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.

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

The 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.

Detection of Large Genomic RNA via DNAzyme-Mediated RNA Cleavage and Rolling Circle Amplification: SARS-CoV-2 as a Model.

A new method for the detection of genomic RNA combines RNA cleavage by the 10-23 DNAzyme and use of the cleavage fragments as primers to initiate rolling circle amplification (RCA). 230 different 10-23 DNAzyme variants were screened to identify those that target accessible RNA sites within the highly structured RNA transcripts of SARS-CoV-2. A total of 28 DNAzymes were identified with >20 % cleavage, 5 with >40 % cleavage and one with >60 % in 10 min. The cleavage fragments from these reactions were then screened for coupling to an RCA reaction, leading to the identification of several cleavage fragments that could efficiently initiate RCA. Using a newly developed quasi-exponential RCA method with a detection limit of 500 aM of RNA, 14 RT-PCR positive and 15 RT-PCR negative patient saliva samples were evaluated for SARS-CoV-2 genomic RNA, achieving a clinical sensitivity of 86 % and specificity of 100 % for detection of the virus in <2.5 h.

Three on Three: Universal and High-Affinity Molecular Recognition of the Symmetric Homotrimeric Spike Protein of SARS-CoV-2 with a Symmetric Homotrimeric Aptamer.

Our previously discovered monomeric aptamer for SARS-CoV-2 (MSA52) possesses a universal affinity for COVID-19 spike protein variants but is ultimately limited by its ability to bind only one subunit of the spike protein. The symmetrical shape of the homotrimeric SARS-CoV-2 spike protein presents the opportunity to create a matching homotrimeric molecular recognition element that is perfectly complementary to its structural scaffold, causing enhanced binding affinity. Here, we describe a branched homotrimeric aptamer with three-fold rotational symmetry, named TMSA52, that not only possesses excellent binding affinity but is also capable of binding several SARS-CoV-2 spike protein variants with picomolar affinity, as well as pseudotyped lentiviruses expressing SARS-CoV-2 spike protein variants with femtomolar affinity. Using Pd-Ir nanocubes as nanozymes in an enzyme-linked aptamer binding assay (ELABA), TMSA52 was capable of sensitively detecting diverse pseudotyped lentiviruses in pooled human saliva with a limit of detection as low as 6.3 × 103 copies/mL. The ELABA was also used to test 50 SARS-CoV-2-positive and 60 SARS-CoV-2-negative patient saliva samples, providing sensitivity and specificity values of 84.0 and 98.3%, respectively, thus highlighting the potential of TMSA52 for the development of future rapid tests.

A sputum bioassay for airway eosinophilia using an eosinophil peroxidase aptamer.

Eosinophils are granulocytes that play a significant role in the pathogenesis of asthma and other airway diseases. Directing patient treatment based on the level of eosinophilia has been shown to be extremely effective in reducing exacerbations and therefore has tremendous potential as a routine clinical test. Herein, we describe the in vitro selection and optimization of DNA aptamers that bind to eosinophil peroxidase (EPX), a protein biomarker unique to eosinophils. Fifteen rounds of magnetic bead aptamer selection were performed prior to high throughput DNA sequencing. The top 10 aptamer candidates were assessed for EPX binding using a mobility shift assay. This process identified a lead aptamer candidate termed EAP1-05 with low nanomolar affinity and high specificity for EPX over other common sputum proteins. This aptamer sequence was further optimized through truncation and used to develop an easy-to-use colourimetric pull-down assay that can detect EPX over a concentration range from 1 - 100 nM in processed sputum. Forty-six clinical samples were processed using a new sputum dispersal method, appropriate for a rapid assessment assay, that avoids centrifugation and lengthy processing times. The assay showed 89% sensitivity and 96% specificity to detect eosinophilia (compared to gold standard sputum cytometry), with results being produced in under an hour. This assay could allow for an easy assessment of eosinophil activity in the airway to guide anti-inflammatory therapy for several airway diseases.

Investigation of discordant SARS-CoV-2 RT-PCR results using minimally processed saliva.

