The protein-only RNase Ps, endonucleases that cleave pre-tRNA: Biological relevance, molecular architectures, substrate recognition and specificity, and protein interactomes.

Protein-only RNase P (PRORP) is an essential enzyme responsible for the 5' maturation of precursor tRNAs (pre-tRNAs). PRORPs are classified into three categories with unique molecular architectures, although all three classes of PRORPs share a mechanism and have similar active sites. Single subunit PRORPs, like those found in plants, have multiple isoforms with different localizations, substrate specificities, and temperature sensitivities. Most recently, Arabidopsis thaliana PRORP2 was shown to interact with TRM1A and B, highlighting a new potential role between these enzymes. Work with At PRORPs led to the development of a ribonuclease that is being used to protect against plant viruses. The mitochondrial RNase P complex, found in metazoans, consists of PRORP, TRMT10C, and SDR5C1, and has also been shown to have substrate specificity, although the cause is unknown. Mutations in mitochondrial tRNA and mitochondrial RNase P have been linked to human disease, highlighting the need to continue understanding this complex. The last class of PRORPs, homologs of Aquifex RNase P (HARPs), is found in thermophilic archaea and bacteria. This most recently discovered type of PRORP forms a large homo-oligomer complex. Although numerous structures of HARPs have been published, it is still unclear how HARPs bind pre-tRNAs and in what ratio. There is also little investigation into the substrate specificity and ideal conditions for HARPs. Moving forward, further work is required to fully characterize each of the three classes of PRORP, the pre-tRNA binding recognition mechanism, the rules of substrate specificity, and how these three distinct classes of PRORP evolved. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems.

Multiple structural flavors of RNase P in precursor tRNA processing.

The precursor transfer RNAs (pre-tRNAs) require extensive processing to generate mature tRNAs possessing proper fold, structural stability, and functionality required to sustain cellular viability. The road to tRNA maturation follows an ordered process: 5'-processing, 3'-processing, modifications at specific sites, if any, and 3'-CCA addition before aminoacylation and recruitment to the cellular protein synthesis machinery. Ribonuclease P (RNase P) is a universally conserved endonuclease in all domains of life, performing the hydrolysis of pre-tRNA sequences at the 5' end by the removal of phosphodiester linkages between nucleotides at position -1 and +1. Except for an archaeal species: Nanoarchaeum equitans where tRNAs are transcribed from leaderless-position +1, RNase P is indispensable for life and displays fundamental variations in terms of enzyme subunit composition, mechanism of substrate recognition and active site architecture, utilizing in all cases a two metal ion-mediated conserved catalytic reaction. While the canonical RNA-based ribonucleoprotein RNase P has been well-known to occur in bacteria, archaea, and eukaryotes, the occurrence of RNA-free protein-only RNase P in eukaryotes and RNA-free homologs of Aquifex RNase P in prokaryotes has been discovered more recently. This review aims to provide a comprehensive overview of structural diversity displayed by various RNA-based and RNA-free RNase P holoenzymes towards harnessing critical RNA-protein and protein-protein interactions in achieving conserved pre-tRNA processing functionality. Furthermore, alternate roles and functional interchangeability of RNase P are discussed in the context of its employability in several clinical and biotechnological applications. This article is categorized under: RNA Processing > tRNA Processing RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

Discovery, structure, mechanisms, and evolution of protein-only RNase P enzymes.

