Rapid Diagnostic Tests (RDTs) for malaria are restricted to a few biomarkers and antibody-mediated detection. However, the expression of commonly used biomarkers varies geographically and the sensibility of immunodetection can be affected by batch-to-batch differences or limited thermal stability. In this study we aimed to overcome these limitations by identifying a potential biomarker and by developing molecular sensors based on aptamer technology. Using gene expression databases, ribosome profiling analysis, and structural modeling, we find that the High Mobility Group Box 1 protein (HMGB1) of Plasmodium falciparum is highly expressed, structurally stable, and present along all blood-stages of P. falciparum infection. To develop biosensors, we used in vitro evolution techniques to produce DNA aptamers for the recombinantly expressed HMG-box, the conserved domain of HMGB1. An evolutionary approach for evaluating the dynamics of aptamer populations suggested three predominant aptamer motifs. Representatives of the aptamer families were tested for binding parameters to the HMG-box domain using microscale thermophoresis and rapid kinetics. Dissociation constants of the aptamers varied over two orders of magnitude between nano- and micromolar ranges while the aptamer-HMG-box interaction occurred in a few seconds. The specificity of aptamer binding to the HMG-box of P. falciparum compared to its human homolog depended on pH conditions. Altogether, our study proposes HMGB1 as a candidate biomarker and a set of sensing aptamers that can be further developed into rapid diagnostic tests for P. falciparum detection.

Ribosome assembly is a multistep process that ensures a functional ribosome structure. The molecular mechanism that ribosome­associated GTPases (RA­GTPases) use to enhance ribosome assembly accuracy remains largely to be elucidated. Here, we use systematic evolution of ligands by exponential enrichment (SELEX), followed by sequencing, comprehensive bioinformatics analysis, and biochemical characterization to identify aptamers that target the RA-GTPase ERA of Staphylococcus aureus. ELONA and thermophoresis assays show that the Apt_ERA 2 interaction with ERA is in the 200 nM range of affinity, displays a high level of specificity, and depends on the target structure. Docking to ERA suggests that Apt_ERA 2 interacts with the protein’s KH domain, consistent with the aptamer’s similarities with helix 45 of the 16S rRNA. Apt_ERA 2 did interact with the isolated KH domain but did not bind to the ∆KH ERA nor to the similar RA-GTPase RbgA, which shares the GTPase core but lacks the KH domain, confirming that the aptamer recognizes and binds the KH domain of ERA. This interaction leads to a significant reduction of 30S-dependent GTP hydrolysis, indicative of allosteric modulation of the enzyme activity or limiting ERA binding or the KH domain interaction with the 3’ end of the 16S rRNA rather than directly blocking GTP binding. Altogether, this work highlights the versatility of aptamers as tools to understand the complex processes of ribosome biogenesis further, offering new insights into bacterial protein synthesis mechanisms.

Bacterial translation initiation factor IF3 is composed of two distinct domains—a well-characterized C-terminal domain (IF3C), which enhances the speed and fidelity of translation initiation, and a less understood N-terminal domain (IF3N). In this study, we developed an aptamer (Apt ³⁴³ ) that targets IF3N with the goal of elucidating its contribution to translation initiation. Rapid kinetics assays revealed that Apt ³⁴³ reduces the rate of IF3 association with the 30S ribosomal subunit by 13-fold, while inducing a pronounced rearrangement of both IF3 domains on the 30S. These changes compromise IF2-, mRNA-, and fMet-tRNA ^fMet -dependent movements of IF3, delaying 30S initiation complex (30S IC) formation by up to two orders of magnitude. Cryo-EM analysis suggests that Apt ³⁴³ may sterically clash with fMet-tRNA ^fMet , thereby perturbing the canonical pathway by which the initiator tRNA is accommodated after IF2-dependent recruitment and prior to start codon decoding. However, once the 30S IC is formed, blocking IF3N does not prevent 50S subunit joining or 70S IC assembly. Collectively, these findings support a role for IF3N in enhancing an efficient path for fMet-tRNA ^fMet accommodation towards the 30S IC and promoting IF3C displacement to unlock 50S recruitment. Moreover, this aptamer-based strategy offers a valuable tool for dissecting domain-specific activities of multidomain factors within complex environments such as the initiating ribosome. GRAPHICAL

Low- and middle-income countries (LMICs) are significantly affected by SARS-CoV-2, partially due to their limited capacity for local development of molecular testing and accentuated by the international supply shortage. Here, we describe a molecular toolkit that can be readily produced and deployed in LMICs using minimal laboratory equipment. Our results show that mid-scale production of enzymes and nucleic acids can supply thousand tests per production batch. One-step RT-PCR was optimized for two SARS-CoV-2 loci and coupled to CRISPR/Cas12a detection. The clinical validation indicated a sensitivity near 100% for mid and high viral load samples (Cq ≤ 33). The specificity was around 100% regardless of viral load. The toolkit was used with the mobile laboratory BentoLab, potentially unlocking LMICs to implement detection services in unattended regions. Altogether, we provide detailed methods and performance evidence of molecular tools aiming to aid LMICs to deploy molecular testing for current or future pathogenic outbreaks. One Sentence Summary We describe a molecular toolkit for the detection of SARS-CoV-2 that is compatible with available facilities in low- and middle-income countries (LMICs).

In prokaryotes, translation initiation orchestrates protein synthesis through a network of dynamic interactions among the ribosome, mRNA, initiator tRNA^fMet, and initiation factors (IFs). Traditional approaches that rely on radioactive labeling or surface immobilization are hindered by inherent safety risks and methodological constraints. We present a fluorescence-based analytical platform that integrates microscale thermophoresis (MST) as a unified, multiparametric toolkit for comprehensive interrogation of bacterial translation initiation at the molecular level. By systematically applying MST to a panel of fluorescently labeled components—initiator tRNA^fMet, mRNAs, and initiation factors—we quantify assembly pathways and equilibria as initiation progresses from simple bimolecular interactions to higher-order, multicomponent complexes. To broaden the fluorescence toolbox for ribosomal studies, we developed a robust BODIPY-labeling protocol for 70S ribosomes and confirmed preservation of structural integrity and function by nano differential scanning fluorimetry, stopped-flow kinetic assays, and peptide-synthesis activity tests. Our microscale fluorescent system facilitates probing initiation at a variety of steps, since the role of magnesium ions and initiation factors upon 30S initiation complex formation. The same platform can be applied to investigate the effects of different compounds on translation initiation, as demonstrated for a number of antibiotics, aptamers, and antimicrobial peptides. Using this approach, we determined the antibiotic streptomycin dissociation constant for both 30S and 70S ribosomes, which proved identical at 0.3 ± 0.1 μM, and demonstrated the effect of the antimicrobial peptide rumicidin-1 on translation initiation. Offering a cost-effective and high-sensitivity alternative to conventional methods, this approach advances mechanistic understanding of prokaryotic translation and provides a versatile framework for the discovery of novel protein synthesis inhibitors.