Saliva is an attractive sample for coronavirus disease 2019 testing due its ease of collection and amenability to detect viral RNA with minimal processing. Using a direct-to-RT-PCR method with saliva self-collected from confirmed COVID-19 positive volunteers, we observed 32% false negative results. Confirmed negative and healthy volunteer samples spiked with 106 genome copies/mL of heat-inactivated severe acute respiratory syndrome coronavirus 2 showed false negative results of 10% and 13%, respectively. Additional sample heating or dilution of the false negative samples conferred only modest improvements. These results highlight the potential to significantly underdiagnose COVID-19 infections when testing directly from minimally processed heterogeneous saliva samples.

A Lateral Flow Test for Staphylococcus aureus in Nasal Mucus Using a New DNAzyme as the Recognition Element.

Detection of pathogenic bacteria in complex biological matrices remains a major challenge. Herein, we report the selection and optimization of a new DNAzyme for Staphylococcus aureus (SA) and the use of the DNAzyme to develop a simple lateral flow device (LFD) for detection of SA in nasal mucus. The DNAzyme was generated by in vitro selection using a crude extra/intracellular mixture derived from SA, which could be used directly for simple solution or paper-based fluorescence assays for SA. The DNAzyme was further modified to produce a DNA cleavage fragment that acted as a bridging element to bind DNA-modified gold nanoparticles to the test line of a LFD, producing a simple colorimetric dipstick test. The LFD was evaluated with nasal mucus samples spiked with SA, and demonstrated that SA detection was possible in minutes with minimal sample processing.

Quantitative Point-of-Care Colorimetric Assay Modeling Using a Handheld Colorimeter.

Colorimetric assays typically offer a rapid and convenient method to assess analytes that span healthcare monitoring to water quality testing. However, such tests can only provide qualitative results when employed in resource-limited settings or require bulky and expensive equipment such as lab spectrophotometers to allow quantitative measurements. In this paper, we report on the use of a handheld colorimeter to quantitatively determine the concentration of analytes in a manner that is independent of ambient lighting or initial sample color. The method combines the response of the sensor with first-principles modeling that better describes the nature of the assay compared to linear-in-parameters regression modeling that is typically performed in other studies. This method was successfully demonstrated using a number of colorimetric assays: (1) determination of solution pH using a universal indicator, (2) quantification of the DNase presence using a DNA-gold nanoparticle assay, and (3) quantification of the concentration of the antibiotic tetracycline using a cell-based assay.

High-Affinity Dimeric Aptamers Enable the Rapid Electrochemical Detection of Wild-Type and B.1.1.7 SARS-CoV-2 in Unprocessed Saliva.

We report a simple and rapid saliva-based SARS-CoV-2 antigen test that utilizes a newly developed dimeric DNA aptamer, denoted as DSA1N5, that specifically recognizes the spike proteins of the wildtype virus and its Alpha and Delta variants with dissociation constants of 120, 290 and 480 pM, respectively, and binds pseudotyped lentiviruses expressing the wildtype and alpha trimeric spike proteins with affinity constants of 2.1 pM and 2.3 pM, respectively. To develop a highly sensitive test, DSA1N5 was immobilized onto gold electrodes to produce an electrochemical impedance sensor, which was capable of detecting 1000 viral particles per mL in 1:1 diluted saliva in under 10 min without any further sample processing. Evaluation of 36 positive and 37 negative patient saliva samples produced a clinical sensitivity of 80.5 % and specificity of 100 % and the sensor could detect the wildtype virus as well as the Alpha and Delta variants in the patient samples, which is the first reported rapid test that can detect any emerging variant of SARS-CoV-2.

Diverse high-affinity DNA aptamers for wild-type and B.1.1.7 SARS-CoV-2 spike proteins from a pre-structured DNA library.

We performed in vitro selection experiments to identify DNA aptamers for the S1 subunit of the SARS-CoV-2 spike protein (S1 protein). Using a pool of pre-structured random DNA sequences, we obtained over 100 candidate aptamers after 13 cycles of enrichment under progressively more stringent selection pressure. The top 10 sequences all exhibited strong binding to the S1 protein. Two aptamers, named MSA1 (Kd = 1.8 nM) and MSA5 (Kd = 2.7 nM), were assessed for binding to the heat-treated S1 protein, untreated S1 protein spiked into 50% human saliva and the trimeric spike protein of both the wildtype and the B.1.1.7 variant, demonstrating comparable affinities in all cases. MSA1 and MSA5 also recognized the pseudotyped lentivirus of SARS-CoV-2 with respective Kd values of 22.7 pM and 11.8 pM. Secondary structure prediction and sequence truncation experiments revealed that both MSA1 and MSA5 adopted a hairpin structure, which was the motif pre-designed into the original library. A colorimetric sandwich assay was developed using MSA1 as both the recognition element and detection element, which was capable of detecting the pseudotyped lentivirus in 50% saliva with a limit of detection of 400 fM, confirming the potential of these aptamers as diagnostic tools for COVID-19 detection.