The endoribonuclease RNase P is responsible for tRNA 5' maturation in all domains of life. A unique feature of RNase P is the variety of enzyme architectures, ranging from dual- to multi-subunit ribonucleoprotein forms with catalytic RNA subunits to protein-only enzymes, the latter occurring as single- or multi-subunit forms or homo-oligomeric assemblies. The protein-only enzymes evolved twice: a eukaryal protein-only RNase P termed PRORP and a bacterial/archaeal variant termed homolog of Aquifex RNase P (HARP); the latter replaced the RNA-based enzyme in a small group of thermophilic bacteria but otherwise coexists with the ribonucleoprotein enzyme in a few other bacteria as well as in those archaea that also encode a HARP. Here we summarize the history of the discovery of protein-only RNase P enzymes and review the state of knowledge on structure and function of bacterial HARPs and eukaryal PRORPs, including human mitochondrial RNase P as a paradigm of multi-subunit PRORPs. We also describe the phylogenetic distribution and evolution of PRORPs, as well as possible reasons for the spread of PRORPs in the eukaryal tree and for the recruitment of two additional protein subunits to metazoan mitochondrial PRORP. We outline potential applications of PRORPs in plant biotechnology and address diseases associated with mutations in human mitochondrial RNase P genes. Finally, we consider possible causes underlying the displacement of the ancient RNA enzyme by a protein-only enzyme in a small group of bacteria.

The specificity landscape of bacterial ribonuclease P.

Developing quantitative models of substrate specificity for RNA processing enzymes is a key step toward understanding their biology and guiding applications in biotechnology and biomedicine. Optimally, models to predict relative rate constants for alternative substrates should integrate an understanding of structures of the enzyme bound to "fast" and "slow" substrates, large datasets of rate constants for alternative substrates, and transcriptomic data identifying in vivo processing sites. Such data are either available or emerging for bacterial ribonucleoprotein RNase P a widespread and essential tRNA 5' processing endonuclease, thus making it a valuable model system for investigating principles of biological specificity. Indeed, the well-established structure and kinetics of bacterial RNase P enabled the development of high throughput measurements of rate constants for tRNA variants and provided the necessary framework for quantitative specificity modeling. Several studies document the importance of conformational changes in the precursor tRNA substrate as well as the RNA and protein subunits of bacterial RNase P during binding, although the functional roles and dynamics are still being resolved. Recently, results from cryo-EM studies of E. coli RNase P with alternative precursor tRNAs are revealing prospective mechanistic relationships between conformational changes and substrate specificity. Yet, extensive uncharted territory remains, including leveraging these advances for drug discovery, achieving a complete accounting of RNase P substrates, and understanding how the cellular context contributes to RNA processing specificity in vivo.

A tRNA-modifying enzyme facilitates RNase P activity in Arabidopsis nuclei.

RNase P is the essential activity that performs the 5' maturation of transfer RNA (tRNA) precursors. Beyond the ancestral form of RNase P containing a ribozyme, protein-only RNase P enzymes termed PRORP were identified in eukaryotes. In human mitochondria, PRORP forms a complex with two protein partners to become functional. In plants, although PRORP enzymes are active alone, we investigate their interaction network to identify potential tRNA maturation complexes. Here we investigate functional interactions involving the Arabidopsis nuclear RNase P PRORP2. We show, using an immuno-affinity strategy, that PRORP2 occurs in a complex with the tRNA methyl transferases TRM1A and TRM1B in vivo. Beyond RNase P, these enzymes can also interact with RNase Z. We show that TRM1A/TRM1B localize in the nucleus and find that their double knockout mutation results in a severe macroscopic phenotype. Using a combination of immuno-detections, mass spectrometry and a transcriptome-wide tRNA sequencing approach, we observe that TRM1A/TRM1B are responsible for the m22G26 modification of 70% of cytosolic tRNAs in vivo. We use the transcriptome wide tRNAseq approach as well as RNA blot hybridizations to show that RNase P activity is impaired in TRM1A/TRM1B mutants for specific tRNAs, in particular, tRNAs containing a m22G modification at position 26 that are strongly downregulated in TRM1A/TRM1B mutants. Altogether, results indicate that the m22G-adding enzymes TRM1A/TRM1B functionally cooperate with nuclear RNase P in vivo for the early steps of cytosolic tRNA biogenesis.

Human RNase P exhibits and controls distinct ribonucleolytic activities required for ordered maturation of tRNA.

