SLAS Discovery

In vitro cytotoxicity assessments frequently rely on staining-based methods that indirectly estimate viable cell numbers indirectly. A major limitation of many such techniques is their endpoint nature, requiring cell lysis or irreversible processing that precludes longitudinal monitoring of cellular responses following treatment. An ideal assay for evaluating cell viability and proliferation should be simple, rapid, cost-effective, reproducible, and highly sensitive, while also enabling accurate quantification with minimal interference from test compounds. The resazurin reduction assay satisfies these criteria, offering a sensitive and economical alternative to conventional tetrazolium-based methods. Although both assay types depend on the metabolic reduction of a dye by viable cells, they differ mechanistically. Tetrazolium salts (e.g., MTT) are reduced by cellular dehydrogenases to insoluble formazan crystals that require solubilization before to detection. In contrast, resazurin--a cell-permeable, non-fluorescent blue dye--is reduced to resorufin, a highly fluorescent compound detectable without additional processing steps. This property renders the resazurin assay broadly applicable to viability testing in eukaryotic cells cultured in both 2D and 3D formats, as well as in bacterial systems. Here, we present a streamlined, universal protocol for implementing the resazurin reduction assay across diverse experimental models, emphasizing its practicality, reproducibility, and adaptability for real-time viability monitoring. Key featuresO_LIReal-time, non-destructive monitoring: Enables longitudinal studies by allowing repeated measurements of the same samples over hours without toxicity or disruption. C_LIO_LIStreamlined workflow: A simple "add-incubate-read" protocol eliminates the need for cell lysis, washing, or extraction, saving time and reducing variability. C_LIO_LIBroad sample compatibility: Versatile and reliable for use with 2D monolayers, 3D spheroids, organoids, and bacterial cultures. C_LIO_LIHigh sensitivity: Fluorescent detection of resorufin provides exceptional sensitivity, enabling accurate quantification of even small viable cell populations. C_LIO_LILow background and minimal interference: A clean fluorescent readout reduces the risk of signal artifacts, offering a more reliable alternative to traditional colorimetric assays. C_LIO_LICost-effective and accessible: Utilizes standard laboratory plate readers and commercially available reagents, making it an economical choice for any lab. C_LIO_LIScalable for high-throughput screening: Easily adaptable to various plate formats, supporting both small-scale experiments and large-scale automated screening applications. C_LI Graphical overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/718248v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@82bcecorg.highwire.dtl.DTLVardef@14164aforg.highwire.dtl.DTLVardef@395118org.highwire.dtl.DTLVardef@fb1349_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundDNA polymerase activity assays are required for enzyme quality control in biotechnology and diagnostics, but standard methods rely on specialist reagents, radioactivity and other hazardous materials, or real-time PCR instruments that are not widely accessible in resource-limited settings. This constrains local production of high quality, validated reagents and increases dependence on imported enzymes. MethodsBased on experiences derived from partnerships with scientists in several low and middle-income countries (LMICs) and stakeholder consultations, we adapted a commercial EvaGreen-based fluorometric DNA polymerase activity assay for isothermal operation using minimal equipment. Assay conditions were optimized using Design of Experiments (DOE) methodology, varying temperature, reaction volume, and MgCl2 concentration. To address reagent cost and supply-chain constraints, we developed detailed protocols for in-house synthesis of the off-patent AOAO-12 DNA dye (sold commercially as EvaGreen) and generation of single-stranded DNA templates via asymmetric PCR. ResultsOptimized isothermal assay conditions (40{degrees}C, 7.75 mM MgCl2) reliably quantified activity across multiple DNA polymerase families. In-house synthesized AOAO-12 dye exhibited comparable DNA-binding performance to commercial alternatives (R{superscript 2} = 0.95), reducing costs by more than an order of magnitude when normalized to working concentrations, enabling assay costs of approximately {pound}0.001 per reaction. The assay is effective across multiple polymerases (Bst-LF, OpenVent, Taq, Q5) and is compatible with both plate readers and qByte, a low-cost, open-source fluorometric device. ConclusionsThis stakeholder-informed assay provides an accessible, cost-effective solution for DNA polymerase quality control in resource-limited settings. The combination of optimized commercial protocols and in-house reagent synthesis offers flexibility for different resource contexts, potentially improving access to molecular biology tools globally.

