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1.
RNA ; 28(2): 227-238, 2022 02.
Article in English | MEDLINE | ID: covidwho-1533393

ABSTRACT

The Bacillus subtilis genome is predicted to encode numerous ribonucleases, including four 3' exoribonucleases that have been characterized to some extent. A strain containing gene knockouts of all four known 3' exoribonucleases is viable, suggesting that one or more additional RNases remain to be discovered. A protein extract from the quadruple RNase mutant strain was fractionated and RNase activity was followed, resulting in the identification of an enzyme activity catalyzed by the YloC protein. YloC is an endoribonuclease and is a member of the highly conserved "YicC family" of proteins that is widespread in bacteria. YloC is a metal-dependent enzyme that catalyzes the cleavage of single-stranded RNA, preferentially at U residues, and exists in an oligomeric form, most likely a hexamer. As such, YloC shares some characteristics with the SARS-CoV Nsp15 endoribonuclease. While the in vivo function of YloC in B. subtilis is yet to be determined, YloC was found to act similarly to YicC in an Escherichia coli in vivo assay that assesses decay of the small RNA, RyhB. Thus, YloC may play a role in small RNA regulation.


Subject(s)
Bacillus subtilis/genetics , Endoribonucleases/genetics , Endoribonucleases/metabolism , Bacillus subtilis/enzymology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Endoribonucleases/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial , Microorganisms, Genetically-Modified , Mutation , RNA Stability , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , Ribonucleases/genetics , Ribonucleases/metabolism , Substrate Specificity , Viral Nonstructural Proteins/metabolism
2.
Nat Commun ; 12(1): 5033, 2021 08 19.
Article in English | MEDLINE | ID: covidwho-1366816

ABSTRACT

Characteristic properties of type III CRISPR-Cas systems include recognition of target RNA and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of the target RNA and production of cyclic oligoadenylate (cOA) molecules. Here we report that an exposed seed region at the 3' end of the crRNA is essential for target RNA binding and cleavage, whereas cOA production requires base pairing at the 5' end of the crRNA. Moreover, we uncover that the variation in the size and composition of type III complexes within a single host results in variable seed regions. This may prevent escape by invading genetic elements, while controlling cOA production tightly to prevent unnecessary damage to the host. Lastly, we use these findings to develop a new diagnostic tool, SCOPE, for the specific detection of SARS-CoV-2 from human nasal swab samples, revealing sensitivities in the atto-molar range.


Subject(s)
Adenine Nucleotides/chemistry , COVID-19/diagnosis , CRISPR-Associated Proteins/metabolism , CRISPR-Cas Systems , Oligoribonucleotides/chemistry , RNA, Bacterial/genetics , Ribonucleases/metabolism , SARS-CoV-2/genetics , COVID-19/genetics , COVID-19/metabolism , COVID-19/virology , Diagnostic Tests, Routine/methods , Humans , SARS-CoV-2/isolation & purification , SARS-CoV-2/pathogenicity
3.
Brief Bioinform ; 22(2): 1215-1224, 2021 03 22.
Article in English | MEDLINE | ID: covidwho-1343625

ABSTRACT

The pandemic of coronavirus disease 2019 (COVID-19) urgently calls for more sensitive molecular diagnosis to improve sensitivity of current viral nuclear acid detection. We have developed an anchor primer (AP)-based assay to improve viral RNA stability by bioinformatics identification of RNase-binding site of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and implementing AP dually targeting the N gene of SARS-CoV-2 RNA and RNase 1, 3, 6. The arbitrarily primed polymerase chain reaction (AP-PCR) improvement of viral RNA integrity was supported by (a) the AP increased resistance of the targeted gene (N gene) of SARS-CoV-2 RNA to RNase treatment; (b) the detection of SARS-CoV-2 RNA by AP-PCR with lower cycle threshold values (-2.7 cycles) compared to two commercially available assays; (c) improvement of the viral RNA stability of the ORF gene upon targeting of the N gene and RNase. Furthermore, the improved sensitivity by AP-PCR was demonstrated by detection of SARS-CoV-2 RNA in 70-80% of sputum, nasal, pharyngeal swabs and feces and 36% (4/11) of urine of the confirmed cases (n = 252), 7% convalescent cases (n = 54) and none of 300 negative cases. Lastly, AP-PCR analysis of 306 confirmed and convalescent cases revealed prolonged presence of viral loading for >20 days after the first positive diagnosis. Thus, the AP dually targeting SARS-CoV-2 RNA and RNase improves molecular detection by preserving SARS-CoV-2 RNA integrity and reveals the prolonged viral loading associated with older age and male gender in COVID-19 patients.


