RNA-cleaving DNAzymes Research Articles

A DNAzyme-Based Nanohybrid for Ultrasound and Enzyme Dual-Controlled mRNA Regulation and Combined Tumor Treatment.

Despite the significant potential of RNA-cleaving DNAzymes for gene regulation, their application is limited by low therapeutic efficacy and lack of cell-specific control. Here, a DNAzyme-based nanohybrid designed for ultrasound (US)-controlled, enzyme-activatable mRNA regulation is presented, enabling tumor cell-selective combination therapy. The nanohybrid is constructed by coordination-directed self-assembly of an enzymatically-triggerable therapeutic DNAzyme (En-Dz) and natural sonosensitizer hemoglobin (Hb). Controlled US exposure induces reactive oxygen species generation from Hb units, which not only facilitates efficient endosomal escape of En-Dz, but also promotes the translocation of specific enzyme from the nucleus to the cytoplasm, thereby enhancing gene regulation efficacy. Notably, the enzyme-triggered, spatiotemporally-controlled activation of En-Dz's catalytic activity results in enhanced cancer-cell selectivity in the therapeutic treatment. Furthermore, the combination of enzyme-activated mRNA regulation and sonodynamic therapy significantly enhances anti-tumor efficacy both in vitro and in vivo. This work highlights the potential of integrating a sonodynamic strategy to overcome the current limitations of DNAzyme-based gene regulators, providing a spatiotemporally-controlled approach for precise tumor treatment.

Protein-Templated Click Ligation Reaction Triggered by Protein-Split Aptamer Interactions.

DNA-templated reactions have found wide applications in sensing and drug discovery. However, this strategy has been limited to the use of nucleic acids as templating elements to direct the proximity effect. Herein, we describe a versatile protein-templated split aptamer click ligation reaction (PT-SpA-CLR) in which the protein template-induced covalent proximity ligation of split aptamer elements enables translating protein/aptamer binding events into the output of ligated DNA products. A ligation yield of >80% is observed for three model protein templates, including VEGF165, PDGF-BB, and SARS-CoV-2 S1. The ligation reaction compensates for the weakness of reduced binding affinity resulting from splitting the aptamer, as evidenced by an approximately 2-fold lower dissociation constant than the non-ligated system. This newly developed PT-SpA-CLR strategy is further integrated with colorimetric or fluorescent reporting mechanisms to achieve easy-to-use and low-cost biosensors utilizing ligation to produce a fully active G-quadruplex or an RNA-cleaving DNAzyme to report protein binding. Both assays can achieve specific detection of an intended protein target with a limit of detection at the picomolar level even when challenged in biological samples. The combined PT-SpA-CLR and versatile sensing strategies offer attractive universal platforms for efficient detection of protein biomarkers.

Fluorescence assay for aflatoxin B1 based on aptamer-binding triggered DNAzyme activity.

As a kind of mycotoxin, aflatoxin B1 (AFB1), which is often found in agricultural products, poses a threat to human health. Developing asimple sensitive method for AFB1 detection is in great demand. Here, we reported an aptamer-based fluorescence assay for AFB1 detection by using DNAzyme to generate and amplify asignal. We redesigned a pair of DNA sequences, which originated from the anti-AFB1 aptamer and RNA-cleaving DNAzyme 10-23. In the absence of AFB1, the aptamer hybridized with the region of thesubstrate-binding arm of the DNAzyme, inhibiting the activity of theDNAzyme. In the presence of AFB1, the binding of AFB1 to theaptamer led to the displacement of theDNAzyme from theaptamer. The substrate-binding arm was unblocked, and the activity of theDNAzyme was restored for the hydrolysis of thefluorophore and quencher-labeled substrate, causing asignificant fluorescence increase. This assay could detect AFB1 in the dynamic range from 0.98 to 2000nmol/L with high selectivity, and the detection limit was 0.98nmol/L. Moreover, the assay was able to detect AFB1 in acomplex sample matrix. This work provides a useful tool for the analysis of AFB1.

Making target sites in large structured RNAs accessible to RNA-cleaving DNAzymes through hybridization with synthetic DNA oligonucleotides.