The p21-activated kinases (PAKs) are a group of serine-threonine kinases central to multiple signaling pathways that govern cell survival and proliferation. Aberrant activity of PAK1, the most well characterized member of the PAK family, drives progression of several malignancies and brain disorders, including Alzheimers disease and neurodevelopmental disorders. Despite growing interest in PAK1 as a drug target for these diseases, there is no assay to evaluate the intracellular target engagement of PAK1 inhibitors. To address this need, we developed first-in-class NanoBRET assays for wild-type PAK1 and a neurodevelopmental disorder-causing gain-of-function PAK1 mutant. Furthermore, we executed our novel PAK1 NanoBRET assay to evaluate target engagement of PAK1 inhibitors in primary hippocampal neurons. To the best of our knowledge, this is the first demonstration of a NanoBRET cellular target engagement assay in primary neurons, thereby increasing the relevance of our work by confirming PAK1 inhibitor binding to the aberrant form of the protein in primary neurons.

BackgroundHigh-content assays (HCAs) have problems distinguishing biologically significant effects from the incidental effects of non-repeatable technical factors. Non-repeatable results are attributed to variations in the cell culture environment and the numerous, heterogeneous descriptors evaluated. The aim here was to determine whether preprocessing operations impacted the reproducibility of class assignments of experimental data. MethodsBatch effects that could affect reproducibility, i.e., signal/noise ratio, instrumental conditions, and segmentation, were controlled variables. The remaining batch effects, variations in materials, personnel, and culture environment could not be controlled. Descriptors values were measured directly from images. Exploratory factor analysis was used to solve the identifiable and interpretable feature, factor 4. In each of five trials, one sample was treated with the same chemical mixture (EXP) and another with the solvent vehicle alone (CON). ResultsRepeated CON and EXP samples showed significant differences among factor 4 means in data regularized within each trial. The mean of Trial 3 CON differed significantly from all other CON samples. These differences disappeared upon regularization to comprehensive databases. Among repeated EXPs, the Trial 2 mean differed from three other EXPs, but regularization to comprehensive databases had little effect. However, classification patterns were unchanged after regularization to any comprehensive database derived by the same protocol. After regularization to datasets derived by two different protocols, the classification pattern differed but only reflected elevation of differences that had been marginal to statistical significance. Outlier removal was deleterious. Even with the most sparing definition of outliers, over 3% of a single samples contents were removed from most trials. Elimination based on the overall within-trial distributions caused type I and type II errors. ConclusionsNon-repeatable factor 4 means in repeated trials had negligible influence on classification outcomes, so repeatability may not be a good indicator of assay quality. Irreducible batch effects, combined with small sample sizes and skewed distributions of descriptors values, may account for non-repeatability. As the current results are based on real-world data, they suggest that non-repeatability is an uncorrectable feature of these assays. Classification patterns are not affected by several irreducible technical factors, namely materials, personnel, and non-repeatable environmental variables.

Malaria remains a major global health challenge, with emerging partial resistance to first-line therapies in Africa threatening current control efforts. Drug combinations are essential to improve treatment efficacy and restrain resistance development. However, in vitro assays that quantify parasite viability after drug exposure and characterize pharmacodynamic drug interactions are labor- and resource-intensive, with standard approaches such as the parasite reduction ratio assay limiting systematic, high-resolution evaluation of drug combinations. We present the MUltidimensional Luminescence Test for integration of interactions (MULT-i2), an in vitro assay that enables scalable, high-resolution assessment of parasite viability across multidimensional drug concentration spaces. For dual drug combinations, the MULT-i2 assay characterizes interaction surfaces while requiring [~]50-fold fewer resources and more than two-fold less time than conventional methods, enabling exploration of broader combination scenarios. The assay combines a highly sensitive chemiluminescence readout with inducible reporter expression in Plasmodium falciparum, supporting potential extension to multidimensional combination testing. Using the general pharmacodynamic interaction (GPDI) model, the MULT-i2 assay quantified interaction potency and directionality, confirming and refining the known synergy between atovaquone and proguanil, and revealing detailed interaction patterns for additional drug combinations. Overall, this approach provides an efficient framework for testing and characterizing pharmacodynamic drug interactions and supports the rational development of antimalarial combination therapies.

