RESUMEN
There is tremendous potential for oligonucleotide (ON) therapeutics, but low cellular penetration due to their polyanionic nature is a major obstacle. We addressed this problem by developing a new approach for ON charge neutralization in which multiple branched charge-neutralizing sleeves (BCNSs) are attached to the internucleoside phosphates of ON by phosphotriester bonds. The BCNSs are terminated with positively charged amino groups, and are optimized to form ion pairs with the neighboring phosphate groups. The new modified ONs can be prepared by standard automated phosphoramidite chemistry in good yield and purity. They possess good solubility and hybridization properties, are not involved in non-standard intramolecular aggregation, have low cytotoxicity, adequate chemical stability, improved serum stability, and above all, display significantly enhanced cellular uptake. Thus, the new ON derivatives exhibit properties that make them promising candidates for the development of novel therapeutics or research tools for modulation of the expression of target genes.
Asunto(s)
Oligonucleótidos/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Cinética , Modelos Moleculares , Estructura Molecular , Oligonucleótidos/química , Solubilidad , Relación Estructura-ActividadRESUMEN
BACKGROUND: The Signal Transducer and Activator of Transcription 3 (STAT3) is activated in tumor cells, and STAT3-inhibitors are able to induce the death of those cells. Decoy oligodeoxynucleotides (dODNs), which bind to the DNA Binding Domain (DBD) of STAT3, are efficient inhibitors. However, they also inhibit STAT1, whose activity is essential not only to resistance to pathogens, but also to cell growth inhibition and programmed cell death processes. The aim of this study was to design STAT3-specific dODNs which do not affect STAT1-mediated processes. RESULTS: New dODNs with a hairpin (hpdODNs) were designed. Modifications were introduced, based on the comparison of STAT3- and STAT1-DBD interactions with DNA using 3D structural analyses. The designed hpdODNs were tested for their ability to inhibit STAT3 but not STAT1 by determining: i) cell death in the active STAT3-dependent SW480 colon carcinoma cell line, ii) absence of inhibition of interferon (IFN) γ-dependent cell death, iii) expression of STAT1 targets, and iv) nuclear location of STAT3 and STAT1. One hpdODN was found to efficiently induce the death of SW480 cells without interfering with IFNγ-activated STAT1. This hpdODN was found in a complex with STAT3 but not with STAT1 using an original in-cell pull-down assay; this hpdODN also did not inhibit IFNγ-induced STAT1 phosphorylation, nor did it inhibit the expression of the STAT1-target IRF1. Furthermore, it prevented the nuclear transfer of STAT3 but not that of IFNγ-activated STAT1. CONCLUSIONS: Comparative analyses at the atomic level revealed slight differences in STAT3 and STAT1 DBDs' interaction with their DNA target. These were sufficient to design a new discriminating hpdODN that inhibits STAT3 and not STAT1, thereby inducing tumor cell death without interfering with STAT1-dependent processes. Preferential interaction with STAT3 depends on oligodeoxynucleotide sequence modifications but might also result from DNA shape changes, known to modulate protein/DNA interactions. The finding of a STAT3-specific hpdODN establishes the first rational basis for designing STAT3 DBD-specific inhibitors.
