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Pesquisa : D08.811.277.352.355.350.700 [Categoria DeCS]
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  1 / 2283 MEDLINE  
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[PMID]:29281624
[Au] Autor:Lafuente-Barquero J; Luke-Glaser S; Graf M; Silva S; Gómez-González B; Lockhart A; Lisby M; Aguilera A; Luke B
[Ad] Endereço:Andalusian Center for Molecular Biology and Regenerative Medicine-CABIMER, Universidad de Sevilla-CSIC-Universidad Pablo de Olavide, Avda. Americo Vespucio 24, Seville, Spain.
[Ti] Título:The Smc5/6 complex regulates the yeast Mph1 helicase at RNA-DNA hybrid-mediated DNA damage.
[So] Source:PLoS Genet;13(12):e1007136, 2017 12.
[Is] ISSN:1553-7404
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:RNA-DNA hybrids are naturally occurring obstacles that must be overcome by the DNA replication machinery. In the absence of RNase H enzymes, RNA-DNA hybrids accumulate, resulting in replication stress, DNA damage and compromised genomic integrity. We demonstrate that Mph1, the yeast homolog of Fanconi anemia protein M (FANCM), is required for cell viability in the absence of RNase H enzymes. The integrity of the Mph1 helicase domain is crucial to prevent the accumulation of RNA-DNA hybrids and RNA-DNA hybrid-dependent DNA damage, as determined by Rad52 foci. Mph1 forms foci when RNA-DNA hybrids accumulate, e.g. in RNase H or THO-complex mutants and at short telomeres. Mph1, however is a double-edged sword, whose action at hybrids must be regulated by the Smc5/6 complex. This is underlined by the observation that simultaneous inactivation of RNase H2 and Smc5/6 results in Mph1-dependent synthetic lethality, which is likely due to an accumulation of toxic recombination intermediates. The data presented here support a model, where Mph1's helicase activity plays a crucial role in responding to persistent RNA-DNA hybrids.
[Mh] Termos MeSH primário: Proteínas de Ciclo Celular/genética
RNA Helicases DEAD-box/genética
RNA Helicases DEAD-box/metabolismo
Dano ao DNA
RNA Fúngico/genética
Proteínas de Saccharomyces cerevisiae/genética
Proteínas de Saccharomyces cerevisiae/metabolismo
[Mh] Termos MeSH secundário: Proteínas de Ciclo Celular/metabolismo
DNA/metabolismo
Reparo do DNA
Replicação do DNA/genética
Replicação do DNA/fisiologia
RNA Helicases/metabolismo
RNA Fúngico/metabolismo
Ribonuclease H/genética
Saccharomyces cerevisiae/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Cell Cycle Proteins); 0 (RNA, Fungal); 0 (SMC5 protein, S cerevisiae); 0 (SMC6 protein, S cerevisiae); 0 (Saccharomyces cerevisiae Proteins); 9007-49-2 (DNA); EC 3.1.26.4 (Ribonuclease H); EC 3.6.1.- (MPH1 protein, S cerevisiae); EC 3.6.4.13 (DEAD-box RNA Helicases); EC 3.6.4.13 (RNA Helicases)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180227
[Lr] Data última revisão:
180227
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171228
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pgen.1007136


  2 / 2283 MEDLINE  
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[PMID]:28450058
[Au] Autor:Edwards TC; Lomonosova E; Patel JA; Li Q; Villa JA; Gupta AK; Morrison LA; Bailly F; Cotelle P; Giannakopoulou E; Zoidis G; Tavis JE
[Ad] Endereço:Department of Molecular Microbiology and Immunology and Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, MO, USA; Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, MO, USA. Electronic address: edwardstc@slu.edu.
[Ti] Título:Inhibition of hepatitis B virus replication by N-hydroxyisoquinolinediones and related polyoxygenated heterocycles.
[So] Source:Antiviral Res;143:205-217, 2017 07.
