To test whether the previously described tHDA system [1
] functions after cycles of freeze/thawing, we subjected aliquots of tHDA to up to 20 cycles of freezing on dry ice followed by thawing at 30°C. Subsequently, the solutions were incubated at 65°C to allow for the thermophilic DNA polymerase to replicate the DNA template. As seen in Figure , up to 20 cycles of freeze/thawing did not inhibit the reaction. This is useful because freeze/thaw cycles are a common method to increase encapsulation efficiency and to facilitate the formation of vesicles [14
Figure 1 The influence of freeze/thaw cycles on tHDA enzymatic activity. Unencapsulated reaction mixtures were subjected to either 0 (lane 2), 5 (lane 3), 10 (lane 4), or 20 (lane 5) cycles of freeze-thawing. Lane 1 is a 50 bp DNA ladder. The 1350 bp, 100 bp, (more ...)
Next, we wanted to ensure that the reaction was controllable by temperature. Since all of the proteins of the tHDA system are from thermophilic microorganisms, we expected a greatly diminished ability to replicate DNA at low temperatures. Therefore, we tested the activity of the tHDA system at 4°C, 23°C, 37°C, and 65°C. As expected, the yield at all of the tested temperatures, except for 65°C, was below the detection limit (<5 ng) of ethidium bromide staining of an agarose gel (Figure ). This not only allows for the control of DNA replication by temperature, but also facilitates preparatory steps, including those of vesicle generation and the enzymatic degradation of extravesicular material.
Figure 2 The effect of temperature on tHDA enzymatic activity. (A) The influence of temperature on the enzymatic activity of the isothermal tHDA system. Samples were incubated at 65°C (lane 2), 37°C (lane 3), 23°C (lane 4), and 4°C (more ...)
Since vesicle production methods typically employ an overnight incubation step, we tested the ability of the tHDA system to survive overnight incubation. The reaction components were mixed on ice and then either incubated overnight at 4°C or 23°C followed by an incubation at 65°C to allow the system to replicate DNA. We could not detect amplification after an overnight incubation at 23°C. Conversely, incubation at 4°C overnight did not observably diminish DNA yields (Figure ).
Having established that the tHDA system is controllable and survives the steps necessary for vesicle formation, we encapsulated the tHDA system in POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) vesicles. The protocol exploited an overnight incubation at 4°C of the tHDA components with phospholipids, 20 freeze/thaw cycles, an incubation with proteinase K that was added to the outside of the vesicles to inhibit extravesicular reactions, and finally incubation at 65°C for 1.5 h. As seen in Figure , the isothermal amplification of DNA occurred within the phospholipid vesicles. The presence of proteinase K outside of the vesicles did not inhibit the reaction, whereas the inclusion of the protease in both the intra- and extra-vesicle environment inhibited DNA amplification. As a further confirmation that the reaction occurred inside of the vesicles, dNTPs were added outside, but not inside, of the vesicles. Since POPC membranes are impermeable to nucleotides [16
], the replication reaction was undetectable. The lower band intensity resulting from the intravesicle reaction shown in Figure reflects the inefficiency of the overall reaction. For example, based on total sample volumes, the vesicle reactions were only ca. 1% as efficient as the solution reactions. However, such a comparison is misleading. The total intravesicle volume is much lower than the total solution volume. Further, a functional compartment requires the simultaneous encapsulation of several components, including a template, two primers, and three proteins. Similar difficulties arising from the Poisson distribution of reaction components within vesicles have been thoroughly described by Luisi [10
] and Yomo [17
]. Nevertheless, the efficiency of the encapsulated tHDA system is sufficient, in the sense that only a single functional compartment is required to build a self-replicating cell-like structure capable of propagation.
Figure 3 Intravesicular isothermal replication. Lanes 1 and 7 contain a 50 bp DNA ladder. Lanes 2 and 3 are from unencapsulated reactions and lanes 4-6 are within phospholipid vesicles. Lanes 2 and 3 are isothermal replication reactions under standard conditions (more ...)
In summary, it is possible to reconstitute bacterial DNA replication machinery capable of copying DNA isothermally inside of phospholipid vesicles.