Asunto(s)
Muerte Celular/efectos de los fármacos , Oligodesoxirribonucleótidos/farmacología , Factor de Transcripción STAT1/antagonistas & inhibidores , Factor de Transcripción STAT3/antagonistas & inhibidores , Línea Celular Tumoral , Humanos , Fosforilación/efectos de los fármacos , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
BACKGROUND: The transcription factor STAT3 (signal transducer and activator of transcription 3) is frequently activated in tumor cells. Activated STAT3 forms homodimers, or heterodimers with other TFs such as NF-κB, which becomes activated. Cytoplasmic STAT3 dimers are activated by tyrosine phosphorylation; they interact with importins via a nuclear localization signal (NLS) one of which is located within the DNA-binding domain formed by the dimer. In the nucleus, STAT3 regulates target gene expression by binding a consensus sequence within the promoter. STAT3-specific decoy oligonucleotides (STAT3-decoy ODN) that contain this consensus sequence inhibit the transcriptional activity of STAT3, leading to cell death; however, their mechanism of action is unclear. RESULTS: The mechanism of action of a STAT3-decoy ODN was analyzed in the colon carcinoma cell line SW 480. These cells' dependence on activated STAT3 was verified by showing that cell death is induced by STAT3-specific siRNAs or Stattic. STAT3-decoy ODN was shown to bind activated STAT3 within the cytoplasm, and to prevent its translocation to the nucleus, as well as that of STAT3-associated NF-κB, but it did not prevent the nuclear transfer of STAT3 with mutations in its DNA-binding domain. The complex formed by STAT3 and the STAT3-decoy ODN did not associate with importin, while STAT3 alone was found to co-immunoprecipitate with importin. Leptomycin B and vanadate both trap STAT3 in the nucleus. They were found here to oppose the cytoplasmic trapping of STAT3 by the STAT3-decoy ODN. Control decoys consisting of either a mutated STAT3-decoy ODN or a NF-κB-specific decoy ODN had no effect on STAT3 nuclear translocation. Finally, blockage of STAT3 nuclear transfer correlated with the induction of SW 480 cell death. CONCLUSIONS: The inhibition of STAT3 by a STAT3-decoy ODN, leading to cell death, involves the entrapment of activated STAT3 dimers in the cytoplasm. A mechanism is suggested whereby this entrapment is due to STAT3-decoy ODN's inhibition of active STAT3/importin interaction. These observations point to the high potential of STAT3-decoy ODN as a reagent and to STAT3 nucleo-cytoplasmic shuttling in tumor cells as a potential target for effective anti-cancer compounds.
Asunto(s)
Nucléolo Celular/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/fisiopatología , Silenciador del Gen , FN-kappa B/metabolismo , Oligonucleótidos/genética , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transporte Activo de Núcleo Celular , Muerte Celular , Línea Celular Tumoral , Nucléolo Celular/genética , Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica , Humanos , FN-kappa B/genética , Unión ProteicaRESUMEN
Three oligodeoxyribonucleotides (ODN) covalently labeled with near-infrared (NIR) fluorochromes were synthesized and characterized with a goal of comparing in vitro a hairpin-based and a duplex-based FRET probe designed for the detection of human recombinant NF-κB p50/p65 heterodimer binding to DNA. Using deoxyguanosine phosphoramidite with a phosphorus-linked aminoethylene (diethylene glycol) hydrophilic linker, we synthesized ODNs with internucleoside reactive sites. The hairpin loop amino linker was modified with IRDye 800CW (FRET acceptor), and the 3'-end was modified with Cy5.5 (FRET donor) using a dithio-linker. To obtain a duplex probe, we conjugated Cy5.5 and 800CW to complementary strands at the distance of ten base pairs in the resultant duplex. No quenching of dyes was observed in either probe. The FRET efficiency was higher in the duplex (71%) than in the hairpin (56%) due to a more favorable distance between the donor and the acceptor. However, the hairpin design allowed more precise ratiometric measurement of fluorescence intensity changes as a result of NF-κB p50/p65 binding to the probe. We determined that as a result of binding there was a statistically significant increase of fluorescence intensity of Cy5.5 (donor) due to a decrease of FRET if normalized by 800CW intensity measured independently of FRET. We conclude that the hairpin based probe design allows for the synthesis of a dual fluorescence imaging probe that renders signal changes that are simple to interpret and stoichiometrically correct for detecting transcription factor-DNA interactions.