[Is] ISSN:1872-9096
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:We previously reported low sensitivity of the hepatitis B virus (HBV) ribonuclease H (RNaseH) enzyme to inhibition by N-hydroxyisoquinolinedione (HID) compounds. Subsequently, our biochemical RNaseH assay was found to have a high false negative rate for predicting HBV replication inhibition, leading to underestimation of the number of HIDs that inhibit HBV replication. Here, 39 HID compounds and structurally related polyoxygenated heterocycles (POH), N-hydroxypyridinediones (HPD), and flutimides were screened for inhibition of HBV replication in vitro. Inhibiting the HBV RNaseH preferentially blocks synthesis of the positive-polarity DNA strand and causes accumulation of RNA:DNA heteroduplexes. Eleven HIDs and one HPD preferentially inhibited HBV positive-polarity DNA strand accumulation. EC s ranged from 0.69 µM to 19 µM with therapeutic indices from 2.4 to 71. Neither the HIDs nor the HPD had an effect on the ability of the polymerase to elongate DNA strands in capsids. HBV RNaseH inhibition by the HIDs was confirmed with an improved RNaseH assay and by detecting accumulation RNA:DNA heteroduplexes in HBV capsids from cells treated with a representative HID. Therefore, the HID scaffold is more promising for anti-HBV drug discovery than we originally reported, and the HPD scaffold may hold potential for antiviral development. The preliminary structure-activity relationship will guide optimization of the HID/HPDs as HBV inhibitors.
[Mh] Termos MeSH primário: Antivirais/antagonistas & inibidores
Antivirais/química
Vírus da Hepatite B/efeitos dos fármacos
Replicação Viral/efeitos dos fármacos
[Mh] Termos MeSH secundário: Animais
Antivirais/administração & dosagem
Proteínas do Capsídeo/genética
Linhagem Celular Tumoral
Cercopithecus aethiops
Replicação do DNA/efeitos dos fármacos
DNA Viral/efeitos dos fármacos
Descoberta de Drogas
Avaliação Pré-Clínica de Medicamentos
Hepatite B/virologia
Vírus da Hepatite B/enzimologia
Vírus da Hepatite B/genética
Vírus da Hepatite B/fisiologia
Seres Humanos
Testes de Sensibilidade Microbiana
Piperazinas/farmacologia
Ribonuclease H/efeitos dos fármacos
Relação Estrutura-Atividade
Células Vero
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T; RESEARCH SUPPORT, N.I.H., EXTRAMURAL
[Nm] Nome de substância:
0 (Antiviral Agents); 0 (Capsid Proteins); 0 (DNA, Viral); 0 (Piperazines); 162666-34-4 (flutimide); EC 3.1.26.4 (Ribonuclease H); EC 3.1.26.4 (ribonuclease HI)
[Em] Mês de entrada:1712
[Cu] Atualização por classe:180213
[Lr] Data última revisão:
180213
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170429
[St] Status:MEDLINE


  3 / 2283 MEDLINE  
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[PMID]:27777304
[Au] Autor:Li A; Gong S; Johnson KA
[Ad] Endereço:From the University of Texas at Austin, Institute for Cell and Molecular Biology, Department of Molecular Biosciences, Austin, Texas 78712.
[Ti] Título:Rate-limiting Pyrophosphate Release by HIV Reverse Transcriptase Improves Fidelity.
[So] Source:J Biol Chem;291(51):26554-26565, 2016 Dec 16.
[Is] ISSN:1083-351X
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Previous measurements of the rates of polymerization and pyrophosphate release with DNA templates showed that pyrophosphate (PP ) dissociation was fast after nucleotide incorporation so that it did not contribute to enzyme specificity (k /K ). Here, kinetic parameters governing nucleotide incorporation and PP release were determined using an RNA template. Compared with a DNA template of the same sequence, the rate of chemistry increased by up to 10-fold (250 versus 24 s ), whereas the rate of PP release decreased to approximately 58 s so that PP release became the rate-limiting step. During processive nucleotide incorporation, the first nucleotide (TTP) was incorporated at a fast rate (152 s ), whereas the rates of incorporation of remaining nucleotides (CGTCG) were much slower with an average rate of 24 s , suggesting that sequential incorporation events were limited by the relatively slow PP release step. The accompanying paper shows that slow PP release allows polymerization and RNase H to occur at comparable rates. Although PP release is the rate-determining step, it is not the specificity-determining step for correct incorporation based on our current estimates of the rate of reversal of the chemistry step (3 s ). In contrast, during misincorporation, PP release became extremely slow, which we estimated to be ∼0.002 s These studies establish the mechanistic basis for DNA polymerase fidelity during reverse transcription and provide a free energy profile. We correct previous underestimates of discrimination by including the slow PP release step. Our current estimate of 2.4 × 10 is >20-fold greater than estimated previously.
