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1.
Nature ; 630(8017): 769-776, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38718836

RESUMEN

Angiogenin, an RNase-A-family protein, promotes angiogenesis and has been implicated in cancer, neurodegenerative diseases and epigenetic inheritance1-10. After activation during cellular stress, angiogenin cleaves tRNAs at the anticodon loop, resulting in translation repression11-15. However, the catalytic activity of isolated angiogenin is very low, and the mechanisms of the enzyme activation and tRNA specificity have remained a puzzle3,16-23. Here we identify these mechanisms using biochemical assays and cryogenic electron microscopy (cryo-EM). Our study reveals that the cytosolic ribosome is the activator of angiogenin. A cryo-EM structure features angiogenin bound in the A site of the 80S ribosome. The C-terminal tail of angiogenin is rearranged by interactions with the ribosome to activate the RNase catalytic centre, making the enzyme several orders of magnitude more efficient in tRNA cleavage. Additional 80S-angiogenin structures capture how tRNA substrate is directed by the ribosome into angiogenin's active site, demonstrating that the ribosome acts as the specificity factor. Our findings therefore suggest that angiogenin is activated by ribosomes with a vacant A site, the abundance of which increases during cellular stress24-27. These results may facilitate the development of therapeutics to treat cancer and neurodegenerative diseases.


Asunto(s)
Microscopía por Crioelectrón , Ribonucleasa Pancreática , Ribosomas , Humanos , Anticodón/química , Anticodón/genética , Anticodón/metabolismo , Anticodón/ultraestructura , Dominio Catalítico , Citosol/metabolismo , Activación Enzimática , Modelos Moleculares , Ribonucleasa Pancreática/química , Ribonucleasa Pancreática/metabolismo , Ribonucleasa Pancreática/ultraestructura , Ribosomas/metabolismo , Ribosomas/química , Ribosomas/ultraestructura , División del ARN , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Especificidad por Sustrato , Sitios de Unión , Estrés Fisiológico
2.
Chembiochem ; 25(11): e202400085, 2024 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-38574237

RESUMEN

Over the last three decades, significant advancements have been made in the development of biosensors and bioassays that use RNA-cleaving DNAzymes (RCDs) as molecular recognition elements. While early examples of RCDs were primarily responsive to metal ions, the past decade has seen numerous RCDs reported for more clinically relevant targets such as bacteria, cancer cells, small metabolites, and protein biomarkers. Over the past 5 years several RCD-based biosensors have also been evaluated using either spiked biological matrixes or patient samples, including blood, serum, saliva, nasal mucus, sputum, urine, and faeces, which is a critical step toward regulatory approval and commercialization of such sensors. In this review, an overview of the methods used to generate RCDs and the properties of key RCDs that have been utilized for in vitro testing is first provided. Examples of RCD-based assays and sensors that have been used to test either spiked biological samples or patient samples are then presented, highlighting assay performance in different biological matrixes. A summary of current prospects and challenges for development of in vitro diagnostic tests incorporating RCDs and an overview of future directions of the field is also provided.


Asunto(s)
Técnicas Biosensibles , ADN Catalítico , ADN Catalítico/metabolismo , ADN Catalítico/química , Humanos , ARN/metabolismo , ARN/análisis , División del ARN
3.
Nucleic Acids Res ; 52(10): 5866-5879, 2024 Jun 10.
Artículo en Inglés | MEDLINE | ID: mdl-38661191

