Cellular Uptake and Metabolism of DNA Frayed Wires

Abstract

DNA frayed wires are a novel, multistranded form of DNA that arises from interactions between single-stranded oligodeoxyribonucleotides with the general sequence d(N(x)G(y)) or d(G(y)N(x)), where y > 10 and x > 5. Frayed wires exhibit greater stability with respect to thermal and chemical denaturation than single- or double-stranded DNA molecules and, thus, may have potential usefulness for DNA drug delivery. However, the stability and uptake of frayed wires have not been investigated in biological systems. Our objective was to examine the cellular uptake and stability of frayed wires in cultured hepatic cells. In these studies, the parent oligonucleotide d(A(15)G(15)) was used to form DNA frayed wires (DNA(FW)) while a random 30-mer oligonucleotide was used as the control nonaggregated DNA (DNA(SS)). Uptake and metabolism studies of DNA(FW) were performed in cultured human hepatoma, HepG2 cells and compared to DNA(SS). Our results indicate that DNA(FW) are not cytotoxic and that their intracellular uptake in HepG2 cells is 2-3.5-fold greater than that of DNA(SS) within the first 2 h (p < 0.05). Similarly, nuclear localization of DNA(FW) is 10-13-fold higher than that of DNA(SS) (p < 0.05). As both internalized and extracellular DNA(FW) appear to be more stable in vitro than DNA(SS), the enhanced uptake may be due to either increased stability or enhanced intracellular transport. These studies also indicate that uptake of DNA(FW) likely occurs via active processes such as receptor-mediated endocytosis similar to mechanisms which have been proposed for DNA(SS). The internalization pathways of DNA(FW) may differ somewhat from that of DNA(SS) insofar as chloroquine does not appear to alter DNA(FW) uptake and degradation, as is the case with DNA(SS).

In vivo disposition and stability of DNA frayed wires in mice

“…As we mentioned above, DNA FW consist of a mixture of species with different aggregation numbers, thus, the molecular weight of these supermolecular structures can be quite large. Despite this, we have shown in a previous study that in comparison to singlestranded DNA, DNA FW exhibit higher intracellular accumulation, increased nuclear localization, and diminished cytotoxicity in the human hepatoma cell line, HepG2 [19]. Given the characteristics of DNA FW , it seemed apparent that they might be potentially useful as an alternative DNA drug delivery system; however, further characterization is required.…”

“…The non-guanine bases branch off from the guanine-rich stem and behave in many respects like single-stranded DNA. Previous studies have shown that DNA FW possess greater chemical, enzymatic, and thermal stability than regular DNA molecules [19,30]. As we mentioned above, DNA FW consist of a mixture of species with different aggregation numbers, thus, the molecular weight of these supermolecular structures can be quite large.…”

“…One limiting factor in the use of these agents stems from their poor stability in biological fluids. For example, the serum and liver contain numerous types of nucleases that rapidly degrade or metabolize ODNs [16][17][18][19]. Hence, it is generally accepted that "protected" ODNs are more stable, efficient, and more effective than "naked" ODNs [1,13,[16][17][18][19][20][21][22][23].…”

“…Encapsulation of a therapeutic ODN in viral capsids and synthetic vesicles, e.g. lipoplexes, and polyplexes have been exploited to enhance the stability of the ODN in vivo and in vitro and to improve transfection efficiency [17][18][19]. The improved efficiency of protected ODNs is presumably due to an increased resistance to nucleases, which enables a higher concentration of ODNs to reach the site of action.…”

“…parent ODNs with the general sequence d(N x G y ) or d(G y N x ), where y > 10 and x > 5 [19,[27][28][29][30]. DNA FW formed in solution consist of a polydisperse mixture of aggregates with 2 to ∼15 associated parent ODN molecules.…”