T-box riboswitches are noncoding RNA elements involved in genetic regulation of most Gram-positive bacteria. They regulate amino acid metabolism by assessing the aminoacylation status of tRNA, subsequently affecting the transcription or translation of downstream amino acid metabolism-related genes. Here we present single-molecule FRET studies of the Mycobacterium tuberculosis IleS T-box riboswitch, a paradigmatic translational T-box. Results support a two-step binding model, where the tRNA anticodon is recognized first, followed by interactions with the NCCA sequence. Furthermore, after anticodon recognition, tRNA can transiently dock into the discriminator domain even in the absence of the tRNA NCCA-discriminator interactions. Establishment of the NCCA-discriminator interactions significantly stabilizes the fully bound state. Collectively, the data suggest high conformational flexibility in translational T-box riboswitches; and supports a conformational selection model for NCCA recognition. These findings provide a kinetic framework to understand how specific RNA elements underpin the binding affinity and specificity required for gene regulation.

The Bifidobacterium bifidum SAM-VI riboswitch undergoes dynamic conformational changes that modulate downstream gene expression. Traditional structural methods such as crystallography capture the bound conformation at high resolution, and additional efforts would reveal details from the dynamic transition. Here, we revealed a transcription-dependent conformation model for Bifidobacterium bifidum SAM-VI riboswitch. In this study, we combine small-angle X-ray scattering, chemical probing, and isothermal titration calorimetry to unveil the ligand-binding properties and conformational changes of the Bifidobacterium bifidum SAM-VI riboswitch and its variants. Our results suggest that the SAM-VI riboswitch contains a pre-organized ligand-binding pocket and stabilizes into the bound conformation upon binding to SAM. Whether the P1 stem formed and variations in length critically influence the conformational dynamics of the SAM-VI riboswitch. Our study provides the basis for artificially engineering the riboswitch by manipulating its peripheral sequences without modifying the SAM-binding core.

Adeno-associated virus (AAV) vectors have become the leading platform for gene delivery in both preclinical research and therapeutic applications, making the production of high-titer AAV preparations essential. To date, most AAV-based studies use constitutive promoters (e.g., CMV, CAG), which are also active in human embryonic kidney (HEK)-293 producer cells, thus leading to the expression of the transgene already during production. Depending on the transgene\'s function, this might negatively impact producer cell performance and result in decreased AAV vector yields. Here, we evaluated a panel of diverse microRNA (miRNA)-based shRNA designs to identify a highly potent artificial miRNA for the transient suppression of transgenes during AAV production. Our results demonstrate that insertion of miRNA target sites into the 3\' UTR of the transgene and simultaneous expression of the corresponding miRNA from the 3\' UTR of conventional AAV production plasmids (rep/cap, pHelper) enabled efficient silencing of toxic transgene expression, thereby increasing AAV vector yields up to 240-fold. This strategy not only allows to maintain the traditional triple-transfection protocol, but also represents a universally applicable approach to suppress toxic transgenes, thereby boosting vector yields with so far unprecedented efficiency.

The rational targeting of RNA with small molecules is hampered by our still limited understanding of RNA structural and dynamic properties. Most in silico tools for binding site identification rely on static structures and therefore cannot face the challenges posed by the dynamic nature of RNA molecules. Here, we present SHAMAN, a computational technique to identify potential small-molecule binding sites in RNA structural ensembles. SHAMAN enables exploring the conformational landscape of RNA with atomistic molecular dynamics simulations and at the same time identifying RNA pockets in an efficient way with the aid of probes and enhanced-sampling techniques. In our benchmark composed of large, structured riboswitches as well as small, flexible viral RNAs, SHAMAN successfully identifies all the experimentally resolved pockets and ranks them among the most favorite probe hotspots. Overall, SHAMAN sets a solid foundation for future drug design efforts targeting RNA with small molecules, effectively addressing the long-standing challenges in the field.

