A rapid and efficient method for the synthesis of 125I-labeled oligodeoxynucleotides ([125I]ODNs) is described. The key intermediates are tributylstannylbenzamide-modified ODNs (Sn-ODNs). Reaction conditions are described for the preparation of 5'-modified Sn-ODNs. Treatment with NaI and chloramine T gave conversion to the desired I-ODN, which was easily isolated by reversed phase chromatography. Thermal denaturation (Tm) studies showed that hybridization properties were not disturbed by the 4-iodobenzamide modification. An [125I]ODN was prepared and characterized by hybridization to 32P-labeled DNA targets. Sequence specific cleavage of the target DNA strand by 125I was measured.
Antigene radiotherapy is based upon damaging selected genes by a high dose of radiation from radionuclides delivered to this gene by a sequence-specific DNA-binding molecule. Here we describe our recent trials of antigene radiotherapy using the human mdr1 gene over-expressed in KB-V1 cells as a model. As a delivery molecule, we used a triplex-forming oligonucleotide (TFO) with a binding site in intron 14 of mdr1. This TFO was labeled with an Auger-electron-emitting radionuclide 125I. Decay of 125I releases a shower of low energy electrons that produce DNA strand breaks mostly within 10 bp from the decay site. Targeting in situ was assessed by restriction enzyme digestion of the DNA recovered from the TFO-treated cells followed by Southern hybridization with DNA probes flanking the target sequence. Double-strand breaks in the target sequence were detected in purified nuclei and digitonin-permeabilized cells, but not in the intact cells when TFO were delivered with liposomes. On the basis of these observations we hypothesized that there are cytoplasmic factors that bind such TFO and deliver them into the nucleus, but do not release them inside the nucleus, thus preventing TFO from binding their genomic targets. To test this hypothesis we (i) delivered TFO along with an excess of unlabeled oligonucleotide with an arbitrary sequence ("ballast") and (ii) conjugated TFO with a nuclear localization sequence peptide (NLS). We have found that TFO/NLS conjugates cleaved 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." These results may provide a new insight into the mechanism of intracellular transport of oligonucleotides.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.