RESUMEN
Synthesis of 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-adenine (7, ara-A2'F) and -guanine (12, ara-G2'F) was accomplished via the condensation of 2,6-dichloropurine (1) with 2-deoxy-2-fluoro-1,3,5-tri-O-benzoyl-alpha-D-arabinofuranose (2) as a key chemical step. Condensation of silylated N6-benzoyladenine (6) with 2 gave, after deblocking and chromatographic separation, ara-A2'F (7) (14%), it's alpha-anomer 8 (14%) and N7-alpha-isomer 9 (25%). The PSEUROT analysis of N9-betaD-arabinosides 7 and 12 manifested slight preference for the S rotamer (64%) for the former, and an equal population of the N and S rotamers for the latter. The arabinosides 7 and 12 were used for the preparation of the respective phosphoamidite building blocks 13 and 14 for automated oligonucleotide synthesis. Four 15-mer oligonucleotides (ONs) complementary to the initiation codon region of firefly luciferase mRNA were prepared: unmodified 2'-deoxy-ON (AS 1) and containing (i) ara-A2'F instead of the only A (AS2), (ii) ara-G2'F vs. 3-G from the 5'-terminus (AS3), and (iii) both arabinosides at the same positions (AS4). All these ONs display practically the same (i) affinity to both complementary DNA and RNA, and (ii) ability to inhibit a luciferase gene expression in a cell-free transcription-translation system.
Asunto(s)
Arabinonucleósidos/química , Hibridación de Ácido Nucleico , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/síntesis química , Oligonucleótidos/química , Secuencia de Bases , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Luciferasas/genética , Espectroscopía de Resonancia Magnética , Oligonucleótidos Antisentido/farmacología , TermodinámicaRESUMEN
Eighteen peptide-oligonucleotide phosphorothioate conjugates were prepared in good yield and thoroughly characterized with electrospray ionization mass spectra. When applied to the living cells, conjugates exhibiting membrane translocation and nuclear localization properties displayed efficient intracellular penetration but failed to show any serious antisense effect. Studies on the intracellular distribution of the fluorescein-labeled conjugates revealed their trapping in endosomes.
Asunto(s)
Núcleo Celular/química , Oligonucleótidos/química , Oligopéptidos/química , Compuestos Organotiofosforados/química , Membrana Celular/química , Reactivos de Enlaces Cruzados , Disulfuros/química , Electroforesis en Gel de Poliacrilamida , Fluoresceína-5-Isotiocianato , Expresión Génica/genética , Hidrólisis , Luciferasas/biosíntesis , Luciferasas/genética , Espectrometría de Masas , Microscopía Confocal , Oligonucleótidos/síntesis química , Oligonucleótidos/metabolismo , Oligonucleótidos Antisentido/síntesis química , Oligonucleótidos Antisentido/química , Oligopéptidos/síntesis química , Oligopéptidos/metabolismo , Compuestos Organotiofosforados/síntesis química , Compuestos Organotiofosforados/metabolismo , Ribonucleasa HRESUMEN
Clodronate (dichloromethylidene-bisphosphonate), a halogen-containing bisphosphonate, can inhibit the release of cytokines from RAW 264 macrophages and has anti-inflammatory properties in rheumatoid arthritis, whilst amino-containing bisphosphonates such as alendronate (4-amino-1-hydroxybutylidene-bisphosphonate), have pro-inflammatory properties and can cause an acute phase response. The basis for these pharmacological properties is unclear. Recently, it was demonstrated that clodronate is metabolised by certain cell lines in vitro to an analogue of ATP, whereas amino-bisphosphonates are not. We therefore investigated whether clodronate can also be metabolised by RAW 264 macrophages and whether intracellular accumulation of the metabolite (AppCCl2p) could account for the anti-inflammatory properties of clodronate. The effect of alendronate and AppCCl2p on the release of cytokines (IL-1beta, IL-6, and TNFalpha) from RAW 264 cells was compared, and the effect of the bisphosphonates and AppCCl2p on the DNA binding activities of transcription factors, NF-kappaB and AP-1, was investigated. Pretreatment of RAW 264 macrophages with alendronate augmented the LPS-stimulated release of IL-1beta and increased the binding of NF-kappaB to DNA in an electrophoretic mobility shift assay. Without LPS-induction, alendronate did not affect cytokine release or NF-kappaB binding. Clodronate was metabolised by RAW 264 cells to AppCCl2p. Like clodronate, AppCCl2p inhibited the LPS-induced release of cytokines and NO from RAW 264 macrophages. Both clodronate and its metabolite also inhibited the LPS-stimulated binding of NF-kappaB to DNA. In conclusion, these results suggest that the metabolite of clodronate may be responsible for the anti-inflammatory properties of clodronate, and that the contrasting effects of different bisphosphonates on the release of cytokines could be mediated partly through changes in the DNA binding activity of NF-kappaB.