RESUMEN

Multivalent recognition and binding of biological molecules is a natural phenomenon that increases the binding stability (avidity) without decreasing the recognition specificity. In this study, we took advantage of this phenomenon to increase the efficiency and maintain high specificity of RNA cleavage by DNAzymes (Dz). We designed a series of DNA constructs containing two Dz agents, named here bivalent Dz devices (BDD). One BDD increased the cleavage efficiency of a folded RNA fragment up to 17-fold in comparison with the Dz of a conventional design. Such an increase was achieved due to both the improved RNA binding and the increased probability of RNA cleavage by the two catalytic cores. By moderating the degree of Dz agent association in BDD, we achieved excellent selectivity in differentiating single-base mismatched RNA, while maintaining relatively high cleavage rates. Furthermore, a trivalent Dz demonstrated an even greater efficiency than the BDD in cleaving folded RNA. The data suggests that the cooperative action of several RNA-cleaving units can significantly improve the efficiency and maintain high specificity of RNA cleavage, which is important for the development of Dz-based gene knockdown agents.


Asunto(s)
ADN Catalítico , División del ARN , Pliegue del ARN , ARN , ADN Catalítico/química , ADN Catalítico/metabolismo , Conformación de Ácido Nucleico , ARN/química , ARN/metabolismo , ADN/química , ADN/metabolismo , Ribonucleasa H/metabolismo , Sitios de Unión , Especificidad por Sustrato
4.
Nat Commun ; 15(1): 3324, 2024 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-38637512

RESUMEN

CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction1-5. Target RNA cleavage at regular intervals is characteristic of type III effector complexes6-8. Here, we determine the structures of the Synechocystis type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors9,10, in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2'-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes.


Asunto(s)
Proteínas Asociadas a CRISPR , ARN Catalítico , ARN/metabolismo , ARN Catalítico/metabolismo , Sistemas CRISPR-Cas/genética , ADN/metabolismo , Dominio Catalítico , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , División del ARN
5.
Sci Adv ; 10(17): eadl0164, 2024 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-38657076

RESUMEN

Type VI CRISPR-Cas systems are among the few CRISPR varieties that target exclusively RNA. The CRISPR RNA-guided, sequence-specific binding of target RNAs, such as phage transcripts, activates the type VI effector, Cas13. Once activated, Cas13 causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from the phage spread. We show here that the principal form of collateral RNA degradation elicited by Leptotrichia shahii Cas13a expressed in Escherichia coli cells is the cleavage of anticodons in a subset of transfer RNAs (tRNAs) with uridine-rich anticodons. This tRNA cleavage is accompanied by inhibition of protein synthesis, thus providing defense from the phages. In addition, Cas13a-mediated tRNA cleavage indirectly activates the RNases of bacterial toxin-antitoxin modules cleaving messenger RNA, which could provide a backup defense. The mechanism of Cas13a-induced antiphage defense resembles that of bacterial anticodon nucleases, which is compatible with the hypothesis that type VI effectors evolved from an abortive infection module encompassing an anticodon nuclease.


Asunto(s)
Anticodón , Sistemas CRISPR-Cas , Escherichia coli , ARN de Transferencia , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Anticodón/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Leptotrichia/genética , Leptotrichia/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/genética , Bacteriófagos/genética , División del ARN
6.
Nucleic Acids Res ; 52(8): 4483-4501, 2024 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-38587191

RESUMEN

Messenger RNA precursors (pre-mRNA) generally undergo 3' end processing by cleavage and polyadenylation (CPA), which is specified by a polyadenylation site (PAS) and adjacent RNA sequences and regulated by a large variety of core and auxiliary CPA factors. To date, most of the human CPA factors have been discovered through biochemical and proteomic studies. However, genetic identification of the human CPA factors has been hampered by the lack of a reliable genome-wide screening method. We describe here a dual fluorescence readthrough reporter system with a PAS inserted between two fluorescent reporters. This system enables measurement of the efficiency of 3' end processing in living cells. Using this system in combination with a human genome-wide CRISPR/Cas9 library, we conducted a screen for CPA factors. The screens identified most components of the known core CPA complexes and other known CPA factors. The screens also identified CCNK/CDK12 as a potential core CPA factor, and RPRD1B as a CPA factor that binds RNA and regulates the release of RNA polymerase II at the 3' ends of genes. Thus, this dual fluorescence reporter coupled with CRISPR/Cas9 screens reliably identifies bona fide CPA factors and provides a platform for investigating the requirements for CPA in various contexts.


