Use of a Pteridine Moiety to Track DNA Uptake in Cells.
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-PK1membrane 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.
Division of Nephrology, Department of Medicine, Mt. Sinai School of Medicine, New York, NY.
Justin A
JA
Costa
Department of Physiology and Biophysics, Virginia Commonwealth University Medical Center, Richmond, VA.
Edgar
E
Leal-Pinto
Department of Anatomy and Neurobiology, Virginia Commonwealth University Medical Center, Richmond, VA.
Scott C
SC
Henderson
South Denver Nephrology, Denver, CO.
Troy
T
Zabel
Laboratory of Receptor Biology and Gene Expression, NCI/NIH, Bethesda, MD.
Mary E
ME
Hawkins
Division of Nephrology, Department of Medicine, Mt. Sinai School of Medicine, New York, NY.
Basil
B
Hanss
DNA uptake monitoring; fluroescence label; methylisoxathopterin