No enhancement of nuclear entry by direct conjugation of a nuclear localization signal peptide to linearized DNA.
Efficient nuclear entry of exogenous DNA is one of the key factors toward gene therapy success with nonviral vectors. To re-address the effects of a nuclear localization signal (NLS) peptide attached directly to DNA, we prepared three dumbbell-shaped, green fluorescent protein (GFP)-encoding DNAs containing one or two NLS peptides. The peptide was conjugated to the loop-forming oligodeoxyribonucleotides by cross-linking reactions between the peptide and a modified uracil base with a dioxaoctylamino linker, and the oligonucleotides were then ligated to the DNA molecules. The NLS-conjugated DNA dumbbells were microinjected into the cytosols and nuclei of simian COS-7 cells. In addition, unconjugated DNA dumbbells, with or without a modified uracil base, were also examined for comparison. The GFP gene was expressed with efficiencies in the order of the unmodified DNA >or= the NLS-conjugated DNA > the unconjugated DNA with the base modification, with both cytosolic and intranuclear microinjections. Thus, we concluded that (i) one or two NLS peptide(s) did not dramatically improve the nuclear entry of DNA and that (ii) chemical modification of DNA reduced the transcription efficiency or stability in the nucleus.
Tanimoto
Mitsuhide
M
Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan.
Kamiya
Hiroyuki
H
Minakawa
Noriaki
N
Matsuda
Akira
A
Harashima
Hideyoshi
H
D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism*; D003587:Cytomegalovirus / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D015870:Gene Expression; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008845:Microinjections; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides / Q000235:genetics*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D009842:Oligopeptides; C402304:SV40 nuclear localization peptide; D049452:Green Fluorescent Proteins; D004247:DNA