quencher strand, culminating in fluorescence, detectable in a plate reader. Notably, the emitted fluorescence is proportional to the amount of detached cleaved-off RNAs, displacing the DNA quencher strand. This method can replace or complement radio-hazardous unstable 32P as a method of measurement of the product release from ribozyme cleavage reactions; it also eliminates the need for polyacrylamide gels, for the same purpose. Critically, this method allows to distinguish between the total amount of cleaved ribozymes and the amount of detached fragments, resulting from that cleavage reaction.

Small RNAs beyond Model Organisms: Have We Only Scratched the Surface?

Small RNAs (sRNAs) are essential regulators in the adaptation of bacteria to environmental changes and act by binding targeted mRNAs through base complementarity. Approximately 550 distinct families of sRNAs have been identified since their initial characterization in the 1980s, accelerated by the emergence of RNA-sequencing. Small RNAs are found in a wide range of bacterial phyla, but they are more prominent in highly researched model organisms compared to the rest of the sequenced bacteria. Indeed, Escherichia coli and Salmonella enterica contain the highest number of sRNAs, with 98 and 118, respectively, with Enterobacteriaceae encoding 145 distinct sRNAs, while other bacteria families have only seven sRNAs on average. Although the past years brought major advances in research on sRNAs, we have perhaps only scratched the surface, even more so considering RNA annotations trail behind gene annotations. A distinctive trend can be observed for genes, whereby their number increases with genome size, but this is not observable for RNAs, although they would be expected to follow the same trend. In this perspective, we aimed at establishing a more accurate representation of the occurrence of sRNAs in bacteria, emphasizing the potential for novel sRNA discoveries.

Identification of 11 candidate structured noncoding RNA motifs in humans by comparative genomics.

BACKGROUND: Only 1.5% of the human genome encodes proteins, while large part of the remaining encodes noncoding RNAs (ncRNA). Many ncRNAs form structures and perform many important functions. Accurately identifying structured ncRNAs in the human genome and discovering their biological functions remain a major challenge. RESULTS: Here, we have established a pipeline (CM-line) with the following features for analyzing the large genomes of humans and other animals. First, we selected species with larger genetic distances to facilitate the discovery of covariations and compatible mutations. Second, we used CMfinder, which can generate useful alignments even with low sequence conservation. Third, we removed repetitive sequences and known structured ncRNAs to reduce the workload of CMfinder. Fourth, we used Infernal to find more representatives and refine the structure. We reported 11 classes of structured ncRNA candidates with significant covariations in humans. Functional analysis showed that these ncRNAs may have variable functions. Some may regulate circadian clock genes through poly (A) signals (PAS); some may regulate the elongation factor (EEF1A) and the T-cell receptor signaling pathway by cooperating with RNA binding proteins. CONCLUSIONS: By searching for important features of RNA structure from large genomes, the CM-line has revealed the existence of a variety of novel structured ncRNAs. Functional analysis suggests that some newly discovered ncRNA motifs may have biological functions. The pipeline we have established for the discovery of structured ncRNAs and the identification of their functions can also be applied to analyze other large genomes.

High-sensitivity small-molecule detection of microcystin-LR cyano-toxin using a terahertz-aptamer biosensor.

We demonstrate the rapid and highly sensitive detection of a small molecule, microcystin-LR (MC-LR) toxin using an aptasensor based on a terahertz (THz) emission technique named the terahertz chemical microscope (TCM). The main component of the TCM is the sensing plate, which consists of a thin silicon layer deposited on a sapphire substrate, with a natural SiO2 layer formed on the top of the Si layer. The DNA aptamer is linked to the oxidized top surface of the silicon layer by a one-step reaction (click chemistry) between the DBCO-labeled aptamer and an azido group that binds to the surface. Using density functional theory (DFT) calculations, the number of active sites on the surface has been estimated to be 3.8 × 1013 cm-2. Aptamer immobilization and MC-LR binding have been optimized by adjusting the aptamer concentration and the binding buffer composition. When MC-LR binds with the DNA aptamer, it causes a change in the chemical potential at the surface of the sensing plate, which leads to a change in the amplitude of the THz signal. Compared with other bio-sensing methods such as surface plasmon resonance (SPR), TCM is a rapid assay that can be completed in 15 min (10 min incubation and 5 min data acquisition). Moreover, our results show that the aptamer-based TCM can detect MC-LR with an excellent detection limit of 50 ng L-1, which is 20 times more sensitive compared with SPR measurements of MC-LR.