The 10-23 DNAzyme is one of the most active DNA-based enzymes, and in theory, can be designed to target any purine-pyrimidine junction within an RNA sequence for cleavage. However, purine-pyrimidine junctions within a large, structured RNA (lsRNA) molecule of biological origin are not always accessible to 10-23, negating its general utility as an RNA-cutting molecular scissor. Herein, we report a generalizable strategy that allows 10-23 to access any purine-pyrimidine junction within an lsRNA. Using three large SARS-CoV-2 mRNA sequences of 566, 584 and 831 nucleotides in length as model systems, we show that the use of antisense DNA oligonucleotides (ASOs) that target the upstream and downstream regions flanking the cleavage site can restore the activity (kobs) of previously poorly active 10-23 DNAzyme systems by up to 2000-fold. We corroborated these findings mechanistically using in-line probing to demonstrate that ASOs reduced 10-23 DNAzyme target site structure within the lsRNA substrates. This approach represents a simple, efficient, cost-effective, and generalizable way to improve the accessibility of 10-23 to a chosen target site within an lsRNA molecule, especially where direct access to the genomic RNA target is necessary.

Exploring the catalytic mechanism of the 10-23 DNAzyme: insights from pH-rate profiles.

The 10-23 DNAzyme, a catalytic DNA molecule with RNA-cleaving activity, has garnered significant interest for its potential therapeutic applications as a gene-silencing agent. However, the lack of a detailed understanding about its mechanism has hampered progress. A recent structural analysis has revealed a highly organized conformation thanks to the stabilization of specific interactions within the catalytic core of the 10-23 DNAzyme, which facilitate the cleavage of RNA. In this configuration, it has been shown that G14 is in good proximity to the cleavage site which suggests its role as a general base, by activating the 2'-OH nucleophile, in the catalysis of the 10-23 DNAzyme. Also, the possibility of a hydrated metal acting as a general acid has been proposed. In this study, through activity assays, we offer evidence of the involvement of general acid-base catalysis in the mechanism of the 10-23 DNAzyme by analyzing its pH-rate profiles and the role of G14, and metal cofactors like Mg2+ and Pb2+. By substituting G14 with its analogue 2-aminopurine and examining the resultant pH-rate profiles, we propose the participation of G14 in a catalytically relevant proton transfer event, acting as a general base. Further analysis, using Pb2+ as a cofactor, suggests the capability of the hydrated metal ion to act as a general acid. These functional results provide critical insights into the catalytic strategies of RNA-cleaving DNAzymes, revealing common mechanisms among nucleic acid enzymes that cleave RNA.

Integration of RCA-Based DNA Nanoscaffold with Target Triggered RNA-Cleaving DNAzyme for Sensitive Detection of miRNA21.

Cascaded amplification showed promising potential for detection of trace target miRNAs in molecular diagnosis and prevention of many diseases. In this study, miRNA21 was chosen as the target, and rolling circle amplification (RCA)-based DNA nanoscaffold was integrated with target triggered RNA-cleaving DNAzyme for sensitive detection of miRNA21. That is, the H1 probe was bound with the long-chain product of RCA to self-assemble into DNA nanoscaffold. Target miRNA21 triggered the hybridization chain reaction (HCR) located on the nanoscaffold, and led to rapid proximity of DNAzyme fragments modified at both ends of the H2 probe, which realized the cyclic cleavage of self-quenching substrate probe efficiently, and the fluorescence signal was restored. The results demonstrated that the proposed assay was sensitive, 0.76pM of miRNA21 can be detected. The proposed assay was specific; only one-base mismatched miRNA21 can be effectively recognized, other nucleic acid sequence and the serum matrix did not cause any interference. The proposed assay was accurate; recoveries from 82.1 to 115.0% can be obtained in the spiked fetal bovine serum (FBS). The flexible and programmable characteristics of DNA nanoscaffold and DNAzyme provide a confident and robust strategy for more sensitive nucleic acid detection, and can be developed to be a universal sensing platform for detecting other miRNAs just needing modification on the corresponding sequence of H1 probe in HCR.

