RESUMEN
TLRs (Toll-like receptors) are a family of innate immune receptors that induce protective immune responses against infections. Single-stranded viral RNA and bacterial DNA containing unmethylated CpG motifs are the ligands for TLR7 and TLR8 and 9 respectively. We have carried out extensive structure-activity relationship studies of DNA- and RNA-based compounds to elucidate the impact of nucleotide motifs and structures on these TLR-mediated immune responses. These studies have led us to design novel DNA- and RNA-based compounds, which act as potent agonists of TLR9 and TLR7 and 8 respectively. These novel synthetic agonists produce different immune response profiles depending on the structures and nucleotide motifs present in them. The ability to modulate TLR-mediated immune responses with these novel DNA- and RNA-based agonists in a desired fashion may allow targeting a broad range of diseases, including cancers, asthma, allergies and infections, alone or in combination with other therapeutic agents, and their use as adjuvants with vaccines. IMO-2055, our first lead candidate, is a TLR9 agonist that is currently in clinical evaluation in oncology patients. A second candidate, IMO-2125, is also a TLR9 agonist that has been shown to induce high and sustained levels of IFN (interferon) in non-human primates and is being evaluated in HepC-infected human subjects.
Asunto(s)
Receptor Toll-Like 7/agonistas , Receptor Toll-Like 8/agonistas , Receptor Toll-Like 9/agonistas , Animales , Islas de CpG/inmunología , ADN Bacteriano/síntesis química , ADN Bacteriano/farmacología , ADN Bacteriano/uso terapéutico , Humanos , Interferones/efectos de los fármacos , Interferones/inmunología , Ligandos , ARN Viral/síntesis química , ARN Viral/farmacología , ARN Viral/uso terapéutico , Receptor Toll-Like 7/inmunología , Receptor Toll-Like 8/inmunología , Receptor Toll-Like 9/inmunologíaRESUMEN
Bacterial and synthetic DNA containing unmethylated 2'-deoxyribo(cytidine-phosphate-guanosine) (CpG) dinucleotides in specific sequence contexts activate the vertebrate innate immune system. A molecular pattern recognition receptor, Toll-like receptor 9 (TLR9), recognizes CpG DNA and initiates the signalling cascade, although a direct interaction between CpG DNA and TLR9 has not been demonstrated yet. TLR9 in different species exhibits sequence specificity. Our extensive structure-immunostimulatory activity relationship studies showed that a number of synthetic pyrimidine (Y) and purine (R) nucleotides are recognized by the receptor as substitutes for the natural nucleotides deoxycytidine and deoxyguanosine in a CpG dinucleotide. These studies permitted development of synthetic YpG, CpR and YpR immunostimulatory motifs, and showed divergent nucleotide motif recognition pattern of the receptor. Surprisingly, we found that synthetic immunostimulatory motifs produce different cytokine induction profiles compared with natural CpG motifs. Importantly, we also found that some of these synthetic immunostimulatory motifs show optimal activity in both mouse and human systems without the need to change sequences, suggesting an overriding of the species-dependent specificity of the receptor by the use of synthetic motifs. In the present paper, we review current understanding of structural recognition and functional modulation of TLR9 receptor by second-generation synthetic CpG DNAs and their potential application as wide-spectrum therapeutic agents.
