SUNY at Albany
June 19-23, 2001
Cellular Uptake Characteristics of DNA Frayed Wires
DNA frayed wires (DNAFW) are novel, multi-stranded form of DNA that arise from interactions between single stranded oligodeoxyribonucleotides. DNAFW have the general sequence d(NxGy) where x * 5 and y * 10. These structures are extremely stable with respect to thermal and chemical denaturation. Although it has been determined that DNAFW are highly resistant to digestion with nucleases, information regarding their cellular transport and biological fate does not exist. Thus we examined the uptake kinetics and mechanisms of DNAFW (A15G15) using two continuous human cell lines; HepG2 hepatoma cells and CNE-1 nasopharangeal carcinomic cells. In these studies, DNA oligonucleotides were 32P-ATP end-labelled and incubated at concentrations of 0.1-0.5 mM with plated cells for periods of 0-120 minutes (25 ºC). In both HepG2 and CNE-1 cells, we observed two to five fold higher 60- minute uptakes of DNAFW relative to conventional single-stranded DNA (DNASS, N30). As compared to DNASS, uptake also occurred more rapidly with DNAFW. Analysis on 10% PAGE denaturing gels indicated that intact DNAFW was present intracellularly in cell lysates. Concentration dependency studies exhibited saturability of DNAFW uptake over the concentration range of 0.1-0.5 mM range while temperature dependency studies indicated that DNAFW uptake is impaired at 0 ºC. Several inhibitors of endocytosis (sodium azide, deoxyglucose, heparin) were found to significantly inhibited DNAFW uptake. Hence our results indicate that DNAFW structures display enhanced cellular uptake relative to DNASS. This may result from either an increased stability against cellular degradation or via enhanced intracellular transport processes. Studies characterizing the transport of DNAFW suggest that active transport mechanisms, including endocytosis contribute to DNAFW uptake. As poor DNA stability and uptake remain major obstacles in DNA-based gene therapy, these findings denote the potential application of DNAFW as novel DNA delivery systems.
Micheline Piquette-Miller, Won-Sang Lee, Robert B. Macgregor, Jr.
Department of Pharmaceutical Sciences
Faculty of Pharmacy
University of Toronto,
19 Russell Street
Toronto, Ontario M5S 2S2 Canada