Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 8.497
Filtrar
2.
Chem Asian J ; 14(19): 3380-3385, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31478313

RESUMO

An oligonucleotide of triazole-linked RNA (TL RNA) was synthesized by performing consecutive copper-catalyzed azide-alkyne cycloaddition reactions for elongation. The reaction conditions that had been optimized for the synthesis of 3-mer TL RNA were found to be inappropriate for longer oligonucleotides, and the conditions were reoptimized for the solid-phase synthesis of an 11-mer TL RNA oligonucleotide. Duplex formation of the 11-mer TL RNA oligonucleotide was examined with the complementary oligonucleotide of natural RNA to reveal the effects of the 2'-OH groups on the duplex stability.


Assuntos
Oligonucleotídeos/química , RNA/química , Triazóis/química , Alquinos/química , Azidas/química , Catálise , Cobre/química , Reação de Cicloadição , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Oligonucleotídeos/síntese química , Técnicas de Síntese em Fase Sólida
3.
Chem Commun (Camb) ; 55(72): 10713-10716, 2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31429427

RESUMO

A red light-triggered reaction based on cyclic oligonucleotide substrates that is accelerated over 30-fold by specific nucleic acid templates and generates a bright fluorescent probe was developed. We confirmed that this reaction is compatible with fluorescence correlation spectroscopy (FCS) thereby allowing detection of nucleic acids down to 1 nM.


Assuntos
Luz , Ácidos Nucleicos/análise , Oligonucleotídeos/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Estrutura Molecular , Espectrometria de Fluorescência
4.
Chem Commun (Camb) ; 55(69): 10300-10303, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31397452

RESUMO

Shorter DNA probes provide better specificity for hybridization, but they may not form stable duplexes at room temperature. In this study, we used thiazole orange to follow DNA hybridization upon freezing and achieved stable 5-mer duplex DNA. Using multiple short probes in tandem, long DNA could also be studied. This study provides insights into DNA hybridization in the frozen state and expands the application of freezing for nucleic acid chemistry.


Assuntos
DNA/química , Hibridização de Ácido Nucleico , Oligonucleotídeos/química , Pareamento Incorreto de Bases , Sequência de Bases , Benzotiazóis/análise , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Corantes Fluorescentes/análise , Congelamento , Oligonucleotídeos/genética , Quinolinas/análise , Espectrometria de Fluorescência
5.
Chem Pharm Bull (Tokyo) ; 67(6): 505-518, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31155555

RESUMO

Nucleic acid therapeutics such as antisense and small interfering RNA (siRNA) have attracted increasing attention as innovative medicines that interfere with and/or modify gene expression systems. We have developed new functional oligonucleotides that can target DNA and RNA with high efficiency and selectivity. This review summarizes our achievements, including (1) the formation of non-natural triplex DNA for sequence-specific inhibition of transcription; (2) artificial receptor molecules for 8-oxidized-guanosine nucleosides; and (3) reactive oligonucleotides with a cross-linking agent or a functionality-transfer nucleoside for RNA pinpoint modification.


Assuntos
DNA/química , RNA/química , Reagentes para Ligações Cruzadas/química , DNA/metabolismo , DNA/farmacologia , Humanos , Nucleosídeos/análogos & derivados , Nucleosídeos/farmacologia , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Polietilenoglicóis/química , RNA/metabolismo , Telomerase/genética , Telomerase/metabolismo , Transcrição Genética/efeitos dos fármacos
6.
Nat Commun ; 10(1): 2704, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221964

RESUMO

Attachment of lipid tails to oligonucleotides has emerged as a powerful technology in constructing cell membrane-anchorable nucleic acid-based probes. In practice, however, conventional lipid-conjugated oligonucleotides fail to distinguish among different cell membranes. Herein, a phosphorylated lipid-conjugated oligonucleotide (DNA-lipid-P) is reported for alkaline phosphatase (ALP)-dependent cell membrane adhesion. In the absence of ALP, DNA-lipid-P with its poor hydrophobicity shows only weak interaction with cell membrane. However, in the presence of the highly expressed plasma membrane-associated ALP, DNA-lipid-P is converted to lipid-conjugated oligonucleotide (DNA-lipid) by enzymatic dephosphorylation. As a result of such conversion, the generated DNA-lipid has greater hydrophobicity than DNA-lipid-P and is thus able to insert into cell membranes in situ. Accordingly, DNA-lipid-P enables selective anchoring on cell membranes with elevated ALP level. Since elevated ALP level is a critical index of some diseases and even cancers, DNA-lipid-P holds promise for cell membrane engineering and disease diagnostics at the molecular level.