Asunto(s)
Colorantes Fluorescentes/química , Imagen Molecular , Subunidad p50 de NF-kappa B/análisis , Oligodesoxirribonucleótidos/química , Factor de Transcripción ReIA/análisis , Sitios de Unión , ADN/química , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/síntesis química , Humanos , Modelos Moleculares , Estructura Molecular , Subunidad p50 de NF-kappa B/química , Subunidad p50 de NF-kappa B/metabolismo , Oligodesoxirribonucleótidos/síntesis química , Proteínas Recombinantes/análisis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Factor de Transcripción ReIA/química , Factor de Transcripción ReIA/metabolismoRESUMEN
Optical imaging in the near-infrared (NIR) range enables detecting ligand-receptor interactions and enzymatic activity in vivo due to lower scattering and absorption of NIR photons in the tissue. We designed and tested prototype NIR fluorescent oligodeoxyribonucleotide (ODN) reporters that can sense transcription factor NF-kappaB p50 protein binding. The reporter duplexes included donor NIR Cy5.5 indodicarbocyanine fluorochrome linked to the 3' end of the first ODN and NIR acceptor fluorochromes (indodicarbocyanine Cy7 or, alternatively, a heptamethine cyanine IRDye 800CW) that were linked at the positions +8 and +12 to the complementary ODN that encoded p50 binding sites. Both Cy7 and 800CW fluorochromes were linked by using hydrophilic internucleoside phosphate linkers that enable interaction between the donor and the acceptor with no base-pairing interference. We observed efficient fluorescence resonance energy transfer (FRET) both in the case of Cy5.5-Cy7 and Cy5.5-800CW pairs of fluorochromes, which was sensitive to the relative position of the dyes. Higher FRET efficiency observed in the case of Cy5.5-Cy7 pair was due to a larger overlap between the ODN-linked Cy5.5 emission and Cy7 excitation spectra. Fluorescent mobility shift assay showed that the addition of human recombinant p50 to ODN duplexes resulted in p50 binding and measurable increase of Cy5.5 emission. In addition, p50 binding provided a concomitant protection of FRET effect from exonuclease-mediated hydrolysis. We conclude that NIR FRET effect can be potentially used for detecting protein-DNA interactions and that the feasibility of detection depends on FRET efficacy and relative fluorochrome positions within ODN binding sites.
Asunto(s)
ADN/química , ADN/metabolismo , Oligonucleótidos/química , Proteínas/química , Proteínas/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Hidrólisis , Estructura Molecular , Unión ProteicaRESUMEN
A novel thymidine phosphoramidite synthon was synthesized and successfully used for incorporation of primary amino groups, attached through a triethylene glycol linker to the internucleoside phosphates, at desired locations during automated oligodeoxynucleotide synthesis. The synthesized amino-linker bearing oligonucleotides are stable under deprotection conditions and exhibit Watson-Crick base-pairing properties. Covalent labeling of oligonucleotides with carbocyanine near-infrared fluorochromes resulted in 2.5 times higher labeling yields when compared with oligonucleotides containing base-attached aminolinkers. We anticipate that the developed synthetic approach will be useful for nucleotide sequence-specific attachment of single or multiple ligands or reporter molecules.
Asunto(s)
Oligodesoxirribonucleótidos/síntesis química , Compuestos Organofosforados/síntesis química , Timidina/síntesis química , Emparejamiento Base , Carbocianinas/síntesis química , Carbocianinas/química , Oligodesoxirribonucleótidos/química , Compuestos Organofosforados/química , Timidina/químicaRESUMEN
The transcription factor NF-kappaB is frequently activated in cancer, and is therefore a valuable target for cancer therapy. Decoy oligodeoxynucleotides (ODNs) inhibit NF-kappaB by preventing its binding to the promoter region of target genes. Few studies have used NF-kappaB-targeting with ODNs in cancer. Using a hairpin NF-kappaB-decoy ODN we found that it induced growth inhibition and cell death in NF-kappaB-dependent tumour cell lines. The ODN colocalized with the p50 subunit of NF-kappaB in cells and directly interacted with it in nuclear extracts. In TNFalpha-treated cells the ODN and the p50 subunit were found in the cytoplasm suggesting that the complex did not translocate to the nucleus. Transcriptional activity of NF-kappaB was efficiently inhibited by the ODN, whereas a scrambled ODN was without effect on transcription. Thus, ODN-mediated inhibition of NF-kappaB can efficiently promote cell death in cancer cells providing a potentially powerful approach to tumour growth inhibition.