Drug candidates that fail in clinical trials for efficacy reasons might still have favorable safety and bioavailability characteristics that could be exploited. A failed drug candidate could be repurposed if a receptor, such as an aptamer, were created that binds the compound with high specificity. Branaplam is a small molecule that was previously in development to treat spinal muscular atrophy and Huntington\'s disease. Here, we report the development of a small (48-nucleotide) RNA aptamer for branaplam with a dissociation constant of ∼150 nM. Starting with a combinatorial RNA pool integrating the secondary and tertiary structural scaffold of a Guanine-I riboswitch aptamer interspersed with regions of random sequence, in vitro selection yielded aptamer candidates for branaplam. Reselection and rational design were employed to improve binding of a representative branaplam aptamer candidate. A resulting variant retains the pseudoknot and two of the paired elements (P2 and P3) from the scaffold but lacks the enclosing paired element (P1) that is essential for the function of the natural Guanine-I riboswitch aptamer. A second combinatorial RNA pool based on the scaffold for TPP (thiamin pyrophosphate) riboswitches also yielded a candidate offering additional opportunities for branaplam aptamer development.

BACKGROUND: RNA design is a key technique to achieve new functionality in fields like synthetic biology or biotechnology. Computational tools could help to find such RNA sequences but they are often limited in their formulation of the search space.
RESULTS: In this work, we propose partial RNA design, a novel RNA design paradigm that addresses the limitations of current RNA design formulations. Partial RNA design describes the problem of designing RNAs from arbitrary RNA sequences and structure motifs with multiple design goals. By separating the design space from the objectives, our formulation enables the design of RNAs with variable lengths and desired properties, while still allowing precise control over sequence and structure constraints at individual positions. Based on this formulation, we introduce a new algorithm, libLEARNA, capable of efficiently solving different constraint RNA design tasks. A comprehensive analysis of various problems, including a realistic riboswitch design task, reveals the outstanding performance of libLEARNA and its robustness.
METHODS: libLEARNA is open-source and publicly available at: https://github.com/automl/learna_tools.

The btuB riboswitch is a regulatory RNA sequence controlling gene expression of the outer membrane B12 transport protein BtuB by specifically binding coenzyme B12 (AdoCbl) as its natural ligand. The B12 sensing riboswitch class is known to accept various B12 derivatives, leading to a division into two riboswitch subclasses, dependent on the size of the apical ligand. Here we focus on the role of side chains b and e on affinity and proper recognition, i. e. correct structural switch of the btuB RNA, which belongs to the AdoCbl-binding class I. Chemical modification of these side chains disturbs crucial hydrogen bonds and/or electrostatic interactions with the RNA, its effect on both affinity and switching being monitored by in-line probing. Chemical modifications at sidechain b of vitamin B12 show larger effects indicating crucial B12-RNA interactions. When introducing the same modification to AdoCbl the influence of any side-chain modification tested is reduced. This renders the impact of the adenosyl-ligand for B12-btuB riboswitch recognition clearly beyond the known role in affinity.

Artificial riboswitches responsive to user-defined analytes can be constructed by successfully inserting in vitro selected aptamers, which bind to the analytes, into untranslated regions of mRNA. Among them, eukaryotic riboswitches are more promising as biosensors than bacterial ones because they function well at ambient temperature. In addition, cell-free expression systems allow the broader use of these riboswitches as cell-free biosensors in an environmentally friendly manner without cellular limitations. The current best cell-free eukaryotic riboswitch regulates eukaryotic canonical translation initiation through self-cleavage mediated by an implanted analyte-responsive ribozyme (i.e., an aptazyme, an aptamer-ribozyme fusion). However, it has critical flaws as a sensor: due to the less-active ribozyme used, self-cleavage and translation reactions must be conducted separately and sequentially, and a different aptazyme has to be selected to change the analyte specificity, even if an aptamer for the next analyte is available. We here stepwise engineered novel types of cell-free eukaryotic riboswitches that harness highly active self-cleavage and thus require no reaction partitioning. Despite the single-step and one-pot reaction, these riboswitches showed higher analyte dose dependency and sensitivities than the current best cell-free eukaryotic riboswitch requiring multistep reactions. In addition, the analyte specificity can be changed in an extremely facile way, simply by aptamer substitution (and the subsequent simple fine-tuning for giant aptamers). Given that cell-free systems can be lyophilized for storage and transport, the present one-pot and thus easy-to-handle cell-free biosensors utilizing eukaryotic riboswitches are expected to be widely used for on-the-spot sensing of analytes at ambient temperature.