Asunto(s)
Proteínas de Unión al ADN/genética , ADN/genética , Receptores de Superficie Celular/genética , Animales , Secuencia de Bases , Citosina , ADN/síntesis química , Proteínas de Unión al ADN/fisiología , Fosfatos de Dinucleósidos/genética , Genes Sintéticos , Guanina , Receptores de Superficie Celular/fisiología , Transducción de Señal , Receptor Toll-Like 9 , VertebradosRESUMEN
Cells from individuals with Cockayne syndrome (CS) have a defect in transcription-coupled DNA repair (TCR), which rapidly corrects certain DNA lesions located on the transcribed strand of active genes. Despite this DNA repair defect, individuals with CS (of which there are two complementation groups, CSA and CSB) do not demonstrate an elevated incidence of cancer. Recently, we demonstrated that disruption of the CSB gene reduces the spontaneous tumor rate in cancer predisposed Ink4a/ARF-/- mice as well as causing their embryo fibroblasts to proliferate more slowly and be more sensitive to UV-induced apoptosis. In the present study we characterized phosphorothioate backbone antisense oligodeoxynucleotides (AOs) that reduced the levels of CSB mRNA in A2780/CP70 ovarian carcinoma cells. The AOs caused the cells to proliferate more slowly and made them more sensitive to either cisplatin or oxaliplatin. The AOs also enhanced the cytotoxicity of hydrogen peroxide and gamma-radiation, both of which can induce oxidative DNA lesions, which are subject to TCR. The AOs did not potentiate the cytotoxicity of topotecan, which induces DNA strand breaks. Chemically modified (
Asunto(s)
ADN Helicasas/genética , Reparación del ADN , Oligonucleótidos Antisentido/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Cisplatino/farmacología , Síndrome de Cockayne/genética , ADN Helicasas/metabolismo , Cartilla de ADN/química , Enzimas Reparadoras del ADN , Resistencia a Medicamentos , Sinergismo Farmacológico , Femenino , Fibroblastos/metabolismo , Expresión Génica , Humanos , Ratones , Ratones Desnudos , Compuestos Organoplatinos/farmacología , Neoplasias Ováricas/patología , Oxaliplatino , Proteínas de Unión a Poli-ADP-Ribosa , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tionucleótidos/uso terapéutico , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/efectos de la radiaciónRESUMEN
Bacterial DNA and synthetic oligodeoxynucleotides containing unmethylated CpG-motifs in a particular sequence context activate vertebrate immune cells. We examined the significance of negatively charged internucleoside linkages in the flanking sequences 5' and 3' to the CpG-motif on immunostimulatory activity. Cell proliferation and secretion of IL-12 and IL-6 in mouse spleen cell cultures, and spleen weights of mice increased significantly when a non-ionic linkage was placed at least four or more internucleoside linkages away from the CpG-motif in the 5'-flanking sequence. When the non-ionic linkage was placed closer than three internucleoside linkages in the 5'-flanking sequence to the CpG-motif, immunostimulatory activity was suppressed compared with that observed with the unmodified parent oligo. In general, the placement of non-ionic linkage in the 3'-flanking sequence to the CpG-motif either did not affect or slightly increased immunostimulatory activity compared with the parent oligo. These results have significance in understanding CpG oligonucleotide-receptor interactions and the development of potent immunomodulatory agents.
Asunto(s)
Adyuvantes Inmunológicos/química , Islas de CpG , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/farmacología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/farmacología , Animales , Secuencia de Bases , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Inmunidad Celular/efectos de los fármacos , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos BALB C , Oligodesoxirribonucleótidos/administración & dosificación , Bazo/citología , Bazo/efectos de los fármacos , Esplenomegalia/inducido químicamente , Relación Estructura-ActividadRESUMEN
Survivin inhibits apoptosis during development and carcinogenesis and is absent in differentiated cells. To determine whether survivin inhibition induces cell death in neural tumor cells, survivin antisense oligonucleotides (SAO) were administered to a human neuroblastoma (MSN) and an oligodendroglioma (TC620) resulting in a dose-dependent reduction in survivin protein. Although 74% of the SAO-treated MSN cells were trypan blue(+), PARP cleavage or activated caspase-3 was not observed. However nuclear translocation of AIF occurred and XIAP increased dramatically. Co-administration of z-Val-Ala-Asp(OMe)-fluoromethyl ketone (zVAD-fmk) with SAO did not inhibit cell death suggesting a caspase-independent mechanism of cell death. Propidium iodide (PI) staining revealed multiple large macronuclei with no apoptotic bodies supporting a role for survivin in cell division. By contrast, while 70% of the SAO-treated TC620 cells were trypan blue(+), PARP was cleaved, cells were TUNEL(+) and PI-staining revealed macronuclei and numerous apoptotic bodies. Co-treatment of the TC620 cells with SAO and zVAD-fmk blocked cell death. While no macronuclei or apoptotic bodies were observed there was a two-fold increase in metaphase cells. Our results suggest that survivin inhibition decreases the viability of human neural tumor cells and as a result of mitotic catastrophe, cell death can be initiated by either a classic apoptotic mechanism or a caspase-independent mechanism.