Assuntos
Fosfatase Alcalina/metabolismo , Membrana Celular/metabolismo , Sondas Moleculares/metabolismo , Oligonucleotídeos/metabolismo , Linhagem Celular Tumoral , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipídeos/química , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Sondas Moleculares/química , Oligonucleotídeos/química , Compostos Organofosforados/química , Fosforilação
7.
Chem Commun (Camb) ; 55(48): 6850-6853, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31123731

RESUMO

PAGE and UV melting analysis revealed that longer LNA-based splice-switching oligonucleotides (SSOs) formed secondary structures by themselves, reducing their effective concentration. To avoid such secondary structure formation, we introduced 7-deaza-2'-deoxyguanosine or 2'-deoxyinosine into the SSOs. These modified SSOs, with fewer secondary structures, showed higher exon skipping activities.


Assuntos
Éxons , Oligonucleotídeos/química , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Inosina/análogos & derivados , Inosina/química , Conformação de Ácido Nucleico , Oxirredução
8.
Nat Methods ; 16(6): 533-544, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110282

RESUMO

Fluorescence in situ hybridization (FISH) reveals the abundance and positioning of nucleic acid sequences in fixed samples. Despite recent advances in multiplexed amplification of FISH signals, it remains challenging to achieve high levels of simultaneous amplification and sequential detection with high sampling efficiency and simple workflows. Here we introduce signal amplification by exchange reaction (SABER), which endows oligonucleotide-based FISH probes with long, single-stranded DNA concatemers that aggregate a multitude of short complementary fluorescent imager strands. We show that SABER amplified RNA and DNA FISH signals (5- to 450-fold) in fixed cells and tissues. We also applied 17 orthogonal amplifiers against chromosomal targets simultaneously and detected mRNAs with high efficiency. We then used 10-plex SABER-FISH to identify in vivo introduced enhancers with cell-type-specific activity in the mouse retina. SABER represents a simple and versatile molecular toolkit for rapid and cost-effective multiplexed imaging of nucleic acid targets.


Assuntos
DNA/análise , Corantes Fluorescentes/metabolismo , Hibridização in Situ Fluorescente/métodos , Oligonucleotídeos/química , Imagem Óptica/métodos , RNA/análise , Retina/metabolismo , Animais , Células Cultivadas , DNA/genética , DNA de Cadeia Simples/química , Humanos , Camundongos , RNA/genética , Retina/diagnóstico por imagem
9.
Nat Commun ; 10(1): 2104, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068591

RESUMO

Protein-induced fluorescence enhancement (PIFE) is a popular tool for characterizing protein-DNA interactions. PIFE has been explained by an increase in local viscosity due to the presence of the protein residues. This explanation, however, denies the opposite effect of fluorescence quenching. This work offers a perspective for understanding PIFE mechanism and reports the observation of a phenomenon that we name protein-induced fluorescence quenching (PIFQ), which exhibits an opposite effect to PIFE. A detailed characterization of these two fluorescence modulations reveals that the initial fluorescence state of the labeled mediator (DNA) determines whether this mediator-conjugated dye undergoes PIFE or PIFQ upon protein binding. This key role of the mediator DNA provides a protocol for the experimental design to obtain either PIFQ or PIFE, on-demand. This makes the arbitrary nature of the current experimental design obsolete, allowing for proper integration of both PIFE and PIFQ with existing bulk and single-molecule fluorescence techniques.


Assuntos
DNA/metabolismo , Corantes Fluorescentes/química , Imagem Individual de Molécula/métodos , DNA/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Endonucleases Flap/química , Endonucleases Flap/isolamento & purificação , Endonucleases Flap/metabolismo , Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Coloração e Rotulagem , Proteínas Virais/química , Proteínas Virais/isolamento & purificação , Proteínas Virais/metabolismo
10.
Artigo em Inglês | MEDLINE | ID: mdl-31071581

RESUMO

Recently, non-coding RNA (ncRNA) became the centerpiece of human genome research. Modern ncRNA-based research has revolutionized disease diagnosis and therapeutics. However, decoding structural/functional information of ncRNA requires large amount of pure RNA, and hence effective RNA preparation and purification protocols. This review focuses on purification schemes of synthetic oligonucleotides, particularly liquid chromatographic (LC) techniques as improved alternatives to urea-polyacrylamide gel electrophoresis (urea-PAGE) purification. Moreover, the review summarizes the shortcomings of urea-PAGE purification method and details the chromatographic purification such as affinity, ion-exchange (IE) or size-exclusion (SE) chromatography. Specifically, we discuss denaturing and native RNA purification schemes with newest developments. In short, the review evaluates nucleic acid purification schemes required for various structural analyses.


