Short Activators and Repressors of RNA Toehold Switches.

RNA toehold switches are a widely used class of molecule to detect specific RNA "trigger" sequences, but their design, intended function, and characterization to date leave it unclear whether they can function properly with triggers shorter than 36 nucleotides. Here, we explore the feasibility of using standard toehold switches with 23-nucleotide truncated triggers. We assess the crosstalk of different triggers with significant homology and identify a highly sensitive trigger region where just one mutation from the consensus trigger sequence can reduce switch activation by 98.6%. However, we also find that triggers with as many as seven mutations outside of this region can still lead to 5-fold induction of the switch. We also present a new approach using 18- to 22-nucleotide triggers as translational repressors for toehold switches and assess the off-target regulation for this strategy as well. The development and characterization of these strategies could help enable applications like microRNA sensors, where well-characterized crosstalk between sensors and detection of short target sequences are critical.

Corrigendum: Effective use of linear DNA in cell-free expression systems.

[This corrects the article DOI: 10.3389/fbioe.2021.715328.].

Effective Use of Linear DNA in Cell-Free Expression Systems.

Cell-free expression systems (CFEs) are cutting-edge research tools used in the investigation of biological phenomena and the engineering of novel biotechnologies. While CFEs have many benefits over in vivo protein synthesis, one particularly significant advantage is that CFEs allow for gene expression from both plasmid DNA and linear expression templates (LETs). This is an important and impactful advantage because functional LETs can be efficiently synthesized in vitro in a few hours without transformation and cloning, thus expediting genetic circuit prototyping and allowing expression of toxic genes that would be difficult to clone through standard approaches. However, native nucleases present in the crude bacterial lysate (the basis for the most affordable form of CFEs) quickly degrade LETs and limit expression yield. Motivated by the significant benefits of using LETs in lieu of plasmid templates, numerous methods to enhance their stability in lysate-based CFEs have been developed. This review describes approaches to LET stabilization used in CFEs, summarizes the advancements that have come from using LETs with these methods, and identifies future applications and development goals that are likely to be impactful to the field. Collectively, continued improvement of LET-based expression and other linear DNA tools in CFEs will help drive scientific discovery and enable a wide range of applications, from diagnostics to synthetic biology research tools.

Enhancing the Thermal Stability of Carbon Nanomaterials with DNA.

Single-walled carbon nanotubes (SWCNTs) have recently been utilized as fillers that reduce the flammability and enhance the strength and thermal conductivity of material composites. Enhancing the thermal stability of SWCNTs is crucial when these materials are applied to high temperature applications. In many instances, SWCNTs are applied to composites with surface coatings that are toxic to living organisms. Alternatively, single-stranded DNA, a naturally occurring biological polymer, has recently been utilized to form singly-dispersed hybrids with SWCNTs as well as suppress their known toxicological effects. These hybrids have shown unrivaled stabilities in both aqueous suspension or as a dried material. Furthermore, DNA has certain documented flame-retardant effects due to the creation of a protective char upon heating in the presence of oxygen. Herein, using various thermogravimetric analytical techniques, we find that single-stranded DNA has a significant flame-retardant effect on the SWCNTs, and effectively enhances their thermal stability. Hybridization with DNA results in the elevation of the thermal decomposition temperature of purified SWCNTs in excess of 200 °C. We translate this finding to other carbon nanomaterials including multi-walled carbon nanotubes (MWCNTs), reduced graphene oxide (RGO) and fullerene (C60), and show similar effects upon complexation with DNA. The rate of thermal decomposition of the SWCNTs was also explored and found to significantly depend upon the sequence of DNA that was used.

Ubiquitination of CD86 is a key mechanism in regulating antigen presentation by dendritic cells.