[Mh] Termos MeSH primário: Difosfatos/química
Transcriptase Reversa do HIV/química
HIV-1/enzimologia
Ribonuclease H/química
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Diphosphates); 4E862E7GRQ (diphosphoric acid); EC 2.7.7.- (reverse transcriptase, Human immunodeficiency virus 1); EC 2.7.7.49 (HIV Reverse Transcriptase); EC 3.1.26.4 (Ribonuclease H)
[Em] Mês de entrada:1705
[Cu] Atualização por classe:171216
[Lr] Data última revisão:
171216
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161026
[St] Status:MEDLINE


  4 / 2283 MEDLINE  
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[PMID]:27777303
[Au] Autor:Li A; Li J; Johnson KA
[Ad] Endereço:From the The University of Texas at Austin, Institute for Cell and Molecular Biology, Department of Molecular Biosciences, Austin, Texas 78712.
[Ti] Título:HIV-1 Reverse Transcriptase Polymerase and RNase H (Ribonuclease H) Active Sites Work Simultaneously and Independently.
[So] Source:J Biol Chem;291(51):26566-26585, 2016 Dec 16.
[Is] ISSN:1083-351X
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:HIV reverse transcriptase plays a central role in viral replication and requires coordination of both polymerase and RNase H activities. Although this coordination is crucial in viral replication, whether a DNA/RNA hybrid can simultaneously engage both active sites has yet to be determined as structural and kinetic analyses have provided contradictory results. Single nucleotide incorporation and RNase H cleavage were examined using presteady-state kinetics with global data analysis. The results revealed three interconverting reverse transcriptase-DNA/RNA species; 43% were active for both sites simultaneously, 27% showed only polymerase activity, and the remaining 30% were nonproductive. Our data clearly demonstrated that the DNA/RNA hybrid could contact both active sites simultaneously, although the single nucleotide incorporation (105 s ) was ∼5-fold faster than the cleavage (23 s ). By using a series of primers with different lengths, we found that a string of at least 4-6 nucleotides downstream of the cleaving site was required for efficient RNA cleavage. This was corroborated by our observations that during processive nucleotide incorporation, sequential rounds of RNA cleavage occurred each time after ∼6 nucleotides were incorporated. More importantly, during processive primer extension, pyrophosphate (PP ) release was rate-limiting so that the average rate of nucleotide incorporation (∼28 s ) was comparable with that of net RNA cleavage (∼27 nucleotides(s)). Although polymerization is efficient and processive, RNase H is inefficient and periodic. This combination allows the two catalytic centers of HIVRT to work simultaneously at similar speeds without being tightly coupled.
[Mh] Termos MeSH primário: DNA/química
Transcriptase Reversa do HIV/química
HIV-1/enzimologia
RNA/química
Ribonuclease H/química
[Mh] Termos MeSH secundário: Domínio Catalítico
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
63231-63-0 (RNA); 9007-49-2 (DNA); EC 2.7.7.- (reverse transcriptase, Human immunodeficiency virus 1); EC 2.7.7.49 (HIV Reverse Transcriptase); EC 3.1.26.4 (Ribonuclease H)
[Em] Mês de entrada:1705
[Cu] Atualização por classe:171216
[Lr] Data última revisão:
171216
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161026
[St] Status:MEDLINE


  5 / 2283 MEDLINE  
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[PMID]:28977560
[Au] Autor:Shen W; Sun H; De Hoyos CL; Bailey JK; Liang XH; Crooke ST
[Ad] Endereço:Department of Core Antisense Research, Ionis Pharmaceuticals, Inc., 2855 Gazelle Court, Carlsbad, CA 92010, USA.
[Ti] Título:Dynamic nucleoplasmic and nucleolar localization of mammalian RNase H1 in response to RNAP I transcriptional R-loops.
