DNA delivery by phage as a strategy for encapsulating toroidal condensates of arbitrary size into liposomes

Abstract

We report a strategy for encapsulating and condensing DNA. When T5 phage binds to its membrane protein receptor, FhuA, its double stranded DNA (120,000 bp) is progressively released base pair after base pair in the surrounding medium. Using cryoelectron microscopy, we have visualized the structures formed after T5 phage DNA is released into neutral unilamellar proteoliposomes reconstituted with the receptor FhuA. In the presence of spermine, toroidal condensates of circumferentially wrapped DNA were formed. Most significantly, the sizes of these toroids were shown to vary, from 90 to 200 nm in their outer diameters, depending on the number of DNA stands transferred. We have also analyzed T5 DNA release in bulk solution containing the detergent-solubilized FhuA receptor. After DNA release in a spermine containing solution, huge DNA condensates with a diameter of about 300 nm were formed containing the DNAs from as many as 10 -20 capsids. At alkaline pH, the condensates appeared as large hollow cylinders with a diameter of 200 nm and a height of 100 -200 nm. Overall, the striking feature of our experiments is that, because of the progressive release of DNA from the phage capsid, the mechanism of toroid formation is fundamentally different from that in the classical studies in which highly dilute, ''naked'' DNA is condensed by direct addition of polyvalent cations; as a consequence, our method leads to toroids of arbitrary size.polyamines ͉ condensation ͉ FhuA ͉ toroids T he condensation of DNA, i.e., the collapse of an extended worm-like random coil DNA molecule into a compact ordered state, has received considerable attention in diverse areas of science (1-3). In biology, it represents a process by which genetic information is packaged and protected. In medicine, it provides a promising means for gene therapy applications through new forms of condensed genes to be delivered to target cells. In polymer and condensed matter physics, DNA condensation is an example of a coil-globule transition or of a selfassembly process in which aggregation of two or more DNA molecules are involved.In vitro condensation of DNA by different chemical agents has provided useful insights into the physical factors governing folding and packaging of DNA. Among these condensing agents, much interest has focused on polycations that modify electrostatic interactions between DNA segments through neutralization of their charges (4) and͞or mediation of attractive forces between them (5). These agents, which induce collapse, aggregation, or complexation by interacting directly with DNA molecules, include small ions (polyamines, cobalt hexamine), cationic polypeptides (polylysine, polyhistidine), or proteins (histones) (6-11).When condensation is induced by addition of polyamines (spermine, spermidine) to very dilute aqueous solution of DNA, striking toroidal structures are the most common morphologies observed by electron microscopy (12-14). Here DNA is circumferentially wrapped as a toroid with a well defined hole in the middle and an outer di...

Finite-size polyelectrolyte bundles at thermodynamic equilibrium

“…For DNA, it has been argued [10,3,11,12] that the observed finite size of the aggregates is a kinetic effect based on the formation of an energy barrier, and not a consequence of equilibrium thermodynamic properties of the system. Experiments performed with viral DNA support this conjecture at least for the investigated systems [13]. However, recently Henle and Pincus [14] argued that, depending on the actual parameters of the system, finite or infinite bundles should be possible in the presence of short-ranged attractions.…”

Membrane proteins: functional and structural studies using reconstituted proteoliposomes and 2-D crystals

“…In the presence of 50 mM spermine-containing liposomes, the DNA strands transferred from the phage capsids appeared condensed into characteristic toroidal structures that occupied a volume smaller than the internal volume of the proteoliposome ( Figure 4B). Increasing the number of DNA strands transferred into the proteoliposomes did not change the number of toroids in the liposomes but increased significantly the size of the toroid formed (37). From another point of view, the approach could be important for gene therapy applications, since the DNA-containing proteoliposomes produced in this way could …”

Kinetics and mechanism of DNA uptake into the cell nucleus

“…A beautiful example of such a process has been seen in the uptake to synthetic vesicles of T5 phage DNA when the former are loaded with condensing concentrations of multivalent ions (48). The relevant energy scale is that of deforming the polymeric molecule, leading to force scales of a magnitude associated with thermal diffusion.…”