Precursor tRNAs are transcribed with flanking and intervening sequences known to be processed by specific ribonucleases. Here, we show that transcription complexes of RNA polymerase III assembled on tRNA genes comprise RNase P that cleaves precursor tRNA and subsequently degrades the excised 5' leader. Degradation is based on a 3'-5' exoribonucleolytic activity carried out by the protein subunit Rpp14, as determined by biochemical and reverse genetic analyses. Neither reconstituted nor purified RNase P displays this magnesium ion-dependent, processive exoribonucleolytic activity. Markedly, knockdown of Rpp14 by RNA interference leads to a wide-ranging inhibition of cleavage of flanking and intervening sequences of various precursor tRNAs in extracts and cells. This study reveals that RNase P controls tRNA splicing complex and RNase Z for ordered maturation of nascent precursor tRNAs by transcription complexes.

Transcriptome analysis reveals a lncRNA-miRNA-mRNA regulatory network in OsRpp30-mediated disease resistance in rice.

BACKGROUND: Long non-coding RNAs (lncRNAs) play critical roles in various biological processes in plants. Extensive studies utilizing high-throughput RNA sequencing have revealed that many lncRNAs are involved in plant disease resistance. Oryza sativa RNase P protein 30 (OsRpp30) has been identified as a positive regulator of rice immunity against fungal and bacterial pathogens. Nevertheless, the specific functions of lncRNAs in relation to OsRpp30-mediated disease resistance in rice remain elusive. RESULTS: We conducted a comprehensive analysis of lncRNAs, miRNAs, and mRNAs expression patterns in wild type (WT), OsRpp30 overexpression (OsRpp30-OE), and OsRpp30 knockout (OsRpp30-KO) rice plants. In total, we identified 91 differentially expressed lncRNAs (DElncRNAs), 1671 differentially expressed mRNAs (DEmRNAs), and 41 differentially expressed miRNAs (DEmiRNAs) across the different rice lines. To gain further insights, we investigated the interaction between DElncRNAs and DEmRNAs, leading to the discovery of 10 trans- and 27 cis-targeting pairs specific to the OsRpp30-OE and OsRpp30-KO samples. In addition, we constructed a competing endogenous RNA (ceRNA) network comprising differentially expressed lncRNAs, miRNAs, and mRNAs to elucidate their intricate interplay in rice disease resistance. The ceRNA network analysis uncovered a set of gene targets regulated by lncRNAs and miRNAs, which were found to be involved in pathogen recognition, hormone pathways, transcription factor activation, and other biological processes related to plant immunity. CONCLUSIONS: Our study provides a comprehensive expression profiling of lncRNAs, miRNAs, and mRNAs in a collection of defense mutants in rice. To decipher the putative functional significance of lncRNAs, we constructed trans- and cis-targeting networks involving differentially expressed lncRNAs and mRNAs, as well as a ceRNA network incorporating differentially expressed lncRNAs, miRNAs, and mRNAs. Together, the findings from this study provide compelling evidence supporting the pivotal roles of lncRNAs in OsRpp30-mediated disease resistance in rice.

Importance of residue 248 in Escherichia coli RNase P RNA mediated cleavage.

tRNA genes are transcribed as precursors and RNase P generates the matured 5' end of tRNAs. It has been suggested that residue - 1 (the residue immediately 5' of the scissile bond) in the pre-tRNA interacts with the well-conserved bacterial RNase P RNA (RPR) residue A248 (Escherichia coli numbering). The way A248 interacts with residue - 1 is not clear. To gain insight into the role of A248, we analyzed cleavage as a function of A248 substitutions and N-1 nucleobase identity by using pre-tRNA and three model substrates. Our findings are consistent with a model where the structural topology of the active site varies and depends on the identity of the nucleobases at, and in proximity to, the cleavage site and their potential to interact. This leads to positioning of Mg2+ that activates the water that acts as the nucleophile resulting in efficient and correct cleavage. We propose that in addition to be involved in anchoring the substrate the role of A248 is to exclude bulk water from access to the amino acid acceptor stem, thereby preventing non-specific hydrolysis of the pre-tRNA. Finally, base stacking is discussed as a way to protect functionally important base-pairing interactions from non-specific hydrolysis, thereby ensuring high fidelity during RNA processing and the decoding of mRNA.