[So] Source:Nucleic Acids Res;45(18):10672-10692, 2017 Oct 13.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:An R-loop is a DNA:RNA hybrid formed during transcription when a DNA duplex is invaded by a nascent RNA transcript. R-loops accumulate in nucleoli during RNA polymerase I (RNAP I) transcription. Here, we report that mammalian RNase H1 enriches in nucleoli and co-localizes with R-loops in cultured human cells. Co-migration of RNase H1 and R-loops from nucleoli to perinucleolar ring structures was observed upon inhibition of RNAP I transcription. Treatment with camptothecin which transiently stabilized nucleolar R-loops recruited RNase H1 to the nucleoli. It has been reported that the absence of Topoisomerase and RNase H activity in Escherichia coli or Saccharomyces cerevisiae caused R-loop accumulation along rDNA. We found that the distribution of RNase H1 and Top1 along rDNA coincided at sites where R-loops accumulated in mammalian cells. Loss of either RNase H1 or Top1 caused R-loop accumulation, and the accumulation of R-loops was exacerbated when both proteins were depleted. Importantly, we observed that protein levels of Top1 were negatively correlated with the abundance of RNase H1. We conclude that Top1 and RNase H1 are partially functionally redundant in mammalian cells to suppress RNAP I transcription-associate R-loops.
[Mh] Termos MeSH primário: Nucléolo Celular/genética
DNA Ribossômico/química
RNA Polimerase I/metabolismo
Ribonuclease H/análise
Transcrição Genética
[Mh] Termos MeSH secundário: Animais
Camptotecina/farmacologia
Nucléolo Celular/efeitos dos fármacos
Nucléolo Celular/enzimologia
Dano ao DNA
DNA Topoisomerases Tipo I/análise
DNA Ribossômico/metabolismo
Células HEK293
Células HeLa
Seres Humanos
Camundongos Knockout
Domínios Proteicos
RNA/química
RNA Polimerase I/análise
Ribonuclease H/química
Ribonuclease H/metabolismo
Transcrição Genética/efeitos dos fármacos
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (DNA, Ribosomal); 63231-63-0 (RNA); EC 2.7.7.- (RPA194 protein, human); EC 2.7.7.6 (RNA Polymerase I); EC 3.1.26.4 (Ribonuclease H); EC 3.1.26.4 (ribonuclease HI); EC 5.99.1.2 (DNA Topoisomerases, Type I); EC 5.99.1.2 (TOP1 protein, human); XT3Z54Z28A (Camptothecin)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171107
[Lr] Data última revisão:
171107
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171005
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx710


  6 / 2283 MEDLINE  
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[PMID]:28934489
[Au] Autor:Liang XH; Sun H; Shen W; Wang S; Yao J; Migawa MT; Bui HH; Damle SS; Riney S; Graham MJ; Crooke RM; Crooke ST
[Ad] Endereço:Department of Core Antisense Research, Ionis Pharmaceuticals, Inc., Carlsbad, CA, USA.
[Ti] Título:Antisense oligonucleotides targeting translation inhibitory elements in 5' UTRs can selectively increase protein levels.
[So] Source:Nucleic Acids Res;45(16):9528-9546, 2017 Sep 19.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:A variety of diseases are caused by deficiencies in amounts or activity of key proteins. An approach that increases the amount of a specific protein might be of therapeutic benefit. We reasoned that translation could be specifically enhanced using trans-acting agents that counter the function of negative regulatory elements present in the 5' UTRs of some mRNAs. We recently showed that translation can be enhanced by antisense oligonucleotides (ASOs) that target upstream open reading frames. Here we report the amount of a protein can also be selectively increased using ASOs designed to hybridize to other translation inhibitory elements in 5' UTRs. Levels of human RNASEH1, LDLR, and ACP1 and of mouse ACP1 and ARF1 were increased up to 2.7-fold in different cell types and species upon treatment with chemically modified ASOs targeting 5' UTR inhibitory regions in the mRNAs encoding these proteins. The activities of ASOs in enhancing translation were sequence and position dependent and required helicase activity. The ASOs appear to improve the recruitment of translation initiation factors to the target mRNA. Importantly, ASOs targeting ACP1 mRNA significantly increased the level of ACP1 protein in mice, suggesting that this approach has therapeutic and research potentials.
