Self-neutralizing oligonucleotides with enhanced cellular uptake.

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.

STAT3 inhibitors for cancer therapy: Have all roads been explored?

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.

The three-dimensional context of a double helix determines the fluorescence of the internucleoside-tethered pair of fluorophores.

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.

A STAT3-inhibitory hairpin decoy oligodeoxynucleotide discriminates between STAT1 and STAT3 and induces death in a human colon carcinoma cell line.

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.

A STAT3-decoy oligonucleotide induces cell death in a human colorectal carcinoma cell line by blocking nuclear transfer of STAT3 and STAT3-bound NF-κB.

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.

Hairpin-like fluorescent probe for imaging of NF-κB transcription factor activity.

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.

Efficient killing of SW480 colon carcinoma cells by a signal transducer and activator of transcription (STAT) 3 hairpin decoy oligodeoxynucleotide--interference with interferon-gamma-STAT1-mediated killing.

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.

Near-infrared fluorescent oligodeoxyribonucleotide reporters for sensing NF-kappaB DNA interactions in vitro.

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).

Fluorescence resonance energy transfer in near-infrared fluorescent oligonucleotide probes for detecting protein-DNA interactions.

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.

A novel thymidine phosphoramidite synthon for incorporation of internucleoside phosphate linkers during automated oligodeoxynucleotide synthesis.

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.

Cell death in NF-kappaB-dependent tumour cell lines as a result of NF-kappaB trapping by linker-modified hairpin decoy oligonucleotide.

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.

Structure-based cross-linking of NF-kappaB p50 homodimer and decoy bearing a novel 2'-disulfide trapping site.

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.

Synthesis and properties of fluorescent NF-kappa B-recognizing hairpin oligodeoxyribonucleotide decoys.

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.

Specific covalent binding of a NF-kappaB decoy hairpin oligonucleotide targeted to the p50 subunit and induction of apoptosis.

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.

PspGI, a type II restriction endonuclease from the extreme thermophile Pyrococcus sp.: structural and functional studies to investigate an evolutionary relationship with several mesophilic restriction enzymes.

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.

Specific conjugation of DNA binding proteins to DNA templates through thiol-disulfide exchange.

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.