Influence of a Hairpin Loop on the Thermodynamic Stability of a DNA Oligomer

DSC was used to evaluate the mechanism of the thermally induced unfolding of the single-stranded hairpin HP = 5′-CGGAATTCCGTCTCCGGAATTCCG-3′ and its core duplex D (5′-CGGAATTCCG-3′)2. The DSC melting experiments performed at several salt concentrations were successfully described for HP and D in terms of a three-state transition model HP↔I (intermediate state) ↔ S (unfolded single-stranded state) and two state transition model D↔2S, respectively. Comparison of the model-based thermodynamic parameters obtained for each HP and D transition shows that in unfolding of HP only the HP↔I transition is affected by the TCTC loop. This observation suggests that in the intermediate state its TCTC loop part exhibits significantly more flexible structure than in the folded state while its duplex part remains pretty much unchanged

Development of a microfluidic platform for R-Phycoerythrin purification using an aqueous micellar two-Phase system

Temperature-dependent aqueous micellar two-phase systems (AMTPSs) have recently been gaining attention in the isolation of high-added-value biomolecules from their natural sources. Despite their sustainability, aqueous two-phase systems, and particularly AMTPSs, have not been extensively applied in the industry, which might be changed by applying process integration and continuous manufacturing. Here, we report for the first time on an integrated microfluidic platform for fast and low-material-consuming development of continuous protein purification using an AMTPS. A system comprised of a microchannel incubated at high temperature, enabling instantaneous triggering of a two-phase system formation, and a microsettler, allowing complete phase separation at the outlets, is reported here. The separation of phycobiliproteins and particularly the purification of R-phycoerythrin from the contaminant proteins present in the aqueous crude extract obtained from fresh cells of Gracilaria gracilis were thereby achieved. The results from the developed microfluidic system revealed that the fractionation performance was maintained while reducing the processing time more than 20-fold when compared with the conventional lab-scale batch process. Furthermore, the integration of a miniaturized ultrafiltration module resulted in the complete removal of the surfactant from the bottom phase containing R-phycoerythrin, as well as in nearly twofold target protein concentration. The process setup successfully exploits the benefits of process intensification along with the integration of various downstream processes. Further transfer to a meso-scale integrated system would make such a system appropriate for the separation and purification of biomolecules with high commercial interest.publishe