[Mh] Termos MeSH primário: Regiões 5´ não Traduzidas
Oligonucleotídeos Antissenso/farmacologia
Proteínas Tirosina Fosfatases/genética
Proteínas Proto-Oncogênicas/genética
Receptores de LDL/genética
Ribonuclease H/genética
[Mh] Termos MeSH secundário: Animais
Seres Humanos
Lipoproteínas LDL/farmacocinética
Masculino
Camundongos Endogâmicos BALB C
Oligonucleotídeos Antissenso/química
Fases de Leitura Aberta
Biossíntese de Proteínas
RNA Mensageiro/química
Receptores de LDL/metabolismo
Ribonuclease H/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (5' Untranslated Regions); 0 (LDLR protein, human); 0 (Lipoproteins, LDL); 0 (Oligonucleotides, Antisense); 0 (Proto-Oncogene Proteins); 0 (RNA, Messenger); 0 (Receptors, LDL); EC 3.1.26.4 (Ribonuclease H); EC 3.1.26.4 (ribonuclease HI); EC 3.1.3.48 (ACP1 protein, human); EC 3.1.3.48 (Acp1 protein, mouse); EC 3.1.3.48 (Protein Tyrosine Phosphatases)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171024
[Lr] Data última revisão:
171024
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170922
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx632


  7 / 2283 MEDLINE  
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[PMID]:28911100
[Au] Autor:Lee N; Le Sage V; Nanni AV; Snyder DJ; Cooper VS; Lakdawala SS
[Ad] Endereço:University of Pittsburgh School of Medicine, Department of Microbiology and Molecular Genetics, 450 Technology Drive, Pittsburgh, PA 15219, USA.
[Ti] Título:Genome-wide analysis of influenza viral RNA and nucleoprotein association.
[So] Source:Nucleic Acids Res;45(15):8968-8977, 2017 Sep 06.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Influenza A virus (IAV) genomes are composed of eight single-stranded RNA segments that are coated by viral nucleoprotein (NP) molecules. Classically, the interaction between NP and viral RNA (vRNA) is depicted as a uniform pattern of 'beads on a string'. Using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP), we identified the vRNA binding profiles of NP for two H1N1 IAV strains in virions. Contrary to the prevailing model for vRNA packaging, NP does not bind vRNA uniformly in the A/WSN/1933 and A/California/07/2009 strains, but instead each vRNA segment exhibits a unique binding profile, containing sites that are enriched or poor in NP association. Intriguingly, both H1N1 strains have similar yet distinct NP binding profiles despite extensive sequence conservation. Peaks identified by HITS-CLIP were verified as true NP binding sites based on insensitivity to DNA antisense oligonucleotide-mediated RNase H digestion. Moreover, nucleotide content analysis of NP peaks revealed that these sites are relatively G-rich and U-poor compared to the genome-wide nucleotide content, indicating an as-yet unidentified sequence bias for NP association in vivo. Taken together, our genome-wide study of NP-vRNA interaction has implications for the understanding of influenza vRNA architecture and genome packaging.
[Mh] Termos MeSH primário: Genoma Viral
Vírus da Influenza A Subtipo H1N1/genética
Nucleoproteínas/química
RNA Viral/química
Proteínas Virais/química
Vírion/genética
[Mh] Termos MeSH secundário: Sequência de Bases
Sítios de Ligação
Sequência Conservada
Expressão Gênica
Sequenciamento de Nucleotídeos em Larga Escala
Vírus da Influenza A Subtipo H1N1/metabolismo
Vírus da Influenza A Subtipo H1N1/ultraestrutura
Modelos Moleculares
Nucleoproteínas/genética
Nucleoproteínas/metabolismo
Oligonucleotídeos Antissenso/química
Ligação Proteica
RNA Viral/genética
RNA Viral/metabolismo
Ribonuclease H/química
Proteínas Virais/genética
Proteínas Virais/metabolismo
Vírion/metabolismo
Vírion/ultraestrutura
Montagem de Vírus/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Nucleoproteins); 0 (Oligonucleotides, Antisense); 0 (RNA, Viral); 0 (Viral Proteins); EC 3.1.26.4 (Ribonuclease H)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171116
[Lr] Data última revisão:
171116
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170916
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx584


  8 / 2283 MEDLINE  
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[PMID]:28854734
[Au] Autor:Mutisya D; Hardcastle T; Cheruiyot SK; Pallan PS; Kennedy SD; Egli M; Kelley ML; Smith AVB; Rozners E
[Ad] Endereço:Department of Chemistry, Binghamton University, The State University of New York, Binghamton, NY 13902, USA.