Assuntos
Cromatografia Líquida/métodos , RNA não Traduzido , Eletroforese em Gel de Poliacrilamida/métodos , Oligonucleotídeos/análise , Oligonucleotídeos/química , Oligonucleotídeos/isolamento & purificação , RNA não Traduzido/análise , RNA não Traduzido/química , RNA não Traduzido/isolamento & purificação
11.
Phys Chem Chem Phys ; 21(20): 10798-10807, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31086926

RESUMO

Although DNA hybridization/melting is one of the most important biochemical reactions, the non-trivial kinetics of the process is not yet fully understood. In this work, we use Förster resonance energy transfer (FRET) to investigate the influence of temperature, ionic strength, and oligonucleotide length on the kinetic and equilibrium constants of DNA oligonucleotide binding and dissociation. We show that at low reagent concentrations and ionic strength, the time needed to establish equilibrium between single and double strand forms may be of the order of days, even for simple oligonucleotides of a length of 20 base pairs or less. We also identify and discuss the possible artifacts related to fluorescence-based experiments conducted in extremely dilute solutions. The results should prove useful for the judicious design of technologies based on DNA-matching, including sensors, DNA multiplication, sequencing, and gene manipulation.


Assuntos
DNA/química , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Cinética , Hibridização de Ácido Nucleico , Temperatura de Transição
12.
Chemistry ; 25(44): 10408-10419, 2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31062885

RESUMO

Nucleoside configuration (α-d vs. ß-d), nucleobase substituents, and the helical DNA environment of silver-mediated 5-aza-7-deazaguanine-cytosine base pairs have a strong impact on DNA stability. This has been demonstrated by investigations on oligonucleotide duplexes with silver-mediated base pairs of α-d and ß-d anomeric 5-aza-7-deaza-2'-deoxyguanosines and anomeric 2'-deoxycytidines incorporated in 12-mer duplexes. To this end, a new synthetic protocol has been developed to access the pure anomers of 5-aza-7-deaza-2'-deoxyguanosine by glycosylation of either the protected nucleobase or its salt followed by separation of the glycosylation products by crystallization and chromatography. Thermal stability measurements were performed on duplexes with α-d/α-d and ß-d/ß-d homo base pairs or α-d/ß-d and ß-d/α-d hybrid pairs within two sequence environments, positions 6 or 7, of oligonucleotide duplexes. The respective Tm stability increases observed after silver ion addition differ significantly. Homo base pairs with ß-d/ß-d or α-d/α-d nucleoside combinations are more stable than α-d/ß-d hybrid base pairs. The positional switch of silver-ion-mediated base pairs has a significant impact on stability. Nucleobase substituents introduced at the 5-position of the dC site of silver-mediated base pairs affect base pair stability to a minor extent. Our investigation might lead to applications in the construction of bioinspired nanodevices, in DNA diagnostics, or metal-DNA hybrid materials.


Assuntos
DNA/química , Desoxiguanosina/análogos & derivados , Prata/química , Pareamento Incorreto de Bases , Pareamento de Bases , Cátions Monovalentes , Citosina/química , Desoxicitidina/química , Desoxiguanosina/química , Glicosilação , Guanina/análogos & derivados , Guanina/química , Modelos Moleculares , Conformação de Ácido Nucleico , Oligonucleotídeos/síntese química , Oligonucleotídeos/química , Relação Estrutura-Atividade , Termodinâmica
13.
Nat Biotechnol ; 37(6): 640-650, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31036929