Fluorescent reporters provide a useful tool for studying degron motifs. Fusing a degron of interest to a fluorescent protein allows to accurately track protein levels overtime to characterise the degradation kinetics of studied degrons. Here we describe a rapid and simple method to study degron peptides in Escherichia coli using plasmid-encoded eGFP-degron fusion constructs. The described methods provide an accessible workflow to evaluate degrons. We provide protocols for generation of pBAD plasmids encoding the studied constructs and two different methods for evaluating degrons - an end-point fluorescence measurement on agar plates and a kinetic measurement in liquid cultures in a 96-well format for high-throughput degron studies.

Powder X-ray diffraction (PXRD) measurements performed on platforms originally designed for single-crystal diffraction are strongly affected by how the powder sample is presented to the X-ray beam, including the delivery configuration and support geometry. Here, we developed a modified Terasaki-plate-based sample-delivery method for PXRD using a laboratory single-crystal diffractometer implemented with the XtalCheck-S plate-reader operational mode at Turkish Light Source. The method was regarded under comparable measurement conditions relative to a standard loop/pin-based and a grease-based Terasaki setup using 5-{[4-(2-Methoxyphenyl)piperazin-1-yl]methyl}-4-ethyl-4H-1,2,4-triazole-3-thiol as a model analyte. The loop-based method allowed only limited powder sampling, whereas the grease-based Terasaki setup enabled multi-well sample delivery but produced higher background and weaker diffraction profiles. Conversely, Kapton-sealed Terasaki ensured secure retention of small amounts powder while providing lower background and clearer diffraction patterns. Within short total data collection times of only 1-2 min, the Kapton-Terasaki method delivered the best overall PXRD performance among the tested methods. Search-match and profile-fitting analyses showed that all three approaches sampled the same crystalline material, while the Kapton-based method gave the lowest profile residual (Rp = 9.6%) and the most reliable whole-pattern profile. These results demonstrate that optimizing sample delivery, rather than modifying the core instrument hardware, can substantially extend PXRD capability on an existing in situ crystallography platform for rapid, laboratory-based screening and comparative multi-sample measurements.

Traditional clonogenic assays remain central to evaluating the self-renewal capacity of tumor cells. However, the assay relies on subjective endpoint measurements, is restricted to two-dimensional monolayer growth, and lacks the single cell resolution required to resolve heterogeneous expansion behaviors. We describe a high-density microwell array-based platform for quantitative assessment of single cell clonogenic growth outcomes, defined by cell count distributions spanning non-dividing, slow-dividing, and fast-dividing three-dimensional colony forming phenotypes. This approach links initial single-cell occupancy to defined growth outcomes across thousands of indexed microwells per well. The platform integrates high-density, low-adhesion microwell arrays within industry standard device plate formats and an automated image analysis pipeline incorporating machine learning, enabling parallel quantification of spatially indexed founder-derived microwells using widely accessible automated imaging systems. The assay was implemented in both 4-well and 96-well plate formats to evaluate reproducibility and scalability across different plate configurations. Using three glioblastoma cell lines as model systems, we demonstrate reproducible single founder occupancy and consistent clonal growth outcome distributions across replicate formats. This integrated microscale assay platform enables systematic quantitative characterization of clonogenic expansion capacity at single cell resolution and is compatible with applications in cancer biology, therapeutic testing, and functional single cell phenotyping. By resolving single-cell persistence, limited expansion and high expansion outcomes within a scalable high-density format, this approach expands the analytical resolution of single cell clonogenic profiling beyond traditional binary colony scoring.