Asunto(s)
Sistemas CRISPR-Cas , Genes Reporteros , Precursores del ARN , Factores de Escisión y Poliadenilación de ARNm , Humanos , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/genética , Genoma Humano , Células HEK293 , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Poliadenilación , División del ARN , ARN Polimerasa II/metabolismo , Precursores del ARN/metabolismo , Precursores del ARN/genética
7.
Nucleic Acids Res ; 52(7): 3896-3910, 2024 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-38340341

RESUMEN

The type III CRISPR-Cas effector complex Csm functions as a molecular Swiss army knife that provides multilevel defense against foreign nucleic acids. The coordinated action of three catalytic activities of the Csm complex enables simultaneous degradation of the invader's RNA transcripts, destruction of the template DNA and synthesis of signaling molecules (cyclic oligoadenylates cAn) that activate auxiliary proteins to reinforce CRISPR-Cas defense. Here, we employed single-molecule techniques to connect the kinetics of RNA binding, dissociation, and DNA hydrolysis by the Csm complex from Streptococcus thermophilus. Although single-stranded RNA is cleaved rapidly (within seconds), dual-color FCS experiments and single-molecule TIRF microscopy revealed that Csm remains bound to terminal RNA cleavage products with a half-life of over 1 hour while releasing the internal RNA fragments quickly. Using a continuous fluorescent DNA degradation assay, we observed that RNA-regulated single-stranded DNase activity decreases on a similar timescale. These findings suggest that after fast target RNA cleavage the terminal RNA cleavage products stay bound within the Csm complex, keeping the Cas10 subunit activated for DNA destruction. Additionally, we demonstrate that during Cas10 activation, the complex remains capable of RNA turnover, i.e. of ongoing degradation of target RNA.


Asunto(s)
Streptococcus thermophilus , Streptococcus thermophilus/genética , Streptococcus thermophilus/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Sistemas CRISPR-Cas , ARN/metabolismo , ARN/química , Proteínas Asociadas a CRISPR/metabolismo , ADN/metabolismo , ADN/química , ADN/genética , Cinética , División del ARN , Hidrólisis , Imagen Individual de Molécula , Unión Proteica
8.
Anal Chim Acta ; 1290: 342218, 2024 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-38246744

RESUMEN

BACKGROUND: Lead (Pb) is one of the most toxic heavy-metal pollutants. Additionally, lead ions (Pb2+) can accumulate in the human body through the food chain, causing irreversible damage through organ damage and system disorders. In the past few years, the detection of Pb2+ has mainly relied on instrumental methods such as atomic absorption spectroscopy (AAS) and inductively coupled plasma mass spectrometry (ICP-MS). Nonetheless, these techniques are complicated in terms of equipment and procedures, along with being time-intensive and expensive in terms of detection. These drawbacks have limited their wide application. Hence, there is a pressing need to develop detection techniques for Pb2+ that are not only cost-efficient but also highly sensitive and specific. RESULTS: A novel "on-off-on" electrochemiluminescence (ECL) sensor for detecting Pb2+ was developed based on the resonance energy transfer (RET) effect between AuNPs and boron nitride quantum dots (BN QDs) and the recognition of Pb2+ by DNAzyme along with the cleavage reaction of the substrate chain. Poly(6-carboxyindole)/stannic sulfide (P6ICA/SnS2) nanocomposite was employed as a co-reaction accelerator to consequently facilitate the production of intermediate SO4•-. This effective enhancement of the reaction led to an improved ECL intensity of BN QDs and enabled the sensor platform to exhibit a higher original ECL response. Benefiting from the combination of the DNAzyme signal amplification strategy with the "on-off-on" design, the ECL sensor showed satisfactory selectivity, good stability, and high sensitivity. This ECL sensor exhibited a linear detection range (LDR) of 10-12-10-5 M and a limit of detection (LOD) of 2.6 × 10-13 M. SIGNIFICANCE: In the present work, an "on-off-on" ECL sensor is constructed based on RET effect for ultrasensitive detection of Pb2+. P6ICA/SnS2 was investigated as the co-reaction accelerator in this sensor. Moreover, this ECL sensor exhibited excellent analytical capability for detecting Pb2+ in actual water samples, providing a method for detecting other heavy metal ions as well.


