Optimization of nuclear localization signal for nuclear transport of DNA-encapsulating particles.
The nuclear membrane is a tight barrier against the delivery of therapeutic genes into non-dividing tissue cells. Overcoming this barrier with the aid of peptidic nuclear localization signals (NLS) is crucial for improving the performance of synthetic gene-delivery vehicles. In this article, we examine the nuclear transport of lambda phage particles displaying various peptides containing the minimum NLS of SV40 T antigen on their surface. As the minimum NLS (PKKKRKV) is a binding domain to importin alpha, recombinant proteins and molecular conjugates containing this peptide accumulate into the nucleus efficiently. However, we find that the C-terminal and N-terminal structures besides the minimum NLS profoundly affect the efficiency of the nuclear transport of the phage particles as well as their binding capacity to importin alpha: either truncation of a few amino acid residues from the C-terminus or the replacement of the N-terminus with a FLAG- or c-myc-tag abolish both of these biological activities. The structure of the optimized NLS is unpredictable from conventional protein transport assay and from the structural analysis in silico. Our results reveal that the objects with 50 nm in diameter can pass through the nuclear pore complex when the optimized NLS is displayed at a sufficient density on their surface.
Eguchi
Akiko
A
Gene Function Research Center, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, 305-8562, Japan.
Furusawa
Hiroyuki
H
Yamamoto
Akitsugu
A
Akuta
Teruo
T
Hasegawa
Mamoru
M
Okahata
Yoshio
Y
Nakanishi
Mahito
M
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics*; D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018014:Gene Transfer Techniques; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D049329:Nanostructures; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D028901:alpha Karyopherins / Q000378:metabolism
D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D028901:alpha Karyopherins; D004247:DNA