Advances in transcriptome profiling have revealed transcriptomic differences across different cellular states. However, functional interpretation requires precise perturbation tools and experimental frameworks. This study benchmarked two widely used modalities: CRISPR interference (CRISPRi) and Cas13d/CasRx. A standardized workflow was established to generate human pluripotent stem cells (PSCs) with inducible ZIM3-dCas9 or CasRx expression. The cell lines were subjected to flow cytometry, copy number, and immunocytochemical analyses. The knockdown performance was validated via robust OCT4 suppression and the expected downstream effects on pluripotency genes. Time-course measurements indicated that CRISPRi produced faster and stronger repression but slower recovery after inducer withdrawal. In contrast, CasRx yielded slower and typically weaker knockdown with rapid reversibility. Furthermore, a key limitation of CRISPRi was demonstrated using the ATF5-NUP62 locus, wherein CRISPRi could co-repress genes with overlapping promoter regions. In contrast, CasRx avoids these limitations and supports isoform-resolved targeting of circular and alternatively spliced transcripts, albeit with variable efficiency. These results provide practical guidance for selecting complementary knockdown tools to improve the interpretability of transcriptomic function studies. MOTIVATIONAdvances in transcriptome profiling have enabled the detection of subtle cell type-specific differences. However, mechanistic interpretation still depends on perturbation tools that can modulate transcripts with high precision and efficiency. Recent CRISPR-based modalities, CRISPRi and Cas13/CasRx, function as robust and orthogonal methods to achieve the knockdown of specific gene targets. However, a standardized approach for cell line preparation and comparative studies on their relative performances and limitations remains unclear. Consequently, this study presents a standardized workflow for generating cell lines that support high-efficiency knockdown using CRISPRi and CasRx. Moreover, it compares the trade-offs in potency, reversibility, and isoform resolution, along with a practical overview of method-specific pitfalls to guide tool selection and data interpretation in future studies. HIGHLIGHTSO_LIDoxycycline-inducible AAVS1 knock-in human PSC platforms for CRISPRi (ZIM3-dCas9) and CasRx (RfxCas13d) were generated to enable standardized RNA perturbation experiments. C_LIO_LIThe prepared cell lines demonstrated strong OCT4 knockdown, with expected downstream effects on the expression of another pluripotency gene, NANOG. C_LIO_LIA comparison of knockdown characteristics and their reversibility revealed rapid and sustained repression with CRISPRi, whereas slow but rapid recovery was observed with CasRx. C_LIO_LIA CRISPRi-specific off-target effect arising from TSS proximity/overlap (ATF5-NUP62) was identified, whereas CasRx achieved ATF5 knockdown without collateral repression of the neighboring NUP62 gene. C_LIO_LICasRx enables isoform-resolved knockdown of structural isoforms (circHIPK3 vs. linear HIPK3 mRNA) and splice isoforms (RAB6A-iso1 vs. RAB6A-iso2). C_LI

IntroductionAccurate in silico evaluation of primers and probes is essential for the rational design of molecular multi-parameter assays. We present Assay-BLAST v2 to automate and simplify this process for extensive assay designs. ResultsA newly integrated strand and proximity check enables precise validation of corresponding oligonucleotides, ensuring correct orientation and spacing for efficient amplification. Based on predicted oligonucleotide interactions, Assay-BLAST v2 estimates amplification outcomes, offering a computational benchmark for downstream wet-lab validation and performance correlation. Additionally, the updated software integrates an adaptive BLAST parameter optimization that dynamically scales with database size, thereby improving both analytical sensitivity and computational performance. These improvements are supported by a comparative evaluation against the previous version of AssayBLAST. ConclusionsCollectively, these enhancements streamline the assay development workflow, reduce costs associated with suboptimal primer and probe synthesis, and increase the robustness and reliability of molecular diagnostics and research applications.

