Size-dependent DNA Mobility in Cytoplasm and Nucleus*

By G. Lukács, Peter M. Haggie, O. Seksek, D. Lechardeur, N. Freedman, A. Verkman. Journal of Biological Chemistry, 2000

780 citations recorded by the source corpus.

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 × 10−8 to 0.81 × 10−8 cm2/s for sizes of 21–6000 bp; D w was related empirically to DNA size:D w = 4.9 × 10−6cm2/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.

Subfields: Ecology, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/85edf196ef33fddc4b37c9351c6738e654ce9d82

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Citations in the corpus

• Nonviral Gene Delivery: Principle, Limitations, and Recent Progress (2009, neutral): “Observations made by direct intracellular microinjection of naked DNA prove that the movement by diffusion is slow and inefficient, and the resulting levels of gene expression are very weak (10).”
• PEGylation significantly affects cellular uptake and intracellular trafficking of non-viral gene delivery particles. (2004, background): “Diffusion of pDNA in cytoplasm is severely restricted (Lukacs et al., 2000; Lechardeur and Lukacs, 2002).”
• Design and development of polymers for gene delivery (2005, background): “— DNA larger than 3,000 base pairs in length is essentially immobil”
• SINGLE-PARTICLE TRACKING (2009, background): “One problem with large labels is that their motion may be hindered by cellular structures such as the extracellular matrix [61], the cytoskeleton [62,63], or chromatin [64].”