Room-Temperature and Selective Triggering of Supramolecular DNA Assembly/Disassembly by Nonionizing Radiation.

Recent observations have suggested that nonionizing radiation in the microwave and terahertz (THz; far-infrared) regimes could have an effect on double-stranded DNA (dsDNA). These observations are of significance owing to the omnipresence of microwave emitters in our daily lives (e.g., food preparation, telecommunication, and wireless Internet) and the increasing prevalence of THz emitters for imaging (e.g., concealed weapon detection in airports, skin cancer screenings) and communication technologies. By examining multiple DNA nanostructures as well as two plasmid DNAs, microwaves were shown to promote the repair and assembly of DNA nanostructures and single-stranded regions of plasmid DNA, while intense THz pulses had the opposite effect (in particular, for short dsDNA). Both effects occurred at room temperature within minutes, showed a DNA length dependence, and did not affect the chemical integrity of the DNA. Intriguingly, the function of six proteins (enzymes and antibodies) was not affected by exposure to either form of radiation under the conditions examined. This particular detail was exploited to assemble a fully functional hybrid DNA-protein nanostructure in a bottom-up manner. This study therefore provides entirely new perspectives for the effects, on the molecular level, of nonionizing radiation on biomolecules. Moreover, the proposed structure-activity relationships could be exploited in the field of DNA nanotechnology, which paves the way for designing a new range of functional DNA nanomaterials that are currently inaccessible to state-of-the-art assembly protocols.

Label-free cocaine aptasensor based on a long-period fiber grating.

In this Letter, we combined a promising bioreceptor, a cocaine aptamer MN6, with an ultrasensitive optical platform long-period fiber grating (LPFG) to create a new cocaine biosensor. The cocaine induces a conformational rearrangement of the aptamer which changes the refractive index around the LPFG producing a measurable shift of the transmission spectrum. We were able to track subtle interaction between the receptor and cocaine molecules over a concentration range of 25 to 100 µM. The presented biosensor does not require labeling or signal enhancement, resulting in a simple user-friendly device.

Search for 5'-leader regulatory RNA structures based on gene annotation aided by the RiboGap database.

The discovery of noncoding RNAs (ncRNAs) and their importance for gene regulation led us to develop bioinformatics tools to pursue the discovery of novel ncRNAs. Finding ncRNAs de novo is challenging, first due to the difficulty of retrieving large numbers of sequences for given gene activities, and second due to exponential demands on calculation needed for comparative genomics on a large scale. Recently, several tools for the prediction of conserved RNA secondary structure were developed, but many of them are not designed to uncover new ncRNAs, or are too slow for conducting analyses on a large scale. Here we present various approaches using the database RiboGap as a primary tool for finding known ncRNAs and for uncovering simple sequence motifs with regulatory roles. This database also can be used to easily extract intergenic sequences of eubacteria and archaea to find conserved RNA structures upstream of given genes. We also show how to extend analysis further to choose the best candidate ncRNAs for experimental validation.

Automated design of hammerhead ribozymes and validation by targeting the PABPN1 gene transcript.

We present a new publicly accessible web-service, RiboSoft, which implements a comprehensive hammerhead ribozyme design procedure. It accepts as input a target sequence (and some design parameters) then generates a set of ranked hammerhead ribozymes, which target the input sequence. This paper describes the implemented procedure, which takes into consideration multiple objectives leading to a multi-objective ranking of the computer-generated ribozymes. Many ribozymes were assayed and validated, including four ribozymes targeting the transcript of a disease-causing gene (a mutant version of PABPN1). These four ribozymes were successfully tested in vitro and in vivo, for their ability to cleave the targeted transcript. The wet-lab positive results of the test are presented here demonstrating the real-world potential of both hammerhead ribozymes and RiboSoft. RiboSoft is freely available at the website http://ribosoft.fungalgenomics.ca/ribosoft/.

Complex autoregulation of the post-transcriptional regulator RsmA in Pseudomonas aeruginosa.

RsmA is a post-transcriptional RNA-binding protein that acts as a pleiotropic global regulator of mRNAs in the opportunistic pathogen Pseudomonas aeruginosa. Upon binding to its target, RsmA impedes the translation of the mRNA by the ribosome. The RsmA regulon affects over 500 genes, many of which have been identified as important in the pathogenicity of P. aeruginosa. Whilst the regulatory function of RsmA is relatively well characterized, the genetic regulation of rsmA itself at the transcriptional and translational levels remains poorly understood. Here, we show that RsmA is capable of self-regulation through an unorthodox mechanism. This regulation occurs via direct interaction of the protein with an RsmA-binding site located in the early portion of its coding sequence. To the best of our knowledge this is the first report of such an unusual regulation in pseudomonads.