Asunto(s)
ADN Catalítico , Nanopartículas del Metal , Humanos , Oro , Plomo , División del ARN , Transferencia de Energía , Iones
9.
PLoS Genet ; 19(9): e1010930, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37729124

RESUMEN

Cas12g is an endonuclease belonging to the type V RNA-guided CRISPR-Cas family. It is known for its ability to cleave RNA substrates using a conserved endonuclease active site located in the RuvC domain. In this study, we determined the crystal structure of apo-Cas12g, the cryo-EM structure of the Cas12g-sgRNA binary complex and investigated conformational changes that occur during the transition from the apo state to the Cas12g-sgRNA binary complex. The conserved zinc finger motifs in Cas12g undergo an ordered-to-disordered transition from the apo to the sgRNA-bound state and their mutations negatively impact on target RNA cleavage. Moreover, we identified a lid motif in the RuvC domain that undergoes transformation from a helix to loop to regulate the access to the RuvC active site and subsequent cleavage of the RNA substrate. Overall, our study provides valuable insights into the mechanisms by which Cas12g recognizes sgRNA and the conformational changes it undergoes from sgRNA binding to the activation of the RNase active site, thereby laying a foundation for the potential repurposing of Cas12g as a tool for RNA-editing.


Asunto(s)
Endonucleasas , ARN Guía de Sistemas CRISPR-Cas , División del ARN , Endonucleasas/genética , Endorribonucleasas , ARN/genética
10.
J Med Virol ; 95(9): e29090, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37695079

RESUMEN

The widespread dissemination of coronavirus 2019 imposes a significant burden on society. Therefore, rapid detection facilitates the reduction of transmission risk. In this study, we proposed a multiplex diagnostic platform for the rapid, ultrasensitive, visual, and simultaneous detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) open reading frame 1ab (ORF1ab) and N genes. A visual diagnostic method was developed using a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a/Cas13a dual-enzyme digestion system integrated with multiplex reverse transcriptase-recombinase polymerase amplification (RT-RPA). Two CRISPR-Cas proteins (Cas12a and Cas13a) were introduced into the system to recognize and cleave the N gene and ORF1ab gene, respectively. We used fluorescent or CRISPR double digestion test strips to detect the digested products, with the N gene corresponding to the FAM channel in the PCR instrument or the T1 line on the test strip, and the ORF1ab gene corresponding to the ROX channel in the PCR instrument or the T2 line on the test strip. The analysis can be completed in less than 20 min. Meanwhile, we assessed the application of the platform and determined a sensitivity of up to 200 copies/mL. Additionally, dual gene validation in 105 clinical nasopharyngeal swab samples showed a 100% positive predictive value agreement and a 95.7% negative predictive value agreement between our method and quantitative reverse transcription-polymerase chain reaction. Overall, our method offered a novel insight into the rapid diagnosis of SARS-CoV-2.


