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1.
Yakugaku Zasshi ; 140(10): 1259-1268, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32999205

RESUMO

RNA interference (RNAi) is the standard method of suppressing gene expression because of its target specificity, potency, and ability to silence the expression of virtually any gene. Using 21-mer small interfering RNA (siRNA) is the general approach for inducing RNAi, as siRNA can be easily prepared using a DNA/RNA synthesizer. Synthetic siRNA can be chemically modified to increase the potency of RNAi activity and abrogate innate immune stimulation. However, designing chemically modified siRNA requires substantial experimentation. A practical method for understanding the interaction of siRNA and RNAi-related proteins and how modifications affect RNA-protein interactions is therefore needed. Plasmid DNA (pDNA) expressing short hairpin RNA (shRNA) can also be used to induce RNAi. pDNA produces numerous shRNAs that induce RNAi with potent and longterm RNAi activity, even if only one pDNA molecule is delivered to the nucleus. However, this approach has some drawbacks with regard to its therapeutic application, such as a low pDNA transfection efficiency due to its huge molecular size and innate immune responses induced by extra genes, such as CpG motifs. To overcome these issues with RNAi inducers (siRNA and pDNA), our group developed some chemical approaches using chemically modified oligonucleotides. This article focuses on our two original approaches. The first involves the groove modification of siRNA duplexes to understand siRNA-protein interactions using 7-bromo-7-deazaadenosine and 3-bromo-3-deazaadenosine as chemical probes, while the second involves the generation of RNAi medicine using chemically modified DNA, known as an intelligent shRNA expression device (iRed).


Assuntos
Desenvolvimento de Medicamentos/métodos , Interferência de RNA , RNA Interferente Pequeno/síntese química , DNA , Imunidade Inata , Oligonucleotídeos/química , Domínios e Motivos de Interação entre Proteínas , RNA Interferente Pequeno/química , Terapêutica com RNAi , Tubercidina/química
2.
Nucleic Acids Res ; 48(13): 7041-7051, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32678885

RESUMO

Z-DNA is known to be a left-handed alternative form of DNA and has important biological roles as well as being related to cancer and other genetic diseases. It is therefore important to investigate Z-DNA structure and related biological events in living cells. However, the development of molecular probes for the observation of Z-DNA structures inside living cells has not yet been realized. Here, we have succeeded in developing site-specific trifluoromethyl oligonucleotide DNA by incorporation of 8-trifluoromethyl-2'-deoxyguanosine (FG). 2D NMR strongly suggested that FG adopted a syn conformation. Trifluoromethyl oligonucleotides dramatically stabilized Z-DNA, even under physiological salt concentrations. Furthermore, the trifluoromethyl DNA can be used to directly observe Z-form DNA structure and interaction of DNA with proteins in vitro, as well as in living human cells by19F NMR spectroscopy for the first time. These results provide valuable information to allow understanding of the structure and function of Z-DNA.


Assuntos
DNA Forma Z/análise , Desoxiguanosina/química , Espectroscopia de Ressonância Magnética/métodos , Oligonucleotídeos/química , Clonagem Molecular , Escherichia coli/genética , Células HeLa , Humanos , Metanol/análogos & derivados , Metanol/química
3.
J Chromatogr A ; 1625: 461338, 2020 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-32709362

RESUMO

Until today, ion-pair reversed-phase chromatography is still the dominating method for analytical characterization of synthetic oligonucleotides. Its hyphenation with mass spectrometry, however, has some drawbacks such as ion-suppression in electrospray ionization. To overcome this problem, we present in this work a multiple heart-cutting (MHC) two-dimensional liquid chromatography (2D-LC) method with ultra-violet (UV) and electrospray ionization (ESI) mass spectrometry (MS) detection. A reversed-phase/weak anion-exchange (RP/WAX) stationary phase in the first dimension (1D) provides the selectivity for separation of structurally closely related oligonucleotide sequences and deletions (shortmers), respectively, using a mixed pH/triethylammonium phosphate buffer gradient at constant organic modifier content. Heart cuts of the oligonucleotide peaks are transferred to the second dimension (2D) via a multiple heart-cutting valve which is equipped with two loop decks. The 2D RP column is used for desalting via a diverter valve. Active solvent modulation enables to refocus the oligonucleotide peak into a sharp zone by 2D RP entirely free of non-volatile buffer components and ion-pair agents. Oligonucleotides can thus be sensitively detected by ESI-QTOF-MS under MS-compatible conditions.


