The effect of lyophilization on plasmid DNA activity.
The effect of lyophilization of plasmid DNA's ability to express an encoded protein was studied. Plasmid DNA, pRL-CMV expressing Renilla luciferase, was purified and stored in Tris-ethylenedi-aminetetraacetic acid (EDTA) buffer. Aliquots of the plasmid were lyophilized using analytical equipment, both alone and in the presence of carbohydrate. Samples were rehydrated and subject to functional and structural analyses. Analytical techniques included transfection efficiency in COS-1 cells, agarose gel electrophoresis, dimethylethylenediamine (DMED) assay for abasic sites, circular dichroism measurement, and UV spectroscopy. The lyophilization of pRL-CMV plasmid DNA resulted in a statistically significant loss of transfection efficiency (p < 0.05). Mono- and disaccharides could completely restore transfection efficiency. Agarose gel electrophoresis and the DMED assay demonstrated no change in gross plasmid structure or increase in abasic sites during lyophilization, respectively. Changes in DNA form, as measured by a change in ellipsisity, were observed on lyophilization. However, these changes were transient and were not shown to be responsible for loss of transfection efficiency. A hyperchromic effect was observed at 260 nm after lyophilization and could be reversed by the presence of carbohydrates. Lyophilization causes a decrease in plasmid DNA activity as measured by an in vitro transfection assay. Carbohydrates can ameliorate this decreased activity, which may be due to structural changes seen during the lyophilization process.
Poxon
S W
SW
Whitehall-Robins Healthcare, Richmond, Virginia 23261, USA.
Hughes
J A
JA
D000818:Animals; D019556:COS Cells; D002623:Chemistry Techniques, Analytical / Q000379:methods; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004492:Edetic Acid / Q000737:chemistry; D005612:Freeze Drying / Q000009:adverse effects*; D008156:Luciferases / Q000235:genetics*; D009005:Monosaccharides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry; D013997:Time Factors; D014162:Transfection
D009005:Monosaccharides; D011134:Polysaccharides; D004247:DNA; D004492:Edetic Acid; D008156:Luciferases