Controllable self-cleaning FET self-assembled RNA-cleaving DNAzyme based DNA nanotree for culture-free Staphylococcus aureus detection

Staphylococcus aureus (SA) poses a serious risk to human and animal health, necessitating a low-cost and high-performance analytical platform for point-of-care diagnostics. Cellulose paper-based field-effect transistors (FETs) with RNA-cleaving DNAzymes (RCDs) can fulfill the low-cost requirements, however, its high hydrophilicity and lipophilicity hinder biochemical modification and result in low sensitivity, poor mechanical stability and poor fouling performance. Herein, we proposed a controllable self-cleaning FET to simplify biochemical modification and improve mechanical stability and antifouling performance. Then, we constructed an RCD-based DNA nanotree to significantly enhance the sensitivity for SA detection. For controllable self-cleaning FET, 1 H,1 H,2 H,2 H-perfluorodecyltrimethoxysilane based-polymeric nanoparticles were synthesized to decorate cellulose paper and whole carbon nanofilm wires. O2 plasma was applied to regulate to reduce fluorocarbon chain density, and then control the hydrophobic-oleophobic property in sensitive areas. Because negatively charged DNA affected the sensitivity of semiconducting FETs, three Y-shaped branches with low-cost were designed and applied to synthesize an RCD-based DNA-Nanotree based on similar DNA-origami technology, which further improved the sensitivity. The trunk of DNA-Nanotree was composed of RCD, and the canopy was self-assembled using multiple Y-shaped branches. The controllable self-cleaning FET biosensor was applied for SA detection without cultivation, which had a wide linear range from 1 to 105 CFU/mL and could detect a low value of 1 CFU/mL.

Nanosized DNA Hydrogel Functionalized with a DNAzyme Tetrahedron for Highly Efficient Gene Silencing.

DNAzymes are DNA oligonucleotides that have catalytic activity without the assistance of protein enzymes. In particular, RNA-cleaving DNAzymes were considered as ideal candidates for gene therapy due to their unique characteristics. Nevertheless, efforts to use DNAzyme as a gene therapeutic agent are limited by issues such as their low physiological stability in serum and intracellular delivery efficiency. In this study, we developed a nanosized synthetic DNA hydrogel functionalized with a DNAzyme tetrahedron (TDz Dgel) to overcome these limitations. We observed remarkable improvement in the gene-silencing effect as well as intracellular uptake without the support of gene transfection reagents using TDz Dgel. The improved catalytic activity of the DNAzyme resulted from the combination of the cell-penetrating DNA tetrahedron structure and high stability of DNA hydrogel. We envision that this approach will become a convenient and efficient strategy for gene-silencing therapy using DNAzyme in the future.

In-vitro Clinical Diagnostics using RNA-Cleaving DNAzymes.

Over the last three decades, significant advancements have been made in the development of biosensors and bioassays that use RNA-cleaving DNAzymes (RCDs) as molecular recognition elements. While early examples of RCDs were primarily responsive to metal ions, the past decade has seen numerous RCDs reported for more clinically relevant targets such as bacteria, cancer cells, small metabolites, and protein biomarkers. Over the past 5 years several RCD-based biosensors have also been evaluated using either spiked biological matrixes or patient samples, including blood, serum, saliva, nasal mucus, sputum, urine, and faeces, which is a critical step toward regulatory approval and commercialization of such sensors. In this review, an overview of the methods used to generate RCDs and the properties of key RCDs that have been utilized for in vitro testing is first provided. Examples of RCD-based assays and sensors that have been used to test either spiked biological samples or patient samples are then presented, highlighting assay performance in different biological matrixes. A summary of current prospects and challenges for development of in vitro diagnostic tests incorporating RCDs and an overview of future directions of the field is also provided.

Colorimetric detection of circulating tumor cells in breast cancer based on ladder-branch hybridization chain reaction and DFs/AuNCs nanozyme

Breast cancer is the most common malignant tumor in women, which accounts for 6.9% of all cancer-related deaths. Early diagnosis is crucial for making the best clinical decision and improving the prognosis of patients. Circulating tumor cells (CTCs) have been regarded as significant tumor biomarkers. Herein, we designed a colorimetric biosensor for breast cancer CTCs quantification based on ladder-branch hybridization chain reaction (HCR) and DNA flowers/gold nanoclusters (DFs/AuNCs) nanozyme. With the assistance of complementary DNA labeled on magnetic beads (MBs), the cleavage products of RNA-cleaving DNAzymes (RCDs) could be rapidly captured, subsequently triggering ladder-branch HCR. In addition, the DFs/AuNCs nanozyme was applied for colorimetric analysis, which further improved the sensitivity for the detection of target CTCs. Benefiting from specific RCDs, ladder-branch HCR and DFs/AuNCs, we achieved a superior detection limit of 3 cells/mL as well as a broad linear range of 10 cells/mL to 104 cells/mL. Conclusively, this colorimetric biosensor achieved sensitively and selectively detection of breast cancer CTCs without the participation of enzymes at room temperature, which might provide new insight into the early detection of breast cancer.

