Single-Molecule Study of the Inhibition of HIV-1 Transactivation Response Region DNA/DNA Annealing by Argininamide

Single-molecule spectroscopy was used to examine how a model inhibitor of HIV-1, argininamide, modulates the nucleic acid chaperone activity of the nucleocapsid protein (NC) in the minus-strand transfer step of HIV-1 reverse transcription, in vitro. In minus-strand transfer, the transactivation response region (TAR) RNA of the genome is annealed to the complementary “TAR DNA” generated during minus-strand strong-stop DNA synthesis. Argininamide and its analogs are known to bind to the hairpin bulge region of TAR RNA as well as to various DNA loop structures, but its ability to inhibit the strand transfer process has only been implied. Here, we explore how argininamide modulates the annealing kinetics and secondary structure of TAR DNA. The studies reveal that the argininamide inhibitory mechanism involves a shift of the secondary structure of TAR, away from the NC-induced “Y” form, an intermediate in reverse transcription, and toward the free closed or “C” form. In addition, more potent inhibition of the loop-mediated annealing pathway than stem-mediated annealing is observed. Taken together, these data suggest a molecular mechanism wherein argininamide inhibits NC-facilitated TAR RNA/DNA annealing in vitro by interfering with the formation of key annealing intermediates

Co-Compartmentation of Terpene Biosynthesis and Storage <i>via</i> Synthetic Droplet

Traditional bioproduct engineering focuses on pathway optimization, yet is often complicated by product inhibition, downstream consumption, and the toxicity of certain products. Here, we present the co-compartmentation of biosynthesis and storage via a synthetic droplet as an effective new strategy to improve the bioproduct yield, with squalene as a model compound. A hydrophobic protein was designed and introduced into the tobacco chloroplast to generate a synthetic droplet for terpene storage. Simultaneously, squalene biosynthesis enzymes were introduced to chloroplasts together with the droplet-forming protein to co-compartmentalize the biosynthesis and storage of squalene. The strategy has enabled a record yield of squalene at 2.6 mg/g fresh weight without compromising plant growth. Confocal fluorescent microscopy imaging, stimulated Raman scattering microscopy, and droplet composition analysis confirmed the formation of synthetic storage droplet in chloroplast. The co-compartmentation of synthetic storage droplet with a targeted metabolic pathway engineering represents a new strategy for enhancing bioproduct yield

Co-Compartmentation of Terpene Biosynthesis and Storage <i>via</i> Synthetic Droplet

Traditional bioproduct engineering focuses on pathway optimization, yet is often complicated by product inhibition, downstream consumption, and the toxicity of certain products. Here, we present the co-compartmentation of biosynthesis and storage <i>via</i> a synthetic droplet as an effective new strategy to improve the bioproduct yield, with squalene as a model compound. A hydrophobic protein was designed and introduced into the tobacco chloroplast to generate a synthetic droplet for terpene storage. Simultaneously, squalene biosynthesis enzymes were introduced to chloroplasts together with the droplet-forming protein to co-compartmentalize the biosynthesis and storage of squalene. The strategy has enabled a record yield of squalene at 2.6 mg/g fresh weight without compromising plant growth. Confocal fluorescent microscopy imaging, stimulated Raman scattering microscopy, and droplet composition analysis confirmed the formation of synthetic storage droplet in chloroplast. The co-compartmentation of synthetic storage droplet with a targeted metabolic pathway engineering represents a new strategy for enhancing bioproduct yield