Asunto(s)
Neoplasias Encefálicas/fisiopatología , Caspasas/fisiología , Proteínas Cromosómicas no Histona/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos , Neuroblastoma/fisiopatología , Oligodendroglioma/fisiopatología , Apoptosis , Neoplasias Encefálicas/patología , Caspasa 3 , Caspasas/metabolismo , Muerte Celular/fisiología , Núcleo Celular/ultraestructura , Proteínas Cromosómicas no Histona/genética , Fase G2 , Humanos , Proteínas Inhibidoras de la Apoptosis , Mitosis , Proteínas de Neoplasias , Neuroblastoma/patología , Oligodendroglioma/patología , Oligonucleótidos Antisentido/farmacología , Proteínas/metabolismo , Survivin , Células Tumorales Cultivadas , Proteína Inhibidora de la Apoptosis Ligada a XRESUMEN
The effect of nucleobase deletion in the 3'- or the 5'-flanking sequence to a CpG-motif on immunostimulatory activity of CpG-containing oligonucleotides was examined by cell proliferation, secretion of IL-12 and IL-6 in mouse spleen cell cultures, and by spleen enlargement in mice. Deletion of one or two nucleobases in the 3'-flanking sequence to a CpG-motif at certain positions did not affect immunostimulatory activity, while similar deletions in the 5'-flanking sequence increased immunostimulatory activity compared with the parent oligo.
Asunto(s)
Islas de CpG/inmunología , Oligonucleótidos/inmunología , Oligonucleótidos/farmacología , Región de Flanqueo 3' , Región de Flanqueo 5' , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos BALB C , Oligonucleótidos/química , Tamaño de los Órganos/efectos de los fármacos , Eliminación de Secuencia , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunología , Relación Estructura-ActividadRESUMEN
We have constructed a series of 22 phosphorothioate 20-mer antisense oligonucleotides directed against different regions of the human (EGFR) mRNA. Treatment with EGFR antisense oligonucleotides showed a dose-dependent inhibition of human GEO colon cancer cell growth in soft agar. Western blot analysis demonstrated a significant reduction in EGFR expression after treatment with each EGFR antisense oligonucleotide. The ability to inhibit GEO anchorage-independent growth, however, varied among the EGFR antisense sequences with an IC(50) ranging between 0.5 and 3.5 microM. Two of these antisense oligonucleotides targeting the regions between 2457-2476 and 614-4633 bases of the human EGFR mRNA have been modified as hybrid DNA/RNA mixed backbone oligonucleotides (MBO) to examine their anticancer properties in vivo. The 2 EGFR antisense MBOs retained the same biological properties of the fully phosphorothioate EGFR antisense oligonucleotides targeting the same EGFR mRNA sequences, such as blocking EGFR synthesis, inhibiting cell growth and enhancing programmed cell death in human cancer cell lines that express functional EGFRs. Furthermore, a potentiation in the growth inhibitory effect on GEO cancer cells was observed after treatment with these EGFR antisense MBOs in combination with cytotoxic drugs, including cisplatin, doxorubicin, paclitaxel, or topotecan. These results show the antiproliferative activity of specific EGFR antisense oligonucleotides and allow to identify novel EGFR antisense MBOs that deserve further evaluation as potential selective anticancer agents alone or in combination with cytotoxic drugs in human carcinomas that express functional EGFRs.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis , Receptores ErbB/antagonistas & inhibidores , Oligonucleótidos Antisentido/farmacología , División Celular/efectos de los fármacos , Cisplatino/farmacología , Doxorrubicina/farmacología , Combinación de Medicamentos , Receptores ErbB/genética , Humanos , Paclitaxel/farmacología , Topotecan/farmacología , Células Tumorales CultivadasRESUMEN
