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Mechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, which impedes selections in the presence of ligands typically needed to populate defined conformational states. To overcome this key limitation, we developed an in vitro selection platform based on synthetic single domain antibodies named sybodies. To target the limited hydrophilic surfaces of membrane proteins, we designed three sybody libraries that exhibit different shapes and moderate hydrophobicity of the randomized surface. A robust binder selection cascade combining ribosome and phage display enabled the generation of conformation-selective, high affinity sybodies against an ABC transporter and two previously intractable human SLC transporters, GlyT1 and ENT1. The platform does not require access to animal facilities and builds exclusively on commercially available reagents, thus enabling every lab to rapidly generate binders against challenging membrane proteins.

Over the past two decades, enormous progress has been made in designing fluorescent sensors or probes for divalent metal ions. In contrast, the development of fluorescent sensors for monovalent metal ions, such as sodium (Na ⁺), has remained underdeveloped, even though Na ⁺ is one the most abundant metal ions in biological systems and plays a critical role in many biological processes. Here, we report the in vitro selection of the first (to our knowledge) Na ⁺-specific, RNA-cleaving deoxyribozyme (DNAzyme) with a fast catalytic rate [observed rate constant ( k ₒbₛ) ∼0.1 min ⁻¹], and the transformation of this DNAzyme into a fluorescent sensor for Na ⁺ by labeling the enzyme strand with a quencher at the 3′ end, and the DNA substrate strand with a fluorophore and a quencher at the 5′ and 3′ ends, respectively. The presence of Na ⁺ catalyzed cleavage of the substrate strand at an internal ribonucleotide adenosine (rA) site, resulting in release of the fluorophore from its quenchers and thus a significant increase in fluorescence signal. The sensor displays a remarkable selectivity (>10,000-fold) for Na ⁺ over competing metal ions and has a detection limit of 135 µM (3.1 ppm). Furthermore, we demonstrate that this DNAzyme-based sensor can readily enter cells with the aid of α-helical cationic polypeptides. Finally, by protecting the cleavage site of the Na ⁺-specific DNAzyme with a photolabile o -nitrobenzyl group, we achieved controlled activation of the sensor after DNAzyme delivery into cells. Together, these results demonstrate that such a DNAzyme-based sensor provides a promising platform for detection and quantification of Na ⁺ in living cells. Significance Monovalent ions, such as Na ⁺, play important roles in biology, yet few sensors that image intracellular Na ⁺ have been reported. Although deoxyribozymes (DNAzymes) have been shown to be a promising platform for detection of metal ions, most reported DNAzymes require multivalent metal ions for catalytic activity. Existing monovalent ion-responsive DNAzymes have poor selectivity for Na ⁺, low catalytic rate, and require high ion concentrations for function. Here, we report in vitro selection of the first (to our knowledge) highly selective, sensitive, and efficient Na ⁺-specific, RNA-cleaving DNAzyme and its conversion into a catalytic beacon sensor for imaging Na ⁺ in living cells, using an efficient cationic polypeptide delivery method, together with a photocaging strategy, to allow controllable activation of the DNAzyme probe inside cells.

Nucleic acid aptamers are novel molecular recognition tools that offer many advantages compared to their antibody and peptide-based counterparts. However, challenges associated with in vitro selection, characterization, and validation have limited their wide-spread use in the fields of diagnostics and therapeutics. Here, we extracted detailed information about aptamer selection experiments housed in the Aptamer Base, spanning over two decades, to perform the first parameter analysis of conditions used to identify and isolate aptamers de novo. We used information from 492 published SELEX experiments and studied the relationships between the nucleic acid library, target choice, selection methods, experimental conditions, and the affinity of the resulting aptamer candidates. Our findings highlight that the choice of target and selection template made the largest and most significant impact on the success of a de novo aptamer selection. Our results further emphasize the need for improved documentation and more thorough experimentation of SELEX criteria to determine their correlation with SELEX success.

