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Wu‐Pong, Susanna; Tl, Weiss; Hunt, C. Anthony

doi:10.1023/a:1015846209681

Cited by 64 publications

(19 citation statements)

References 28 publications

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“…The different rates of accumulation suggest that more than one uptake mechanism exists. These data support the hypothesis of other investigators, notably Wu-Pong and colleagues (35), that nucleic acid uptake in cells and tissues results from at least two mechanisms: endocytosis and an endocytosis-independent “channel-like” process. To date NACh is the only nucleic acid channel described in LLC-PK 1 cells which leads us to propose that NACh is this “channel-like” transporter previously postulated by other groups.…”

Section: Discussionsupporting

confidence: 91%

Use of a Pteridine Moiety to Track DNA Uptake in Cells

Costa¹,

Leal-Pinto

Henderson

et al. 2012

Pteridines

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Fluorescence labeled oligonucleotides have a long history of being used to monitor nucleic acid transport and uptake. However, it is not known if the fluorescent moiety itself physically limits the number of pathways that can be used by the cell due to steric, hydrophobic, or other chemical characteristics. Here, we report a method for comparing the uptake kinetics of oligonucleotides labeled either with the fluorescent pteridine, 3-methyl-8-(2-deoxy-β-D-ribofuranosyl) isoxanthopterin (3MI), or the common fluorophore 5-carboxyfluorescein (5-FAM). We use a multiphoton microscopic technique to monitor nucleic acid uptake LLC-PK1, a pig renal tubular cell line that is known to have multiple uptake pathways. We find that the two fluorophores enter the cells at different rates, suggesting that choice of fluorescent moiety influences the uptake pathway used by a cell. Finally, we reconstituted an LLC-PK1 membrane channel that is selective for nucleic acids in planar lipid bilayers, and tested the ability of the labeled nucleic acids to permeate the channel. We find that 3MI, and not 5-FAM labeled oligonucleotides can traverse the plasma membrane through the channel. These results have implications for future studies aimed at delivering pteridine moieties to cells and for tracking nucleic acid transport into tissues.

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Section: Discussionsupporting

confidence: 91%

Use of a Pteridine Moiety to Track DNA Uptake in Cells

Costa¹,

Leal-Pinto

Henderson

et al. 2012

Pteridines

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show abstract

“…43 Therefore, these results support the notion that ODN was taken up by the cells not solely through a receptormediated process, but probably also by adsorptive or fluidphase endocytosis. [44][45][46][47] In addition, the observation that the association of ODN with cells was temperature dependent in our preliminary study supports the involvement of an endocytotic mechanism of ODN uptake.…”

Section: Resultssupporting

confidence: 71%

Effects of Oligodeoxynucleotides on the Physicochemical Characteristics and Cellular Uptake of Liposomes

Arima¹,

Aramaki²,

Tsuchiya³

1997

Journal of Pharmaceutical Sciences

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We studied the effects of a phosphodiester oligodeoxynucleotide (ODN), which is antisense to the site in the neighborhood of the AUG initiation codon of the mouse tumor necrosis factor alpha gene (TNF-alpha), on the physicochemical characteristics and the cellular association of three types of liposomes with different surface charges. The physicochemical characteristics of the liposomes changed after adding ODN. When the ODN/lipid molar ratio was approximately 0.15 in cationic (TMAG) liposomes [consisting of N-(alpha-trimethylammonioacetyl)didodecyl-D-glutamate chloride (TMAG), dilauroylphosphatidylcholine (DLPC), and dioleolylphosphatidylethanolamine (DOPE) in a 1:2:2 ratio], but not in neutral and negatively charged liposomes, then the liposomes aggregated and fused. At higher molar ratios, these changes in TMA liposomes were not evident. In addition, ODN inverted the zeta-potential of TMAG liposomes from positive to negative at an ODN/lipid molar ratio of approximately 0.15. Therefore, the aggregation and fusion induced by ODN could be explained by a lower surface charge repulsion between TMAG liposomes. On the other hand, the association of ODN with RAW264.7 cells, a mouse macrophage-like cell line, was very slight. The cellular association of ODN was significantly enhanced compared with neutral and negatively charged liposomes by encapsulation in TMAG liposomes. The ODN added to liposome suspensions did not affect the rate and extent of TMAG liposome cellular association, even at an ODN/lipid molar ratio of approximately 0.15. These results indicate that the lipid composition and ODN/lipid molar ratio are critical for the physicochemical characteristics of cationic liposomes. However, the changes had less influence on the cellular uptake properties of cationic liposomes.

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“…Although oligonucleotides do not readily cross the blood-brain barrier, they accumulate in neuronal tissue when injected directly into cerebral spinal fluid. Previous studies have established that short single-stranded DNAs are rapidly internalized by a variety of cultured cells (8)(9)(10)(11)(12)(13)(14)(15)(16). Several putative DNA binding͞transport proteins have been isolated from cell membranes (7,9,10,13,(17)(18)(19), and recently an integrin, Mac-1 (19), has been found to potentially mediate endocytosis of nucleic acid on the surface of macrophages.…”

Section: Introductionmentioning

confidence: 99%

Identification and characterization of a cell membrane nucleic acid channel

Hanss

Leal-Pinto

Bruggeman

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

118

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We have identified a 45-kDa protein purified from rat renal brush border membrane that binds short single-stranded nucleic acid sequences. This activity was purified, reconstituted in proteoliposomes, and then fused with model planar lipid bilayers. In voltage-clamp experiments, the reconstituted 45-kDa protein functioned as a gated channel that allows the passage of nucleic acids. Channel activity was observed immediately after addition of oligonucleotide. Channel activity was not observed in the absence of purified protein or of oligonucleotide or when protein was heat-inactivated prior to forming proteoliposomes. In the presence of symmetrical buffered solution and oligonucleotide, current passed linearly over the range of holding potentials tested. Conductance was 10.4 ؎ 0.4 picosiemens (pS) and reversal potential was 0.2 ؎ 1.7 mV. There was no difference in channel conductance or reversal potential between phosphodiester and phosphorothioate oligonucleotides. Ionsubstitution experiments documented a shift in reversal potential only when a concentration gradient for oligonucleotide was established, indicating that movement of oligonucleotide alone was responsible for current. Movement of oligonucleotide across the bilayer was confirmed by using 32 P-labeled oligonucleotides. Channel open probability decreased significantly in the presence of heparan sulfate. These studies provide evidence for a cell surface channel that conducts nucleic acids.

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Cited by 64 publications

References 28 publications

Use of a Pteridine Moiety to Track DNA Uptake in Cells

Use of a Pteridine Moiety to Track DNA Uptake in Cells

Effects of Oligodeoxynucleotides on the Physicochemical Characteristics and Cellular Uptake of Liposomes

Identification and characterization of a cell membrane nucleic acid channel

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