Exceptional structured noncoding RNAs revealed by bacterial metagenome analysis.

Estimates of the total number of bacterial species indicate that existing DNA sequence databases carry only a tiny fraction of the total amount of DNA sequence space represented by this division of life. Indeed, environmental DNA samples have been shown to encode many previously unknown classes of proteins and RNAs. Bioinformatics searches of genomic DNA from bacteria commonly identify new noncoding RNAs (ncRNAs) such as riboswitches. In rare instances, RNAs that exhibit more extensive sequence and structural conservation across a wide range of bacteria are encountered. Given that large structured RNAs are known to carry out complex biochemical functions such as protein synthesis and RNA processing reactions, identifying more RNAs of great size and intricate structure is likely to reveal additional biochemical functions that can be achieved by RNA. We applied an updated computational pipeline to discover ncRNAs that rival the known large ribozymes in size and structural complexity or that are among the most abundant RNAs in bacteria that encode them. These RNAs would have been difficult or impossible to detect without examining environmental DNA sequences, indicating that numerous RNAs with extraordinary size, structural complexity, or other exceptional characteristics remain to be discovered in unexplored sequence space.

The ubiquitous hammerhead ribozyme.

The hammerhead ribozyme is a small catalytic RNA motif capable of endonucleolytic (self-) cleavage. It is composed of a catalytic core of conserved nucleotides flanked by three helices, two of which form essential tertiary interactions for fast self-scission under physiological conditions. Originally discovered in subviral plant pathogens, its presence in several eukaryotic genomes has been reported since. More recently, this catalytic RNA motif has been shown to reside in a large number of genomes. We review the different approaches in discovering these new hammerhead ribozyme sequences and discuss possible biological functions of the genomic motifs.

Expression strategy of Aedes albopictus densovirus.

The transcription map of the Aedes albopictus densovirus (AalDNV) brevidensovirus was identified by Northern blotting, rapid amplification of cDNA ends (RACE) analysis, and RNase protection assays. AalDNV produced mRNAs of 3,359 (NS1), 3,345 (NS2), and 1,246 (VP) nucleotides. The two overlapping P7/7.4 NS promoters employed closely located alternate transcription initiation sites, positioned at either side of the NS1 initiation codon. All NS mRNAs coterminated with VP mRNA. All promoters, explored using luciferase assays, were functional in insect and human cell lines.

Electrochemically Exfoliated Graphene Oxide for Simple Fabrication of Cocaine Aptasensors.

Transducers made from graphene-type materials are widely used in sensing applications. However, utilization of graphene oxide obtained from electrochemical exfoliation of graphite (EGO) has remained relatively unexplored. In this study, electrochemical cocaine aptasensors based on large-size EGO flakes were investigated. In particular, the influence of the following parameters on the sensor performance was examined: (i) aptamer's terminal group (-NH2 vs -OH), (ii) functionalization of EGO with the aptamer via physical adsorption and covalent immobilization, and (iii) intrinsic electrochemical properties of EGO such as the electrochemical surface area (ESA) and standard rate constant of electron transfer (k0). The results demonstrate that EGO-based electrochemical aptasensors fabricated by physical adsorption with an NH2-modified aptamer have very good reproducibility, shelf-life stability, and high sensitivity for detecting cocaine with a detection limit of 50 nM. Their performance is comparable to that of the aptasensors prepared using the covalent immobilization. Additionally, it is shown that EGO materials with high ESA and k0 can enhance the sensing performance. The fast (less than 10 min) and strong adsorption of the NH2-modified cocaine aptamer on the surface of large EGO flakes makes the fabrication of the sensing platform simple and rapid. This simple approach has the potential to simplify the fabrication of sensors.

Identification of hammerhead ribozymes in all domains of life reveals novel structural variations.

Hammerhead ribozymes are small self-cleaving RNAs that promote strand scission by internal phosphoester transfer. Comparative sequence analysis was used to identify numerous additional representatives of this ribozyme class than were previously known, including the first representatives in fungi and archaea. Moreover, we have uncovered the first natural examples of "type II" hammerheads, and our findings reveal that this permuted form occurs in bacteria as frequently as type I and III architectures. We also identified a commonly occurring pseudoknot that forms a tertiary interaction critical for high-speed ribozyme activity. Genomic contexts of many hammerhead ribozymes indicate that they perform biological functions different from their known role in generating unit-length RNA transcripts of multimeric viroid and satellite virus genomes. In rare instances, nucleotide variation occurs at positions within the catalytic core that are otherwise strictly conserved, suggesting that core mutations are occasionally tolerated or preferred.