The Gram-positive bacterium Bacillus anthracis is the causative agent of anthrax and a bioterrorism threat worldwide. As a crucial second messenger in many bacterial species, cyclic di-AMP (c-di-AMP) modulates various key processes for bacterial homeostasis and pathogenesis. Overaccumulation of c-di-AMP alters cellular growth and reduces anthrax toxin expression as well as virulence in Bacillus anthracis by unresolved underlying mechanisms. In this report, we discovered that c-di-AMP binds to a series of receptors involved in potassium uptake in B. anthracis. By analyzing Kdp and Ktr mutants for osmotic stress, gene expression, and anthrax toxin expression, we also showed that c-di-AMP inhibits Kdp operon expression through binding to the KdpD and ydaO riboswitch; up-regulating intracellular potassium promotes anthrax toxin expression in c-di-AMP accumulated B. anthracis. Decreased anthrax toxin expression at high c-di-AMP occurs through the inhibition of potassium uptake. Understanding the molecular basis of how potassium uptake affects anthrax toxin has the potential to provide new insight into the control of B. anthracis.IMPORTANCEThe bacterial second messenger cyclic di-AMP (c-di-AMP) is a conserved global regulator of potassium homeostasis. How c-di-AMP regulates bacterial virulence is unknown. With this study, we provide a link between potassium uptake and anthrax toxin expression in Bacillus anthracis. c-di-AMP accumulation might inhibit anthrax toxin expression by suppressing potassium uptake.

Cells use transition metal ions as structural components of biomolecules and cofactors in enzymatic reactions, making transition metal ions integral cellular components. Organisms optimize metal ion concentration to meet cellular needs by regulating the expression of proteins that import and export that metal ion, often in a metal ion concentration-dependent manner. One such regulation mechanism is via riboswitches, which are 5\'-untranslated regions of an mRNA that undergo conformational changes to promote or inhibit the expression of the downstream gene, commonly in response to a ligand. The yybP-ykoY family of bacterial riboswitches shares a conserved aptamer domain that binds manganese ions (Mn2+). In Escherichia coli, the yybP-ykoY riboswitch precedes and regulates the expression of two different genes: mntP, which based on genetic evidence encodes an Mn2+ exporter, and alx, which encodes a putative metal ion transporter whose cognate ligand is currently in question. The expression of alx is upregulated by both elevated concentrations of Mn2+ and alkaline pH. With metal ion measurements and gene expression studies, we demonstrate that the alkalinization of media increases the cytoplasmic manganese pool, which, in turn, enhances alx expression. The Alx-mediated Mn2+ export prevents the toxic buildup of the cellular manganese, with the export activity maximal at alkaline pH. We pinpoint a set of acidic residues in the predicted transmembrane segments of Alx that play a critical role in Mn2+ export. We propose that Alx-mediated Mn2+ export serves as a primary protective mechanism that fine tunes the cytoplasmic manganese content, especially during alkaline stress.IMPORTANCEBacteria use clever ways to tune gene expression upon encountering certain environmental stresses, such as alkaline pH in parts of the human gut and high concentration of a transition metal ion manganese. One way by which bacteria regulate the expression of their genes is through the 5\'-untranslated regions of messenger RNA called riboswitches that bind ligands to turn expression of genes on/off. In this work, we have investigated the roles and regulation of alx and mntP, the two genes in Escherichia coli regulated by the yybP-ykoY  riboswitches, in alkaline pH and high concentration of Mn2+. This work highlights the intricate ways through which bacteria adapt to their surroundings, utilizing riboregulatory mechanisms to maintain Mn2+ levels amidst varying environmental factors.