Assuntos
Cromatografia de Fase Reversa/métodos , Oligonucleotídeos/química , Oligonucleotídeos/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/métodos , Ânions , Cromatografia por Troca Iônica , Oligonucleotídeos/análise , Polímeros/química
4.
Nucleic Acids Res ; 48(15): e89, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32544247

RESUMO

Understanding the thermodynamics of DNA motifs is important for prediction and design of probes and primers, but melt curve analyses are low-throughput and produce inaccurate results for motifs such as bulges and mismatches. Here, we developed a new, accurate and high-throughput method for measuring DNA motif thermodynamics called TEEM (Toehold Exchange Energy Measurement). It is a refined framework of comparing two toehold exchange reactions, which are competitive strand displacement between oligonucleotides. In a single experiment, TEEM can measure over 1000 ΔG° values with standard error of roughly 0.05 kcal/mol.


Assuntos
DNA/isolamento & purificação , Ensaios de Triagem em Larga Escala/métodos , Oligonucleotídeos/genética , Termodinâmica , DNA/química , Humanos , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , Oligonucleotídeos/química
5.
Proc Natl Acad Sci U S A ; 117(24): 13428-13436, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32493746

RESUMO

The recent advent of immune checkpoint inhibitor (CPI) antibodies has revolutionized many aspects of cancer therapy, but the efficacy of these breakthrough therapeutics remains limited, as many patients fail to respond for reasons that still largely evade understanding. An array of studies in human patients and animal models has demonstrated that local signaling can generate strongly immunosuppressive microenvironments within tumors, and emerging evidence suggests that delivery of immunostimulatory molecules into tumors can have therapeutic effects. Nanoparticle formulations of these cargoes offer a promising way to maximize their delivery and to enhance the efficacy of checkpoint inhibitors. We developed a modular nanoparticle system capable of encapsulating an array of immunostimulatory oligonucleotides that, in some cases, greatly increase their potency to activate inflammatory signaling within immune cells in vitro. We hypothesized that these immunostimulatory nanoparticles could suppress tumor growth by activating similar signaling in vivo, and thereby also improve responsiveness to immune checkpoint inhibitor antibody therapies. We found that our engineered nanoparticles carrying a CpG DNA ligand of TLR9 can suppress tumor growth in several animal models of various cancers, resulting in an abscopal effect on distant tumors, and improving responsiveness to anti-CTLA4 treatment with combinatorial effects after intratumoral administration. Moreover, by incorporating tumor-homing peptides, immunostimulatory nucleotide-bearing nanoparticles facilitate antitumor efficacy after systemic intravenous (i.v.) administration.


Assuntos
Adjuvantes Imunológicos , Antineoplásicos Imunológicos/uso terapêutico , Imunoterapia/métodos , Nanopartículas , Oligonucleotídeos/uso terapêutico , Animais , Antígeno CTLA-4/antagonistas & inibidores , Linhagem Celular , Sistemas de Liberação de Medicamentos , Quimioterapia Combinada , Inflamação , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Nanopartículas/química , Neoplasias Experimentais/terapia , Oligonucleotídeos/química , Oligonucleotídeos/imunologia
6.
PLoS One ; 15(6): e0232603, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32530964

RESUMO

Drug discovery with phosphorothioate oligonucleotides is an area of intensive research. In this study we have controlled the stereochemistry of the phosphorothioate backbone of LNA oligonucleotides to investigate the differences in safety profile, target mRNA knock down, and cellular uptake in vitro. The study reveals that controlling only four stereocenters in an isomeric phosphorothioate mixture can improve the therapeutic index significantly by improving safety without compromising activity.


