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.

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.

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.

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.

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.

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.

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.

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.

Novel Strategies to Optimize the Amplification of Single-Stranded DNA.

The generation of single stranded DNA plays a key role in in vitro selection of DNA aptamers and in other molecular techniques such as DNA sequencing and microarrays. Here we describe three novel methodologies for ssDNA production and amplification. Furthermore, we describe some previously unnoticed aspects of random DNA amplification. Our results showed that in asymmetric PCR the addition of a high melting temperature reverse primer blocked at its 3' end by a dideoxy nucleotide drives the reaction further toward ssDNA production. We demonstrated also that incorporation of internally inverted nucleotide/(s) in one primer can be used as a new method of polymerization termination. Using such modified primer, the PCR product includes two complementary DNA strands having different lengths and separable from one another by denaturing gel electrophoresis. In addition, we showed that nicking enzymes can be used to cleave the undesirable strand allowing the isolation of the target ssDNA strand.

Single Mutation in Hammerhead Ribozyme Favors Cleavage Activity with Manganese over Magnesium.

Hammerhead ribozymes are one of the most studied classes of ribozymes so far, from both the structural and biochemical point of views. The activity of most hammerhead ribozymes is cation-dependent. Mg2+ is one of the most abundant divalent cations in the cell and therefore plays a major role in cleavage activity for most hammerhead ribozymes. Besides Mg2+, cleavage can also occur in the presence of other cations such as Mn2+. The catalytic core of hammerhead ribozymes is highly conserved, which could contribute to a preference of hammerhead ribozymes toward certain cations. Here, we show a naturally occurring variation in the catalytic core of hammerhead ribozymes, A6C, that can favor one metallic ion, Mn2+, over several other cations.

Simple rolling circle amplification colorimetric assay based on pH for target DNA detection.

Detection and identification of DNA by PCR has opened tremendous possibilities and allows detection of minute quantities of DNA highly specifically. However, PCR remains confined to laboratory settings because of the need of thermocyclers and other analytical equipment. This led to development of isothermal amplification techniques, among which Pad Lock Probe (PLP)-based Rolling Circle Amplification (RCA) has several advantages, but typically also requires a laboratory apparatus of some sort to measure DNA amplification. To circumvent this limitation, while still taking advantage of PLP-based RCA, we developed a colorimetric assay that relies on pH change. Using this assay, we can detect DNA in the low picomolar range and obtain results observable with the naked eye in only 20 min without any requirement for a thermocycler or other complex device, making it a particularly portable assay.

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.

RNA-Based Therapy Utilizing Oculopharyngeal Muscular Dystrophy Transcript Knockdown and Replacement.

Oculopharyngeal muscular dystrophy (OPMD) is caused by a small expansion of a short polyalanine (polyAla) tract in the poly(A)-binding protein nuclear 1 protein (PABPN1). Despite the monogenic nature of OPMD, no treatment is currently available. Here we report an RNA replacement strategy that has therapeutic potential in cell and C. elegans OPMD models. We develop selective microRNAs (miRNAs) against PABPN1, and we report that miRNAs and our previously developed hammerhead ribozymes (hhRzs) are capable of reducing the expression of both the mRNA and protein levels of PABPN1 by as much as 90%. Since OPMD derives from a very small expansion of GCG within the polyAla tract, our hhRz and miRNA molecules cannot distinguish between the wild-type and mutant mRNAs of PABPN1. Therefore, we designed an optimized-codon wild-type PABPN1 (opt-PABPN1) that is resistant to cleavage by hhRzs and miRNAs. Co-expression of opt-PABPN1 with either our hhRzs or miRNAs restored the level of PABPN1, concomitantly with a reduction in expanded PABPN1-associated cell death in a stable C2C12 OPMD model. Interestingly, knockdown of the PABPN1 by selective hhRzs in the C. elegans OPMD model significantly improved the motility of the PABPN1-13Ala worms. Taken together, RNA replacement therapy represents an exciting approach for OPMD treatment.

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.

Peptide modification results in the formation of a dimer with a 60-fold enhanced antimicrobial activity.

Cationic antimicrobial peptides (CAMPs) occur naturally in numerous organisms and are considered as a class of antibiotics with promising potential against multi-resistant bacteria. Herein, we report a strategy that can lead to the discovery of novel small CAMPs with greatly enhanced antimicrobial activity and retained antibiofilm potential. We geared our efforts towards i) the N-terminal cysteine functionalization of a previously reported small synthetic cationic peptide (peptide 1037, KRFRIRVRV-NH2), ii) its dimerization through a disulfide bond, and iii) a preliminary antimicrobial activity assessment of the newly prepared dimer against Pseudomonas aeruginosa and Burkholderia cenocepacia, pathogens responsible for the formation of biofilms in lungs of individuals with cystic fibrosis. This dimer is of high interest as it does not only show greatly enhanced bacterial growth inhibition properties compared to its pep1037 precursor (up to 60 times), but importantly, also displays antibiofilm potential at sub-MICs. Our results suggest that the reported dimer holds promise for its use in future adjunctive therapy, in combination with clinically-relevant antibiotics.

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.

Transcriptional pausing at the translation start site operates as a critical checkpoint for riboswitch regulation.

On the basis of nascent transcript sequencing, it has been postulated but never demonstrated that transcriptional pausing at translation start sites is important for gene regulation. Here we show that the Escherichia coli thiamin pyrophosphate (TPP) thiC riboswitch contains a regulatory pause site in the translation initiation region that acts as a checkpoint for thiC expression. By biochemically probing nascent transcription complexes halted at defined positions, we find a narrow transcriptional window for metabolite binding, in which the downstream boundary is delimited by the checkpoint. We show that transcription complexes at the regulatory pause site favour the formation of a riboswitch intramolecular lock that strongly prevents TPP binding. In contrast, cotranscriptional metabolite binding increases RNA polymerase pausing and induces Rho-dependent transcription termination at the checkpoint. Early transcriptional pausing may provide a general mechanism, whereby transient transcriptional windows directly coordinate the sensing of environmental cues and bacterial mRNA regulation.