ABSTRACT
Using a fluorescence complementation assay, Delivered Complementation in Planta (DCIP), we demonstrate cell-penetrating peptide-mediated cytosolic delivery of peptides and recombinant proteins in Nicotiana benthamiana. We show that DCIP enables quantitative measurement of protein delivery efficiency and enables functional screening of cell-penetrating peptides for in-planta protein delivery. Finally, we demonstrate that DCIP detects cell-penetrating peptide-mediated delivery of recombinantly expressed proteins such as mCherry and Lifeact into intact leaves. We also demonstrate delivery of a recombinant plant transcription factor, WUSCHEL (AtWUS), into N. benthamiana. RT-qPCR analysis of AtWUS delivery in Arabidopsis seedlings also suggests delivered WUS can recapitulate transcriptional changes induced by overexpression of AtWUS. Taken together, our findings demonstrate that DCIP offers a new and powerful tool for interrogating cytosolic delivery of proteins in plants and highlights future avenues for engineering plant physiology.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Cell-Penetrating Peptides , Cell-Penetrating Peptides/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Fluorescence , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolismABSTRACT
Deep eutectic solvents (DESs) are an emerging class of non-aqueous solvents that are potentially scalable, easy to prepare and functionalize for many applications ranging from biomass processing to energy storage technologies. Predictive understanding of the fundamental correlations between local structure and macroscopic properties is needed to exploit the large design space and tunability of DESs for specific applications. Here, we employ a range of computational and experimental techniques that span length-scales from molecular to macroscopic and timescales from picoseconds to seconds to study the evolution of structure and dynamics in model DESs, namely Glyceline and Ethaline, starting from the parent compounds. We show that systematic addition of choline chloride leads to microscopic heterogeneities that alter the primary structural relaxation in glycerol and ethylene glycol and result in new dynamic modes that are strongly correlated to the macroscopic properties of the DES formed.
ABSTRACT
Solvation and transport properties of methly viologen dichloride (MVCl2) in 1:2, 1:4, and 1:6 molar mixtures of choline chloride (ChCl) and ethylene glycol (EG), including the deep eutectic solvent (DES) ethaline (1:2 mixture), were studied through the application of the hole theory to measured physical properties, cyclic voltammetry, and Raman spectroscopy. The ChCl:EG mixtures were compared to the ionic liquid (IL) 1-methyl-1-propylpyrrolidinium bis(trifluoromethylsulfonyl) imide ([PYR13][TFSI]) and choline bis(trifluoromethylsulfonyl)imide (ChTFSI) EG mixtures with the same molar ratios in order to understand the impact of the anion and hydrogen bond donor on solvation. Exchanging the chloride anion with TFSI is found to increase the fluidity of the solvent and promote stronger solute-solvent interactions. Raman spectroscopy suggests MVCl2 is strongly solvated by EG in ChTFSI:EG solutions and interstitially accommodated in holes in ChCl:EG mixtures and [PYR13][TFSI]. Complex solvents such as ILs and DESs are regarded as "designer solvents", and it is demonstrated here that the physical properties and solvation characteristics of these fluids strongly depend on the choice of the anion.
ABSTRACT
A range of techniques including physical property measurements, neutron scattering experiments, ab initio molecular dynamics, and classical molecular dynamics simulations are used to probe the structural, thermodynamic, and transport properties of a deep eutectic solvent comprised of a 1:2 molar ratio of choline chloride and ethylene glycol. This mixture, known as Ethaline, has many desirable properties for use in a range of applications, and therefore, understanding its liquid structure and transport properties is of interest. Simulation results are able to capture experimental densities, diffusivities, viscosities, and structure factors extremely well. The solvation environment is dynamic and dominated by different hydrogen bonding interactions. Dynamic heterogeneities resulting from hydrogen bonding interactions are quantified. Rotational dynamics of molecular dipole moments of choline and ethylene glycol are computed and found to exhibit a fast and slow mode.
ABSTRACT
In the past decade, reports detailing the preparation, characterization, and application of deep eutectic solvents (DESs) have grown in number significantly, showing signs of increased interest and attention. Indeed, these systems provide tunable polar environments attractive for their ease of synthesis and lack of need for purification. DESs are homogeneous systems composed of two or more components having a significantly depressed melting point compared to either constituent material. As interest and application of DESs grow, the need for a common understanding of their preparation and characterization is required. In this Perspective, we discuss metal-free DESs, focusing on their preparation, characterization of physical properties, and considerations for their application. We highlight inconsistencies or omissions in literature reports as well as factors for researchers to consider when investigating these systems.