In order to provide a structural basis for the unusual properties of 2',5' nucleic acids, especially their unsuitability as information molecules, we report here a high resolution NMR structure of a 2',5' RNA fragment r(GCCGCGGC). It forms an A type duplex with C2'endo compact nucleotide repeat, instead of the familiar C3'endo compact nucleotide (seen in RNA) supporting the deductions made earlier from stereochemical considerations. This data together with the observation that 2',5' nucleic acids require mandatory slide and displacement for duplex and triplex structure formation suggest their reluctance to form the biologically relevant B type duplex. It is argued that this lack of flexibility for helical polymorphism and other inadequacies as a consequence of this may be a contributing factor for the rejection of 2',5' links by nature. The structure exhibits interesting features such as the syn glycosyl conformation for the terminal guanine and a hydrogen bond between O3' hydroxyl and anionic oxygen of the phosphate.
Asunto(s)
ARN/química , Secuencia de Bases , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación de Ácido Nucleico , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Co-administration of synthetic chemically modified oligonucleotides with irinotecan, a selective topoisomerase I inhibitor, provided a significant enhancement in the antitumor activity of irinotecan. The enhancement of antitumor activity of irinotecan with co-administration of chemically modified oligonucleotides was observed in several tumor models--pancreatic cancer (Panc-1), colon cancer (HCT-116) and melanoma (A375). Inhibition of tumor growth in all three models required the co-administration of irinotecan and chemically modified oligonucleotides, but was independent of the nucleotide sequence of the oligonucleotides. The potentiation of antitumor activity was dependent on the dose of irinotecan and chemically modified oligonucleotides administered. The enhancement of antitumor activity of irinotecan was also observed by co-administration of a phosphorothioate oligonucleotide, however, to a lesser extent than did chemically modified oligonucleotides, suggesting that metabolic stability of the oligonucleotide contributes to the enhancement of antitumor activity seen with irinotecan. The co-administration of dextran sulfate sodium with irinotecan showed insignificant potentiation of antitumor activity of irinotecan, suggesting that the enhancement of antitumor activity of irinotecan observed was not a result of polyanionic characteristic of oligonucleotides. Co-administration of irinotecan and chemically modified oligonucleotides did not result in increased toxicity in the tumor models studied. Potentiation of antitumor activity of irinotecan observed with co-administration of oligonucleotides suggests that the oligonucleotides affect the pharmacokinetics and/or metabolism of irinotecan. The use of chemically modified oligonucleotides together with irinotecan may increase the therapeutic index of irinotecan in cancer patients and continued development of such agents should be considered.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Camptotecina/análogos & derivados , Camptotecina/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Neoplasias/tratamiento farmacológico , Proteínas Nucleares , Oligonucleótidos Antisentido/uso terapéutico , Proteínas Proto-Oncogénicas/genética , Inhibidores de Topoisomerasa I , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Sinergismo Farmacológico , Femenino , Humanos , Irinotecán , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Resultado del TratamientoRESUMEN
Oligodeoxynucleotides containing unmethylated CpG-motifs stimulate the innate immune system, including inducing B-cell proliferation and cytokine production. However, the mechanism of immunostimulation by CpG-oligonucleotides and the precise structural requirements and specific functional groups of cytosine and guanine necessary for recognition of and interaction with protein/receptor factors that are responsible for immune stimulation have not been elucidated. We sought to understand the critical role of each functional group of the cytosine and guanine moieties in a CpG-motif in inducing immunostimulatory activity. To this end, we examined structure-immunostimulatory activity relationships of phosphorothioate oligodeoxynucleotides (PS-oligos) containing YpG- and CpR-motifs (Y and R stand for pyrimidine and