The JmjC histone demethylases (KDMs) are linked to tumour cell proliferation and are current cancer targets; however, very few highly selective inhibitors for these are available. Here we report cyclic peptide inhibitors of the KDM4A-C with selectivity over other KDMs/2OG oxygenases, including closely related KDM4D/E isoforms. Crystal structures and biochemical analyses of one of the inhibitors (CP2) with KDM4A reveals that CP2 binds differently to, but competes with, histone substrates in the active site. Substitution of the active site binding arginine of CP2 to N -ɛ-trimethyl-lysine or methylated arginine results in cyclic peptide substrates, indicating that KDM4s may act on non-histone substrates. Targeted modifications to CP2 based on crystallographic and mass spectrometry analyses results in variants with greater proteolytic robustness. Peptide dosing in cells manifests KDM4A target stabilization. Although further development is required to optimize cellular activity, the results reveal the feasibility of highly selective non-metal chelating, substrate-competitive inhibitors of the JmjC KDMs. JmjC histone demethylases (KDMs) are cancer targets due to their links to cell proliferation, but selective inhibition remains a challenge. Here the authors identify potent inhibitors of KDM4A-C—via in vitro selection from a vast library of cyclic peptides—that show selectivity over other KDMs.

Aptamer therapeutics represent a class of target-based therapies that leverage their high specificity and affinity for diverse molecular targets. As single-stranded DNA or RNA oligonucleotides, aptamers offer advantages in therapeutic applications. A critical aspect of aptamer drug development is the selection process, which has seen significant advancements through various in vitro selection methods, including Systematic Evolution of Ligands by Exponential Enrichment and its emerging variations. Recent progress has also introduced functional screening strategies that directly identify pharmacologically active aptamers, accelerating drug discovery. The applications of aptamers in disease treatment are expanding across oncology, neurodegenerative disorders, infectious diseases and other diseases. Aptamers exhibit versatile mechanisms of action, including blocking interactions, recruiting protein machinery, and inhibiting target functions. By addressing key limitations and presenting future directions, this review provides a comprehensive perspective on the recent evolving landscape of aptamer technology and its transformative potential in modern medicine. [Display omitted] •Aptamers exhibit high specificity and affinity, offering advantages in drug discovery, disease treatment, and diagnostics.•Modern in vitro selection methods have enhanced the identification of high-affinity and functional aptamers.•Aptamer therapeutics show potential in treating various diseases by offering high specificity and reduced off-target effects.•Despite their potential, aptamer-based drugs struggle with many barriers, with only a few reaching clinical use.•Future directions in aptamer research: Intracellular therapeutics, targeted delivery, and AI-assisted screening.

Increased production of sugarcane in Indonesia can be done with extensification sugarcane plantations which largely dominated by acidic upland red-yellow podzolic soil. High aluminium (Al) content and low pH of the soil can inhibit plant growth and development. Tolerant sugarcane in acid soil is the most efficient way, but the adaptive variety is still limited. In vitro culture technique can increase genetic variability to assemble new varieties through somaclonal variation combined with mutation using ethyl methane sulphonate (EMS). The new characters was directed by in vitro selection using AlCl3.6H2O with pH = 4 as a component of selection for resistance to high aluminium. VMC 7616 and PS 862 varieties were used as materials. Mutation induced using EMS at concentrations of 0.1%, 0.3%, and 0.5% for 30, 60 and 120 minutes. Plantlets mutant obtained through callus formation, immersion callus in EMS, in vitro selection, and regeneration of callus. Result of study showed that the long immersion in the EMS solution caused greater damage to the cells, as indicated by the change in callus colour. Callus immersion time in EMS gave greater influence to regeneration compared to concentration of EMS. PS 862 had higher Al tolerance than VMC 7616. Rooting of shoot induced using indole-3-butyric acid (IBA) 3 mg/L.

