RESUMO
RNA-RNA interactions are essential for biology, but they can be difficult to study due to their transient nature. While crosslinking strategies can in principle be used to trap such interactions, virtually all existing strategies for crosslinking are poorly reversible, chemically modifying the RNA and hindering molecular analysis. We describe a soluble crosslinker design (BINARI) that reacts with RNA through acylation. We show that it efficiently crosslinks noncovalent RNA complexes with mimimal sequence bias and establish that the crosslink can be reversed by phosphine reduction of azide trigger groups, thereby liberating the individual RNA components for further analysis. The utility of the new approach is demonstrated by reversible protection against nuclease degradation and trapping transient RNA complexes of E. coli DsrA-rpoS derived bulge-loop interactions, which underlines the potential of BINARI crosslinkers to probe RNA regulatory networks.
Assuntos
Azidas/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , Escherichia coli/química , Fosfinas/metabolismo , RNA Bacteriano/metabolismo , Acilação , Azidas/química , Reagentes de Ligações Cruzadas/química , Escherichia coli/metabolismo , Fosfinas/química , RNA Bacteriano/químicaRESUMO
Recent advances in SHAPE technology have converted the classic primer extension method to next-generation sequencing platforms, allowing transcriptome-level analysis of RNA secondary structure. In particular, icSHAPE and SHAPE-MaP, using NAI-N3 and 1M7 reagents, respectively, are methods that claim to measure in vivo structure with high-throughput sequencing. However, these compounds have not been compared on an unbiased, raw-signal level. Here, we directly compare several in vivo SHAPE acylation reagents using the simple primer extension assay. We conclude that while multiple SHAPE technologies are effective at measuring purified RNAs in vitro, acylimidazole reagents NAI and NAI-N3 give markedly greater signals with lower background than 1M7 for in vivo measurement of the RNA structurome.
Assuntos
Bioensaio/normas , Imidazóis/química , Oxazinas/química , RNA/química , Acilação , Animais , Escherichia coli/genética , Escherichia coli/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Indicadores e Reagentes , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Nitrosaminas/química , Conformação de Ácido Nucleico , Cultura Primária de CélulasRESUMO
We describe a selective and mild chemical approach for controlling RNA hybridization, folding, and enzyme interactions. Reaction of RNAs in aqueous buffer with an azide-substituted acylating agent (100-200â mm) yields several 2'-OH acylations per RNA strand in as little as 10â min. This poly-acylated ("cloaked") RNA is strongly blocked from hybridization with complementary nucleic acids, from cleavage by RNA-processing enzymes, and from folding into active aptamer structures. Importantly, treatment with a water-soluble phosphine triggers a Staudinger reduction of the azide groups, resulting in spontaneous loss of acyl groups ("uncloaking"). This fully restores RNA folding and biochemical activity.
Assuntos
Azidas/farmacologia , RNA/efeitos dos fármacos , Acilação/efeitos dos fármacos , Azidas/antagonistas & inibidores , Azidas/química , Estrutura Molecular , Fosfinas/química , Fosfinas/farmacologia , Dobramento de RNA/efeitos dos fármacosRESUMO
A bone imaging toolkit of 21 fluorescent probes with variable spectroscopic properties, bone mineral binding affinities, and antiprenylation activities has been created, including a novel linking strategy. The linking chemistry allows attachment of a diverse selection of dyes fluorescent in the visible to near-infrared range to any of the three clinically important heterocyclic bisphosphonate bone drugs (risedronate, zoledronate, and minodronate or their analogues). The resultant suite of conjugates offers multiple options to "mix and match" parent drug structure, fluorescence emission wavelength, relative bone affinity, and presence or absence of antiprenylation activity, for bone-related imaging applications.
