Programmable RNA-binding protein composed of repeats of a single modular unit

The ability to monitor and perturb RNAs in living cells would benefit greatly from a modular protein architecture that targets unmodified RNA sequences in a programmable way. We report that the RNA-binding protein PumHD (Pumilio homology domain), which has been widely used in native and modified form for targeting RNA, can be engineered to yield a set of four canonical protein modules, each of which targets one RNA base. These modules (which we call Pumby, for Pumilio-based assembly) can be concatenated in chains of varying composition and length, to bind desired target RNAs. The specificity of such Pumby–RNA interactions was high, with undetectable binding of a Pumby chain to RNA sequences that bear three or more mismatches from the target sequence. We validate that the Pumby architecture can perform RNA-directed protein assembly and enhancement of translation of RNAs. We further demonstrate a new use of such RNA-binding proteins, measurement of RNA translation in living cells. Pumby may prove useful for many applications in the measurement, manipulation, and biotechnological utilization of unmodified RNAs in intact cells and systems.United States. National Institutes of Health (1R01NS075421)National Science Foundation (U.S.) (1344219)United States. National Institutes of Health (1U01MH106011)United States. National Institutes of Health (1R01MH103910)United States. National Institutes of Health (1DP1NS087724

Enabling community-based metrology for wood-degrading fungi

Background: Lignocellulosic biomass could support a greatly-expanded bioeconomy. Current strategies for using biomass typically rely on single-cell organisms and extensive ancillary equipment to produce precursors for downstream manufacturing processes. Alternative forms of bioproduction based on solid-state fermentation and wood-degrading fungi could enable more direct means of manufacture. However, basic methods for cultivating wood-degrading fungi are often ad hoc and not readily reproducible. Here, we developed standard reference strains, substrates, measurements, and methods sufficient to begin to enable reliable reuse of mycological materials and products in simple laboratory settings. Results: We show that a widely-available and globally-regularized consumer product (Pringles™) can support the growth of wood-degrading fungi, and that growth on Pringles™-broth can be correlated with growth on media made from a fully-traceable and compositionally characterized substrate (National Institute of Standards and Technology Reference Material 8492 Eastern Cottonwood Whole Biomass Feedstock). We also establish a Relative Extension Unit (REU) framework that is designed to reduce variation in quantification of radial growth measurements. So enabled, we demonstrate that five laboratories were able to compare measurements of wood-fungus performance via a simple radial extension growth rate assay, and that our REU-based approach reduced variation in reported measurements by up to ~ 75%. Conclusions: Reliable reuse of materials, measures, and methods is necessary to enable distributed bioproduction processes that can be adopted at all scales, from local to industrial. Our community-based measurement methods incentivize practitioners to coordinate the reuse of standard materials, methods, strains, and to share information supporting work with wood-degrading fungi

Ser-His catalyses the formation of peptides and PNAs

AbstractThe dipeptide seryl-histidine (Ser-His) catalyses the condensation of esters of amino acids, peptide fragments, and peptide nucleic acid (PNA) building blocks, bringing to the formation of peptide bonds. Di-, tri- or tetra-peptides can be formed with yields that vary from 0.5% to 60% depending on the nature of the substrate and on the conditions. Other simpler peptides as Gly-Gly, or Gly-Gly-Gly are also effective, although less efficiently. We discuss the results from the viewpoint of primitive chemistry and the origin of long macromolecules by stepwise fragment condensations

Enabling community-based metrology for wood-degrading fungi

Background: Lignocellulosic biomass could support a greatly-expanded bioeconomy. Current strategies for using biomass typically rely on single-cell organisms and extensive ancillary equipment to produce precursors for downstream manufacturing processes. Alternative forms of bioproduction based on solid-state fermentation and wood-degrading fungi could enable more direct means of manufacture. However, basic methods for cultivating wood-degrading fungi are often ad hoc and not readily reproducible. Here, we developed standard reference strains, substrates, measurements, and methods sufficient to begin to enable reliable reuse of mycological materials and products in simple laboratory settings. Results: We show that a widely-available and globally-regularized consumer product (Pringles™) can support the growth of wood-degrading fungi, and that growth on Pringles™-broth can be correlated with growth on media made from a fully-traceable and compositionally characterized substrate (National Institute of Standards and Technology Reference Material 8492 Eastern Cottonwood Whole Biomass Feedstock). We also establish a Relative Extension Unit (REU) framework that is designed to reduce variation in quantification of radial growth measurements. So enabled, we demonstrate that five laboratories were able to compare measurements of wood-fungus performance via a simple radial extension growth rate assay, and that our REU-based approach reduced variation in reported measurements by up to ~ 75%. Conclusions: Reliable reuse of materials, measures, and methods is necessary to enable distributed bioproduction processes that can be adopted at all scales, from local to industrial. Our community-based measurement methods incentivize practitioners to coordinate the reuse of standard materials, methods, strains, and to share information supporting work with wood-degrading fungi