Fluoride-Controlled Riboswitch-Based Dampening of Gene Expression for Cloning Potent Promoters.

Bioreporter systems based on detectable enzyme activity, such as that of beta-galactosidase or luciferase, are key in novel bacterial promoter discovery and study. While these systems permit quantification of gene expression, their use is limited by the toxicity of the expressed reporter enzymes in a given host. Indeed, the most potent promoters may be overlooked if their activity causes a lethal overproduction of the reporter genes when screening for transcriptional activity of potential promoter sequences with the luxCDABE cassette. To overcome this limitation, a variation of the mini-CTX-lux plasmid has been designed which allows reduction of promoter activity via the addition of an adjacent fluoride riboswitch. The riboswitch adds a layer of regulation between the promoter and the reporter gene, allowing cloning of stronger promoters by weakening expression, while giving the potential to induce with fluoride to provide a good signal for weaker promoters, thus circumventing limitations associated with reporter toxicity. We noticed the riboswitch potential portability issues between species, suggesting caution when using riboswitches non-native to the species where it is being used. This study introduces a new molecular biology tool which will allow for the identification of previously unverifiable or uncharacterized potent promoters and also provides a cloning vector for translational fusion with luciferase in a plasmid compatible with many species such as from the genera Burkholderia and Pseudomonas.

Computer-Aided Design of Active Pseudoknotted Hammerhead Ribozymes.

Pseudoknots are important motifs for stabilizing the structure of functional RNAs. As an example, pseudoknotted hammerhead ribozymes are highly active compared to minimal ribozymes. The design of new RNA sequences that retain the function of a model RNA structure includes taking in account pseudoknots presence in the structure, which is usually a challenge for bioinformatics tools. Our method includes using "Enzymer," a software for designing RNA sequences with desired secondary structures that may include pseudoknots. Enzymer implements an efficient stochastic search and optimization algorithm to sample RNA sequences from low ensemble defect mutational landscape of an initial design template to generate an RNA sequence that is predicted to fold into the desired target structure.

Real-time isothermal DNA amplification monitoring in picoliter volumes using an optical fiber sensor.

Rolling circle amplification (RCA) of DNA can be considered as a great alternative to the gold standard polymerase chain reaction (PCR), especially during this pandemic period, where rapid, sensitive, and reliable test results for hundreds of thousands of samples are required daily. This work presents the first research to date on direct, real-time and label-free isothermal DNA amplification monitoring using a microcavity in-line Mach-Zehnder interferometer (µIMZI) fabricated in an optical fiber. The solution based on µIMZI offers a great advantage over many other sensing concepts - making possible optical analysis in just picoliter sample volumes. The selectivity of the biosensor is determined by DNA primers immobilized on the microcavity's surface that act as selective biorecognition elements and trigger initiation of the DNA amplification process. In this study, we verified the sensing concept using circular DNA designed to target the H5N1 influenza virus. The developed biosensor exhibits an ultrahigh refractive index sensitivity reaching 14 000 nm per refractive index unit and a linear detection range between 9.4 aM and 94 pM of the target DNA sequence. Within a 30 min period, the amplification of as little as 9.4 aM DNA can be effectively detected, with a calculated limit of detection of as low as 0.2 aM DNA, suggesting that this methodology holds great promise in practical disease diagnosis applications in the future.

Digital Microfluidics Chips for the Execution and Real-Time Monitoring of Multiple Ribozymatic Cleavage Reactions.

In this paper, we describe the design and performance of two digital microfluidics (DMF) chips capable of executing multiple ribozymatic reactions, with proper controls, in response to short single-stranded DNA inducers. Since the fluorescence output of a reaction is measurable directly from the chip, without the need for gel electrophoresis, a complete experiment involving up to eight reactions (per chip) can be carried out reliably, relatively quickly, and efficiently. The ribozymes can also be used as biosensors of the concentration of oligonucleotide inputs, with high sensitivity, low limits of quantification and of detection, and excellent signal-to-noise ratio. The presented chips are readily usable devices that can be used to automate, speed up, and reduce the costs of ribozymatic reaction experiments.