There is considerable interest in novel cell imaging tools that avoid the use of fluorescent proteins. One widely used class of such reagents are “pro-fluorescent” biarsenical dyes such as FlAsH, ReAsH, CrAsH, and Cy3As. Despite their utility, biarsenicals are plagued by high background labeling and cytotoxicity, and are challenging to apply in oxidizing cellular locale. Here we demonstrate that [(3-oxospiro[isobenzofuran-1(3H),9′-[9H]xanthene]-3′,6′-diyl)bis(iminomethylene-2,1-phenylene)]bis-(9CI), a rhodamine-derived bisboronic acid (RhoBo) described initially as a monosaccharide sensor, functions as a cell-permeable, turn-on fluorescent sensor for a tetraserine-motifs in recombinant proteins. RhoBo binds peptides or proteins containing Ser-Ser-Pro-Gly-Ser-Ser with affinities in the nanomolar concentration range, and prefers this sequence to simple monosaccharides by >10,000-fold. RhoBo fails to form fluorescent complexes with constituents of the mammalian cell surface, as judged by epifluorescent, confocal, and TIRF microscopy, but fluoresces brightly within the Ser-Ser-Pro-Gly-Ser-Ser-rich cell interior. These results suggest that current efforts to identify optimal serine-rich sequences for RhoBo will allow it to function effectively as a selective small-molecule label for appropriately tagged proteins either upon or within living cells.

Some anaerobic bacteria use insoluble minerals as terminal electron acceptors and discovering the ways in which electrons move through the membrane barrier to the exterior acceptor forms an active field of research with implications for both bacterial physiology and bioenergy. A previous study suggested that Shewanella oneidensis MR-1 utilizes a small, polar, redox active molecule that serves as an electron shuttle between the bacteria and insoluble acceptors, but the shuttle itself has never been identified. Through isolation and synthesis, we identify it as ACNQ (2-amino-3-carboxy-1,4-naphthoquinone), a soluble analog of menaquinone. ACNQ is derived from DHNA (1,4-dihydroxy-2-naphthoic acid) in a non-enzymatic process that frustrated genetic approaches to identify the shuttle. Both ACNQ and DHNA restore reduction of AQDS under anaerobic growth in menaquinone-deficient mutants. Bioelectrochemistry analyses reveal that ACNQ (−0.32 VAg/AgCl) contributes to the extracellular electron transfer (EET) as an electron shuttle, without altering menaquinone generation or EET related cytochrome c expression.

In vitro transcription (IVT) is a DNA-templated process for synthesizing long RNA transcripts, including messenger RNA (mRNA). For many research and commercial applications, IVT of mRNA is typically performed using bacteriophage T7 RNA polymerase (T7 RNAP) owing to its ability to produce full-length RNA transcripts with high fidelity; however, T7 RNAP can also produce immunostimulatory byproducts such as double-stranded RNA that can affect protein expression. Such byproducts require complex purification processes, using methods such as reversed-phase high-performance liquid chromatography, to yield safe and effective mRNA-based medicines. To minimize the need for downstream purification processes, we rationally and computationally engineered a double mutant of T7 RNAP that produces substantially less immunostimulatory RNA during IVT compared with wild-type T7 RNAP. The resulting mutant allows for a simplified production process with similar mRNA potency, lower immunostimulatory content and quicker manufacturing time compared with wild-type T7 RNAP. Herein, we describe the computational design and development of this improved T7 RNAP variant.

Signaling cascades are managed in time and space by interactions between and among proteins. These interactions are often aided by adaptor proteins, which guide enzyme-substrate pairs into proximity. Miniature proteins are a class of small, well-folded protein domains possessing engineered binding properties. Here we make use of two miniature proteins with complementary binding properties to create a synthetic adaptor protein that effectively redirects a ubiquitous signaling event: tyrosine phosphorylation. We report that miniature-protein-based adaptor 3 uses templated catalysis to redirect the Src family kinase Hck to phosphorylate hDM2, a negative regulator of the p53 tumor suppressor and a poor Hck substrate. Phosphorylation occurs with multiple turnover and at a single site targeted by c-Abl kinase in the cell.