DNAzymes are catalytically active single-stranded DNAs that fold into metal-ion-assisted architectures to mediate diverse reactions. Addressing the performance gap in biological settings, we establish a novel conceptual framework based on a continuous iteration workflow of selection, enhancement, and application. This paradigm integrates selection constraints, molecular engineering, and clinical context into a unified cycle. We summarize the evolution of SELEX toward application-driven selection incorporating functional/environmental constraints, deep-sequencing-enabled high-throughput activity readouts, droplet compartmentalization and structure- and computation-guided design. We further consolidate engineering strategies to improve stability, kinetics and controllability, including 2′-sugar modifications and XNA substitution, backbone and nucleobase functionalization, arm and secondary-structure engineering for switchable or split architectures and multivalent organization on nanocarriers or nucleic acid scaffolds to enhance local concentration, protection and targeted delivery. Finally, we survey applications in ultrasensitive biosensing and portable diagnostics, activatable and multimodal in vivo imaging, and therapies for cancer, inflammatory diseases and airway disorders, and outline translational priorities: data-driven design, next-generation delivery, standardized safety/PK-PD evaluation and scalable manufacturing, ultimately for clinical and point-of-care deployment.

selection technology has transformed the development of therapeutic monoclonal antibodies. Using methods such as phage, ribosome, and yeast display, high affinity binders can be selected from diverse repertoires. Here, we review strategies for the next-generation sequencing (NGS) of phage- and other antibody-display libraries, as well as NGS platforms and analysis tools. Moreover, we discuss recent examples relating to the use of NGS to assess library diversity, clonal enrichment, and affinity maturation.

Osteoporosis is caused by a disequilibrium between bone resorption and bone formation. Therapeutics for osteoporosis can be divided into antiresorptives that suppress bone resorption and anabolics which increase bone formation. Currently, the only anabolic treatment options are parathyroid hormone mimetics or an anti-sclerostin monoclonal antibody. With the current global increases in demographics at risk for osteoporosis, development of therapeutics that elicit anabolic activity through alternative mechanisms is imperative. Blockade of the PlexinB1 and Semaphorin4D interaction on osteoblasts has been shown to be a promising mechanism to increase bone formation. Here we report the discovery of cyclic peptides by a novel RaPID (Random nonstandard Peptides Integrated Discovery) system-based affinity maturation methodology that generated the peptide PB1m6A9 which binds with high affinity to both human and mouse PlexinB1. The chemically dimerized peptide, PB1d6A9, showed potent inhibition of PlexinB1 signaling in mouse primary osteoblast cultures, resulting in significant enhancement of bone formation even compared to non-Semaphorin4D–treated controls. This high anabolic activity was also observed in vivo when the lipidated PB1d6A9 (PB1d6A9-Pal) was intravenously administered once weekly to ovariectomized mice, leading to complete rescue of bone loss. The potent osteogenic properties of this peptide shows great promise as an addition to the current anabolic treatment options for bone diseases such as osteoporosis.

Cellular senescence is an irreversible form of cell‐cycle arrest caused by excessive stress or damage. While various biomarkers of cellular senescence have been proposed, there are currently no universal, stand‐alone indicators of this condition. The field largely relies on the combined detection of multiple biomarkers to differentiate senescent cells from non‐senescent cells. Here we introduce a new approach: unbiased cell culture selections to identify senescent cell‐specific folded DNA aptamers from vast libraries of trillions of random 80‐mer DNAs. Senescent mouse adult fibroblasts and their non‐senescent counterparts were employed for selection. We demonstrate aptamer specificity for senescent mouse cells in culture, identify a form of fibronectin as the molecular target of two selected aptamers, show increased aptamer staining in naturally aged mouse tissues, and demonstrate decreased aptamer staining when p16 expressing cells are removed in a transgenic INK‐ATTAC mouse model. This work demonstrates the value of unbiased cell‐based selections to identify new senescence‐specific DNA reagents. Pearson et al. report the selection of DNA aptamers against senescent mouse cells, demonstrating broad binding specificity for multiple senescent mouse cell types and induction methods. Two of the aptamers bind a form of fibronectin with sub‐nanomolar affinity even in complex protein mixtures. One aptamer detects age‐ and senescence‐associated changes in mouse lung tissue, highlighting the ability of DNA aptamer selection against a senescence phenotype to generate powerful new reagents with the potential to detect or target senescent cells.