Towards the development of a continuous-flow semi-biotic device: transcription from DNA-loaded liquid crystalline phases

Abstract

Research published by Corsi et al. (2008) has reported how linearized dsDNA encoding for the firefly luciferase gene can be contained within the inverse hexagonal phase of the phospholipid 1,2 dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), whilst remaining transcriptionally active. The work presented herein builds upon the novel discovery reported by Corsi et al., by fully characterizing and then modifying the reported system to allow for batch, and then continuous-flow, production of mRNA from dsDNA-containing liquid crystalline phases, in the context of establishing proof of concept for a continuous-flow transcription-translation capable ‘semi-biotic’ device. To give a better understanding of the lipid-based method of template dsDNA containment for transcription, experiments were performed to assess the transcriptional activity and characterize the partitioning of nucleic acids (dsDNA and mRNA) in DOPE-based transcription assays; these experiments included varying the ionic, reagent concentration and incubation conditions. A number of experiments were also carried out investigating the effect of doping the liquid crystalline phase with the positively charged lipid species 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and replacing DOPE with the unsaturated monoglyceride monoolein (MO). Investigations have shown that DOPE (95%):DOTAP (5%) liquid crystalline phases were able to achieve similar levels of dsDNA partitioning as pure DOPE lipid buttons; however, these DOTAP-containing phases were transcriptionally inactive. Substitution of DOPE for monoolein did not yield comparable levels of dsDNA partitioning. A batch transcription protocol was developed as a logical progression from one-off transcription towards continuous-flow transcription from dsDNA-containing liquid crystalline phases. Individual liquid crystalline phases, preloaded with dsDNA, were subjected to multiple transcriptions in an effort to establish the maximum lifespan of the phase and the optimal batch transcription parameters. Polarizing light microscopy, small-angle x-ray scattering (SAXS) and mass spectrometry were used assess the effect upon the liquid crystalline phase of multiple cycles of batch transcription, whilst quantitative DNA gel electrophoresis was performed to assess the level of dsDNA partitioning over successive batch transcription assays from individual lipid phases. Investigations into batch transcription from dsDNA-containing DOPE liquid crystalline phases have shown that the partitioned dsDNA leaches out from within the phase during the course of successive transcription reactions. In order to assess the viability of a transcription-translation capable semi biotic device that utilizes lyotropic liquid crystalline phases, investigations were made into the suitability of the optimal batch transcription system when incorporated into a thermostatically controlled polydimethylsiloxane (PDMS) continuous-flow transcription device. Finally, a critical comparison was made between the initially proposed mechanism of dsDNA containment using lipid based liquid crystalline phases and some of the recently reported alternative methods of dsDNA immobilization.

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ORCID for George S. Attard: orcid.org/0000-0001-8304-0742

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