Asunto(s)
Proteínas Bacterianas , COVID-19 , Proteínas Asociadas a CRISPR , Proteínas de la Nucleocápside de Coronavirus , Endodesoxirribonucleasas , Fosfoproteínas , Poliproteínas , SARS-CoV-2 , Proteínas Virales , División del ARN , División del ADN , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , COVID-19/diagnóstico , Proteínas Virales/genética , Poliproteínas/genética , Proteínas Asociadas a CRISPR/química , Proteínas Bacterianas/química , Endodesoxirribonucleasas/química , Proteínas de la Nucleocápside de Coronavirus/genética , Fosfoproteínas/genética , Humanos
11.
J Biol Chem ; 299(6): 104787, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-37149147

RESUMEN

Understanding the functional properties of severe acute respiratory syndrome coronavirus 2 nonstructural proteins is essential for defining their roles in the viral life cycle, developing improved therapeutics and diagnostics, and countering future variants. Coronavirus nonstructural protein Nsp15 is a hexameric U-specific endonuclease whose functions, substrate specificity, mechanism, and dynamics are not fully defined. Previous studies report that Nsp15 requires Mn2+ ions for optimal activity; however, the effects of divalent ions on Nsp15 reaction kinetics have not been investigated in detail. Here, we analyzed the single- and multiple-turnover kinetics for model ssRNA substrates. Our data confirm that divalent ions are dispensable for catalysis and show that Mn2+ activates Nsp15 cleavage of two different ssRNA oligonucleotide substrates but not a dinucleotide. Biphasic kinetics of ssRNA substrates demonstrates that Mn2+ stabilizes alternative enzyme states that have faster substrate cleavage on the enzyme. However, we did not detect Mn2+-induced conformational changes using CD and fluorescence spectroscopy. The pH-rate profiles in the presence and absence of Mn2+ reveal active-site ionizable groups with similar pKas of ca. 4.8 to 5.2. An Rp stereoisomer phosphorothioate modification at the scissile phosphate had minimal effect on catalysis supporting a mechanism involving an anionic transition state. However, the Sp stereoisomer is inactive because of weak binding, consistent with models that position the nonbridging phosphoryl oxygen deep in the active site. Together, these data demonstrate that Nsp15 employs a conventional acid-base catalytic mechanism passing through an anionic transition state, and that divalent ion activation is substrate dependent.


Asunto(s)
Endonucleasas , Iones , División del ARN , SARS-CoV-2 , Catálisis , COVID-19/microbiología , Endonucleasas/genética , Endonucleasas/metabolismo , Cinética , Metales/química , División del ARN/genética , SARS-CoV-2/enzimología , Iones/metabolismo , Activación Enzimática , Manganeso/química , Concentración de Iones de Hidrógeno , Animales , Ratones , Escherichia coli/genética
12.
Spectrochim Acta A Mol Biomol Spectrosc ; 298: 122787, 2023 Oct 05.
Artículo en Inglés | MEDLINE | ID: mdl-37150075

RESUMEN

Heavy metal pollution can pose a threat to food safety and human health, and accurate quantification of heavy metal ions is a vital requirement. Emerging DNA nanostructures-based biosensors offer attractive tools toward ultra-sensitive or rapid analysis of heavy metal ions. However, the problems including complex design, severe reaction conditions and undesirable reliability are inevitable obstacle in advancing their extension and application. Herein, a ratiometric fluorescent platform was established for monitoring lead ion (Pb2+) in food based on dual Förster resonance energy transfer (FRET) and RNA cleavage-inhibited self-assembly of three-arm branched junction (TBJ). GR-5 DNAzyme was employed for Pb2+ recognition, and enzyme-free amplification technique catalytic hairpin assembly (CHA) served to form FRET probes-carried TBJ. The substrate strand (S) of DNAzyme triggered the generation of CHA-TBJ, and Pb2+-responsive cleavage of S hindered the assembly of CHA-TBJ, causing opposite changes in the FRET states of FAM/BHQ1 and ROX/BHQ2 pairs. The fluorescence responses were recorded through synchronous fluorescence spectrometry to indicate Pb2+ concentration, allowing sensitive and reliable identification of Pb2+ in the linear range of 0.05-5 ng mL-1 with the detection limit of 0.03 ng mL-1. The Pb2+ detection can be achieved under conventional reaction conditions, simple mixing procedures and one-step measurement operation. The approach can afford excellent specificity for Pb2+ against competing metal ions, and can be applied to analyze Pb2+ in tea samples with satisfactory results. This facile fluorescence platform shows a capable method for Pb2+ detection, and provides new avenue in the development of ratiometric approaches and DNAzyme strategies for monitoring heavy metal pollution, facilitating the transformation of DNAzyme-based biosensors for food safety control.