Subject(s)
COVID-19/virology , Polymerase Chain Reaction/methods , Ribonucleases/metabolism , SARS-CoV-2/metabolism , Aged , Binding Sites , Female , Humans , Male , RNA, Viral/genetics , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Viral Load
4.
Microbiome ; 9(1): 25, 2021 01 22.
Article in English | MEDLINE | ID: covidwho-1043251

ABSTRACT

BACKGROUND: Determining the role of fomites in the transmission of SARS-CoV-2 is essential in the hospital setting and will likely be important outside of medical facilities as governments around the world make plans to ease COVID-19 public health restrictions and attempt to safely reopen economies. Expanding COVID-19 testing to include environmental surfaces would ideally be performed with inexpensive swabs that could be transported safely without concern of being a source of new infections. However, CDC-approved clinical-grade sampling supplies and techniques using a synthetic swab are expensive, potentially expose laboratory workers to viable virus and prohibit analysis of the microbiome due to the presence of antibiotics in viral transport media (VTM). To this end, we performed a series of experiments comparing the diagnostic yield using five consumer-grade swabs (including plastic and wood shafts and various head materials including cotton, synthetic, and foam) and one clinical-grade swab for inhibition to RNA. For three of these swabs, we evaluated performance to detect SARS-CoV-2 in twenty intensive care unit (ICU) hospital rooms of patients including COVID-19+ patients. All swabs were placed in 95% ethanol and further evaluated in terms of RNase activity. SARS-CoV-2 was measured both directly from the swab and from the swab eluent. RESULTS: Compared to samples collected in VTM, 95% ethanol demonstrated significant inhibition properties against RNases. When extracting directly from the swab head as opposed to the eluent, RNA recovery was approximately 2-4× higher from all six swab types tested as compared to the clinical standard of testing the eluent from a CDC-approved synthetic (SYN) swab. The limit of detection (LoD) of SARS-CoV-2 from floor samples collected using the consumer-grade plastic (CGp) or research-grade plastic The Microsetta Initiative (TMI) swabs was similar or better than the SYN swab, further suggesting that swab type does not impact RNA recovery as measured by the abundance of SARS-CoV-2. The LoD for TMI was between 0 and 362.5 viral particles, while SYN and CGp were both between 725 and 1450 particles. Lastly microbiome analyses (16S rRNA gene sequencing) of paired samples (nasal and floor from same patient room) collected using different swab types in triplicate indicated that microbial communities were not impacted by swab type, but instead driven by the patient and sample type. CONCLUSIONS: Compared to using a clinical-grade synthetic swab, detection of SARS-CoV-2 from environmental samples collected from ICU rooms of patients with COVID was similar using consumer-grade swabs, stored in 95% ethanol. The yield was best from the swab head rather than the eluent and the low level of RNase activity and lack of antibiotics in these samples makes it possible to perform concomitant microbiome analyses. Video abstract.


Subject(s)
COVID-19 Nucleic Acid Testing/instrumentation , COVID-19 Nucleic Acid Testing/methods , Microbiota , RNA, Viral/analysis , SARS-CoV-2/isolation & purification , Specimen Handling/methods , Biological Transport , Ethanol/chemistry , Feasibility Studies , Humans , Intensive Care Units , Limit of Detection , RNA, Ribosomal, 16S/genetics , RNA, Viral/genetics , Ribonucleases/metabolism
5.
Pharmacol Rev ; 72(4): 862-898, 2020 10.
Article in English | MEDLINE | ID: covidwho-767668