Asunto(s)
FN-kappa B/antagonistas & inhibidores , Neoplasias/patología , Oligodesoxirribonucleótidos/farmacología , Sitios de Unión , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Citoplasma/química , Humanos , Subunidad p50 de NF-kappa B/antagonistas & inhibidores , Conformación de Ácido Nucleico , Unión Proteica/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
We present here the first detailed biochemical analysis of an archaeal restriction enzyme. PspGI shows sequence similarity to SsoII, EcoRII, NgoMIV and Cfr10I, which recognize related DNA sequences. We demonstrate here that PspGI, like SsoII and unlike EcoRII or NgoMIV and Cfr10I, interacts with and cleaves DNA as a homodimer and is not stimulated by simultaneous binding to two recognition sites. PspGI and SsoII differ in their basic biochemical properties, viz. stability against chemical denaturation and proteolytic digestion, DNA binding and the pH, MgCl(2) and salt-dependence of their DNA cleavage activity. In contrast, the results of mutational analyses and cross-link experiments show that PspGI and SsoII have a very similar DNA binding site and catalytic center as NgoMIV and Cfr10I (whose crystal structures are known), and presumably also as EcoRII, in spite of the fact that these enzymes, which all recognize variants of the sequence -/CC-GG- (/ denotes the site of cleavage), are representatives of different subgroups of type II restriction endonucleases. A sequence comparison of all known restriction endonuclease sequences, furthermore, suggests that several enzymes recognizing other DNA sequences also share amino acid sequence similarities with PspGI, SsoII and EcoRII in the region of the presumptive active site. These results are discussed in an evolutionary context.
Asunto(s)
Proteínas Arqueales/química , Proteínas Arqueales/genética , Enzimas de Restricción del ADN/genética , Desoxirribonucleasas de Localización Especificada Tipo II/química , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Pyrococcus/enzimología , Secuencia de Aminoácidos , Proteínas Arqueales/metabolismo , Azidas/química , Sitios de Unión , Dominio Catalítico , Cromatografía en Gel , Reactivos de Enlaces Cruzados/química , Cisteína/química , ADN/química , ADN/metabolismo , Análisis Mutacional de ADN , Enzimas de Restricción del ADN/química , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Disulfuros/química , Estabilidad de Enzimas , Escherichia coli/genética , Evolución Molecular , Concentración de Iones de Hidrógeno , Magnesio/química , Magnesio/metabolismo , Microscopía Electrónica/métodos , Modelos Moleculares , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Fotoquímica/métodos , Desnaturalización Proteica , Sales (Química)/química , Homología de Secuencia de AminoácidoRESUMEN
The double-stranded oligodeoxyribonucleotides with single internucleotide disulfide linkages were successfully used for covalent trapping of cysteine containing protein. In particular, an efficient conjugation of DNA methyltransferase SsoII to sequence-specific decoys was demonstrated. The obtained results assume that synthetic oligodeoxyribonucleotides bearing a new trapping site can be used as new tools to study and manipulate biological systems.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Disulfuros/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Moldes Genéticos , Secuencia de Bases , Electroforesis en Gel de PoliacrilamidaRESUMEN
The NF-kappaB transcriptional factor regulates various functions such as immune responses, cellular growth and development, and is frequently activated in tumor cells. Thus, inhibition of NF-kappaB could suppress tumor cell growth. Using a decoy synthetic hairpin-shaped oligodeoxyribonucleotide (ODN) containing the kappaB site with an integrated single diphosphoryldisulfide linkage, we demonstrate its covalent binding to the p50 subunit of NF-kappaB. Furthermore, this decoy ODN induces apoptosis in a lymphoblastoma cell line. Thus, such chemically modified decoys could be valuable tools for blocking nuclear factors and tumor cell growth.
Asunto(s)
Apoptosis/fisiología , Oligodesoxirribonucleótidos/metabolismo , Péptidos/metabolismo , Secuencia de Bases , Sitios de Unión , División Celular/fisiología , Núcleo Celular/metabolismo , Humanos , Oligodesoxirribonucleótidos/química , Péptidos/química , Leucemia-Linfoma Linfoblástico de Células Precursoras , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
The signal transducer and activator of transcription STAT3 is a transcription factor which plays a key role in normal cell growth and is constitutively activated in about 70% of solid and hematological cancers. Activated STAT3 is phosphorylated on tyrosine and forms a dimer through phosphotyrosine/src homology 2 (SH2) domain interaction. The dimer enters the nucleus via interaction with importins and binds target genes. Inhibition of STAT3 results in the death of tumor cells, this indicates that it is a valuable target for anticancer strategies; a view that is corroborated by recent findings of activating mutations within the gene. Yet, there is still only a small number of STAT3 direct inhibitors; in addition, the high similarity of STAT3 with STAT1, another STAT family member mostly oriented toward apoptosis, cell death and defense against pathogens, requires that STAT3-inhibitors have no effect on STAT1. Specific STAT3 direct inhibitors consist of SH2 ligands, including G quartet oligodeoxynucleotides (ODN) and small molecules, they induce cell death in tumor cells in which STAT3 is activated. STAT3 can also be inhibited by decoy ODNs (dODN), which bind STAT3 and induce cell death. A specific STAT3 dODN which does not interfere with STAT1-mediated interferon-induced cell death has been designed pointing to the STAT3 DBD as a target for specific inhibition. Comprehensive analysis of this region is in progress in the laboratory to design DBD-targeting STAT3 inhibitors with STAT3/STAT1 discriminating ability.