[Ti] Título:Amide linkages mimic phosphates in RNA interactions with proteins and are well tolerated in the guide strand of short interfering RNAs.
[So] Source:Nucleic Acids Res;45(14):8142-8155, 2017 Aug 21.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:While the use of RNA interference (RNAi) in molecular biology and functional genomics is a well-established technology, in vivo applications of synthetic short interfering RNAs (siRNAs) require chemical modifications. We recently found that amides as non-ionic replacements for phosphodiesters may be useful modifications for optimization of siRNAs. Herein, we report a comprehensive study of systematic replacement of a single phosphate with an amide linkage throughout the guide strand of siRNAs. The results show that amides are surprisingly well tolerated in the seed and central regions of the guide strand and increase the silencing activity when placed between nucleosides 10 and 12, at the catalytic site of Argonaute. A potential explanation is provided by the first crystal structure of an amide-modified RNA-DNA with Bacillus halodurans RNase H1. The structure reveals how small changes in both RNA and protein conformation allow the amide to establish hydrogen bonding interactions with the protein. Molecular dynamics simulations suggest that these alternative binding modes may compensate for interactions lost due to the absence of a phosphodiester moiety. Our results suggest that an amide can mimic important hydrogen bonding interactions with proteins required for RNAi activity and may be a promising modification for optimization of biological properties of siRNAs.
[Mh] Termos MeSH primário: Amidas/química
Fosfatos/química
RNA Interferente Pequeno/química
Ribonuclease H/química
[Mh] Termos MeSH secundário: Amidas/metabolismo
Sequência de Bases
Cristalografia por Raios X
Seres Humanos
Simulação de Dinâmica Molecular
Conformação de Ácido Nucleico
Fosfatos/metabolismo
Ligação Proteica
Estrutura Terciária de Proteína
Interferência de RNA
RNA Interferente Pequeno/genética
RNA Interferente Pequeno/metabolismo
Ribonuclease H/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Amides); 0 (Phosphates); 0 (RNA, Small Interfering); EC 3.1.26.4 (Ribonuclease H)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171023
[Lr] Data última revisão:
171023
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170901
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx558


  9 / 2283 MEDLINE  
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[PMID]:28854731
[Au] Autor:Credle JJ; Itoh CY; Yuan T; Sharma R; Scott ER; Workman RE; Fan Y; Housseau F; Llosa NJ; Bell WR; Miller H; Zhang SX; Timp W; Larman HB
[Ad] Endereço:Division of Immunology, Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
[Ti] Título:Multiplexed analysis of fixed tissue RNA using Ligation in situ Hybridization.
[So] Source:Nucleic Acids Res;45(14):e128, 2017 Aug 21.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Clinical tissues are prepared for histological analysis and long-term storage via formalin fixation and paraffin embedding (FFPE). The FFPE process results in fragmentation and chemical modification of RNA, rendering it less suitable for analysis by techniques that rely on reverse transcription (RT) such as RT-qPCR and RNA-Seq. Here we describe a broadly applicable technique called 'Ligation in situ Hybridization' ('LISH'), which is an alternative methodology for the analysis of FFPE RNA. LISH utilizes the T4 RNA Ligase 2 to efficiently join adjacent chimeric RNA-DNA probe pairs hybridized in situ on fixed RNA target sequences. Subsequent treatment with RNase H releases RNA-templated ligation products into solution for downstream analysis. We demonstrate several unique advantages of LISH-based assays using patient-derived FFPE tissue. These include >100-plex capability, compatibility with common histochemical stains and suitability for analysis of decade-old materials and exceedingly small microdissected tissue fragments. High-throughput DNA sequencing modalities, including single molecule sequencing, can be used to analyze ligation products from complex panels of LISH probes ('LISH-seq'), which can be amplified efficiently and with negligible bias. LISH analysis of FFPE RNA is a novel methodology with broad applications that range from multiplexed gene expression analysis to the sensitive detection of infectious organisms.
