On-column capping of poly dT media-tethered mRNA accomplishes high capping efficiency, enhanced mRNA recovery, and improved stability against RNase.

The messenger RNA (mRNA) 5'-cap structure is indispensable for mRNA translation initiation and stability. Despite its importance, large-scale production of capped mRNA through in vitro transcription (IVT) synthesis using vaccinia capping enzyme (VCE) is challenging, due to the requirement of tedious and multiple pre-and-post separation steps causing mRNA loss and degradation. Here in the present study, we found that the VCE together with 2'-O-methyltransferase can efficiently catalyze the capping of poly dT media-tethered mRNA to produce mRNA with cap-1 structure under an optimized condition. We have therefore designed an integrated purification and solid-based capping protocol, which involved capturing the mRNA from the IVT system by using poly dT media through its affinity binding for 3'-end poly-A in mRNA, in situ capping of mRNA 5'-end by supplying the enzymes, and subsequent eluting of the capped mRNA from the poly dT media. Using mRNA encoding the enhanced green fluorescent protein as a model system, we have demonstrated that the new strategy greatly simplified the mRNA manufacturing process and improved its overall recovery without sacrificing the capping efficiency, as compared with the conventional process, which involved at least mRNA preseparation from IVT, solution-based capping, and post-separation and recovering steps. Specifically, the new process accomplished a 1.76-fold (84.21% over 47.79%) increase in mRNA overall recovery, a twofold decrease in operation time (70 vs. 140 min), and similar high capping efficiency (both close to 100%). Furthermore, the solid-based capping process greatly improved mRNA stability, such that the integrity of the mRNA could be well kept during the capping process even in the presence of exogenously added RNase; in contrast, mRNA in the solution-based capping process degraded almost completely. Meanwhile, we showed that such a strategy can be operated both in a batch mode and in an on-column continuous mode. The results presented in this work demonstrated that the new on-column capping process developed here can accomplish high capping efficiency, enhanced mRNA recovery, and improved stability against RNase; therefore, can act as a simple, efficient, and cost-effective platform technology suitable for large-scale production of capped mRNA.

Interfacial Ions Sieving for Ultrafast and Complete Desalination through 2D Nanochannel Defined Graphene Composite Membranes.

The layered graphene membrane has high potential for efficient desalination owing to its frictionless surface and hydrophobic nature. However, it has not been demonstrated so far due to the challenges related to controlling membrane microstructure. Herein, we develop a facile and simple thiol-ene click method to prepare a perfluoro-alkyl grafted graphene (fGraphene) membrane on porous ceramic, which features an ultrahigh antiwetting surface, oriented mesoporous surface entrances, and a well-defined interlamellar spacing of ∼1.1 nm. With vacuum membrane distillation, the fGraphene membranes post ∼100% rejections to the small ions of seawater, at least 1 order of magnitude higher water fluxes than those of commercial membranes and graphene-oxide-based membranes, as well as robust stability in the desalination. Fast NaCl desalinations on the fGraphene membrane were also confirmed by the reverse/forward osmosis tests. The complete rejection of ions and high flux are attributed to the interfacial sieving effect over the 2D nanochannels as well as the vapor-phase transport in the mesoscale channels, which is fundamentally different from the solution-diffusion mechanism of dense polymeric membranes and the size-sieving mechanism of microporous membranes. This work not only demonstrates a special separation effect for complete desalination over the layered graphene-based membrane but also offers a reliable method to functionalize and structure graphene membranes for other potential applications.

The rational design of Li-doped nitrogen adsorbents for natural gas purification.

Separation of nitrogen (N2) and methane (CH4) is one of the most challenging and energy-intensive processes in the natural gas industry, due to their close physico-chemical properties. The quest for an effective N2-selective adsorbent has long been the focus of research; however, the results have been sparse. In this work, a first-principle study has been used to construct and investigate Li-doped polycyclic aromatic hydrocarbons (PAHs) for N2 rejection in natural gas purification. We doped lithium on a series of linear/nonlinear PAHs consisting of two to six benzene rings. The adsorption affinity of the Li-doped organic molecular systems toward N2 and CH4 was evaluated by calculating the interaction energy using density functional theory. From the gas adsorption selectivities for different Li-doped PAHs, Li-doped phenanthrene and chrysene showed the highest N2 over CH4 equilibrium selectivities, with values of 119.7 and 80.8, respectively. It was found that the Li atom enabled the π bond of the aromatic substrate to interfere with the N2 lowest unoccupied molecular orbital, resulting in strong physisorption of N2. These results indicate the high potential of Li-doped phenanthrene and chrysene for N2 removal from natural gas.