Asunto(s)
Técnicas Biosensibles , ADN Catalítico , Humanos , ADN Catalítico/química , Plomo , División del ARN , Reproducibilidad de los Resultados , Iones , Técnicas Biosensibles/métodos , Límite de Detección
13.
Science ; 380(6643): 410-415, 2023 04 28.
Artículo en Inglés | MEDLINE | ID: mdl-37104586

RESUMEN

Type VI CRISPR-Cas systems use RNA-guided ribonuclease (RNase) Cas13 to defend bacteria against viruses, and some of these systems encode putative membrane proteins that have unclear roles in Cas13-mediated defense. We show that Csx28, of type VI-B2 systems, is a transmembrane protein that assists to slow cellular metabolism upon viral infection, increasing antiviral defense. High-resolution cryo-electron microscopy reveals that Csx28 forms an octameric pore-like structure. These Csx28 pores localize to the inner membrane in vivo. Csx28's antiviral activity in vivo requires sequence-specific cleavage of viral messenger RNAs by Cas13b, which subsequently results in membrane depolarization, slowed metabolism, and inhibition of sustained viral infection. Our work suggests a mechanism by which Csx28 acts as a downstream, Cas13b-dependent effector protein that uses membrane perturbation as an antiviral defense strategy.


Asunto(s)
Proteínas Bacterianas , Bacteriófagos , Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Endodesoxirribonucleasas , Prevotella , División del ARN , ARN Viral , Microscopía por Crioelectrón , Proteínas de la Membrana/metabolismo , ARN Viral/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/metabolismo , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/metabolismo , Bacteriófagos/metabolismo , Bacteriófago lambda/metabolismo , Escherichia coli/enzimología , Escherichia coli/virología , Prevotella/enzimología , Prevotella/virología
14.
mSphere ; 8(2): e0011923, 2023 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-36897078

RESUMEN

When humans experience a new, devastating viral infection such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), significant challenges arise. How should individuals as well as societies respond to the situation? One of the primary questions concerns the origin of the SARS-CoV-2 virus that infected and was transmitted efficiently among humans, resulting in a pandemic. At first glance, the question appears straightforward to answer. However, the origin of SARS-CoV-2 has been the topic of substantial debate primarily because we do not have access to some relevant data. At least two major hypotheses have been suggested: a natural origin through zoonosis followed by sustained human-to-human spread or the introduction of a natural virus into humans from a laboratory source. Here, we summarize the scientific evidence that informs this debate to provide our fellow scientists and the public with the tools to join the discussion in a constructive and informed manner. Our goal is to dissect the evidence to make it more accessible to those interested in this important problem. The engagement of a broad representation of scientists is critical to ensure that the public and policy-makers can draw on relevant expertise in navigating this controversy.


Asunto(s)
COVID-19 , Pandemias , SARS-CoV-2 , Zoonosis Virales , Humanos , COVID-19/etiología , COVID-19/transmisión , COVID-19/virología , SARS-CoV-2/genética , Zoonosis Virales/etiología , Zoonosis Virales/transmisión , Zoonosis Virales/virología , Furina/metabolismo , División del ARN/genética , Genoma Viral , Quirópteros/virología , Animales
15.
ChemMedChem ; 18(7): e202300040, 2023 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-36734637