RESUMO

The molecular mechanisms of toxicity of chemically modified phosphorothioate antisense oligonucleotides (PS-ASOs) are not fully understood. Here, we report that toxic gapmer PS-ASOs containing modifications such as constrained ethyl (cEt), locked nucleic acid (LNA) and 2'-O-methoxyethyl (2'-MOE) bind many cellular proteins with high avidity, altering their function, localization and stability. We show that RNase H1-dependent delocalization of paraspeckle proteins to nucleoli is an early event in PS-ASO toxicity, followed by nucleolar stress, p53 activation and apoptotic cell death. Introduction of a single 2'-O-methyl (2'-OMe) modification at gap position 2 reduced protein-binding, substantially decreasing hepatotoxicity and improving the therapeutic index with minimal impairment of antisense activity. We validated the ability of this modification to generally mitigate PS-ASO toxicity with more than 300 sequences. Our findings will guide the design of PS-ASOs with optimal therapeutic profiles.


Assuntos
Oligonucleotídeos Antissenso/química , Oligonucleotídeos/química , Oligonucleotídeos Fosforotioatos/química , Humanos , Fígado/efeitos dos fármacos , Oligonucleotídeos/uso terapêutico , Oligonucleotídeos Antissenso/uso terapêutico , Oligonucleotídeos Fosforotioatos/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Ribonuclease H/química , Ribonuclease H/genética , Índice Terapêutico
14.
Anal Chim Acta ; 1068: 1-10, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31072469

RESUMO

Circulating tumor DNA (ctDNA) is a source of mutant DNA found in plasma and holds great promise in guiding cancer diagnostics, prognostics, and treatment. However, ctDNA fragments are challenging to detect in plasma due to their low abundance compared to wild-type DNA. In this study, a series of ion-tagged oligonucleotides (ITO) were synthesized using thiol-ene click chemistry and designed to selectively anneal target DNA. The ITO-DNA duplex was subsequently captured using a hydrophobic magnetic ionic liquid (MIL) as a liquid support. Extracted target DNA was quantified by adding the DNA-enriched MIL to the quantitative polymerase chain reaction (qPCR) buffer to streamline the extraction procedure. Clinically relevant concentrations of the mutation prone KRAS fragment, which has been linked to colorectal, lung, and bladder cancer, were preconcentrated using the ITO-MIL strategy allowing for enrichment factors as high as 19.49 ±â€¯1.44 from pure water and 4.02 ±â€¯0.50 from 10-fold diluted plasma after a 1 min extraction. Preconcentration could only be achieved when adding the ITO probe to the sample validating the selectivity of the ITO in the capture process. In addition, the amplification efficiency of qPCR was not affected when performing extractions from a diluted-plasma matrix demonstrating that the ITO-MIL approach coupled to direct-qPCR can be used to quantitate DNA from complex matrices. In comparison, commercially available steptavidin-coated magnetic beads were observed to lose selectivity when performing extractions from a 10-fold diluted plasma matrix. The selectivity of the ITO-MIL method, coupled with the ability to rapidly preconcentrate clinically relevant concentrations of target DNA from 10-fold diluted plasma, suggests that this method has the potential to be applied towards the extraction of ctDNA fragments from clinical samples.


Assuntos
Líquidos Iônicos/química , Oligonucleotídeos/química , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas p21(ras)/sangue , Humanos , Campos Magnéticos , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Solventes/química
15.
Methods Mol Biol ; 1973: 1-13, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016692

RESUMO

Chemical modification of nucleic acids can be achieved by the enzymatic polymerization of modified nucleoside triphosphates (dN*TPs). This approach obviates some of the requirements and drawbacks imposed by the more traditional solid-phase synthesis of oligonucleotides. Here, we describe the protocol that is necessary to synthesize dN*TPs and evaluate their substrate acceptance by polymerases for their subsequent use in various applications including selection experiments to identify aptamers. The protocol is exemplified for a sugar-constrained nucleoside analog, 7',5'-bc-TTP.


Assuntos
Compostos Bicíclicos com Pontes/química , DNA/biossíntese , Nucleotídeos/química , Açúcares/química , DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Oligonucleotídeos/química , Técnicas de Síntese em Fase Sólida
16.
Methods Mol Biol ; 1973: 131-145, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016699

RESUMO

Displaying ligands in a succinct and predictable manner is essential for elucidating multivalent molecular-level binding events. Organizing ligands with high precision and accuracy provides a distinct advantage over other ligand-display systems, such as polymers, because the number and position of the ligand(s) can be accurately and fully characterized. Here we describe the synthesis of peptide nucleic acids (PNAs), which are oligonucleotide mimics with a pseudopeptide backbone that can hybridize to oligonucleotides through Watson-Crick base pair to form highly predictable and organized scaffold for organizing a ligand. The ligand(s) are covalently attached to the PNA through a squarate coupling reaction that occurs between a free amine on the ligand and a free amine appended to the pseudopeptide backbone of the PNA. In this chapter we describe the synthesis of a LKγT monomer, which ultimately yields the free amine off the backbone of the PNA, incorporation of the monomer in a PNA oligomer, and the sequential squarate coupling to conjugate the ligand.