purine analogues, respectively). The PS-oligos containing a YpG-motif in which the natural deoxycytidine was replaced with deoxy-5-hydroxycytidine or deoxy-N4-ethylcytidine showed immunostimulatory activity. Substitution of deoxycytidine with a deoxy-5-methylisocytidine, deoxyuridine, or deoxy-P-base-nucleoside in the YpG-motif completely abolished the immunostimulatory activity, similar to the results observed with deoxy-5-methylcytidine. In the case of PS-oligos containing a CpR-motif, 7-deazaguanine substitution for natural guanine showed immunostimulatory activity similar to that of a parent PS-oligo. These studies suggest that the 2-keto, 3-imino and 4-amino groups of cytosine, and the 1-imino, 2-amino and 6-keto groups of guanine in a CpG-motif are important for the immunostimulatory activity of CpG-PS-oligos. The absence of N7 on guanine of the CpG-motif does not affect immunostimulatory activity significantly. These studies suggest that it is possible to develop YpG- and CpR-motifs as an alternative to CpG-motifs in PS-oligos for immunostimulatory studies.
Asunto(s)
Activación de Linfocitos/efectos de los fármacos , Oligonucleótidos/inmunología , Animales , Secuencia de Bases , Citosina/química , Didesoxinucleósidos/química , Didesoxinucleósidos/inmunología , Diseño de Fármacos , Femenino , Guanina/química , Inyecciones Intraperitoneales , Linfocitos/citología , Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Oligonucleótidos/química , Oligonucleótidos/farmacología , Bazo/citología , Bazo/efectos de los fármacos , Esplenomegalia/inducido químicamente , Relación Estructura-ActividadRESUMEN
Antisense technology, which is based on a simple and rational principle of Watson-Crick complementary base pairing of a short oligonucleotide with the targeted mRNA to downregulate the disease-causing gene product, has progressed tremendously in the last two decades. Antisense oligonucleotides targeted to a number of cancer-causing genes are being evaluated in human clinical trials. While the first-generation phosphorothioate antisense oligonucleotides are in clinical trials, a number of factors, including sequence motifs that could lead to unwanted mechanisms of action and side effects, have been identified. The severity of the side effects of first-generation antisense oligonucleotides is mostly dependent on the presence of certain sequence motifs, such as CpG dinucleotides. A number of second-generation chemical modifications have been proposed to overcome the limitations of the first-generation antisense oligonucleotides. The safety and efficacy of several second-generation mixed-backbone antisense oligonucleotides are being evaluated in clinical trials. The immune stimulation affects observed with CpG-containing antisense oligonucleotides are being exploited as a novel therapeutic modality, with several CpG oligonucleotides being evaluated in clinical trials. A number of medicinal chemistry studies performed to date suggest that the immunomodulatory activity of CpG oligonucleotides can be fine-tuned by site-specific incorporation of chemical modifications in order to design disease-specific oligonucleotide therapeutics.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Antineoplásicos/uso terapéutico , Islas de CpG/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Oligodesoxirribonucleótidos/inmunología , Oligonucleótidos Antisentido/uso terapéutico , Tionucleótidos/química , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Drogas en Investigación/farmacología , Regulación Neoplásica de la Expresión Génica , Oligodesoxirribonucleótidos/farmacología , ARN MensajeroRESUMEN