RESUMEN

Therapeutic nucleic acid agents (TNA) can be activated by a marker RNA sequence followed by initiation of targeted RNA cleavage. This property can be used in conditional cell suppression, e. g., cancer marker-dependent cell death. However, healthy cells often express lower levels of cancer markers, thus jeopardizing TNA activation exclusively in cancer cells. Earlier, we developed a conditionally activated split deoxyribozyme construct (DNA thresholder or DTh) that can be activated by high but not by low concentrations of cancer markers. It's activity, however, was suppressed by very high marker concentrations. Herein, we combine the DTh functional units in a single DNA association (Thresholding DNA nanomachine or Th-DNM). Th-DNM maintains a high level of RNA cleavage activity in the presence of marker concentrations above the threshold level. Th-DNM differentiated fully complementary miR17 markers sequence from double base mismatched miR-20. Th-DNM can become a building block of DNA nanorobots for cancer treatment.


Asunto(s)
ADN Catalítico , ADN Catalítico/metabolismo , División del ARN , ADN
16.
Chemistry ; 29(27): e202300075, 2023 May 11.
Artículo en Inglés | MEDLINE | ID: mdl-36790320

RESUMEN

A new method for the detection of genomic RNA combines RNA cleavage by the 10-23 DNAzyme and use of the cleavage fragments as primers to initiate rolling circle amplification (RCA). 230 different 10-23 DNAzyme variants were screened to identify those that target accessible RNA sites within the highly structured RNA transcripts of SARS-CoV-2. A total of 28 DNAzymes were identified with >20 % cleavage, 5 with >40 % cleavage and one with >60 % in 10 min. The cleavage fragments from these reactions were then screened for coupling to an RCA reaction, leading to the identification of several cleavage fragments that could efficiently initiate RCA. Using a newly developed quasi-exponential RCA method with a detection limit of 500 aM of RNA, 14 RT-PCR positive and 15 RT-PCR negative patient saliva samples were evaluated for SARS-CoV-2 genomic RNA, achieving a clinical sensitivity of 86 % and specificity of 100 % for detection of the virus in <2.5 h.


Asunto(s)
Técnicas Biosensibles , COVID-19 , ADN Catalítico , Humanos , ADN Catalítico/metabolismo , ARN , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , División del ARN , COVID-19/diagnóstico , Técnicas de Amplificación de Ácido Nucleico/métodos , Genómica , Técnicas Biosensibles/métodos
17.
J Chem Theory Comput ; 19(4): 1322-1332, 2023 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-36753428

RESUMEN

RNA strand cleavage by 2'-O-transphosphorylation is catalyzed not only by numerous nucleolytic RNA enzymes (ribozymes) but also by hydroxide or hydronium ions. In experiments, both cleavage of the 5'-linked nucleoside and isomerization between 3',5'- and 2',5'-phosphodiesters occur under acidic conditions, while only the cleavage reaction is observed under basic conditions. An ab initio path-integral approach for simulating kinetic isotope effects is used to reveal the reaction mechanisms for RNA cleavage and isomerization reactions under acidic conditions. Moreover, the proposed mechanisms can also be combined through the experimental pH-rate profiles.


Asunto(s)
ARN Catalítico , ARN , Isomerismo , División del ARN , Nucleósidos , Cinética , Catálisis
18.
J Mater Chem B ; 11(7): 1568-1579, 2023 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-36722940

