Gene transfer to eukaryotic cells requires the uptake of exogenous DNA into the cell nucleus. Except during mitosis, molecular access to the nuclear interior is limited to passage through the nuclear pores. Here we demonstrate the nuclear uptake of extended linear DNA molecules by a combination of fluorescence microscopy and single-molecule manipulation techniques, using the latter to follow uptake kinetics of individual molecules in real time. The assays were carried out on nuclei reconstituted in vitro from extracts of Xenopus eggs, which provide both a complete complement of biochemical factors involved in nuclear protein import, and unobstructed access to the nuclear pores. We find that uptake of DNA is independent of ATP or GTP hydrolysis, but is blocked by wheat germ agglutinin. The kinetics are much slower than would be expected from hydrodynamic considerations. A fit of the data to a simple model suggests femto-Newton forces and a large friction relevant to the uptake process.A common component in many types of viral infection (1, 2), strategies for genetic therapy (3), and direct DNA vaccination (4) is the accumulation of exogenous DNA in a host cell nucleus. While much attention has been paid to viral invasion mechanisms for cellular entry, relatively little is known about the uptake of DNA into the nucleus itself, particularly as it is not a part of normal cellular physiology. In nondividing cells, molecular exchange across the nuclear envelope normally takes place through the nuclear pore complexes (NPCs). These large channels, whose physiological role is primarily in regulating traffic of proteins and protein-RNA complexes between the nucleus and cytoplasm (5-8), exhibit distinct modes of passive exchange for small molecules and peptide signal-mediated selective transport for larger ones (9). Several pathways for the latter have been identified, based on affinity for receptors of the karyopherin ␤͞importin ␤ (10, 11) and related families (12). Less is known about the biochemistry related to DNA uptake, even whether such exists at all. Indeed, a prominent feature of the viral mechanisms is their ability to co-opt the native protein import machinery by expression of appropriate nuclear localization signals (NLS; ref. 1).The size scales of the NPC make it an unlikely transporter for DNA. The upper cutoff for diffusive passage of colloidal gold particles is Ϸ9 nm, whereas the NLS-mediated mechanism displays an apparent cutoff at Ϸ25 nm diameter (13,14). This scale easily encompasses the size of most protein transport substrates, for which the molecular weight of the cargo-receptor complex will be in the low hundreds of kDa. A recent study of transport rates finds the passage of several molecules per second in the NLS-mediated mode (15). Tightly compacted messenger ribonucleoprotein (mRNP) particles have been observed to unwind during their export through NPCs, conforming to the restricted space within the pore (16). The virulence proteins VirD2 and VirE2 of Agrobacterium tumefaciens mediate the import o...

We have developed a biochip platform technology suitable for controlled cell-free gene expression at the micrometer scale. A new hybrid molecule, "Daisy", was designed and synthesized to form in a single step a biocompatible lithographic interface on silicon dioxide. A protocol is described for the immobilization of linear DNA molecules thousands of base pairs long on Daisy-coated surfaces with submicrometer spatial resolution and up to high densities. On-chip protein synthesis can be obtained with a dynamic range of up to four orders of magnitude and minimal nonspecific activity. En route to on-chip artificial gene circuits, a simple two-stage gene cascade was built, in which the protein synthesized at the first location diffuses to regulate the synthesis of another protein at a second location. We demonstrate the capture of proteins from crude extract onto micrometer-scale designated traps, an important step for the formation of miniaturized self-assembled protein chips. Our biochip platform can be combined with elastomeric microfluidic devices, thereby opening possibilities for isolated and confined reaction chambers and artificial cells in which the transport of products and reagents is done by diffusion and flow. The Daisy molecule and described approach enables groups not proficient in surface chemistry to construct active biochips based on cell-free gene expression.