Sequence requirements for plasmid nuclear import

Abstract

The nuclear envelope is a major barrier for nuclear uptake of plasmids and represents one of the most significant unsolved problems of nonviral gene delivery. We have previously shown that the nuclear entry of plasmid DNA is sequence-specific, requiring a 366-bp fragment containing the SV40 origin of replication and early promoter. In this report, we show that, although fragments throughout this region can support varying degrees of nuclear import, the 72-bp repeats of the SV40 enhancer facilitate maximal transport. The functions of the promoter and the origin of replication are not needed for nuclear localization of plasmid DNA. In contrast to the import activity of the SV40 enhancer, two other strong promoter and enhancer sequences, the human cytomegalovirus (CMV) immediate-early promoter and the Rous sarcoma virus LTR, were unable to direct nuclear localization of plasmids. The inability of the CMV promoter to mediate plasmid nuclear import was confirmed by measurement of the CMV promoter-driven expression of green fluorescent protein (GFP) in microinjected cells. At times before cell division, as few as 3 to 10 copies per cell of cytoplasmically injected plasmids containing the SV40 enhancer gave significant GFP expression, while no expression was obtained with more than 1000 copies per cell of plasmids lacking the SV40 sequence. However, the levels of expression were the same for both plasmids after cell division in cytoplasmically injected cells and at all times in nuclear injected cells. Thus, the inclusion this SV40 sequence in nonviral vectors may greatly increase their ability to be transported into the nucleus, especially in nondividing cells.

Figure 1. Constructs used in these studies

Cartoons of the plasmids used in these studies are shown. Restriction endonuclease sites are abbreviated as follows: A, AflIII; Av, AvaI; B, BamHI; E, EcoRI; H, HindIII; K, KpnI; N, NcoI; S, SmaI; St, StuI; V, EcoRV; X, XbaI.

Figure 2. Functional organization of the SV40 origin/promoter region and map of DNA constructs

The functional elements of the origin of replication, early and late promoters, and enhancer are shown (36). All SV40 DNA fragments shown were cloned into the pBR322 derived plasmid pOR(5171-43), replacing the original SV40 sequences present (4). pOR(5208-43)_20C and pOR(5208-43)_5215G are site-directed mutants of pOR(5208-43) that contain single point mutations in T antigen binding sites and are indicated by an “X” (9).

Figure 3. Nuclear localization of plasmids containing viral promoters and enhancers

Several viral promoter/enhancers were cloned into luciferase or ß-galactosidase-expressing vectors. Plasmids were microinjected into the cytoplasm of TC7 cells and visualized 8 hours later by in situ hybridization. (A) pOR(5171-300) containing the 366 bp SV40 origin/promoter DNA fragment; (B) pBR-CMV; (C) pBR-RSV; (D) pRSVneo; (E) pBR-CMV-SV40 (pBR-CMV containing the 366 bp SV40 origin/promoter DNA fragment); and (F) pRSVneo-SV40 (pRSVneo containing the SV40 origin/promoter DNA fragment).

Figure 4. Sequence-specific nuclear localization and GFP expression

(A) Nuclear injected pGFP and pGFPΔSV40 display similar patterns and efficiencies of GFP expression. TC7 cells were synchronized by treatment with 60 μM lovastatin for 16 hours, washed and then incubated in medium containing serum and 5 mM mevalonic acid for two hours before microinjection. Thirty copies of either plasmid were injected into each nucleus of approximately 100–150 cells and assayed for GFP expression at later times. GFP expression which was recorded on an intensity scale of 1 to 5 and relative GFP expression is given as the total intensity divided by the total number of injected cells. (B) Plasmids with or without the SV40 enhancer differ greatly in their ability to express GFP when injected into the cytoplasm. Thirty copies of either pGFP or pGFPΔSV40 were microinjected into the cytoplasm of approximately 100 cells, synchronized as in (A), and relative GFP expression was measured. The results shown (A and B) are representative of 4 independent experiments.

Figure 5. Nuclear import of plasmid DNA is rate limiting for gene expression at early times after microinjection

pGFP was microinjected into the nucleus or cytoplasm of TC7 cells at the indicated copy number per cell and assayed for gene expression at (A) 4 and 8 hours, and (B) 24 hours post-injection. Between 100 and 150 cells were injected for each concentration shown. The results shown (A and B) are representative of 4 similar experiments.

Figure 6. Model for sequence specific nuclear import of plasmid DNA

(A) Sequence elements and binding sites for transcription factors are shown for the 366 bp SV40 DNA nuclear targeting sequence. (B) Since these transcription factors are synthesized in the cytoplasm, once plasmid DNA has entered the cytoplasm by transfection, injection, or infection, the newly synthesized proteins can bind to the plasmid DNA to form a DNA-protein complex. Thus, the DNA is coated with NLSs from the transcription factors, allowing the NLS-mediated import machinery to recognize DNA as a substrate and target it into the nucleus.