In our ongoing efforts to decipher the sequence and structural requirements in the flanking region of the CpG motif in phosphorothioate oligodeoxynucleotides (PS-oligos), we have examined the requirement of free 5'- and 3'-ends of PS-oligos on immune stimulation. Our model studies using 3'-3'-linked (containing two free 5'-ends) and 5'-5'-linked (containing two free 3'-ends) CpG-containing PS-oligos demonstrate that immunostimulatory activity is significantly reduced when the 5'-end of the PS-oligo is not accessible, rather than the 3'-end, suggesting that the 5'-end plays a critical role in immunostimulatory activity.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Islas de CpG , Tionucleótidos/farmacología , Adyuvantes Inmunológicos/síntesis química , Adyuvantes Inmunológicos/química , Animales , Secuencia de Bases , División Celular/efectos de los fármacos , Linfocitos/citología , Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Compuestos Organofosforados/química , Bazo/citología , Bazo/efectos de los fármacos , Esplenomegalia/inducido químicamente , Tionucleótidos/síntesis química , Tionucleótidos/químicaRESUMEN
Stereo-enriched [Rp] and [Sp]-phosphorothioate oligodeoxynucleotides are synthesized using oxazaphospholidine derivatized monomers. Three different designs of phosphorothioate oligodeoxynucleotides (PS-oligos), (i) stereo-enriched all-[Rp] or all-[Sp] PS-linkages, (ii) stereo-random mixture of PS-linkages, and (iii) segments containing certain number of stereo-enriched [Rp] and [Sp] PS-linkages ([Sp-Rp-Sp] or [Rp-Sp-Rp]), have been studied. Thermal melting studies of these PS-oligos with RNA complementary strands showed that the binding affinities are in the order [Rp] > [Sp-Rp-Sp]-[Rp-Sp-Rp] > stereo-random > [Sp]. Circular dichroism (CD) studies suggest that the stereochemistry of the PS-oligo does not affect the global conformation of the duplex. The in vitro nuclease stability of these PS-oligos is in the order [Sp] > [Sp-Rp-Sp] > stereo-random > [Rp]. The RNase H activation is in the order [Rp] > stereo-random > [Rp-Sp-Rp] > [Sp] > [Sp-Rp-Sp]. Studies in a cancer cell line of PS-oligos targeted to MDM2 mRNA showed that all oligos had similar biological activity under the experimental conditions employed. Protein- and enzyme-binding studies showed insignificant stereo-dependent binding to proteins. The [Sp] and [Sp-Rp-Sp] chimeric and stereo-random PS-oligos that contained a CpG motif showed higher cell proliferation than [Rp] PS-oligo of the same sequence.
Asunto(s)
Proteínas Nucleares , Compuestos Organofosforados/química , Tionucleótidos/síntesis química , Animales , Secuencia de Bases , División Celular/efectos de los fármacos , Dicroismo Circular , Masculino , Ratones , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2 , Espectrofotometría Ultravioleta , Tionucleótidos/química , Tionucleótidos/farmacologíaRESUMEN
We have studied the use of 'pseudocyclic oligonucleotides' (PCOs) (Jiang et al. Bioorg. Med. Chem. 1999, 7, 2727) as hybridization-based fluorescent probes. The resulting fluorescent tag-attached PCOs are called 'cyclicons'. Cyclicons consist of two oligonucleotides linked to each other through 3'-3' or 5'-5' ends. One of the oligos is the probe or primer-probe sequence that is complementary to a target nucleic acid (mRNA/DNA), and the other is a modifier oligo that is complementary to one of the ends of the probe oligo. A fluorescence molecule and a quencher molecule are attached at an appropriate position in the cyclicons. In the absence of the target nucleic acid, the fluorophore and the quencher are brought in close proximity to each other because of the formation of an intramolecular cyclic structure, resulting in fluorescence quenching. When the cyclicon hybridizes to the complementary target nucleic acid strand, the intramolecular cyclic structure of the cyclicon is destabilized and opened up, separating the fluorophore and quencher groups, resulting in spontaneous fluorescence emission. Fluorescent studies in the presence and absence of a target nucleic acid suggest that cyclicons exist in intramolecular cyclic structure form in the absence of the target and form the duplex with the target sequence when present. Both the cyclicons are useful for nucleic acid detection. The studies with DNA polymerase on 5'-5'-attached cyclicons suggest that the presence of quencher moiety in the probe sequence does not inhibit chain elongation by polymerase. The experiments with a 5'-5'-attached cyclicon suggest the new design serves as an efficient unimolecular primer-probe in real-time PCR experiments.