Assuntos
Oligonucleotídeos/química , Animais , Sobrevivência Celular , Células Cultivadas , Química Farmacêutica , Células Epiteliais/metabolismo , Hepatócitos/metabolismo , Humanos , Túbulos Renais/metabolismo , Camundongos , Estrutura Molecular , Oligonucleotídeos/administração & dosagem , Oligonucleotídeos/toxicidade , Oligonucleotídeos Fosforotioatos/química , RNA Mensageiro/antagonistas & inibidores
7.
RNA ; 26(9): 1283-1290, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32482894

RESUMO

Isothermal, cell-free, synthetic biology-based approaches to pathogen detection leverage the power of tools available in biological systems, such as highly active polymerases compatible with lyophilization, without the complexity inherent to live-cell systems, of which nucleic acid sequence based amplification (NASBA) is well known. Despite the reduced complexity associated with cell-free systems, side reactions are a common characteristic of these systems. As a result, these systems often exhibit false positives from reactions lacking an amplicon. Here we show that the inclusion of a DNA duplex lacking a promoter and unassociated with the amplicon fully suppresses false positives, enabling a suite of fluorescent aptamers to be used as NASBA tags (Apta-NASBA). Apta-NASBA has a 1 pM detection limit and can provide multiplexed, multicolor fluorescent readout. Furthermore, Apta-NASBA can be performed using a variety of equipment, for example, a fluorescence microplate reader, a qPCR instrument, or an ultra-low-cost Raspberry Pi-based 3D-printed detection platform using a cell phone camera module, compatible with field detection.


Assuntos
Aptâmeros de Nucleotídeos/química , Corantes Fluorescentes/química , Oligonucleotídeos/química , Reação em Cadeia da Polimerase/métodos , Replicação de Sequência Autossustentável/métodos , Sistema Livre de Células , Fluorescência , Humanos , Regiões Promotoras Genéticas/genética , Sensibilidade e Especificidade
8.
Food Chem ; 326: 126813, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32438234

RESUMO

Herein, we developed a multi-analyte fluorescence immunoassay for detection of three organophosphate pesticides (triazophos, parathion, and chlorpyrifos) in various agro-products (rice, wheat, cucumber, cabbage, and apple) using fluorescently labeled oligonucleotide and gold nanoparticle (AuNP) signal amplification technology. The AuNP probes for the three analytes were constructed by simultaneously modifying the corresponding antibodies and fluorescently labeled oligonucleotides on the probe surface. Three fluorophores (6-FAM, Cy3, and Texas red) with high fluorescence intensity and little overlap of excitation/emission wavelengths were selected. The method showed satisfactory linearity for triazophos, parathion, and chlorpyrifos in the ranges of 0.01-20, 0.05-50, and 0.5-1000 µg/L, respectively. For the 3 analytes, the limits of detection (LODs) were 0.007, 0.009, and 0.087 µg/L, respectively. The average recoveries were 77.7-113.6%, with relative standard deviations (RSDs) of 7.1-17.1% in various food matrices. The proposed method offers great potential in food safety surveillance, and could be used as well as a reference for multi-residue analysis of other small-molecule contaminants.


Assuntos
Ouro/química , Inseticidas/análise , Nanopartículas Metálicas/química , Oligonucleotídeos/química , Clorpirifos/análise , Fluorescência , Análise de Alimentos , Imunoensaio/métodos , Limite de Detecção , Organotiofosfatos/análise , Paration/análise , Praguicidas/análise , Triazóis/análise
9.
Nat Commun ; 11(1): 2174, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358582

RESUMO

Poly-ADP-ribosylation (PARylation) is a fully reversible post-translational modification with key roles in cellular physiology. Due to the multi-domain structure of poly(ADP-ribose) polymerase-1 (PARP1) and the highly dynamic nature of the PARylation reaction, studies on the biochemical mechanism and structural dynamics remain challenging. Here, we report label-free, time-resolved monitoring of PARP1-dependent PARylation using ATR-FTIR spectroscopy. This includes PARP1 activation by binding to DNA strand break models, NAD+ substrate binding, PAR formation, and dissociation of automodified PARP1 from DNA. Analyses of PARP1 activation at different DNA models demonstrate a strong positive correlation of PARylation and PARP1 dissociation, with the strongest effects observed for DNA nicks and 3' phosphorylated ends. Moreover, by examining dynamic structural changes of PARP1, we reveal changes in the secondary structure of PARP1 induced by NAD+ and PARP inhibitor binding. In summary, this approach enables holistic and dynamic insights into PARP1-dependent PARylation with molecular and temporal resolution.