The expansion of short tandem repeats is a feature of over 60 different human diseases. Ongoing somatic instability throughout a patients lifetime can influence disease progression and has emerged as a therapeutic target. Understanding its mechanism is essential for the identification of both drug targets and therapeutic interventions. A major obstacle towards this translational goal has been to measure changes in repeat size distribution in a timely manner. To address this, here we present Single Clone-based Instability Assay (SCIA), a streamlined experimental design that saves weeks in assessing the effect of a gene knockout on repeat instability. The approach avoids bulk cultures and does not require a reporter cell line. It uses targeted long-read sequencing as a readout for repeat instability. We have validated the approach using FAN1, PMS1, and MLH1 knockouts in HEK293-derived cells. We provide a visualization software that generates delta plots, extracts the instability frequency, the bias towards expansion or contraction, and the average size of the changes. Using SCIA, we find that although FAN1 knockout clones showed increased frequency of expansions, the size of the expansions were smaller. This highlights the wealth of information that can be extracted and the potential for novel insights into the mechanism of repeat instability.

Currently there is a lack of high-throughput, low material-input methods to screen early-stage product quality of viral and non-viral gene therapy products. Here we propose using multiplex droplet digital PCR (dPCR) to screen and characterize vector sequences. We describe the adaptation of a Poisson-multinomial model to quantitate integrity of any combination of 4 targets in multiplexed ddPCR. We show the success and limitations of model employment and provide some suggested best practices.

Antimicrobial resistance remains a global existential threat. Given that antimicrobial therapy commonly starts before pathogen identification, rapid and scalable methods capable of determining effective antimicrobial compounds are needed. In this paper, we demonstrate a 2 x 2 array of parallelised microscopes that uses low numerical aperture (NA=0.25) detection optics and LED excitation to determine bacterial viability based on their fluorescence response to an electrical stimulus. Following a 2-hour incubation, the fluorescent viability readout requires less than one minute. We use K-means clustering to classify pixels in a time lapse sequence of widefield fluorescence images and extract changes seen within bacterial clusters. We demonstrate sufficient sensitivity to measure fluorescence changes after electrical stimulation in a bacterial monolayer. To capture these subtle fluorescence changes at high signal-to-background ratios, we place a limit on the minimum optical density of the bacterial sample. This novel approach is scalable to 96-well formats using a suitable consumable electrode array.

Massively multiplexed qPCR is primarily constrained by increasing primer dimer formation as the number of distinct primers in a single reaction increases. Previous multiplex primer design algorithms either fail to sufficiently suppress primer dimers at 100+ plex, or take exceedingly high amounts of computational resources to complete. Here, we present DIMPLE, a linear-runtime primer design algorithm that effectively generates 10,000+ primers to amplify thousands of potential amplicons in a single qPCR reaction. As one clinical demonstration of this algorithm, we designed an assay to detect 2,302 distinct KMT2A gene fusion subtypes using 204 primers in a single tube. In contrast to FISH and convention NGS approaches with 2% variant allele frequency (VAF) limit of detection, our DIMPLE qPCR assay was able to analytically detect gene fusions down to 0.05% VAF. We also constructed proof-of-concept multiplex qPCR panels for additional oncology gene fusions, multiplex pathogen detection, and DNA methylation markers. The scalability and low computational cost DIMPLE are complementary to new instrument platforms for massively multiplex qPCR readout for enabling rapid, point-of-care nucleic acid testing.

Validation of somatic mutation burden assays is fundamentally constrained by the absence of a robust ground truth, limiting the interpretability of performance metrics. To address this, we propose a framework based primarily on relative validation, complemented by a suite of secondary metrics aligned to common failure modes. We implement this approach in SomaticCODEC, a ready-to-run assay for quantifying SNV burden in primary human samples, demonstrating strong linearity across mixtures of sperm and blood samples (R2 = 0.91) and high intra-batch precision (CV = 3.3%). This framework provides a practical approach for validating somatic mutation burden assays without requiring a ground truth.