Asunto(s)
Colorantes Fluorescentes/metabolismo , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos/metabolismo , p-Dimetilaminoazobenceno/análogos & derivados , Autorradiografía , Proteínas Portadoras , ADN/metabolismo , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Humanos , Sondas de Oligonucleótidos/síntesis química , Sondas de Oligonucleótidos/química , Sondas de Oligonucleótidos/genética , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Polimerasa Taq/metabolismo , Células Tumorales Cultivadas , p-Dimetilaminoazobenceno/metabolismoRESUMEN
Antisense oligonucleotides provide a simple and efficient approach for developing target-selective drugs because they can modulate gene expression sequence-specifically. Antisense oligonucleotides have also become efficient molecular biological tools to investigate the function of any protein in the cell. As the application of antisense oligonucleotides has expanded, multiple mechanisms of oligonucleotides have been characterized that impede their routine use. Here, we discuss different mechanisms of action of oligonucleotides and the possible ways of minimizing non-antisense-related [corrected] effects to improve their specificity.
Asunto(s)
Emparejamiento Base/genética , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Antisentido/uso terapéutico , Animales , Secuencia de Bases , Sitios de Unión , Ensayos Clínicos como Asunto , Islas de CpG/genética , Diseño de Fármacos , Humanos , Inmunización , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/genética , Unión Proteica , Especificidad por SustratoRESUMEN
We have designed and studied antisense oligodeoxynucleotides (oligonucleotides; oligos) which we call 'pseudo-cyclic oligonucleotides' (PCOs). PCOs contain two oligonucleotide segments attached through their 3'-3'- or 5'-5'-ends. One of the segments of the PCO is an antisense oligo complementary to a target mRNA, and the other is a short protective oligo that is 5-8 nucleotides long and complementary to the 3'- or 5'-end of the antisense oligo. As a result of complementarity between the antisense and protective oligo segments, PCOs form intramolecular pseudo-cyclic structures in the absence of the target RNA. The antisense oligo segment of PCOs used for the studies described here is complementary to an 18-nucleotide-long site on the mRNA of the protein kinase A regulatory subunit RIalpha (PKA-RIalpha). Thermal melting studies of PCOs in the absence and presence of the complementary RNA suggest that the pseudo-cyclic structures formed in the absence of the target RNA dissociate, bind to the target RNA, and form heteroduplexes. The results of RNase H cleavage assays suggest that PCOs bind to complementary RNA and activate RNase H in a manner similar to that of an 18-mer conventional antisense PS-oligo. In snake venom (a 3'-exonuclease) or spleen (a 5'-exonuclease) phosphodiesterase digestion studies, PCOs are more stable than conventional antisense oligos because of the presence of 3'-3'- or 5'-5'-linkages and the formation of intramolecular pseudo-cyclic structures. PCOs with a phosphorothioate antisense oligo segment inhibited cell growth of MDA-MB-468 and GEO cancer cell lines similar to that of the conventional antisense PS-oligo, suggesting efficient cellular uptake and target binding. The nuclease stability studies in mice suggest that PCOs have higher in vivo stability than antisense PS-oligos. The studies in mice showed similar pharmacokinetic and tissue distribution profiles for PCOs to those of antisense PS-oligos in general, but rapid elimination from selected tissues.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Oligodesoxirribonucleótidos Antisentido/química , Oligodesoxirribonucleótidos Antisentido/farmacología , Animales , Antineoplásicos/farmacocinética , Secuencia de Bases , División Celular/efectos de los fármacos , Diseño de Fármacos , Estabilidad de Medicamentos , Humanos , Masculino , Ratones , Desnaturalización de Ácido Nucleico , Oligodesoxirribonucleótidos Antisentido/genética , ARN Mensajero/genética , Temperatura , Distribución Tisular , Células Tumorales Cultivadas , Ensayo de Tumor de Célula MadreRESUMEN