Chemically Inducible DNAzyme Sensor for Controllable Imaging of Metal Ions.

RNA-cleaving DNAzymes have emerged as a promising tool for metal ion detection. Achieving spatiotemporal control over their catalytic activity is essential for understanding the role of metal ions in various biological processes. While photochemical and endogenous stimuli-responsive approaches have shown potential for controlled metal ion imaging using DNAzymes, limitations such as photocytotoxicity, poor tissue penetration, or off-target activation have hindered their application for safe and precise detection of metal ions in vivo. We herein report a chemically inducible DNAzyme in which the catalytic core is modified to contain chemical caging groups at the selected backbone sites through systematic screening. This inducible DNAzyme exhibits minimal leakage of catalytic activity and can be reactivated by small molecule selenocysteines, which effectively remove the caging groups and restore the activity of DNAzyme. Benefiting from these findings, we designed a fluorogenic chemically inducible DNAzyme sensor for controlled imaging of metal ions with tunable activity and high selectivity in live cells and in vivo. This chemically inducible DNAzyme design expands the toolbox for controlling DNAzyme activity and can be easily adapted to detect other metal ions in vivo by changing the DNAzyme module, offering opportunities for precise biomedical diagnosis.

In vitro selection of N 1-methyladenosine-sensitive RNA-cleaving deoxyribozymes with 105-fold selectivity over unmethylated RNA.

RNA-cleaving DNAzymes (RCDs) are catalytically active DNA molecules that cleave a wide range of RNA targets with extremely high sequence-selectivity, but none is able to faithfully discriminate methylated from unmethylated RNA (typically <30-fold). We report the first efforts to isolate RCDs from a random-sequence DNA pool by in vitro selection that cleave RNA/DNA chimera containing N 1-methyladenosine (m1A), one of the most prevalent RNA modifications that plays important regulatory roles in gene expression and human cancers. A cis-acting deoxyribozyme, RCD1-S2m1A, exhibits an observed rate constant (k obs) of 5.3 × 10-2 min-1, resulting in up to 105-fold faster cleavage of the m1A-modified versus unmethylated RNA. Furthermore, a trans-acting fluorogenic deoxyribozyme was constructed by labeling a fluorophore and a quencher at the 5' and 3' ends of the chimeric substrate, respectively. It permits the synchronization of RNA-cleaving with real-time fluorescence signaling, thus allowing the selective monitoring of ALKBH3-mediated demethylation and inhibitor screening in living cells.

Recent progress on DNAzyme-based biosensors for pathogen detection.

Pathogens endanger food safety, agricultural productivity, and human health. Those pathogens are spread through direct/indirect contact, airborne transmission and food/waterborne transmission, and some cause severe health consequences. As the population grows and global connections intensify, the transmission of infectious diseases expands. Traditional detection methods for pathogens still have some shortcomings, such as time-consuming procedures and high operational costs. To fulfil the demands for simple and effective detection, numerous biosensors have been developed. DNAzyme, a unique DNA structure with catalytic activity, is gradually being applied in the field of pathogen detection owing to its ease of preparation and use. In this review, we concentrated on the two main types of DNAzyme, hemin/G-quadruplex DNAzyme (HGD) and RNA-cleaving DNAzyme (RCD), explaining their research progress in pathogen detection. Furthermore, we introduced two additional novel DNAzymes, CLICK 17 DNAzyme and Supernova DNAzyme, which showed promising potential in pathogen detection. Finally, we summarize the strengths and weaknesses of these four DNAzymes and offer feasible recommendations for the development of biosensors.

A high-fidelity DNAzyme-assisted CRISPR/Cas13a system with single-nucleotide resolved specificity.