Novel homozygous variants in PRORP expand the genotypic spectrum of combined oxidative phosphorylation deficiency 54.

Biallelic hypomorphic variants in PRORP have been recently described as causing the autosomal recessive disorder combined oxidative phosphorylation deficiency type 54 (COXPD54). COXPD54 encompasses a phenotypic spectrum of sensorineural hearing loss and ovarian insufficiency (Perrault syndrome) to leukodystrophy. Here, we report three additional families with homozygous missense PRORP variants with pleiotropic phenotypes. Each missense variant altered a highly conserved residue within the metallonuclease domain. In vitro mitochondrial tRNA processing assays with recombinant TRMT10C, SDR5C1 and PRORP indicated two COXPD54-associated PRORP variants, c.1159A>G (p.Thr387Ala) and c.1241C>T (p.Ala414Val), decreased pre-tRNAIle cleavage, consistent with both variants impacting tRNA processing. No significant decrease in tRNA processing was observed with PRORP c.1093T>C (p.Tyr365His), which was identified in an individual with leukodystrophy. These data provide independent evidence that PRORP variants are associated with COXPD54 and that the assessment of 5' leader mitochondrial tRNA processing is a valuable assay for the functional analysis and clinical interpretation of novel PRORP variants.

Mapping of RNase P Ribozyme Regions in Proximity with a Human RNase P Subunit Protein Using Fe(II)-EDTA Cleavage and Nuclease Footprint Analyses.

Ribonuclease P (RNase P), which may consist of both protein subunits and a catalytic RNA part, is responsible for 5' maturation of tRNA by cleaving the 5'-leader sequence. In Escherichia coli, RNase P contains a catalytic RNA subunit (M1 RNA) and a protein factor (C5 protein). In human cells, RNase P holoenzyme consists of an RNA subunit (H1 RNA) and multiple protein subunits that include human RPP29 protein. M1GS, a sequence specific targeting ribozyme derived from M1 RNA, can be constructed to target a specific mRNA to degrade it in vitro. Recent studies have shown that M1GS ribozymes are efficient in blocking the expression of viral mRNAs in cultured cells and in animals. These results suggest that RNase P ribozymes have the potential to be useful in basic research and in clinical applications. It has been shown that RNase P binding proteins, such as C5 protein and RPP29, can enhance the activities of M1GS RNA in processing a natural tRNA substrate and a target mRNA. Understanding how RPP29 binds to M1GS RNA and enhances the enzyme's catalytic activity will provide great insight into developing more robust gene-targeting ribozymes for in vivo application. In this chapter, we describe the methods of using Fe(II)-ethylenediaminetetraacetic acid (EDTA) cleavage and nuclease footprint analyses to determine the regions of a M1GS ribozyme that are in proximity to RPP29 protein.

Development and clinical validation of a one-step pentaplex real-time reverse transcription PCR assay for detection of hepatitis virus B, C, E, Treponema pallidum, and a human housekeeping gene.