RESUMEN

Herein, we designed a novel and highly sensitive fluorescence multicomponent detachable platform for MDA-MB-231 breast cancer cell detection as a model. The RNA cleavage DNAzyme was used as a central operator of the multicomponent probe through which compilation and induced detachment of probe was done. During the compilation step, the dsDNA-Sybr green 1 complexes on gold nanoparticles (GNP@dsDNA@SG1) were assembled. The intercalated Sybr green in the DNA structure has been used as an amplified signal generator on one site of DNAzyme and magnetic nanoparticles (MNP) act as a biological carrier and probe collector on the opposite side. The enzyme activator co-factor (MDA-MB-231 cell cytoplasmic protein) provokes the activation of the catalytic core of enzyme sequence in the DNAzyme molecule, followed by cleavage reaction in the substrate sequence and releasing GNP@ dsDNA@SG1 into the solution. The results indicate that the Sybr green emission fluorescence (520 nm) increases with the increment of MDA-MB-231 protein concentration in the linear dynamic range of 8.10 × 10-2 to 1.95 ng ml-1 (0.77 × 10-3-0.019 cell ml-1) with a detection limit (LOD) of 1/72 × 10-2 pg ml-1 under optimal conditions. The proposed immunosensor has great potential in developing ultrasensitive and rapid diagnostic platforms.


Asunto(s)
Técnicas Biosensibles , Neoplasias de la Mama , ADN Catalítico , Nanopartículas del Metal , Humanos , Femenino , ADN Catalítico/química , Biomarcadores de Tumor , Oro , Técnicas Biosensibles/métodos , División del ARN , Neoplasias de la Mama/diagnóstico , Inmunoensayo , ADN/química , ARN
19.
Nat Microbiol ; 8(3): 522-532, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36702942

RESUMEN

The recently discovered type III-E CRISPR-Cas effector Cas7-11 shows promise when used as an RNA manipulation tool, but its structure and the mechanisms underlying its function remain unclear. Here we present four cryo-EM structures of Desulfonema ishimotonii Cas7-11-crRNA complex in pre-target and target RNA-bound states, and the cryo-EM structure of DiCas7-11-crRNA bound to its accessory protein DiCsx29. These data reveal structural elements for pre-crRNA processing, target RNA cleavage and regulation. Moreover, a 3' seed region of crRNA is involved in regulating RNA cleavage activity of DiCas7-11-crRNA-Csx29. Our analysis also shows that both the minimal mismatch of 4 nt to the 5' handle of crRNA and the minimal matching of the first 12 nt of the spacer by the target RNA are essential for triggering the protease activity of DiCas7-11-crRNA-Csx29 towards DiCsx30. Taken together, we propose that target RNA recognition and cleavage regulate and fine-tune the protease activity of DiCas7-11-crRNA-Csx29, thus preventing auto-immune responses.


Asunto(s)
Sistemas CRISPR-Cas , ARN , Microscopía por Crioelectrón , ARN/genética , División del ARN , ARN Guía de Sistemas CRISPR-Cas , Péptido Hidrolasas/genética
20.
Cell Rep ; 41(4): 111533, 2022 10 25.
Artículo en Inglés | MEDLINE | ID: mdl-36288702

RESUMEN

Argonaute proteins are widespread in prokaryotes and eukaryotes with diversified catalytic activities. Here, we describe an Argonaute from Marinitoga hydrogenitolerans (MhAgo) with all eight cleavage activities. Utilization of all four types of guides and efficient cleavage of single-stranded DNA (ssDNA) and RNA targets are revealed. The preference for the 5'-terminus nucleotides of 5'P guides, but no obvious preferences for that in 5'OH guides, is further uncovered. Moreover, the cleavage efficiency is heavily impaired by mismatches in the central and 3'-supplementary regions of guides, and the affinity between guides or guides/target duplex and MhAgo is proved as one of the factors affecting cleavage efficiency. Structural and mutational analyses imply some unknown distinctive structural features behind the cleavage activity of MhAgo. Meanwhile, 5'OH-guide RNA (gRNA)-mediated plasmid cleavage activity is unveiled. Conclusively, MhAgo is versatile, and its biochemical characteristics improve our understanding of pAgos and the pAgo-based techniques.


Asunto(s)
Proteínas Argonautas , ARN Guía de Kinetoplastida , Proteínas Argonautas/metabolismo , ARN Guía de Kinetoplastida/genética , ARN , ADN de Cadena Simple , División del ARN , ADN/metabolismo , Nucleótidos/metabolismo
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