Sequence-Specific Gene Cleavage in Intact Mammalian Cells by ¹²⁵I-Labeled Triplex-Forming Oligonucleotides Conjugated with Nuclear Localization Signal Peptide

Abstract

Triplex-forming oligonucleotides (TFO) are designed to bind sequence specifically to their DNA targets without a significant disturbance of the double helix. They have been proposed to deliver DNA-reactive agents to specific DNA sequences for gene targeting applications. We suggested the use of 125I-labeled TFO for delivery of the energy of radioiodine decay to specific genes. This approach is called antigene radiotherapy. Here we demonstrate the ability of 125I-labeled TFO to produce sequence-specific breaks within a target in the human mdrl gene in cultured cells. TFO and TFO conjugated with a nuclear localization signal peptide (NLS) were delivered into cells using cationic liposomes. This was done either alone or in the presence of an excess of a "ballast" oligonucleotide with an unrelated sequence. In all cases, nuclear localization of TFO and survival of the cells after treatment has been confirmed. Breaks in the gene target were analyzed by restriction enzyme digestion of the DNA recovered from the TFO-treated cells followed by Southern hybridization with DNA probes flanking the target sequence. We have found that TFO/NLS conjugates cleave the target in a concentration-dependent manner regardless of the presence of the "ballast" oligonucleotide. In contrast, TFO without NLS cleaved the target only in the presence of an excess of the "ballast." We hypothesize that TFO and TFO/NLS are delivered into the nucleus by different pathways. These results provide a new insight into the mechanism of intracellular transport of oligonucleotides and open new avenues for improvement of the efficacy of antigene therapies.

Coupling Strategies for the Synthesis of Peptide-Oligonucleotide Conjugates for Patterned Synthetic Biomineralization

“…Peptides, in particular, are an increasingly popular choice for conjugation with oligonucleotides because of their ability to aid in targeting specific biological functions while improving permeability through the cell membrane and stability against intracellular degradation. Peptide-oligonucleotide conjugates (POCs) have been used to target mRNA, double-stranded DNA, and proteins, in antisense, triple-helix, and aptamer-based therapy, respectively [3–5]. Our group has recently explored another use of POCs by incorporating peptides used in artificial biomineralization [6–8] into self-assembling DNA nanostructures [9–11] for the demonstration of controlled biomineralization [12].…”

In vivo imaging with oligonucleotides for diagnosis and drug development

“…103 Targeted radiotherapy with oligonucleotide has been proposed by using sequence specific triplex forming oligonucleotides carrying Auger-electron-emitters such as iodine-125 104 to induce localised radio damage to specific sites in the genome. 105 Oligonucleotides as immune modulators The immune stimulation induced by specific oligonucleotide sequences containing unmethylated CpG motifs has already been mentioned. The induction of an immune response by oligonuclotides may find useful applications to protect against infection or trigger immunotherapeutic responses for cancer, allergic or non-conventional infectious diseases.…”

Targeting and Regulation of the HER-2/neu Oncogene Promoter with Bis-Peptide Nucleic Acids

“…Further evidence of the ability of TFOs to bind to target sequences within living cells comes from the ability of TFOs to mediate mutagenesis at the target site in yeast cells (Barre et al, 2000), cultured mammalian cells (Vasquez et al, 1999(Vasquez et al, , 2001Wang et al, 1996), and mice (Vasquez et al, 2000). TFO binding to the target sequence in intact mammalian cells was also demonstrated by site-specific, TFO-directed strand cleavage (Sedelnikova et al, 2002). Although mutagenesis or cleavage events are infrequent, they have unambiguously demonstrated that a TFO can bind to a target sequence in living Arizona Cancer Center,Tucson,AZ 85724. cells.…”