All phosphorothioate mixed-backbone oligonucleotides (MBOs) composed of deoxyribonucleotide and 2'-O-methylribonucleotide segments were studied for their target binding affinity, specificity, and RNase H activation properties. The 2'-O-methylribonucleotide segment, which does not activate RNase H, serves as a high affinity target-binding domain and the deoxyribonucleotide (DNA) segment, which binds to the target with a lower affinity than the former domain, serves as an RNase H-activation or target-cleaving domain. In order to understand the influence of the size and position of the DNA segment of MBOs on RNase H-mediated cleavage of the RNA target, we designed and synthesized a series of 18-mer MBOs with the DNA segment varying from a stretch of two to eight deoxyribonucleotides in the middle, at the 5'-end, or at the 3'-end, of the MBOs. UV absorbance melting experiments of the duplexes of the MBOs with the complementary and singly mismatched RNA targets suggest that the target binding affinity of the MBOs increases as the number of 2'-O-methylribonucleotides increases, and that the binding specificity is influenced by the size and position of the DNA segment. Analysis of RNase H assay results indicates that the minimum substrate cleavage site and cleavage efficiency of RNase H are influenced by the position of the DNA segment in the MBO sequence. RNA cleavage efficiency decreases as the position of the DNA segment of the MBO.RNA heteroduplex is changed from the 3'-end to the middle and to the 5'-end of the target strand. Studies with singly mismatched targets indicate that the RNase H-dependent point mutation selectivity of the MBOs is affected by both the position and size of the DNA segment in the MBO sequence.
Asunto(s)
Escherichia coli/enzimología , Oligonucleótidos/química , Oligonucleótidos/metabolismo , ARN/metabolismo , Ribonucleasa H/metabolismo , Sitios de Unión , ADN/química , Densitometría , Oligodesoxirribonucleótidos Antisentido/química , Oligodesoxirribonucleótidos Antisentido/metabolismo , Organotiofosfatos/química , ARN/química , Ribonucleasa H/química , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Pharmacokinetic studies of phosphorothioate oligodeoxynucleotides (PS-oligonucleotides) in animals show that following intravenous administration, PS-oligonucleotide clears out rapidly from the plasma and is distributed to majority of the organs. PS-oligonucleotides are bound to plasma proteins extensively. This study was aimed to determine the effect of aspirin, a commonly used drug, on pharmacokinetics of PS-oligonucleotides. In the present study, PS-oligonucleotide was administered to rats that had received aspirin by gavage. Pharmacokinetic study shows that if PS-oligonucleotide was administered following aspirin administration in rats, a) plasma pharmacokinetic parameters (t1/2alpha?, t1/2beta, AUC, etc.) had lower values, b) tissue disposition was different, and c) rate and route of elimination was affected in animals compared to rats receiving PS-oligonucleotide alone. This finding suggests that pharmacokinetics of PS-oligonucleotides can be affected with certain class of drugs, which may have direct impact on biological activity and safety.
Asunto(s)
Aspirina/farmacología , Tionucleótidos/farmacocinética , Animales , Área Bajo la Curva , Secuencia de Bases , Proteínas Sanguíneas/metabolismo , Heces/química , Semivida , Masculino , Unión Proteica , Ratas , Ratas Sprague-Dawley , Tionucleótidos/metabolismo , Tionucleótidos/orina , Distribución TisularRESUMEN
We have synthesized and studied the effects of phosphorothioate (PS) oligodeoxyribonucleotide (DNA) and oligoribonucleotides (RNA, 2'-O-methyl-RNA and 2'-5'-RNA) on complement activation and prolongation of activated partial thromboplastin time (aPTT) in vitro. These results suggest that a PS-DNA prolongs aPTT, and inhibits complement lysis more than do the PS-RNA analogs.