BACKGROUND: With the safety of blood transfusion being a major public health concern, the development of a rapid, sensitive, specific, and cost-effective multiplex PCR assay for simultaneous detection of hepatitis B virus(HBV), hepatitis C virus (HCV), hepatitis E virus (HEV), and Treponema pallidum(T. pallidum) in blood is crucial. METHODS: Five primer pairs and probes were designed towards conserved regions of target genes and used to establish a one-step pentaplex real-time reverse transcription PCR(qRT-PCR) assay for simultaneous detection of HBV, HCV, HEV, T. pallidum, and RNase P(housekeeping gene), providing sample quality check. The clinical performance of the assay was further determined with 2400 blood samples from blood donors and patients in Zhejiang province, and compared the results with commercial singleplex qPCR and serological assays. RESULTS: The 95% limit of detection(LOD) of HBV, HCV, HEV, and T. pallidum were 7.11 copies/µL, 7.65 copies/µL, 8.45 copies/µL, and 9.06 copies/µL, respectively. Moreover, the assay has good specificity and precision. Compared to the singleplex qPCR assay, the novel assay for detecting HBV, HCV, HEV, and T. pallidum presented 100% clinical sensitivity, specificity, and consistency. Several discrepant results between serological and pentaplex qRT-PCR assays were found. Of 2400 blood samples, there were 2(0.08%) HBsAg positive samples, 3(0.13%) anti-HCV positive samples, 29(1.21%) IgM anti-HEV positive samples and 6(0.25%) anti-T. pallidum positive samples proven negative in nucleic acid detection. 1(0.04%) HBV DNA positive sample and 1(0.04%) HEV RNA positive sample were detected negative by serological testing. CONCLUSIONS: The developed pentaplex qRT-PCR is the first assay on simultaneous, sensitive, specific, and reproducible detection of HBV, HCV, HEV, T. pallidum, and RNase P in a single tube. It could detect pathogens in blood during the window period of infection and is a good tool for effectively screening blood donors and early clinical diagnosis.

Proteins Rpr2 and Pop3 increase the activity and thermal stability of yeast RNase P.

RNA-based enzyme RNase P is a ribonucleoprotein complex responsible primarily for 5'-maturation of tRNAs. S. cerevisiae RNase P comprises a catalytic RNA component and nine proteins. The assembly and maturation of S. cerevisiae RNase P involves an abundant and catalytically active precursor form, which includes all components except for proteins Rpr2 and Pop3. Rpr2 and Pop3 are essential proteins, but their roles in RNase P were not clear. Here we use a step-wise in vitro assembly of yeast RNase P to show that the addition of proteins Rpr2 and Pop3 increases the activity and thermal stability of the RNase P complex, similar to the effects previously observed for archaeal RNases P.

Suppressing Kaposi's Sarcoma-Associated Herpesvirus Lytic Gene Expression and Replication by RNase P Ribozyme.

Kaposi's sarcoma, an AIDS-defining illness, is caused by Kaposi's sarcoma-associated herpesvirus (KSHV), an oncogenic virus. In this study, we engineered ribozymes derived from ribonuclease P (RNase P) catalytic RNA with targeting against the mRNA encoding KSHV immediate early replication and transcription activator (RTA), which is vital for KSHV gene expression. The functional ribozyme F-RTA efficiently sliced the RTA mRNA sequence in vitro. In cells, KSHV production was suppressed with ribozyme F-RTA expression by 250-fold, and RTA expression was suppressed by 92-94%. In contrast, expression of control ribozymes hardly affected RTA expression or viral production. Further studies revealed both overall KSHV early and late gene expression and viral growth decreased because of F-RTA-facilitated suppression of RTA expression. Our results indicate the first instance of RNase P ribozymes having potential for use in anti-KSHV therapy.

Human RNase P: overview of a ribonuclease of interrelated molecular networks and gene-targeting systems.

The seminal discovery of ribonuclease P (RNase P) and its catalytic RNA by Sidney Altman has not only revolutionized our understanding of life, but also opened new fields for scientific exploration and investigation. This review focuses on human RNase P and its use as a gene-targeting tool, two topics initiated in Altman's laboratory. We outline early works on human RNase P as a tRNA processing enzyme and comment on its expanding nonconventional functions in molecular networks of transcription, chromatin remodeling, homology-directed repair, and innate immunity. The important implications and insights from these discoveries on the potential use of RNase P as a gene-targeting tool are presented. This multifunctionality calls to a modified structure-function partitioning of domains in human RNase P, as well as its relative ribonucleoprotein, RNase MRP. The role of these two catalysts in innate immunity is of particular interest in molecular evolution, as this dynamic molecular network could have originated and evolved from primordial enzymes and sensors of RNA, including predecessors of these two ribonucleoproteins.