RESUMEN
We report a general phenomenon of the formation of either a fluorescent or an entirely quenched oligodeoxynucleotide (ODN) duplex system by hybridizing pairs of complementary ODNs with identical chemical composition. The ODNs carried internucleoside tether-linked cyanines, where the cyanines were chosen to form a Förster's resonance energy transfer (FRET) donor-acceptor pair. The fluorescent and quenched ODN duplex systems differed only in that the cyanines linked to the respective ODNs were linked either closer to the 5'- or 3'-ends of the molecule. In either case, however, the dyes were separated by an identical number (7 or 8) of base pairs. Characterization by molecular modeling and energy minimization using a conformational search algorithm in a molecular operating environment (MOE) revealed that linking of the dyes closer to the 5'-ends resulted in their reciprocal orientation across the major groove which allowed a closely interacting dye pair to be formed. This overlap between the donor and acceptor dye molecules resulted in changes in absorbance spectra consistent with the formation of H-aggregates. Conversely, dyes linked closer to 3'-ends exhibited emissive FRET and formed a pair of dyes that interacted with the DNA helix only weakly. Induced CD spectra analysis suggested that interaction with the double helix was weaker than in the case of the closely interacting cyanine dye pair. Linking the dyes such that the base pair separation was 10 or 0 favored energy transfer with subsequent acceptor emission. Our results suggest that when interpreting FRET measurements from nucleic acids, the use of a "spectroscopic ruler" principle which takes into account the 3D helical context of the double helix will allow more accurate interpretation of fluorescence emission.
Asunto(s)
Emparejamiento Base , Colorantes Fluorescentes/química , Modelos Moleculares , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Secuencia de Bases , Transferencia Resonante de Energía de FluorescenciaRESUMEN
The signal transducers and activators of transcription (STATs) convey signals from the membrane to the nucleus in response to cytokines or growth factors. STAT3 is activated in response to cytokines involved mostly in cell proliferation; STAT1 is activated by cytokines, including interferon-gamma, involved in defence against pathogens and the inhibition of cell proliferation. STAT3, which is frequently activated in tumour cells, is a valuable target with respect to achieving inhibition of tumour cell proliferation. Indeed, its inhibition results in cell death. We previously observed that inhibition of the transcription factor nuclear factor-kappaB, a key regulator of cell proliferation, with decoy oligodeoxynucleotides results in cell death. We used a similar approach for STAT3. A hairpin STAT3 oligodeoxynucleotide was added to a colon carcinoma cell line in which it induced cell death as efficiently as the STAT3 inhibitor stattic. The hairpin STAT3 oligodeoxynucleotide co-localized with STAT3 within the cytoplasm, prevented STAT3 localization to the nucleus, blocked a cyclin D1 reporter promoter and associated with STAT3 in pull-down assays. However, the same cells were efficiently killed by interferon-gamma. This effect was counteracted by the STAT3 oligodeoxynucleotide, which was found to efficiently inhibit STAT1. Thus, although it can inhibit STAT3, the hairpin STAT3 oligodeoxynucleotide appears also to inhibit STAT1-mediated interferon-gamma cell killing, highlighting the need to optimize STAT3-targeting oligodeoxynucleotides.
