title: Size-dependent DNA mobility in cytoplasm and nucleus.
abstract: The diffusion of DNA in cytoplasm is thought to be an important determinant of the efficacy of gene delivery and antisense therapy. We have measured the translational diffusion of fluorescein-labeled double-stranded DNA fragments (in base pairs (bp): 21, 100, 250, 500, 1000, 2000, 3000, 6000) after microinjection into cytoplasm and nucleus of HeLa cells. Diffusion was measured by spot photobleaching using a focused argon laser spot (488 nm). In aqueous solutions, diffusion coefficients of the DNA fragments in water (D(w)) decreased from 53 x 10(-8) to 0.81 x 10(-8) cm(2)/s for sizes of 21-6000 bp; D(w) was related empirically to DNA size: D(w) = 4.9 x 10(-6) cm(2)/s.[bp size](-0.72). DNA diffusion coefficients in cytoplasm (D(cyto)) were lower than D(w) and depended strongly on DNA size. D(cyto)/D(w) decreased from 0.19 for a 100-bp DNA fragment to 0.06 for a 250-bp DNA fragment and was <0.01 for >2000 bp. Diffusion of microinjected fluorescein isothiocyanate (FITC) dextrans was faster than that of comparably sized DNA fragments of 250 bp and greater. In nucleus, all DNA fragments were nearly immobile, whereas FITC dextrans of molecular size up to 580 kDa were fully mobile. These results suggest that the highly restricted diffusion of DNA fragments in nucleoplasm results from extensive binding to immobile obstacles and that the decreased lateral mobility of DNAs >250 bp in cytoplasm is because of molecular crowding. The diffusion of DNA in cytoplasm may thus be an important rate-limiting barrier in gene delivery utilizing non-viral vectors.
authors.affiliation: Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521, USA.
authors.forename: G L
authors.initials: GL
authors.lastname: Lukacs
authors.forename: P
authors.initials: P
authors.lastname: Haggie
authors.forename: O
authors.initials: O
authors.lastname: Seksek
authors.forename: D
authors.initials: D
authors.lastname: Lechardeur
authors.forename: N
authors.initials: N
authors.lastname: Freedman
authors.forename: A S
authors.initials: AS
authors.lastname: Verkman
mesh_terms: D002467:Cell Nucleus / Q000378:metabolism*; D003287:Contrast Media / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D003911:Dextrans / Q000378:metabolism; D019793:Fluorescein / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008845:Microinjections; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D014176:Protein Biosynthesis
chemical_list: D003287:Contrast Media; D003911:Dextrans; D004247:DNA; D019793:Fluorescein