A CRISPR/Cas system represents an innovative tool for developing a new-generation biosensing and diagnostic strategy. However, the off-target issue (i.e., mistaken cleavage of nucleic acid targets and reporters) remains a great challenge for its practical applications. We hypothesize that this issue can be overcome by taking advantage of the site-specific cleavage ability of RNA-cleaving DNAzymes. To test this idea, we propose a DNAzyme Operation Enhances the Specificity of CRISPR/Cas13a strategy (termed DOES-CRISPR) to overcome the problem of relatively poor specificity that is typical of the traditional CRISPR/Cas13a system. The key to the design is that the partial hybridization of the CRISPR RNA (crRNA) with the cleavage fragment of off-target RNA was not able to activate the collateral cleavage activity of Cas13a. We showed that DOES-CRISPR can significantly improve the specificity of traditional CRISPR/Cas13a-based molecular detection by up to ∼43-fold. The broad utility of the strategy is illustrated through engineering three different systems for the detection of microRNAs (miR-17 and let-7e), CYP2C19*17 gene, SARS-Cov-2 variants (Gamma, Delta, and Omicron) and Omicron subtypes (BQ.1 and XBB.1) with single-nucleotide resolved specificity. Finally, clinical evaluation of this assay using 10 patient blood samples demonstrated a clinical sensitivity of 100% and specificity of 100% for genotyping CYP2C19*17, and analyzing 20 throat swab samples provided a diagnostic sensitivity of 95% and specificity of 100% for Omicron detection, and a clinical sensitivity of 92% and specificity of 100% for XBB.1 detection.

Amplification-free detection of Escherichia coli using an acidic deoxyribozyme-based paper device.

We reported a colorimetric paper-based device by integrating the modified acid RNA-cleaving DNAzymes (MaRCD-EC1) for highly sensitive (detection limit = 102 CFU mL-1), and rapid (within 30 min) detection of E. coli without amplification. This device exhibited a clinical sensitivity of 100% and a specificity of 100% in identifying E. coli-associated urinary tract infections (UTIs) using the clinical urine samples.

An RNA-Cleaving DNAzyme That Requires an Organic Solvent to Function.

Engineering functional nucleic acids active under unusual conditions will not only reveal the hidden talents of nucleic acids but also lay groundwork for pursuing them for unique applications. Although many DNAzymes have been derived to catalyze diverse chemical reactions in aqueous solutions, no prior study has been set up to purposely derive DNAzymes that require an organic solvent to function. Herein, we utilized in vitro selection to isolate RNA-cleaving DNAzymes from a random-sequence DNA pool that were "compelled" to accept 35% dimethyl sulfoxide (DMSO) as a cosolvent, via counter selection in a purely aqueous solution followed by positive selection in the same solution containing 35% DMSO. This experiment indeed led to the discovery of a new DNAzyme that requires 35% DMSO for its catalytic activity but exhibits drastically reduced activity without DMSO. This DNAzyme also requires divalent metal ions for catalysis and uses monovalent ions for activity enhancement. A minimized, more efficient DNAzyme was also derived. This work demonstrates that highly functional organic solvent-dependent DNAzymes can be isolated from random-sequence DNA libraries via forced in vitro selection, thus expanding the capability and potential utility of DNA as enzymes.

In Vitro Selection of M2+-Independent, Fast-Responding Acidic Deoxyribozymes for Bacterial Detection.

We report on the first efforts to isolate acidic RNA-cleaving DNAzymes (aRCDs) from a random-sequence DNA pool by in vitro selection that are activated by a microbe Escherichia coli (E. coli), at pH 5.3. Importantly, these E. coli-responsive aRCDs only require monovalent metal ions as cofactors for cleaving a fluorogenic chimeric DNA/RNA substrate. Such characteristics can be used to efficiently protect RCDs from both intrinsic chemical instability and external enzymatic degradation. One remarkable DNAzyme, aRCD-EC1, is specific for E. coli, and its target is likely a protein. Furthermore, truncated aRCD-EC1 had significantly improved catalytic activity with an observed rate constant (kobs) of 1.18 min-1, making it the fastest bacteria-responding RCD reported to date. Clinical evaluation of this aRCD-based fluorescent assay using 40 patient urine samples demonstrated a diagnostic sensitivity of 100% and a specificity of 100% at a total analysis time of 50 min without a bacterial culture. This work can expand the repertoire of DNAzymes that are active under nonphysiological conditions, thus facilitating the development of diverse DNAzyme-based biosensors in clinical diagnosis.

Unraveling the Kinetics of the 10-23 RNA-Cleaving DNAzyme.