Asunto(s)
Carcinoma/metabolismo , Neoplasias del Colon/metabolismo , Interferón gamma/antagonistas & inhibidores , Oligodesoxirribonucleótidos/metabolismo , Factor de Transcripción STAT1/antagonistas & inhibidores , Factor de Transcripción STAT3/antagonistas & inhibidores , Sitios de Unión , Muerte Celular , Línea Celular Tumoral , Proliferación Celular , Técnica del Anticuerpo Fluorescente , Humanos , Oligodesoxirribonucleótidos/química , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Transcripción Genética , TransfecciónRESUMEN
Two types of reporters for optical sensing of NF-kappaB p50 protein-oligodeoxyribonucleotide (ODN) duplex interactions were designed and compared in vitro. The reporters were based on the effect of fluorescence resonance energy transfer (FRET) between the pair donor Cy5.5 near-infrared (NIR) fluorochrome and either 800CW emitting fluorescence dye acceptor (800CW-Cy), or a nonemitting QSY 21 dye quencher (QSY-Cy). The donor and the acceptor dyes were covalently linked to the complementary oligonucleotides, respectively: Cy dye was conjugated to 3'-thiol, whereas 800CW or QSY21 were conjugated to a hydrophilic internucleoside phosphate amino linker. The reporters were tested initially using recombinant NF-kappaB p50 protein binding assays. Both reporters were binding p50 protein, which protected oligonucleotide duplex from degradation in the presence of exonuclease.The incubation of 800CW-Cy reporter in the presence of control or IL-1beta treated human endothelial cells showed the uptake of the reporter in the cytoplasm and the nucleus. The measurement of NIR fluorescence ratio (i.e. Cy5.5/800CW) showed a partial loss of FRET and the increased Cy5.5 fluorescence in nontreated, control cells. Thus, the specific p50 binding to ODN duplex reporters affected the donor-acceptor fluorochrome pair. NF-kappaB p50 exhibited the protective effect on FRET between NIR fluorochromes linked to the complementary strands of the reporter duplex.
Asunto(s)
ADN/metabolismo , Células Endoteliales/metabolismo , Subunidad p50 de NF-kappa B/metabolismo , Oligodesoxirribonucleótidos/metabolismo , Línea Celular , Células Endoteliales/citología , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes , Genes Reporteros , Humanos , Subunidad p50 de NF-kappa B/síntesis química , Oligodesoxirribonucleótidos/química , Proteínas Recombinantes , Venas Umbilicales/citologíaRESUMEN
Double-stranded oligodeoxyribonucleotides with engineered disulfide units were successfully used for covalent trapping of cysteine containing proteins. In particular, an efficient cross-linking of NF-kappaB p50 homodimer to a sequence-specific decoy was demonstrated. The results suggest that the synthetic oligonucleotides bearing a novel 2'-disulfide trapping site can be used as new tools to study and manipulate biological systems.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Disulfuros/química , Subunidad p50 de NF-kappa B/química , Dimerización , Oligodesoxirribonucleótidos/químicaRESUMEN
Intramolecular fluorescence quenching of cyanine dyes was investigated using a model hairpin oligonucleotide decoy encoding a NF-kappaB p50 subunit binding site. Two types of hairpin oligonucleotides were synthesized: (1) 5'-(6-aminohexyl)- and 3'-(3-aminopropyl)-linked (I); (2) 5'-(6-aminohexyl)- and 3'-[3-(3-hydroxypropyldithio)propyl]-linked (II). Oligonucleotide I was covalently modified using monofunctional either Cy3- or Cy5.5-N-hydroxysuccinimide esters. Using reverse-phase HPLC, mono-and dicyanineamide derivatives of I were isolated. Mono-Cy3-modified derivatives of I, but not the mono-Cy5.5-modified derivatives, showed a 2-fold higher Cy3 fluorescence intensity compared to the free dye. There was no detectable difference in fluorescence between the di-Cy3 derivative of I and the free dye at the same concentration. However, there was a 4-fold quenching of fluorescence in the case of the di-Cy5.5 derivative of the same hairpin oligonucleotide. The quenching of Cy5.5 fluorescence could not be explained by the interaction of Cy5.5 with nucleotide bases as demonstrated by incubating free Cy5.5 dye with oligonuclotides. The quenching effect was further investigated using an oligonucleotide bearing a cleavable 3'-amino-terminated linker bearing an S-S bond (III). After modification of the 5'- and 3'-end of oligonucleotide III with a Cy5.5 monofunctional hydroxysuccinimide ester, a 70-75% quenching of fluorescence was observed. Fluorescence was 100% dequenched after the reduction of S-S bond. The obtained result unequivocally demonstrates that the formation of intramolecular Cy5.5 dimers is the dominant mechanism of fluorescence quenching in symmetric dye-dye hairpin decoy beacons.