Catalytic RNA Oligomers Formed by Co-Oligomerization of a Pair of Bimolecular RNase P Ribozymes.

Naturally occurring ribozymes with a modular architecture are promising platforms for construction of RNA nanostructures because modular redesign enables their oligomerization. The resulting RNA nanostructures can exhibit the catalytic function of the parent ribozyme in an assembly dependent manner. In this study, we designed and constructed open-form oligomers of a bimolecular form of an RNase P ribozyme. The ribozyme oligomers were analyzed biochemically and by atomic force microscopy (AFM).

Processing of the alaW alaX operon encoding the Ala2 tRNAs in Escherichia coli requires both RNase E and RNase P.

The alaW alaX operon encodes the Ala2 tRNAs, one of the two alanine tRNA isotypes in Escherichia coli. Our previous RNA-seq study showed that alaW alaX dicistronic RNA levels increased significantly in the absence of both RNase P and poly(A) polymerase I (PAP I), suggesting a role of polyadenylation in its stability. In this report, we show that RNase E initiates the processing of the primary alaW alaX precursor RNA by removing the Rho-independent transcription terminator, which appears to be the rate limiting step in the separation and maturation of the Ala2 pre-tRNAs by RNase P. Failure to separate the alaW and alaX pre-tRNAs by RNase P leads to poly(A)-mediated degradation of the dicistronic RNAs by polynucleotide phosphorylase (PNPase) and RNase R. Surprisingly, the thermosensitive RNase E encoded by the rne-1 allele is highly efficient in removing the terminator (>99%) at the nonpermissive temperature suggesting a significant caveat in experiments using this allele. Together, our data present a comprehensive picture of the Ala2 tRNA processing pathway and demonstrate that unprocessed RNase P substrates are degraded via a poly(A) mediated decay pathway.

An ADP-ribosyltransferase toxin kills bacterial cells by modifying structured non-coding RNAs.

ADP-ribosyltransferases (ARTs) were among the first identified bacterial virulence factors. Canonical ART toxins are delivered into host cells where they modify essential proteins, thereby inactivating cellular processes and promoting pathogenesis. Our understanding of ARTs has since expanded beyond protein-targeting toxins to include antibiotic inactivation and DNA damage repair. Here, we report the discovery of RhsP2 as an ART toxin delivered between competing bacteria by a type VI secretion system of Pseudomonas aeruginosa. A structure of RhsP2 reveals that it resembles protein-targeting ARTs such as diphtheria toxin. Remarkably, however, RhsP2 ADP-ribosylates 2'-hydroxyl groups of double-stranded RNA, and thus, its activity is highly promiscuous with identified cellular targets including the tRNA pool and the RNA-processing ribozyme, ribonuclease P. Consequently, cell death arises from the inhibition of translation and disruption of tRNA processing. Overall, our data demonstrate a previously undescribed mechanism of bacterial antagonism and uncover an unprecedented activity catalyzed by ART enzymes.

Pandemic driven innovation: A pilot evaluation of an alternative respiratory pathogen collection device.

