DNA nanostructures can be tailored to perform a wide variety of functions, with continued interest in biological applications. Some aspects of DNA nanostructure assembly can hinder the ability of nanostructures to be useful in physiological environments. Typical assembly methods employ magnesium ions to stabilize the structure, which leave the structure susceptible to damage by nucleases in body fluids. Further, DNA nanostructure assembly typically involves a thermal annealing protocol in which DNA strands are heated in a specific buffer to a high temperature and cooled slowly at specific rates, preventing convenient encapsulation of temperature-sensitive guest molecules. In this work, we demonstrate the assembly of a wide variety of DNA nanostructures and 3D crystals in a hydrated ionic liquid (choline dihydrogen phosphate, CDHP) instead of magnesium at constant moderate temperatures, thus avoiding thermal annealing. CDHP-assembled structures show enhanced biostability against a variety of nucleases. Molecular dynamics simulations show that choline ions stabilize DNA nanostructures by a direct and close-range interaction in contrast to the predominantly water-mediated interactions of Mg2+, leading to enhanced nuclease resistance in CDHP-containing environments. CDHP-assembled structures do not affect the viability of HepG2 cells and show higher cell internalization. Overall, this work develops a potential method to construct more biostable DNA nanostructures and 3D crystals in a simple one-tube process. Assembly of DNA nanostructures under isothermal conditions is desirable for scaffolding biomolecules and to reduce the need for thermal annealing instruments, allowing nanostructure preparation in low-resource settings.
Pichia pastoris is a widely used host for recombinant protein production because it combines the advantages of microbial cultivation with eukaryotic protein folding and secretion. However, secretion efficiency is often limited by the folding capacity of the endoplasmic reticulum (ER), where recombinant proteins must be translocated, folded, and processed prior to export. When ER folding capacity is exceeded, proteins may be retained, degraded, or secreted in non-native conformations, reducing both yield and product quality. Chaperone engineering and codon optimization represent two promising strategies to address these limitations. Here, we generated stable Pichia strains expressing four model secreted proteins (human serum albumin, interleukin-2, thaumatin-I, and thaumatin-II) using either conventional codon optimization or Epi-MAX codon engineering, which adapts transgene codon usage to stress-responsive translational programs. We also engineered strains containing an additional chromosomal copy of either the ER Hsp70 chaperone Kar2 or protein disulfide isomerase (Pdi1). To assess protein quality, we applied limited proteolysis mass spectrometry (LiP-MS), a structural proteomics approach that can detect subtle conformational differences to secreted proteins. Increased Pdi1 levels improved secretion of all four proteins tested, whereas Kar2 overexpression generally reduced yield. For thaumatin-II, Pdi1 enhanced secretion but promoted release of a non-native conformation, which we could correct through codon engineering. Together, these results demonstrate that maximizing recombinant protein production requires optimization of both yield and structural quality and establish complementary strategies for improving secreted protein expression in Pichia.