ABSTRACT

RNA-based therapies, including RNA molecules as drugs and RNA-targeted small molecules, offer unique opportunities to expand the range of therapeutic targets. Various forms of RNAs may be used to selectively act on proteins, transcripts, and genes that cannot be targeted by conventional small molecules or proteins. Although development of RNA drugs faces unparalleled challenges, many strategies have been developed to improve RNA metabolic stability and intracellular delivery. A number of RNA drugs have been approved for medical use, including aptamers (e.g., pegaptanib) that mechanistically act on protein target and small interfering RNAs (e.g., patisiran and givosiran) and antisense oligonucleotides (e.g., inotersen and golodirsen) that directly interfere with RNA targets. Furthermore, guide RNAs are essential components of novel gene editing modalities, and mRNA therapeutics are under development for protein replacement therapy or vaccination, including those against unprecedented severe acute respiratory syndrome coronavirus pandemic. Moreover, functional RNAs or RNA motifs are highly structured to form binding pockets or clefts that are accessible by small molecules. Many natural, semisynthetic, or synthetic antibiotics (e.g., aminoglycosides, tetracyclines, macrolides, oxazolidinones, and phenicols) can directly bind to ribosomal RNAs to achieve the inhibition of bacterial infections. Therefore, there is growing interest in developing RNA-targeted small-molecule drugs amenable to oral administration, and some (e.g., risdiplam and branaplam) have entered clinical trials. Here, we review the pharmacology of novel RNA drugs and RNA-targeted small-molecule medications, with a focus on recent progresses and strategies. Challenges in the development of novel druggable RNA entities and identification of viable RNA targets and selective small-molecule binders are discussed. SIGNIFICANCE STATEMENT: With the understanding of RNA functions and critical roles in diseases, as well as the development of RNA-related technologies, there is growing interest in developing novel RNA-based therapeutics. This comprehensive review presents pharmacology of both RNA drugs and RNA-targeted small-molecule medications, focusing on novel mechanisms of action, the most recent progress, and existing challenges.


Subject(s)
RNA/drug effects , RNA/pharmacology , Aptamers, Nucleotide/pharmacology , Aptamers, Nucleotide/therapeutic use , Betacoronavirus , COVID-19 , Chemistry Techniques, Analytical/methods , Chemistry Techniques, Analytical/standards , Clustered Regularly Interspaced Short Palindromic Repeats , Coronavirus Infections/drug therapy , Drug Delivery Systems/methods , Drug Development/organization & administration , Drug Discovery , Humans , MicroRNAs/pharmacology , MicroRNAs/therapeutic use , Oligonucleotides, Antisense/pharmacology , Oligonucleotides, Antisense/therapeutic use , Pandemics , Pneumonia, Viral/drug therapy , RNA/adverse effects , RNA, Antisense/pharmacology , RNA, Antisense/therapeutic use , RNA, Guide/pharmacology , RNA, Guide/therapeutic use , RNA, Messenger/drug effects , RNA, Messenger/pharmacology , RNA, Ribosomal/drug effects , RNA, Ribosomal/pharmacology , RNA, Small Interfering/pharmacology , RNA, Small Interfering/therapeutic use , RNA, Viral/drug effects , Ribonucleases/metabolism , Riboswitch/drug effects , SARS-CoV-2
6.
Curr Opin Struct Biol ; 65: 175-183, 2020 12.
Article in English | MEDLINE | ID: covidwho-752884

ABSTRACT

Many viruses, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and Human Immunodeficiency Virus (HIV), use RNA as their genetic material. How viruses harness RNA structure and RNA-protein interactions to control their replication remains obscure. Recent advances in the characterization of HIV-1 reverse transcriptase, the enzyme that converts its single-stranded RNA genome into a double-stranded DNA copy, reveal how the reverse transcription complex evolves during initiation. Here we highlight these advances in HIV-1 structural biology and discuss how they are furthering our understanding of HIV and related ribonucleoprotein complexes implicated in viral disease.


Subject(s)
HIV-1/genetics , Reverse Transcription , Drug Design , HIV-1/drug effects , RNA, Transfer/genetics , Reverse Transcription/drug effects , Ribonucleases/metabolism
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