DNA-based enzymes, or DNAzymes, are single-stranded DNA sequences with the ability to catalyze various chemical reactions, including the cleavage of the bond between two RNA nucleotides. Lately, an increasing interest has been observed in these RNA-cleaving DNAzymes in the biosensing and therapeutic fields for signal generation and the modulation of gene expression, respectively. Additionally, multiple efforts have been made to study the effects of the reaction environment and the sequence of the catalytic core on the conversion of the substrate into product. However, most of these studies have only reported alterations of the general reaction course, but only a few have focused on how each individual reaction step is affected. In this work, we present for the first time a mathematical model that describes and predicts the reaction of the 10-23 RNA-cleaving DNAzyme. Furthermore, the model has been employed to study the effect of temperature, magnesium cations and shorter substrate-binding arms of the DNAzyme on the different kinetic rate constants, broadening the range of conditions in which the model can be exploited. In conclusion, this work depicts the prospects of such mathematical models to study and anticipate the course of a reaction given a particular environment.

Integration of Photoelectrochemical Signal Transduction with RNA-Cleaving Dnazymes for Culture-Free Detection of Bacteria

There is a growing interest in developing ultrasensitive biological sensors that can be used for rapid testing at the point-of-need.1 A major hurdle in developing such sensors for detecting pathogens such as bacteria is that they require target enrichment or amplification to deliver the required limit-of-detection.2 Among signal transduction strategies, photoelectrochemical (PEC) signal readout, built on the use of light for enhancing electrochemical reactions, is emerging as an ultrasensitive signal transduction mechanism.3 However, the existing PEC platforms fail to deliver single step testing due to the existence of multiple manual steps, including the addition of biological materials labelled with inorganic photoactive nanoparticles, for signal transduction.3 RNA-cleaving DNAzymes (RCDs), a class of synthetic nucleic acids, have been selected for precisely identifying specific bacterial species without the need for sample processing.4 RCDs are molecular switches that cleave a segment of themselves in response to a particular bacterial target, combining biological recognition with signal transduction.4 We developed photoactive RCDs by tagging them with TiO2 nanomaterials for combining these molecular switches with PEC signal readout. We designed these molecular switches to make and then break semiconductive heterostructures in response to bacterial targets. These photoactive RCDs were the foundational basis for the design novel and highly sensitive PEC bacterial sensor.We developed two photoactive materials for use in the PEC bacterial assay: TiO2 nanorod clusters (rutile) that form high surface area photoelectrodes and sub-nanometer sized TiO2-nanoparticles (anatase) that link to RCDs to create photoactive reporter probes. Combining TiO2-assemblies and TiO2-nanoparticles gives rise to a semiconductor heterostructure that massively improves the photoexcitation efficiency of the combined material system and improves photocurrent generation. Our PEC bacterial sensor makes use of this phenomenon for bacterial detection by utilizing photoactive RCDs to modulate photocurrent by breaking and then rebuilding the TiO2 heterostructures, as a signaling mechanism. The assay consists of a release electrode – modified with photoactive RCDs and a capture electrode – modified with single-stranded DNA probes. Upon target interaction, RCDs release photoactive reporters which are captured by the probes, decreasing the release electrode signal while raising the capture electrode signal.The resulting biosensor can detect E. coli bacterial contamination with high specificity and has achieved a very low limit of detection of 21 CFU/mL in buffer and 18 CFU/mL in lake water samples. These results have set a new record for amplification-free detection of bacteria, that does not rely on target enrichment, reagent addition, or sample processing. This presents a new tool for rapid and in-field water testing.References Nat. Microbiol., 1, 16089 (2016).L. Castillo-Henríquez et al., Sensors, 20, 6926 (2020).A. Victorious, S. Saha, R. Pandey, T. F. Didar, and L. Soleymani, Front. Chem., 7, 617 (2019).I. Cozma, E. M. McConnell, J. D. Brennan, and Y. Li, Biosens. Bioelectron., 177, 112972 (2021).

A Rapid Sputum‐based Lateral Flow Assay for Airway Eosinophilia using an RNA‐cleaving DNAzyme Selected for Eosinophil Peroxidase

AbstractThe first protein‐binding allosteric RNA‐cleaving DNAzyme (RCD) obtained by direct in vitro selection against eosinophil peroxidase (EPX), a validated marker for airway eosinophilia, is described. The RCD has nanomolar affinity for EPX, shows high selectivity against related peroxidases and other eosinophil proteins, and is resistant to degradation by mammalian nucleases. An optimized RCD was used to develop both fluorescence and lateral flow assays, which were evaluated using 38 minimally processed patient sputum samples (23 non‐eosinophilic, 15 eosinophilic), producing a clinical sensitivity of 100 % and specificity of 96 %. This RCD‐based lateral flow assay should allow for rapid evaluation of airway eosinophilia as an aid for guiding asthma therapy.