Assuntos
Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli ADP Ribosilação/genética , Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Humanos , Cinética , NAD/análogos & derivados , NAD/biossíntese , NAD/metabolismo , Oligonucleotídeos/química , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/genética , Poli ADP Ribosilação/efeitos dos fármacos , Poli ADP Ribosilação/fisiologia , Ligação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier/métodos
10.
Nat Commun ; 11(1): 2185, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32366822

RESUMO

Signal amplification in biological systems is achieved by cooperatively recruiting multiple copies of regulatory biomolecules. Nevertheless, the multiplexing capability of artificial fluorescent amplifiers is limited due to the size limit and lack of modularity. Here, we develop Cayley tree-like fractal DNA frameworks to topologically encode the fluorescence states for multiplexed detection of low-abundance targets. Taking advantage of the self-similar topology of Cayley tree, we use only 16 DNA strands to construct n-node (n = 53) structures of up to 5 megadalton. The high level of degeneracy allows encoding 36 colours with 7 nodes by site-specifically anchoring of distinct fluorophores onto a structure. The fractal topology minimises fluorescence crosstalk and allows quantitative decoding of quantized fluorescence states. We demonstrate a spectrum of rigid-yet-flexible super-multiplex structures for encoded fluorescence detection of single-molecule recognition events and multiplexed discrimination of living cells. Thus, the topological engineering approach enriches the toolbox for high-throughput cell imaging.


Assuntos
DNA/química , Fluorescência , Fractais , Oligonucleotídeos/química , Algoritmos , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Células HeLa , Humanos , Microscopia de Força Atômica/métodos , Microscopia Confocal/métodos , Nanoestruturas/química
11.
Int J Nanomedicine ; 15: 2809-2828, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32368056

RESUMO

Introduction: Glioblastoma (GBM) is the most common and lethal of the central nervous system (CNS) malignancies. The initiation, progression, and infiltration ability of GBMs are attributed in part to the dysregulation of microRNAs (miRNAs). Thus, targeting dysregulated miRNAs with RNA oligonucleotides (RNA interference, RNAi) has been proposed for GBM treatment. Despite promising results in the laboratory, RNA oligonucleotides have clinical limitations that include poor RNA stability and off-target effects. RNAi therapies against GBM confront an additional obstacle, as they need to cross the blood-brain barrier (BBB). Methods: Here, we developed gold-liposome nanoparticles conjugated with the brain targeting peptides apolipoprotein E (ApoE) and rabies virus glycoprotein (RVG). First, we functionalized gold nanoparticles with oligonucleotide miRNA inhibitors (OMIs), creating spherical nucleic acids (SNAs). Next, we encapsulated SNAs into ApoE, or RVG-conjugated liposomes, to obtain SNA-Liposome-ApoE and SNA-Liposome-RVG, respectively. We characterized each nanoparticle in terms of their size, charge, encapsulation efficiency, and delivery efficiency into U87 GBM cells in vitro. Then, they were administered intravenously (iv) in GBM syngeneic mice to evaluate their delivery efficiency to brain tumor tissue. Results: SNA-Liposomes of about 30-50 nm in diameter internalized U87 GBM cells and inhibited the expression of miRNA-92b, an aberrantly overexpressed miRNA in GBM cell lines and GBM tumors. Conjugating SNA-Liposomes with ApoE or RVG peptides increased their systemic delivery to the brain tumors of GBM syngeneic mice. SNA-Liposome-ApoE demonstrated to accumulate at higher extension in brain tumor tissues, when compared with non-treated controls, SNA-Liposomes, or SNA-Liposome-RVG. Discussion: SNA-Liposome-ApoE has the potential to advance the translation of miRNA-based therapies for GBM as well as other CNS disorders.


Assuntos
Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Lipossomos/administração & dosagem , Interferência de RNA , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Técnicas de Transferência de Genes , Glioblastoma/genética , Glioblastoma/patologia , Ouro/química , Humanos , Lipossomos/química , Masculino , Nanopartículas Metálicas/química , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Ácidos Nucleicos/química , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos/farmacocinética , Proteínas do Envelope Viral/genética , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Nucleic Acid Ther ; 30(3): 129-132, 2020 06.
Artigo em Inglês | MEDLINE | ID: covidwho-73985

RESUMO

The present global health emergency involving the emergence and rapid spread of a novel coronavirus has prompted the world scientific community to consider how it can help to fight this growing viral pandemic. With few safe and effective drugs available to combat this threat to humanity and the normal functioning of our society, the oligonucleotide research community is uniquely positioned to apply its technology and expertise to help alleviate the crisis, thanks to its capacity for rational drug design, swift development cycles, and pursuing targets undruggable by conventional treatment strategies.