Dendritic cells (DCs) require costimulatory molecules such as CD86 to efficiently activate T cells for the induction of adaptive immunity. DCs maintain minimal levels of CD86 expression at rest, but upregulate levels upon LPS stimulation. LPS-stimulated DCs produce the immune suppressive cytokine IL-10 that acts in an autocrine manner to regulate CD86 levels. Interestingly, the underlying molecular mechanism behind the tight control of CD86 is not completely understood. In this study, we report that CD86 is ubiquitinated in DCs via MARCH1 E3 ubiquitin ligase and that this ubiquitination plays a key role in CD86 regulation. Ubiquitination at lysine 267 played the most critical role for this regulation. CD86 is ubiquitinated in MARCH1-deficient DCs to a much lesser degree than in wild-type DCs, which also correlated with a significant increase in CD86 expression. Importantly, CD86 is continuously ubiquitinated in DCs following activation by LPS, and this was due to the autocrine IL-10 inhibition of MARCH1 downregulation. Accordingly, DCs lacking MARCH1 and DCs expressing ubiquitination-resistant mutant CD86 both failed to regulate CD86 in response to autocrine IL-10. DCs expressing ubiquitination-resistant mutant CD86 failed to control their T cell-activating abilities at rest as well as in response to autocrine IL-10. These studies suggest that ubiquitination serves as an important mechanism by which DCs control CD86 expression and regulate their Ag-presenting functions.

Early, H+-V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates.

Biased left-right asymmetry is a fascinating and medically important phenomenon. We provide molecular genetic and physiological characterization of a novel, conserved, early, biophysical event that is crucial for correct asymmetry: H+ flux. A pharmacological screen implicated the H+-pump H+-V-ATPase in Xenopus asymmetry, where it acts upstream of early asymmetric markers. Immunohistochemistry revealed an actin-dependent asymmetry of H+-V-ATPase subunits during the first three cleavages. H+-flux across plasma membranes is also asymmetric at the four- and eight-cell stages, and this asymmetry requires H+-V-ATPase activity. Abolishing the asymmetry in H+ flux, using a dominant-negative subunit of the H+-V-ATPase or an ectopic H+ pump, randomized embryonic situs without causing any other defects. To understand the mechanism of action of H+-V-ATPase, we isolated its two physiological functions, cytoplasmic pH and membrane voltage (Vmem) regulation. Varying either pH or Vmem, independently of direct manipulation of H+-V-ATPase, caused disruptions of normal asymmetry, suggesting roles for both functions. V-ATPase inhibition also abolished the normal early localization of serotonin, functionally linking these two early asymmetry pathways. The involvement of H+-V-ATPase in asymmetry is conserved to chick and zebrafish. Inhibition of the H+-V-ATPase induces heterotaxia in both species; in chick, H+-V-ATPase activity is upstream of Shh; in fish, it is upstream of Kupffer's vesicle and Spaw expression. Our data implicate H+-V-ATPase activity in patterning the LR axis of vertebrates and reveal mechanisms upstream and downstream of its activity. We propose a pH- and Vmem-dependent model of the early physiology of LR patterning.

Localization and loss-of-function implicates ciliary proteins in early, cytoplasmic roles in left-right asymmetry.

Left-right asymmetry is a crucial feature of the vertebrate body plan. While much molecular detail of this patterning pathway has been uncovered, the embryonic mechanisms of the initiation of asymmetry, and their evolutionary conservation among species, are still not understood. A popular recent model based on data from mouse embryos suggests extracellular movement of determinants by ciliary motion at the gastrulating node as the initial step. An alternative model, driven by findings in the frog and chick embryo, focuses instead on cytoplasmic roles of motor proteins. To begin to test the latter hypothesis, we analyzed the very early embryonic localization of ciliary targets implicated in mouse LR asymmetry. Immunohistochemistry was performed on frog and chick embryos using antibodies that have (KIF3B, Polaris, Polycystin-2, acetylated alpha-tubulin) or have not (LRD, INV, detyrosinated alpha-tubulin) been shown to detect in frog embryos only the target that they detect in mammalian tissue. Immunohistochemistry revealed localization signals for all targets in the cytoplasm of cleavage-stage Xenopus embryos, and in the base of the primitive streak in chick embryos at streak initiation. Importantly, several left-right asymmetries were detected in both species, and the localization signals were dependent on microtubule and actin cytoskeletal organization. Moreover, loss-of-function experiments implicated very early intracellular microtubule-dependent motor protein function as an obligate aspect of oriented LR asymmetry in Xenopus embryos. These data are consistent with cytoplasmic roles for motor proteins in patterning the left-right axis that do not involve ciliary motion.