BACKGROUND: The nasopharyngeal swab is the gold standard collection method for COVID-19, but is invasive and painful, subsequently resulting in poor patient acceptance. This investigation explores the process of developing and validating an alternative respiratory pathogen collection device that relies on a nasopharyngeal irrigation mechanic. The primary objective was to determine if sufficient pathological sampling can be achieved by mechanism of nasopharyngeal irrigation that is proportionate to the nasopharyngeal swab method. METHODS: The study device was designed using Shapr3D modeling software and fabricated on a fused deposition modeling printer. Fifteen participants were enrolled with each receiving a saline nasopharyngeal washing using the study device. Specimen adequacy was evaluated by two real-time reverse transcriptase polymerase chain reaction (PCR) testing methods to identify the presence of the human RNase P gene. Results were evaluated quantitatively through interpretation of the PCR cycle threshold (Ct). RESULTS: All 15 specimens tested positive for the presence of RNaseP, demonstrating specimen cellularity, adequate extraction of nucleic acids, and the absence of inhibitors to amplification. The mean Ct value was 29.5 (Applied Biosystems TaqPath RT-qPCR) and 30.7 (NECoV19). All participants felt the study device irrigation procedure was faster than the nasopharyngeal swab, with none experiencing any discomfort from the irrigation mechanism. CONCLUSION: The importance of early diagnostic testing and its role in countermeasures for communicable diseases such as COVID-19 is well established in the literature. Innovation to bolster our testing infrastructure is more important now than ever. This study was successful in developing and validating an alternative nasopharyngeal respiratory pathogen collection device that utilizes fluid debridement as its core mechanic. Data from this pilot study demonstrated the study device was successful in producing high-quality specimens for PCR testing. Feedback from the study participants was also in favor of the study device when compared to the nasopharyngeal swab.

An improved, simple and field-deployable CRISPR-Cas12a assay for the detection of SARS-CoV-2.

AIMS: The RT-PCR is the most popular confirmatory test for SARS-CoV-2. It is sensitive, but high instrumentation cost makes it difficult for use outside routine clinical setup. This has necessitated the development of alternative methods such as CRISPR-based DETECTR method which uses lateral flow technology. Although accurate and sensitive, this method is limited by complex steps and recurrent cost of high-quality lateral flow strips. The main goal of this study was to improve the Cas12a-based SARS-CoV-2 DETECTR method and develop a portable and field-deployable system to reduce the recurring consumable cost. METHODS AND RESULTS: Specific regions of N and E genes from SARS-CoV-2 virus and human RNase P (internal control) were reverse transcribed (RT) and amplified by loop-mediated isothermal amplification (LAMP). The amplified products were detected by a Cas12a-based trans-cleavage reaction that generated a fluorescent signal which could be easily visualized by naked eye. Detection of internal control, RNase P gene was improved and optimized by redesigning RT-LAMP primers. A number of steps were reduced by combining the reagents related to the detection of Cas12a trans-cleavage reaction into a single ready-to-use mix. A portable, cost-effective battery-operated instrument, CRISPR-CUBE was developed to run the assay and visualize the outcome. The method and instrument were validated using both contrived and patient samples. CONCLUSIONS: The simplified CRISPR-based SARS-CoV-2 detection and instrument developed in this study, along with improved design for internal control detection allows for easier, more definitive viral detection requiring only reagents, consumables and the battery operable CRISPR-CUBE. SIGNIFICANCE AND IMPACT OF STUDY: Significant improvement in Cas12 method, coupled with simple visualization of end point makes the method and instrument deployable at the point-of-care (POC) for SARS-CoV-2 detection, without any recurrent cost for the lateral flow strips which is used in other POC methods.

RNA Design Principles for Riboswitches that Regulate RNase P-Mediated tRNA Processing.

Riboswitches are an attractive target for the directed design of RNA-based regulators by in silico prediction. These noncoding RNA elements consist of an aptamer platform for the highly selective ligand recognition and an expression platform which controls gene activity typically at the level of transcription or translation. In previous work, we could successfully apply RNA folding prediction to implement a new riboswitch mechanism regulating processing of a tRNA by RNase P. In this contribution, we present detailed information about our pipeline consisting of in silico design combined with the biochemical analysis for the verification of the implemented mechanism. Furthermore, we discuss the applicability of the presented biochemical in vivo and in vitro methods for the characterization of other artificial riboswitches.