Assuntos
Oligonucleotídeos/química , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/síntese química , Ligantes , Modelos Moleculares , Hibridização de Ácido Nucleico
17.
Methods Mol Biol ; 1973: 213-236, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016705

RESUMO

The methodology enabling enzymatic and nonenzymatic information transfer with FNAs is described. This methodology includes the chemical synthesis of fNTPs and fN phosphoramidites, in addition to protocols for the enzymatic and nonenzymatic transfer of information.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Ácidos Nucleicos/química , RNA/química , Replicação do DNA , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Polimerização , Moldes Genéticos
18.
Methods Mol Biol ; 1973: 281-297, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016709

RESUMO

We developed a new technique suitable for improved detection of low-copy dsRNA using modified oligonucleotides as primers in RT-qPCR. Insertion of G8AE-clamp residues into primers significantly improves thermal stability of duplexes with RNA without decrease of hybridization selectivity. The applicability of modified primers is demonstrated for detection of low-copy Kemerovo virus dsRNA.


Assuntos
Primers do DNA/química , Oligonucleotídeos/química , RNA de Cadeia Dupla/análise , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , RNA Viral/química , RNA Viral/genética
19.
Anal Chim Acta ; 1063: 57-63, 2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-30967186

RESUMO

Glass capillary nanopore has been developed as a promising sensing platform for bioassay with single-molecule resolution. Although the diameter of glass capillary nanopore can be easily tuned, direct event-readouts of small biomacromolecules, like short-chain oligonucleotide fragments (within ∼20 nucleotides) remain great challenge, which limited by the configuration of the conical-shaped nanopore and the instrumental temporal resolution. Here, we exploit a smart strategy for glass nanopore detection of short-chain oligonucleotides by using relatively big-sized tetrahedral DNA nanostructures as a signal amplifier, which can amplify the signals and retard the translocation speed meanwhile. The tetrahedral DNA nanostructure with a hairpin loop sequence in one edge, undergoes a shape transformation upon the complementary combination of the target oligonucleotides, in which the presence of short-chain target oligonucleotide can be readout due to obvious variation in amplitude of ion current pulse that caused by volume change of the DNA tetrahedral. Therefore, this strategy is promising for extending glass nanopore sensing platform for sensitive detection of short-chain oligonucleotides.


Assuntos
DNA/análise , DNA/química , Vidro/química , Nanoestruturas/química , Oligonucleotídeos/análise , Oligonucleotídeos/química , Oligonucleotídeos/síntese química
20.
Molecules ; 24(7)2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30974914

RESUMO

The pharmacological relevance of ODNs forming G-quadruplexes as anti-HIV agents has been extensively reported in the literature over the last few years. Recent detailed studies have elucidated the peculiar arrangement adopted by many G-quadruplex-based aptamers and provided insight into their mechanism of action. In this review, we have reported the history of a strong anti-HIV agent: the 6-mer d(TGGGAG) sequence, commonly called "Hotoda's sequence". In particular, all findings reported on this sequence and its modified sequences have been discussed considering the following research phases: (i) discovery of the first 5'-modified active d(TGGGAG) sequences; (ii) synthesis of a variety of end-modified d(TGGGAG) sequences; (iii) biophysical and NMR investigations of natural and modified Hotoda's sequences; (iv); kinetic studies on the most active 5'-modified d(TGGGAG) sequences; and (v) extensive anti-HIV screening of G-quadruplexes formed by d(TGGGAG) sequences. This review aims to clarify all results obtained over the years on Hotoda's sequence, revealing its potentiality as a strong anti-HIV agent (EC50 = 14 nM).


Assuntos
Fármacos Anti-HIV , Quadruplex G , Infecções por HIV , HIV-1 , Oligonucleotídeos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , HIV-1/genética , HIV-1/metabolismo , Humanos , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos/uso terapêutico
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA