RESUMO
The infectious disease coronavirus 2019 (SARS-CoV-2) is caused by a virus that has RNA as its genetic material. To understand the detailed structural features of SARS-COV-2 RNA, we probed the RNA structure by NMR. Two RNA sequences form a duplex and self-associate to form a dimeric G-quadruplex. The FrG nucleoside was employed as a 19F sensor to confirm the RNA structure in cells by 19F NMR. A FRET assay further demonstrated that the dimeric G-quadruplex resulted in RNA dimerization in cells. These results provide the basis for the elucidation of SARS-COV-2 RNA function, which provides new insights into developing novel antiviral drugs against SARS-COV-2.
Assuntos
COVID-19 , Quadruplex G , Humanos , SARS-CoV-2 , RNA Viral/genética , DimerizaçãoRESUMO
Zalpha (Zα) domains bind to left-handed Z-DNA and Z-RNA. The Zα domain protein family includes cellular (ADAR1, ZBP1 and PKZ) and viral (vaccinia virus E3 and cyprinid herpesvirus 3 (CyHV-3) ORF112) proteins. We studied CyHV-3 ORF112, which contains an intrinsically disordered region and a Zα domain. Genome editing of CyHV-3 indicated that the expression of only the Zα domain of ORF112 was sufficient for normal viral replication in cell culture and virulence in carp. In contrast, its deletion was lethal for the virus. These observations revealed the potential of the CyHV-3 model as a unique platform to compare the exchangeability of Zα domains expressed alone in living cells. Attempts to rescue the ORF112 deletion by a broad spectrum of cellular, viral, and artificial Zα domains showed that only those expressing Z-binding activity, the capacity to induce liquid-liquid phase separation (LLPS), and A-to-Z conversion, could rescue viral replication. For the first time, this study reports the ability of some Zα domains to induce LLPS and supports the biological relevance of dsRNA A-to-Z conversion mediated by Zα domains. This study expands the functional diversity of Zα domains and stimulates new hypotheses concerning the mechanisms of action of proteins containing Zα domains.
Assuntos
DNA Forma Z , Herpesviridae , Animais , Adenosina Desaminase/metabolismo , Herpesviridae/genética , Herpesviridae/metabolismo , RNA de Cadeia Dupla , Carpas/virologiaRESUMO
This protocol describes a method for the preparation of ribonucleoside phosphoramidite bearing a bioreductive protecting group on the 2'-OH group and its application in the synthesis of bioreduction-responsive oligonucleotides. The protecting group used in this method consists of the modified 4-nitrobenzyl skeleton, which has gem-dimethyl groups at benzylic positions to enable deprotection under physiological conditions. Applying the synthesized ribonucleoside phosphoramidite to solid-phase synthesis of oligonucleotides, a 2'-O-protected oligonucleotide was obtained without any undesirable cleavages under standard oligonucleotide synthesis conditions. The 2'-O-protected oligonucleotide was then treated with a combination of nitroreductase (Escherichia coli) and NADH as a bioreduction system for cleavage of the 2'-O-protecting group. After reduction of the nitro group, the protecting group was deprotected in a time-dependent manner. Thus, this protection technology is a potential new tool for production of reduction-responsive RNA-based materials that can be used in life and medical sciences. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of ribonucleoside phosphoramidite bearing a bioreductive protecting group Basic Protocol 2: Synthesis of 2'-O-protected oligonucleotides and their deprotection properties under bioreduction.
Assuntos
Ribonucleosídeos , Técnicas de Síntese em Fase Sólida , Nitrorredutases , Oligonucleotídeos , RNARESUMO
Protection and deprotection of the 2'-hydroxyl group of RNAs are critical for RNA-based drug discovery. This paper reports development of a bioreduction labile protecting group of the 2'-hydroxyl group in RNA. After the reduction of the nitro group in a chemical or enzymatic manner, the protecting groups were removed spontaneously. The attachment of electron-donating groups to the benzene ring or benzylic carbon enabled fast and controllable deprotection of the 2'-hydroxyl protecting group under physiological conditions.
Assuntos
RNA , Radical Hidroxila , Estrutura MolecularRESUMO
Pro-fluorescent labeled oligonucleotides are potential alternative tools to classical fluorescently labeled oligonucleotides for monitoring cellular uptake. Here, we report the design and synthesis of a thiol-responsive pro-fluorophore labeled oligonucleotide, and its fluorescence responsivity to glutathione in the test tube and live cells.
Assuntos
Corantes Fluorescentes/farmacocinética , Glutationa/metabolismo , Oligonucleotídeos/farmacocinética , Rodaminas/farmacocinética , Transporte Biológico , Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Oligonucleotídeos/síntese química , Oligonucleotídeos/metabolismo , Rodaminas/síntese química , Rodaminas/metabolismoRESUMO
Numerous applications of metal-mediated base pairs (metallo-base-pairs) to nucleic acid based nanodevices and genetic code expansion have been extensively studied. Many of these metallo-base-pairs are formed in DNA and RNA duplexes containing Watson-Crick base pairs. Recently, a crystal structure of a metal-DNA nanowire with an uninterrupted one-dimensional silver array was reported. We now report the crystal structure of a novel DNA helical wire containing HgII -mediated T:T and T:G base pairs and water-mediated C:C base pairs. The Hg-DNA wire does not contain any Watson-Crick base pairs. Crystals of the Hg-DNA wire, which is the first DNA wire structure driven by HgII ions, were obtained by mixing the short oligonucleotide d(TTTGC) and HgII ions. This study demonstrates the potential of metallo-DNA to form various structural components that can be used for functional nanodevices.
Assuntos
DNA/química , Guanina/química , Mercúrio/química , Nanofios/química , Timina/química , Pareamento de Bases , Humanos , Modelos Moleculares , Conformação de Ácido NucleicoRESUMO
Glutathione-labile protecting groups for phosphodiester moieties in oligonucleotides were designed, synthesized, and incorporated into oligonucleotides. The protecting groups on the phosphodiester moieties were cleaved in a buffer containing 10 mM glutathione, which was used as a model of intracellular fluid. Cellular uptake of oligonucleotides bearing glutathione-labile protecting groups was strongly affected by the location and number of the protecting groups.
Assuntos
Desenho de Fármacos , Glutationa/química , Oligonucleotídeos/farmacocinética , Células HeLa , Humanos , Estrutura Molecular , Oligonucleotídeos/síntese química , Oligonucleotídeos/químicaRESUMO
Herein, we determined the crystal structure of a DNA duplex containing consecutive 6-thioguanine-6-thioguanine disulfides. The disulfide bonds were reversibly formed and cleaved in the presence of Cu(ii) ions and glutathione. To our knowledge, this is the first reaction in which metal ions efficiently accelerated disulfide bond formation between thio-bases in duplexes.
RESUMO
In recent years, nucleic acid-based drug therapeutics have gained considerable attention for their potential in the treatment of various diseases. However, their therapeutic value is greatly hindered by the challenge of delivering them into cells. One possible strategy to improve cellular uptake is the use of "prodrug-type oligonucleotide medicine" in which negatively charged phosphodiester moieties are masked by bio-labile protecting groups. In this review, we describe our recent studies related to bio-labile protecting groups for phosphodiester moieties in the development of prodrug-type oligonucleotide medicines.
Assuntos
Oligonucleotídeos/química , Pró-Fármacos/química , Técnicas de Transferência de Genes , Humanos , Pró-Fármacos/farmacologia , Propriedades de SuperfícieRESUMO
Herein, we determined a high-resolution crystal structure of a B-form DNA duplex containing consecutive dinuclear metal ion-mediated base pairs, namely, 4-thiothymine-2Ag(i)-4-thiothymine (S-2Ag(i)-S), and four Ag(i) ions form a rectangular network and the distances between the Ag(i) ions are 2.8-3.2 Å, which may indicate the existence of metallophilic attractions.
RESUMO
The double-helix structure of DNA, in which complementary strands reversibly hybridize to each other, not only explains how genetic information is stored and replicated, but also has proved very attractive for the development of nanomaterials. The discovery of metal-mediated base pairs has prompted the generation of short metal-DNA hybrid duplexes by a bottom-up approach. Here we describe a metallo-DNA nanowire-whose structure was solved by high-resolution X-ray crystallography-that consists of dodecamer duplexes held together by four different metal-mediated base pairs (the previously observed C-Ag-C, as well as G-Ag-G, G-Ag-C and T-Ag-T) and linked to each other through G overhangs involved in interduplex G-Ag-G. The resulting hybrid nanowires are 2â nm wide with a length of the order of micrometres to millimetres, and hold the silver ions in uninterrupted one-dimensional arrays along the DNA helical axis. The hybrid nanowires are further assembled into three-dimensional lattices by interactions between adenine residues, fully bulged out of the double helix.
Assuntos
DNA/química , Nanofios/química , Prata/química , Cristalografia por Raios X , Modelos MolecularesRESUMO
A versatile conjugatable/bioreduction-responsive protecting group for phosphodiester moieties was designed, synthesized and incorporated into oligonucleotide strands. Subsequently, controlled pore glass-supported oligonucleotides were conjugated to a variety of functional molecules using a copper-catalyzed azide-alkyne cycloaddition reaction. The functionalized protecting groups were deprotected by a nitroreductase/NADH reduction system to give "naked" oligonucleotides. This method allowed the synthesis of oligonucleotide prodrugs bearing the functionalized protecting group at the desired sites and desired residues on oligodeoxyribonucleotide (ODN) backbones.
Assuntos
Alcinos/química , Oligonucleotídeos/síntese química , Pró-Fármacos/síntese química , Alcinos/metabolismo , Estrutura Molecular , NAD/química , NAD/metabolismo , Nitrorredutases/química , Nitrorredutases/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Oxirredução , Pró-Fármacos/química , Pró-Fármacos/metabolismoRESUMO
An efficient conjugatable and bioreduction cleavable linker was designed and synthesized for the 5'-terminal ends of oligonucleotides. A phosphoramidite reagent bearing this linker was successfully applied to solid phase synthesis and incorporated at the 5'-terminal ends of oligonucleotides. The controlled pore glass (CPG)-supported oligonucleotides were subsequently conjugated to a diverse range of functional molecules using a CuAAC reaction. The synthesized oligonucleotide conjugates were then cleaved using a nitroreductase/NADH bioreduction system to release the naked oligonucleotides.
Assuntos
Oligonucleotídeos/química , Estrutura Molecular , OxirreduçãoRESUMO
Cell-permeable oligodeoxyribonucleotides (ODNs) bearing reduction-activated protecting groups were synthesized as oligonucleotide pro-drugs. Although these oligonucleotides were amenable to solid-phase DNA synthesis and purification, the protecting group on their phosphodiester moiety could be readily cleaved by nitroreductase and NADH. Moreover, these compounds exhibited good nuclease resistance against 3'-exonuclease and endonuclease and good stability in human serum. Fluorescein-labeled ODNs modified with reduction-activated protecting groups showed better cellular uptake compared with that of naked ODNs.
Assuntos
Oligonucleotídeos/síntese química , Oligonucleotídeos/metabolismo , Sequência de Bases , Técnicas de Química Sintética , Estabilidade de Medicamentos , Humanos , NAD/metabolismo , Nitrorredutases/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oxirredução , PermeabilidadeRESUMO
Glutathione transferases (GSTs) are often overexpressed in tumors and frequently correlated to bad prognosis and resistance against a number of different anticancer drugs. To selectively target these cells and to overcome this resistance we previously have developed prodrugs that are derivatives of existing anticancer drugs (e.g., doxorubicin) incorporating a sulfonamide moiety. When cleaved by GSTs, the prodrug releases the cytostatic moiety predominantly in GST overexpressing cells, thus sparing normal cells with moderate enzyme levels. By modifying the sulfonamide it is possible to control the rate of drug release and specifically target different GSTs. Here we show that the newly synthesized compounds, 4-acetyl-2-nitro-benzenesulfonyl etoposide (ANS-etoposide) and 4-acetyl-2-nitro-benzenesulfonyl doxorubicin (ANS-DOX), function as prodrugs for GSTA1 and MGST1 overexpressing cell lines. ANS-DOX, in particular, showed a desirable cytotoxic profile by inducing toxicity and DNA damage in a GST-dependent manner compared to control cells. Its moderate conversion of 500 nmol/min/mg, as catalyzed by GSTA1, seems hereby essential since the more reactive 2,4-dinitrobenzenesulfonyl doxorubicin (DNS-DOX) (14000 nmol/min/mg) did not display a preference for GSTA1 overexpressing cells. DNS-DOX, however, effectively killed GSTP1 (20 nmol/min/mg) and MGST1 (450 nmol/min/mg) overexpressing cells as did the less reactive 4-mononitrobenzenesulfonyl doxorubicin (MNS-DOX) in a MGST1-dependent manner (1.5 nmol/min/mg) as shown previously. Furthermore, we show that the mechanism of these prodrugs involves a reduction in GSH levels as well as inhibition of the redox regulatory enzyme thioredoxin reductase 1 (TrxR1) by virtue of their electrophilic sulfonamide moiety. TrxR1 is upregulated in many tumors and associated with resistance to chemotherapy and poor patient prognosis. Additionally, the prodrugs potentially acted as a general shuttle system for DOX, by overcoming resistance mechanisms in cells. Here we propose that GST-dependent prodrugs require a conversion rate "window" in order to selectively target GST overexpressing cells, while limiting their effects on normal cells. Prodrugs are furthermore a suitable system to specifically target GSTs and to overcome various drug resistance mechanisms that apply to the parental drug.