[Mh] Termos MeSH primário: Hibridização In Situ/métodos
Inclusão em Parafina/métodos
RNA/genética
Fixação de Tecidos/métodos
[Mh] Termos MeSH secundário: Perfilação da Expressão Gênica/métodos
Sequenciamento de Nucleotídeos em Larga Escala/métodos
Seres Humanos
Microscopia de Fluorescência
RNA/análise
RNA/metabolismo
RNA Ligase (ATP)/metabolismo
Reação em Cadeia da Polimerase em Tempo Real
Reprodutibilidade dos Testes
Ribonuclease H/metabolismo
Proteínas Virais/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Viral Proteins); 63231-63-0 (RNA); EC 3.1.26.4 (Ribonuclease H); EC 6.5.1.3 (RNA Ligase (ATP)); EC 6.5.1.3 (bacteriophage T4 RNA ligase 2)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171023
[Lr] Data última revisão:
171023
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170901
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx471


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[PMID]:28829437
[Au] Autor:Iwamoto N; Butler DCD; Svrzikapa N; Mohapatra S; Zlatev I; Sah DWY; Meena; Standley SM; Lu G; Apponi LH; Frank-Kamenetsky M; Zhang JJ; Vargeese C; Verdine GL
[Ad] Endereço:Wave Life Sciences, Cambridge, Massachusetts, USA.
[Ti] Título:Control of phosphorothioate stereochemistry substantially increases the efficacy of antisense oligonucleotides.
[So] Source:Nat Biotechnol;35(9):845-851, 2017 Sep.
[Is] ISSN:1546-1696
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Whereas stereochemical purity in drugs has become the standard for small molecules, stereoisomeric mixtures containing as many as a half million components persist in antisense oligonucleotide (ASO) therapeutics because it has been feasible neither to separate the individual stereoisomers, nor to synthesize stereochemically pure ASOs. Here we report the development of a scalable synthetic process that yields therapeutic ASOs having high stereochemical and chemical purity. Using this method, we synthesized rationally designed stereopure components of mipomersen, a drug comprising 524,288 stereoisomers. We demonstrate that phosphorothioate (PS) stereochemistry substantially affects the pharmacologic properties of ASOs. We report that Sp-configured PS linkages are stabilized relative to Rp, providing stereochemical protection from pharmacologic inactivation of the drug. Further, we elucidated a triplet stereochemical code in the stereopure ASOs, 3'-SpSpRp, that promotes target RNA cleavage by RNase H1 in vitro and provides a more durable response in mice than stereorandom ASOs.
[Mh] Termos MeSH primário: Terapia Genética/métodos
Oligonucleotídeos Antissenso/química
Oligonucleotídeos Antissenso/farmacocinética
Oligonucleotídeos Fosforotioatos/química
[Mh] Termos MeSH secundário: Animais
Estabilidade de Medicamentos
Feminino
Seres Humanos
Interações Hidrofóbicas e Hidrofílicas
Masculino
Camundongos
Camundongos Endogâmicos C57BL
Camundongos Transgênicos
Oligonucleotídeos
Oligonucleotídeos Antissenso/uso terapêutico
Ratos
Ratos Sprague-Dawley
Ribonuclease H/metabolismo
Estereoisomerismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Oligonucleotides); 0 (Oligonucleotides, Antisense); 0 (Phosphorothioate Oligonucleotides); 9GJ8S4GU0M (mipomersen); EC 3.1.26.4 (Ribonuclease H); EC 3.1.26.4 (ribonuclease HI)
[Em] Mês de entrada:1709
[Cu] Atualização por classe:170927
[Lr] Data última revisão:
170927
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170823
[St] Status:MEDLINE
[do] DOI:10.1038/nbt.3948



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