In vitro methods to characterize drug combinations typically involve phenotypic screenings using checkerboard assays (CBA) or, more recently, DiaMOND. Such approaches rely on the Fractional Inhibitory Concentration Index (FICI), a fixed-time measurement of growth inhibition that, nonetheless, necessitates secondary validation by time-kill assays (TKA). Longitudinal time-kinetics of bacterial killing are considered the gold standard in vitro proxy for antimicrobial activity, but they required increased assay complexity, particularly against the slow growing Mycobacterium tuberculosis. Here, we developed a new methodology named OPTIKA (Optimized Time Kill Assays) that enhances the capacity of traditional TKA by over 1000-fold. This allows for easy and dynamic examination of n-way drug interactions by simultaneously monitoring bactericidal and sterilizing capacities in a longitudinal manner. We then replicated previous DiaMOND studies and performed comparisons using CBA and OPTIKA methodologies. We demonstrate that selection of the efficacy parameters (either routed on bacteriostatic, bactericidal or sterilizing properties) affects the interpretation of in vitro drug interactions and, consequently, its potential translational value. The increased assay throughput provided by OPTIKA offers a novel framework for developing tuberculosis treatment regimens. TeaserOPTIKA is a new methodology that increases time-kill assay performance against Mycobacterium tuberculosis by over 1,000-fold

Leukocyte immunoglobulin-like receptor B4 (LILRB4, ILT3) is an inhibitory immune checkpoint expressed on myeloid cells, where it contributes to immunosuppression within the tumor microenvironment. Secretogranin 2 (SCG2) has recently been identified as a functional ligand of LILRB4, yet small molecule modulators of this interaction remain unexplored. Here, we report the development of a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay to interrogate the LILRB4 (ILT3)-SCG2 interaction. The assay demonstrated robust performance and was validated using a blocking anti-LILRB4 antibody, consistent with orthogonal ELISA measurements. Pilot screening of chemical libraries identified 23 primary hits, of which two compounds, BMS-813160 and PSB-603, showed reproducible, dose-dependent inhibition with TR-FRET IC50 values of 26.7 {+/-} 1.03 {micro}M and 37.2 {+/-} 2.14 {micro}M, respectively. Activity was confirmed by ELISA, supporting the robustness of the assay. This platform enables high-throughput discovery of first-in-class small molecule modulators of the LILRB4-SCG2 immune checkpoint and provides a foundation for targeting myeloid-driven immunosuppression.

Three-dimensional microtumour models such as spheroids are increasingly used in cancer research as they better capture tumour architecture, growth and invasion than conventional two-dimensional cultures. However, robust and accessible tools for quantitative analysis remain limited. Here we present SImBA-SiQuAl, an integrated open-source workflow for high-throughput quantitative phenotyping of 3D spheroids and organoids. The pipeline combines SImBA, an automated image-analysis framework for performant quality-controlled image segmentation and multi-feature extraction from spheroid assays, with SiQuAl, a downstream analysis platform that automatically performs comprehensive statistical and multivariate analyses to reveal phenotypic differences between experimental conditions. In a first case study, SImBA-SiQuAl resolves intrinsic invasion phenotypes between cancer cell lines. In a second case study, the workflow quantifies both uniform and heterogeneous responses in a spheroid drug screening assay. Together, SImBA-SiQuAl provides a new, timely tool for high-throughput, high-content microtumour phenomics in cancer research. MOTIVATION3D-microtumour assays such as spheroids and organoids are increasingly used in preclinical research. These assays generate rich phenotypic imaging data, but quantitative automated analysis remains a major bottleneck. This limits reproducibility, scalability, and broad adoption for large-scale, high-content phenomics studies, but also implies biologically relevant phenotypic (heterogeneous) responses in e.g. perturbation studies may not be comprehensively addressed. SImBA-SiQuAl is developed to address this gap by providing an open-source, integrated workflow offering solutions in both the image processing and downstream analysis. Together, this enables in-depth quantitative analysis of 3D microtumour phenotypes across experimental settings. HIGHLIGHTSO_LISImBA-SiQuAl provides a complete end-to-end workflow for high-throughput, high-content, quantitative 3D microtumour analysis, from quality-controlled image segmentation to statistical, multivariate and cluster-based biological interpretation. C_LIO_LISImBA-SiQuAl is broadly applicable across multiple 3D systems and assay types. C_LIO_LIWe demonstrate the workflow can capture biologically meaningful heterogeneity and treatment response at scale, supporting robust and unbiased analysis. C_LIO_LIBy combining accessibility, flexibility and analytical depth, SImBA-SiQuAl addresses a key unmet need for accessible advanced open-source tools in 3D preclinical research. C_LI

