Intravesicle Isothermal DNA Replication

Torino et al. BMC Research Notes 2011, 4:128 http://www.biomedcentral.com/1756-0500/4/128 SHORT REPORT Open Access Intravesicle Isothermal DNA Replication Domenica Torino1, Cristina Del Bianco1, Lindsey A Ross1,3, Jennifer L Ong2 and Sheref S Mansy1* Abstract Background: Bacterial and viral DNA replication was previously reconstituted in vitro from component parts [1-4]. Significant advances in building minimal cell-like structures also have been made recently [5-7]. Combining the two approaches would further attempts to build a minimal cell-like structure capable of undergoing evolution by combining membrane encapsulation and genome replication. Towards this end, we attempted to use purified genomic replication protein components from thermophilic bacterial sources to copy strands of DNA isothermally within lipid vesicles. Findings: Bacterial replication components (such as helicases and DNA polymerases) are compatible with methods for the generation of lipid vesicles. Encapsulation inside phospholipid vesicles does not inhibit the activity of bacterial DNA genome replication machinery. Further the described system is efficient at isothermally amplifying short segments of DNA within phospholipid vesicles. Conclusions: Herein we show that bacterial isothermal DNA replication machinery is functional inside of phospholipid vesicles, suggesting that replicating cellular mimics can be built from purified bacterial components. Findings Much interest has centred on building encapsulated replicating genetic systems capable of Darwinian evolution [5,6]. Typically, the exploited methods are based on PCR, including DNA replication within water-in-oil (w/ o) emulsions and phospholipid vesicles [8-10]. Thermocycling methods are undesirable for the construction of cell-like structures since they generate non-autonomous systems, i.e. intervention is required for the cycling of temperature. An alternative is to exploit previously constructed in vitro isothermal DNA amplification methods [1,2,11]. Since extant life uses compartments defined by lipid bilayers, we sought to reconstitute an isothermal replication system inside of phospholipid vesicles. We did not exploit fatty acid vesicles, because fatty acid vesicles are less stable [12] and previous work has shown an incompatibility between fatty acids and some DNA polymerases [13]. To build an isothermal DNA replication system inside of vesicles, several features are desirable. The system must survive mechanisms of vesicle generation, be functional within the microenvironment of the vesicle * Correspondence: 1 Centre for Integrative Biology, University of Trento, Via delle Regole, 101 Mattarello, Italy Full list of author information is available at the end of the article compartment, and be controllable so that reactions only occur after encapsulation. We find that the previously described tHDA (thermophilic helicase-dependent amplification) system [1,2] possesses all of these features, suggesting that the reconstitution of bacterial machinery may provide for a facile route towards the building of replicating cell-like structures. The tHDA mix of thermostable proteins includes a UvrD helicase, a single-strand binding protein, and a DNA polymerase. Results and discussion To test whether the previously described tHDA system [1,2] 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 1, 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 [14,15]. 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 © 2011 Mansy et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Torino et al. BMC Research Notes 2011, 4:128 http://www.biomedcentral.com/1756-0500/4/128 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, and 50 bp bands are labeled. Reaction products were visualized by ethidium bromide staining of a 1.8% agarose gel. The full length reaction product is 85 bp. 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 2A). 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. Since vesicle production methods typically employ an overnight incubation step, we tested the ability of the Page 2 of 4 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 2B). 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 3, 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 3 reflects the (...truncated)