Building a community to engineer synthetic cells and organelles from the bottom-up

Employing concepts from physics, chemistry and bioengineering, 'learning-by-building' approaches are becoming increasingly popular in the life sciences, especially with researchers who are attempting to engineer cellular life from scratch. The SynCell2020/21 conference brought together researchers from different disciplines to highlight progress in this field, including areas where synthetic cells are having socioeconomic and technological impact. Conference participants also identified the challenges involved in designing, manipulating and creating synthetic cells with hierarchical organization and function. A key conclusion is the need to build an international and interdisciplinary research community through enhanced communication, resource-sharing, and educational initiatives

Hypotetical role of autocatalytic properties of nucleic acids in biogenesis

Ribozymes are catalytic RNA molecules. In this article their various types and activities in contrasts with corresponding DNA molecules are briefly characterized. The revolutionary discovery of the autocatalytic properties of nucleic acids by theNobel Prizewinners, Sidney Altman, Thomas R. CECH and their co-workers, led to the hypothesis of RNA world and to speculation on the role of RNA in the origin of life and the early stages of its evolution on Earth. The possible role of the autocatalytic properties of nucleic acids in prebiotic evolution is, therefore, discussed

Nonenzymatic template-directed RNA synthesis inside model protocells, Science 342

Efforts to recreate a prebiotically plausible protocell, in which RNA replication occurs within a fatty acid vesicle, have been stalled by the destabilizing effect of Mg 2+ on fatty acid membranes. Here we report that the presence of citrate protects fatty acid membranes from the disruptive effects of high Mg 2+ ion concentrations while allowing RNA copying to proceed, while also protecting single-stranded RNA from Mg 2+ -catalyzed degradation. This combination of properties has allowed us to demonstrate the chemical copying of RNA templates inside fatty acid vesicles, which in turn allows for an increase in copying efficiency by bathing the vesicles in a continuously refreshed solution of activated nucleotides. T he RNA world hypothesis suggests that the primordial catalysts were ribozymes (1, 2), whereas biophysical considerations suggest that the primordial replicating compartments were membranous vesicles composed of fatty acids and related amphiphiles (3, 4). However, the conditions required for RNA replication chemistry and fatty acid vesicle integrity have appeared to be fundamentally incompatible (5) ( We developed a screen for small molecules that protect oleate fatty acid vesicles from disruption by Mg 2+ . We used two assays to monitor the leakage of either a small charged molecule (calcein) or a larger oligonucleotide, allowing us to distinguish between increased membrane permeability (faster calcein release with oligonucleotide retention) and generalized membrane disruption (rapid release of both calcein and the oligonucleotide) (figs. S2 to S4). We identified several chelators, including citrate, isocitrate, oxalate, nitrilotriacetic acid (NTA), and EDTA, that protect oleate vesicles in the presence of at least 10 mM Mg 2+ (figs. S5 and S6). In the presence of chelated Mg 2+ , oleate vesicles remained intact but exhibited a modest increase in the permeability of a small polar molecule ( We then asked whether these chelators were compatible with the Mg 2+ catalysis of nonenzymatic template-directed RNA primer extension. We measured the rate at which an RNA primer was elongated when annealed to an oligonucleotide with a templating region of C nucleotides, in the presence of an excess of the activated G monomer guanosine 5′-phosphor(2-methyl)imidazolide (2MeImpG) . For comparison, isocitric acid does not fully protect vesicles (figs. S16 and S17) but also does not affect the primer extension reaction. To see whether citrate would allow nonenzymatic RNA copying to proceed within fatty acid vesicles, we encapsulated an RNA primertemplate complex inside oleate vesicles, added Mg 2+ and citrate, and removed unencapsulated RNA by size exclusion chromatography. We then added the activated G monomer 2MeImpG, heated the sample briefly to allow for rapid monomer permeation (7), and then incubated it at room temperature for times up to 24 hours to allo

Engineering Ribosomes to Alleviate Abiotic Stress in Plants: A Perspective

As the centerpiece of the biomass production process, ribosome activity is highly coordinated with environmental cues. Findings revealing ribosome subgroups responsive to adverse conditions suggest this tight coordination may be grounded in the induction of variant ribosome compositions and the differential translation outcomes they might produce. In this perspective, we go through the literature linking ribosome heterogeneity to plants’ abiotic stress response. Once unraveled, this crosstalk may serve as the foundation of novel strategies to custom cultivars tolerant to challenging environments without the yield penalty