Natural products are a rich source of bioactive molecules and undiscovered chemical scaffolds with significant potential for novel drug discovery. Among these, fungi are particularly promising, offering diverse metabolites and undiscovered structural motifs. Large, well-curated collections of crude extracts, or "libraries", are central to fungal natural product discovery, serving as starting material for bioassay-guided isolation of new compounds. However, the systematic influence of fungal selection strategies, culturing methods, and environmental factors on chemical diversity remains underexplored. In this study, we analyzed several large fungal libraries to assess how geographic origin, and phylogenetic classification shape fungal chemical profiles. We also evaluated whether culturing conditions that more closely mimic natural environments can enhance metabolite diversity. Our findings offer practical guidelines for optimizing fungal natural product library design, improving drug development efficiency and access to novel chemotypes for future drug discovery. Summary Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/709592v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@70a0e0org.highwire.dtl.DTLVardef@51f84eorg.highwire.dtl.DTLVardef@184dd90org.highwire.dtl.DTLVardef@1ee2813_HPS_FORMAT_FIGEXP M_FIG C_FIG

RNase R is a processive 3 to 5 exoribonuclease that degrades a broad array of linear RNA species while preserving RNA lariats and circular RNAs (circRNA). In recent years, this enzyme has become pivotal for the field of circRNA research, serving as a key step for circRNA enrichment, purification, and identification. Despite this growing importance, the effects of mutations and truncations in RNase R have been incompletely studied. We make several point mutations and assay their effects on the ability of RNase R to bind and/or degrade RNA substrates. Our data show that selected active site mutations have varying effects on RNA binding and degradation. Furthermore, the increasing interest in circRNA-based RNA therapeutic platforms highlights an urgent need for RNase R in RNA molecular biology labs. However, the substantial cost of commercial RNase R remains a bottleneck, particularly for large-scale studies or the development of circRNA-based technologies. In this protocol, we offer a solution to that problem, namely a more accessible and cost-effective means of purifying high-quality and low-cost RNase R. We provide a highly detailed yet simplified, high-yield protocol that produces recombinant RNase R from Escherichia coli. The method uses a single-step Ni-NTA affinity chromatography procedure without proteolytic tag removal and is optimized for entry-level FPLC systems such as the AKTA Start, ensuring that high-purity enzyme production does not require specialized, high-end instrumentation. A second key feature is the establishment of an optimized reaction framework, including specific buffer compositions and defined enzyme-to-substrate ratios for the purified RNase R. The protocol achieves functional equivalence to premium commercial RNase R, ensuring complete linear RNA digestion without compromising the integrity of circRNA. The combination of a simplified purification workflow and a robust reaction protocol provides an accessible, cost-effective, and reliable solution for any molecular biology laboratory requiring high volumes of RNase R. Key FeaturesO_LIRNase R mutations can block RNase activity, RNA binding or both C_LIO_LIThis protocol purifies [~]40 mg of active RNase R per liter of E. coli culture C_LIO_LIThe protocol avoids medium and high end FPLC systems C_LIO_LIRNase R expression constructs (WT and mutants) will be available on Addgene C_LIO_LIThe protocol includes an optimized reaction buffer to pair with this RNase R C_LIO_LIOptional endotoxin removal step is also included C_LI