Asunto(s)
Alendronato/farmacología , Analgésicos no Narcóticos/farmacología , Artritis Reumatoide/patología , Ácido Clodrónico/farmacología , Difosfonatos/metabolismo , Macrófagos/efectos de los fármacos , Animales , Células Cultivadas , Citocinas/metabolismo , Portadores de Fármacos , Electroforesis en Gel de Poliacrilamida , Liposomas , Macrófagos/metabolismo , Ratones , FN-kappa B/metabolismo , Óxido Nítrico/metabolismo , Biosíntesis de Proteínas , Factor de Transcripción AP-1/metabolismoRESUMEN
Several circular oligonucleotides were synthesized and characterized by electrospray ionization mass spectrometry. Experiments on termination of primer extension catalysed by DNA polymerases, Klenow fragment and Tth have demonstrated that a double helix forming circular 2'-deoxyribooligomer containing a 25mer sequence complementary to the target single-stranded DNA along with a 34mer random mismatching stretch appears to be a potent inhibitor of replication in vitro. Studies on inhibition of luciferase gene expression in a cell-free transcription-translation system have shown that a duplex forming circular 2'-deoxyribooligonucleotide containing a 25mer sequence complementary to the target mRNA and a 14mer random mismatching stretch can serve as an effective antisense compound as a standard linear complementary oligomer. Features of double helix forming circular oligonucleotides composed of 2'-deoxyribonucleosides seem to be useful for the design of new antigene and antisense agents.
Asunto(s)
Replicación del ADN/efectos de los fármacos , Marcación de Gen/métodos , Conformación de Ácido Nucleico , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos/farmacología , ARN Mensajero/antagonistas & inhibidores , Secuencia de Bases , Cromatografía Líquida de Alta Presión , ADN Polimerasa I/metabolismo , ADN de Cadena Simple/genética , Genes Reporteros , Luciferasas/biosíntesis , Luciferasas/genética , Espectrometría de Masas , Datos de Secuencia Molecular , Oligonucleótidos/síntesis química , Oligonucleótidos/química , Oligonucleótidos Antisentido/síntesis química , Oligonucleótidos Antisentido/química , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Moldes GenéticosRESUMEN
Complexing of looped and circular oligonucleotides, composed of either 2'-deoxyribo- or 2'-O-methylribonucleoside units, with completely matching or partially mismatching complementary DNA sequences was studied. Melting experiments revealed considerable differences among the stabilities of these hybrid complexes. Maximum stability and selectivity was displayed by oligomers 2 and 5. It was concluded that a linear stretch, attached to 1'-O- of 3'-deoxypsicothymidine unit (Z) increases the selectivity of hybridisation and stability of the complex as a whole. This allows one to aim the target DNA very precisely at its polyadenine part as well as at adjacent sequence simultaneously. Experiments on termination of primer extension catalysed by different DNA-polymerases--Sequenase, Klenow fragment and Tth--have demonstrated that looped oligomer 5, composed of 2'-O-methylribonucleosides appears to be a highly selective and potent inhibitor of replication in vitro. Features of looped oligonucleotides, composed of 2'-O-methylribonucleosides seem to be useful for design of highly specific antigene oligonucleotides.
Asunto(s)
Replicación del ADN , ADN de Cadena Simple/metabolismo , Conformación de Ácido Nucleico , Oligonucleótidos/metabolismo , Secuencia de Bases , ADN Circular/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Datos de Secuencia Molecular , Desnaturalización de Ácido Nucleico , Hibridación de Ácido Nucleico , Oligodesoxirribonucleótidos/metabolismo , Oligorribonucleótidos/metabolismo , TermodinámicaRESUMEN
Several new branched (1, 2), circular (9) and looped oligonucleotides (14-17) were synthesized. 3'-Deoxypsicothymidine was employed to create the site of branching when required. The circular and looped structures were obtained by oxidative disulfide bond formation between mercaptoalkyl tether groups. All the oligonucleotides prepared contained two T11 sequences, and the branched and looped oligomers an additional alternating CT sequence. The melting experiments revealed that the branched oligonucleotides form relatively weak hybrid (double/triple helix) complexes with the single-stranded oligodeoxyribonucleotide, showing a considerable destabilizing effect produced by the structure at the point of branching. The data obtained with looped oligonucleotides demonstrated considerable stabilization of the hybrid (double/triple helix) complexes with the complement. The data reported may be useful in attempting to design new antisense or antigene oligonucleotides capable of forming selective and stable bimolecular hybrid complexes with nucleic acids.
Asunto(s)
ADN de Cadena Simple/química , Oligodesoxirribonucleótidos/química , Secuencia de Bases , Sitios de Unión , ADN de Cadena Simple/genética , Técnicas In Vitro , Datos de Secuencia Molecular , Estructura Molecular , Conformación de Ácido Nucleico , Desnaturalización de Ácido Nucleico , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/genéticaRESUMEN
Several analogues of the standard M13 sequencing primer that contain up to five 3'-deoxypsicothymidines, or one or two such units labeled with fluorescein at the 1'-position, have been prepared. All these oligonucleotides have been shown to prime the DNA-polymerase-catalyzed synthesis of DNA.