Assuntos
Betacoronavirus/efeitos dos fármacos , Betacoronavirus/fisiologia , Infecções por Coronavirus/tratamento farmacológico , Oligonucleotídeos/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Antivirais/química , Betacoronavirus/química , Betacoronavirus/genética , Sistemas de Liberação de Medicamentos , Humanos , Oligonucleotídeos/química , Pandemias
13.
Protein Sci ; 29(7): 1596-1605, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32304108

RESUMO

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS-CoVs and Middle East Respiratory Syndrome coronavirus (MERS-CoVs), the detailed information about SARS-CoV-2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies, and antivirals. We applied high-throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS-CoV-2 proteins and structures. Here we report two high-resolution crystal structures of endoribonuclease Nsp15/NendoU. We compare these structures with previously reported homologs from SARS and MERS coronaviruses.


Assuntos
Betacoronavirus/química , Endorribonucleases/química , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Oligonucleotídeos/química , Vírus da SARS/química , Proteínas não Estruturais Virais/química , Sequência de Aminoácidos , Betacoronavirus/genética , Betacoronavirus/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Endorribonucleases/genética , Endorribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Modelos Moleculares , Oligonucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vírus da SARS/genética , Vírus da SARS/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
14.
Nucleic Acids Res ; 48(9): 4658-4671, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32313951

RESUMO

Erythropoietic protoporphyria (EPP) is a rare genetic disease in which patients experience acute phototoxic reactions after sunlight exposure. It is caused by a deficiency in ferrochelatase (FECH) in the heme biosynthesis pathway. Most patients exhibit a loss-of-function mutation in trans to an allele bearing a SNP that favors aberrant splicing of transcripts. One viable strategy for EPP is to deploy splice-switching oligonucleotides (SSOs) to increase FECH synthesis, whereby an increase of a few percent would provide therapeutic benefit. However, successful application of SSOs in bone marrow cells is not described. Here, we show that SSOs comprising methoxyethyl-chemistry increase FECH levels in cells. We conjugated one SSO to three prototypical targeting groups and administered them to a mouse model of EPP in order to study their biodistribution, their metabolic stability and their FECH splice-switching ability. The SSOs exhibited distinct distribution profiles, with increased accumulation in liver, kidney, bone marrow and lung. However, they also underwent substantial metabolism, mainly at their linker groups. An SSO bearing a cholesteryl group increased levels of correctly spliced FECH transcript by 80% in the bone marrow. The results provide a promising approach to treat EPP and other disorders originating from splicing dysregulation in the bone marrow.


Assuntos
Ferroquelatase/genética , Oligonucleotídeos/administração & dosagem , Protoporfiria Eritropoética/metabolismo , Processamento de RNA , Albuminas/metabolismo , Animais , Medula Óssea/metabolismo , Células COS , Chlorocebus aethiops , Modelos Animais de Doenças , Ferroquelatase/metabolismo , Humanos , Células K562 , Camundongos , Oligonucleotídeos/sangue , Oligonucleotídeos/química , Oligonucleotídeos/farmacocinética , Polimorfismo de Nucleotídeo Único , Protoporfiria Eritropoética/genética , Protoporfiria Eritropoética/terapia , Sítios de Splice de RNA , Distribuição Tecidual
15.
Nucleic Acids Res ; 48(11): e61, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32313962

RESUMO

High-throughput investigation of structural diversity of nucleic acids is hampered by the lack of suitable label-free methods, combining fast and cheap experimental workflow with high information content. Here, we explore the use of intrinsic fluorescence emitted by nucleic acids for this scope. After a preliminary assessment of suitability of this phenomenon for tracking conformational changes of DNA, we examined steady-state emission spectra of an 89-membered set of oligonucleotides with reported conformation (G-quadruplexes (G4s), i-motifs, single- and double-strands) by means of multivariate analysis. Principal component analysis of emission spectra resulted in successful clustering of oligonucleotides into three corresponding conformational groups, without discrimination between single- and double-stranded structures. Linear discriminant analysis was exploited for the assessment of novel sequences, allowing the evaluation of their G4-forming propensity. Our method does not require any labeling agent or dye, avoiding the related bias, and can be utilized to screen novel sequences of interest in a high-throughput and cost-effective manner. In addition, we observed that left-handed (Z-) G4 structures were systematically more fluorescent than most other G4 structures, almost reaching the quantum yield of 5'-d[(G3T)3G3]-3' (G3T, the most fluorescent G4 structure reported to date).


Assuntos
DNA/química , Fluorescência , Conformação de Ácido Nucleico , Conjuntos de Dados como Assunto , Análise Discriminante , Oligonucleotídeos/química , Análise de Componente Principal , Teoria Quântica
16.
Chemistry ; 26(24): 5441-5448, 2020 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-32271495

RESUMO

N6 -(2-Deoxy-α,ß-d-erythropentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy⋅dG) is a major DNA lesion produced from 2'-deoxyguanosine under oxidizing conditions. Fapy⋅dG is produced from a common intermediate that leads to 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-OxodGuo), and in greater quantities in cells. The impact of Fapy⋅dG on DNA structure and function is much less well understood than that of 8-OxodGuo. This is largely due to the significantly greater difficulty in synthesizing oligonucleotides containing Fapy⋅dG than 8-OxodGuo. We describe a synthetic approach for preparing oligonucleotides containing Fapy⋅dG that will facilitate intensive studies of this lesion in DNA. A variety of oligonucleotides as long as 30 nucleotides are synthesized. We anticipate that the chemistry described herein will provide an impetus for a wide range of studies involving Fapy⋅dG.


Assuntos
8-Hidroxi-2'-Desoxiguanosina/síntese química , DNA/química , Desoxiguanosina/química , Formamidas/síntese química , Furanos/síntese química , Oligonucleotídeos/síntese química , Pirimidinas/síntese química , 8-Hidroxi-2'-Desoxiguanosina/química , Formamidas/química , Furanos/química , Oligonucleotídeos/química , Oxirredução , Estresse Oxidativo , Pirimidinas/química
17.
Exp Parasitol ; 212: 107885, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32234306

RESUMO

A phage-display library was generated using a Bus thalamus scorpion toxin (BTK-2) as a peptide scaffold. BTK-2 belongs to the disulfide-rich family of proteins with pronounced structural stability due to the presence of three disulfide bridges that connects antiparallel beta-sheets and one alpha helix. Using BTK-2 as a phage display scaffold, we introduced mutations in five residues located in the alpha-helix and two residues located in the smaller loop, keeping intact the disulfide bridges to create a peptide phage-displayed library with disulfide-rich family properties. The library was subjected to in vivo and in vitro phage display selections against Trypanosoma evansi, the etiological agent of "Surra", a disease that affects a wide range of mammals. The development of T. evansi specific biomarkers is essential to improve diagnostic methods and epidemiological studies leading to a more accurate clinical decision for the treatment of this disease of economic impact for commercial livestock production. In this study, we identified two disulfide-rich peptides targeting T. evansi parasites. Further specificity studies are necessary to investigate the potential of selected peptides as new biomarkers to aid diagnostic and treatment procedures of T. evansi infections.


Assuntos
Dissulfetos , Peptídeos , Trypanosoma/química , Tripanossomíase/diagnóstico , Tripanossomíase/terapia , Sequência de Aminoácidos , Animais , Biomarcadores , Clonagem Molecular , Dissulfetos/química , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese , Oligonucleotídeos/química , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/genética , Venenos de Escorpião/química , Venenos de Escorpião/genética
18.
Nucleic Acid Ther ; 30(3): 129-132, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32297843

RESUMO

The present global health emergency involving the emergence and rapid spread of a novel coronavirus has prompted the world scientific community to consider how it can help to fight this growing viral pandemic. With few safe and effective drugs available to combat this threat to humanity and the normal functioning of our society, the oligonucleotide research community is uniquely positioned to apply its technology and expertise to help alleviate the crisis, thanks to its capacity for rational drug design, swift development cycles, and pursuing targets undruggable by conventional treatment strategies.


Assuntos
Betacoronavirus/efeitos dos fármacos , Betacoronavirus/fisiologia , Infecções por Coronavirus/tratamento farmacológico , Oligonucleotídeos/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Antivirais/química , Betacoronavirus/química , Betacoronavirus/genética , Sistemas de Liberação de Medicamentos , Humanos , Oligonucleotídeos/química , Pandemias
19.
AAPS PharmSciTech ; 21(3): 108, 2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-32215761

RESUMO

The oral bioavailability of therapeutic proteins is limited by the gastrointestinal barriers. Encapsulation of labile proteins into nanoparticles is a promising strategy. In order to improve the stability of nanoparticles, lyophilisation has been used to remove water molecules from the suspension. Although various cryoprotections were employed in the preparation of lyophilised nanoparticles, the selection of cryoprotectant type and concentration in majority of the developed formulation was not justified. In this study, nanoparticles were fabricated by cationic chitosan and anionic Dz13Scr using complex coacervation. The effect of cryoprotectant types (mannitol, sorbitol, sucrose and trehalose) and their concentrations (1, 3, 5, 7, 10% w/v) on physiochemical properties of nanoparticles were measured. Cellular assays were performed to investigate the impact of selected cryoprotectant on cytotoxicity, glucose consumption, oral absorption mechanism and gastrointestinal permeability. The obtained results revealed that mannitol (7% w/v) could produce nanoparticles with small size (313.2 nm), slight positive charge and uniform size distribution. The addition of cryoprotectant could preserve the bioactivity of entrapped insulin and improve the stability of nanoparticles against mechanical stress during lyophilisation. The gastrointestinal absorption of nanoparticles is associated with both endocytic and paracellular pathways. With the use of 7% mannitol, lyophilised nanoparticles induced a significant glucose uptake in C2C12 cells. This work illustrated the importance of appropriate cryoprotectant in conservation of particle physiochemical properties, structural integrity and bioactivity. An incompatible cryoprotectant and inappropriate concentration could lead to cake collapse and formation of heterogeneous particle size populations.


Assuntos
Diabetes Mellitus/tratamento farmacológico , Insulina/química , Nanopartículas/química , Oligonucleotídeos/química , Animais , Crioprotetores/administração & dosagem , Estabilidade de Medicamentos , Liofilização/métodos , Insulina/administração & dosagem , Polímeros/química
20.
Proc Natl Acad Sci U S A ; 117(13): 7276-7283, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32188786

RESUMO

All known polymerases copy genetic material by catalyzing phosphodiester bond formation. This highly conserved activity proceeds by a common mechanism, such that incorporated nucleoside analogs terminate chain elongation if the resulting primer strand lacks a terminal hydroxyl group. Even conservatively substituted 3'-amino nucleotides generally act as chain terminators, and no enzymatic pathway for their polymerization has yet been found. Although 3'-amino nucleotides can be chemically coupled to yield stable oligonucleotides containing N3'→P5' phosphoramidate (NP) bonds, no such internucleotide linkages are known to occur in nature. Here, we report that 3'-amino terminated primers are, in fact, slowly extended by the DNA polymerase from B. stearothermophilus in a template-directed manner. When its cofactor is Ca2+ rather than Mg2+, the reaction is fivefold faster, permitting multiple turnover NP bond formation to yield NP-DNA strands from the corresponding 3'-amino-2',3'-dideoxynucleoside 5'-triphosphates. A single active site mutation further enhances the rate of NP-DNA synthesis by an additional 21-fold. We show that DNA-dependent NP-DNA polymerase activity depends on conserved active site residues and propose a likely mechanism for this activity based on a series of crystal structures of bound complexes. Our results significantly broaden the catalytic scope of polymerase activity and suggest the feasibility of a genetic transition between native nucleic acids and NP-DNA.


Assuntos
Amidas/química , DNA Polimerase Dirigida por DNA/química , DNA/química , Ácidos Fosfóricos/química , Amidas/síntese química , Amidas/metabolismo , DNA/síntese química , DNA Polimerase Dirigida por DNA/metabolismo , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Ácidos Fosfóricos/síntese química , Ácidos Fosfóricos/metabolismo , Polimerização , RNA/química
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