Assuntos
Doenças Ósseas/diagnóstico , Osso e Ossos/patologia , Difosfonatos/química , Corantes Fluorescentes/química , Imagem Óptica/métodos , Animais , Linhagem Celular , Humanos , Masculino , Ratos Sprague-DawleyRESUMO
SAMHD1 is a GTP-activated nonspecific dNTP triphosphohydrolase that depletes dNTP pools in resting CD4+ T cells and macrophages and effectively restricts infection by HIV-1. We have designed a nonsubstrate dUTP analogue with a methylene bridge connecting the α phosphate and 5' carbon that potently inhibits SAMHD1. Although pppCH2dU shows apparent competitive inhibition, it acts by a surprising allosteric mechanism that destabilizes active enzyme tetramer.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Proteínas Monoméricas de Ligação ao GTP/antagonistas & inibidores , Desenho de Fármacos , Ativação Enzimática/efeitos dos fármacos , Guanosina Trifosfato/farmacologia , Proteínas Monoméricas de Ligação ao GTP/química , Proteína 1 com Domínio SAM e Domínio HD , Bibliotecas de Moléculas PequenasRESUMO
Although single-nucleotide variants (SNVs) make up the majority of cancer-associated genetic changes and have been comprehensively catalogued, little is known about their impact on tumor initiation and progression. To enable the functional interrogation of cancer-associated SNVs, we developed a mouse system for temporal and regulatable in vivo base editing. The inducible base editing (iBE) mouse carries a single expression-optimized cytosine base editor transgene under the control of a tetracycline response element and enables robust, doxycycline-dependent expression across a broad range of tissues in vivo. Combined with plasmid-based or synthetic guide RNAs, iBE drives efficient engineering of individual or multiple SNVs in intestinal, lung and pancreatic organoids. Temporal regulation of base editor activity allows controlled sequential genome editing ex vivo and in vivo, and delivery of sgRNAs directly to target tissues facilitates generation of in situ preclinical cancer models.
Assuntos
Edição de Genes , Neoplasias , Camundongos , Animais , Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas , Neoplasias/genética , Neoplasias/terapia , PulmãoRESUMO
Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36 kilobases at multiple genomic loci across three human cell lines, primary T cells and non-dividing primary human hepatocytes. To augment PASTE, we discovered 25,614 serine integrases and cognate attachment sites from metagenomes and engineered orthologs with higher activity and shorter recognition sequences for efficient programmable integration. PASTE has editing efficiencies similar to or exceeding those of homology-directed repair and non-homologous end joining-based methods, with activity in non-dividing cells and in vivo with fewer detectable off-target events. PASTE expands the capabilities of genome editing by allowing large, multiplexed gene insertion without reliance on DNA repair pathways.
Assuntos
Sistemas CRISPR-Cas , Integrases , Humanos , Sistemas CRISPR-Cas/genética , Clivagem do DNA , Edição de Genes , DNA/genética , Reparo do DNA por Junção de Extremidades/genéticaRESUMO
The influence of water: crystallization of (R/S)-α,ß-CHF-dATP with the preorganized pol ß-DNA complex shows that (S)-α,ß-CHF-dATP is preferentially bound to the active site with the C=F fluorine proximal to a structural water bound to Asp276.
Assuntos
DNA Polimerase beta/química , Nucleotídeos de Desoxiadenina/química , Cristalografia por Raios X , DNA Polimerase beta/metabolismo , Nucleotídeos de Desoxiadenina/metabolismo , Modelos Moleculares , Estrutura Molecular , EstereoisomerismoRESUMO
Intramolecular 1,3-dipolar cycloaddition of alkoxycarbonyl-substituted azomethine ylides to ester carbonyl was realized for the first time in the reaction of imines of O-acylsalicylic aldehyde with ethyl diazoacetate in the presence of Cu(tfacac)(2). The stereoselectivity of the cycloaddition is explained using DFT calculations.
RESUMO
RNA-targeting CRISPR-Cas13 proteins have recently emerged as a powerful platform to modulate gene expression outcomes. However, protein and CRISPR RNA (crRNA) delivery in human cells can be challenging with rapid crRNA degradation yielding transient knockdown. Here we compare several chemical RNA modifications at different positions to identify synthetic crRNAs that improve RNA targeting efficiency and half-life in human cells. We show that co-delivery of modified crRNAs and recombinant Cas13 enzyme in ribonucleoprotein (RNP) complexes can alter gene expression in primary CD4+ and CD8+ T cells. This system represents a robust and efficient method to modulate transcripts without genetic manipulation.
Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , RNA Guia de Cinetoplastídeos/genética , Células Cultivadas , Edição de Genes , Humanos , RNA Guia de Cinetoplastídeos/síntese química , RNA Guia de Cinetoplastídeos/químicaRESUMO
A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Antivirais/farmacologia , Humanos , Lipossomos , Nanopartículas , SARS-CoV-2/genéticaRESUMO
BACKGROUND: Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice over a TFBS called a CArG box in the Tspan2 promoter. RESULTS: Quantitative RT-PCR showed loss of Tspan2 mRNA in aorta and bladder, but not heart or brain, of mice homozygous for an HDR-mediated three base pair substitution in the Tspan2 CArG box. Using the same protospacer, mice homozygous for a PE2-mediated single-base substitution in the Tspan2 CArG box displayed similar cell-specific loss of Tspan2 mRNA; expression of an overlapping long noncoding RNA was also nearly abolished in aorta and bladder. Immuno-RNA fluorescence in situ hybridization validated loss of Tspan2 in vascular smooth muscle cells of HDR and PE2 CArG box mutant mice. Targeted sequencing demonstrated variable frequencies of on-target editing in all PE2 and HDR founders. However, whereas no on-target indels were detected in any of the PE2 founders, all HDR founders showed varying levels of on-target indels. Off-target analysis by targeted sequencing revealed mutations in many HDR founders, but none in PE2 founders. CONCLUSIONS: PE2 directs high-fidelity editing of a single base in a TFBS leading to cell-specific loss in expression of an mRNA/long noncoding RNA gene pair. The PE2 platform expands the genome editing toolbox for modeling and correcting relevant noncoding SNVs in the mouse.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Regulação da Expressão Gênica , Mutação Puntual , Animais , Sequência de Bases , Sítios de Ligação , Imunofluorescência/métodos , Edição de Genes/métodos , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas , Ligação Proteica , Reparo de DNA por Recombinação , Tetraspaninas/genéticaRESUMO
Most known pathogenic point mutations in humans are Câ¢G to Tâ¢A substitutions, which can be directly repaired by adenine base editors (ABEs). In this study, we investigated the efficacy and safety of ABEs in the livers of mice and cynomolgus macaques for the reduction of blood low-density lipoprotein (LDL) levels. Lipid nanoparticle-based delivery of mRNA encoding an ABE and a single-guide RNA targeting PCSK9, a negative regulator of LDL, induced up to 67% editing (on average, 61%) in mice and up to 34% editing (on average, 26%) in macaques. Plasma PCSK9 and LDL levels were stably reduced by 95% and 58% in mice and by 32% and 14% in macaques, respectively. ABE mRNA was cleared rapidly, and no off-target mutations in genomic DNA were found. Re-dosing in macaques did not increase editing, possibly owing to the detected humoral immune response to ABE upon treatment. These findings support further investigation of ABEs to treat patients with monogenic liver diseases.
Assuntos
Adenina , LDL-Colesterol , Edição de Genes/métodos , Pró-Proteína Convertase 9/genética , Animais , LDL-Colesterol/sangue , LDL-Colesterol/genética , Fígado/metabolismo , Macaca , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Following introduction of CRISPR-Cas9 components into a cell, genome editing occurs unabated until degradation of its component nucleic acids and proteins by cellular processes. This uncontrolled reaction can lead to unintended consequences including off-target editing and chromosomal translocations. To address this, we develop a method for light-induced degradation of sgRNA termed CRISPRoff. Here we show that light-induced inactivation of ribonucleoprotein attenuates genome editing within cells and allows for titratable levels of editing efficiency and spatial patterning via selective illumination.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Luz , Estabilidade de RNA/efeitos da radiação , RNA Guia de Cinetoplastídeos/metabolismo , Sistemas CRISPR-Cas/efeitos da radiação , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Estudos de Viabilidade , Células HEK293 , Humanos , RNA Guia de Cinetoplastídeos/efeitos da radiação , Ribonucleoproteínas/metabolismo , Translocação GenéticaRESUMO
Novel α,ß-CH2 and ß,γ-NH (1a) or α,ß-NH and ß,γ-CH2 (1b) "Met-Im" dTTPs were synthesized via monodemethylation of triethyl-dimethyl phosphorimido-bisphosphonate synthons (4a, 4b), formed via a base-induced [1,3]-rearrangement of precursors (3a, 3b) in a reaction with dimethyl or diethyl phosphochloridate. Anomerization during final bromotrimethylsilane (BTMS) deprotection after Mitsunobu conjugation with dT was avoided by microwave conditions. 1a was 9-fold more potent in inhibiting DNA polymerase ß, attributed to an NH-group interaction with R183 in the active site.