Assuntos
Glutationa Transferase/metabolismo , Pró-Fármacos/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Citostáticos/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Etoposídeo/farmacologia , Glutationa/metabolismo , Humanos , Células MCF-7 , Sulfonamidas/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
We have examined substituted benzyl protecting groups for the phosphodiester in oligodeoxyribonucleotides. Stability of the protecting groups in buffer and rates of deprotection by glutathione (GSH) were strongly influenced by benzyl ring substituents.
Assuntos
Compostos de Benzil/metabolismo , Glutationa/metabolismo , Oligodesoxirribonucleotídeos/metabolismo , Pró-Fármacos/metabolismo , Compostos de Benzil/química , Oligodesoxirribonucleotídeos/química , Pró-Fármacos/químicaRESUMO
Oligonucleotides containing 4-O-(4-NO2-benzyl)thymine residues were synthesized to assess potential prodrug-type action against hypoxic cells. These modified oligonucleotides were incapable of stable duplex formation under non-hypoxic conditions. However, following deprotection of the thymine residues under bioreductive conditions, the deprotected oligonucleotides were able to form stable duplexes with target oligonucleotides.
Assuntos
Nitrofenóis/química , Oligonucleotídeos/química , Timina/química , Cromatografia Líquida de Alta Pressão , Hipóxia , Estrutura Molecular , Oligonucleotídeos/síntese química , OxirreduçãoRESUMO
Recently, metal-mediated base-pairs (metallo-base-pairs) have been studied extensively with the aim of exploring novel base-pairs; their structures, physicochemical properties, and applications have been studied. This trend has become more evident after the discovery of Hg(II)-mediated thymine-thymine (T-Hg(II)-T) and Ag(I)-mediated cytosine-cytosine (C-Ag(I)-C) base-pairs. In this article, we focus on the basic science and applications of these metallo-base-pairs, which are composed of natural bases.
Assuntos
Complexos de Coordenação/química , Mercúrio/química , Prata/química , Pareamento de Bases , Citosina/química , Timina/químicaRESUMO
Metallo-base pairs have been extensively studied for applications in nucleic acid-based nanodevices and genetic code expansion. Metallo-base pairs composed of natural nucleobases are attractive because nanodevices containing natural metallo-base pairs can be easily prepared from commercially available sources. Previously, we have reported a crystal structure of a DNA duplex containing T-Hg(II)-T base pairs. Herein, we have determined a high-resolution crystal structure of the second natural metallo-base pair between pyrimidine bases C-Ag(I)-C formed in an RNA duplex. One Ag(I) occupies the center between two cytosines and forms a C-Ag(I)-C base pair through N3-Ag(I)-N3 linear coordination. The C-Ag(I)-C base pair formation does not disturb the standard A-form conformation of RNA. Since the C-Ag(I)-C base pair is structurally similar to the canonical Watson-Crick base pairs, it can be a useful building block for structure-based design and fabrication of nucleic acid-based nanodevices.
Assuntos
Pareamento de Bases , RNA/química , Prata/química , Cristalografia por Raios X , Modelos Moleculares , Pirimidinas/químicaRESUMO
A photolabile protecting group, consisting of an o-nitrobenzyl group and a 3-(2'-hydroxy-3',6'-dimethylphenyl)-2,2-dimethylpropyl moiety, was developed for phosphodiesters in oligodeoxyribonucleotides. Deprotection was triggered by photoirradiation and subsequent spontaneous cyclization to release the naked oligonucleotide.