Metabolomic-guided discovery of cyclic nonribosomal peptides from Xylaria ellisii sp. nov., a leaf and stem endophyte of Vaccinium angustifolium.

Fungal endophytes are sources of novel bioactive compounds but relatively few agriculturally important fruiting plants harboring endophytes have been carefully studied. Previously, we identified a griseofulvin-producing Xylaria species isolated from Vaccinium angustifolium, V. corymbosum, and Pinus strobus. Morphological and genomic analysis determined that it was a new species, described here as Xylaria ellisii. Untargeted high-resolution LC-MS metabolomic analysis of the extracted filtrates and mycelium from 15 blueberry isolates of this endophyte revealed differences in their metabolite profiles. Toxicity screening of the extracts showed that bioactivity was not linked to production of griseofulvin, indicating this species was making additional bioactive compounds. Multivariate statistical analysis of LC-MS data was used to identify key outlier features in the spectra. This allowed potentially new compounds to be targeted for isolation and characterization. This approach resulted in the discovery of eight new proline-containing cyclic nonribosomal peptides, which we have given the trivial names ellisiiamides A-H. Three of these peptides were purified and their structures elucidated by one and two-dimensional nuclear magnetic resonance spectroscopy (1D and 2D NMR) and high-resolution tandem mass spectrometry (HRMS/MS) analysis. The remaining five new compounds were identified and annotated by high-resolution mass spectrometry. Ellisiiamide A demonstrated Gram-negative activity against Escherichia coli BW25113, which is the first reported for this scaffold. Additionally, several known natural products including griseofulvin, dechlorogriseofulvin, epoxy/cytochalasin D, zygosporin E, hirsutatin A, cyclic pentapeptides #1-2 and xylariotide A were also characterized from this species.

A paper-based biosensor for visual detection of glucose-6-phosphate dehydrogenase from whole blood.

Screening for a deficiency of glucose-6-phosphate dehydrogenase (G6PD) in red blood cells is vital for determining the potentially life-threatening presence of congenital, hereditary or induced hemolytic anemias. In this study, a "sample-to-readout" paper-based point-of-care (POC) colorimetric biosensor was developed for direct detection of G6PD in whole blood by simple visual comparison to a color card. The G6PD paper sensor was highly stable with no observable loss in performance after room temperature storage for at least 6 weeks, and worked equally well at room temperature and 37 °C. The simple printed paper format and the stability of the colorimetric reagents facilitates scalable manufacturing. The ability to utilize well established sample collection and preparation protocols along with a colorimetric visual readout should facilitate future transfer of this proof-of-concept POC biosensor to remote or resource-poor locations.

Epoxynemanione A, nemanifuranones A-F, and nemanilactones A-C, from Nemania serpens, an endophytic fungus isolated from Riesling grapevines.

Ten polyketide specialized metabolites, epoxynemanione A, nemanifuranones A-F, and nemanilactones A-C, were isolated from the culture filtrate of Nemania serpens (Pers.) Grey (1821), an endophytic fungus from a Riesling grapevine (Vitis vinifera) found in Canada's Niagara region. Additionally, four known metabolites 2-(hydroxymethyl)-3-methoxy-benzoic acid, phyllostine, 5-methylmellein and a nordammarane triterpenoid were isolated. A related known metabolite 2,3-dihydro-2-hydroxy-2,4-dimethyl-5-trans-propenylfuran-3-one has also been included for structural and biological comparison to the nemanifuranones. The latter was isolated from the culture filtrates of Mollisia nigrescens, an endophytic fungus from the leaves and stems of lowbush blueberry (Vaccinium angustifolium) found in the Acadian forest of Nova Scotia, Canada. Their structures were elucidated based on 1D and 2D NMR, HRESIMS measurements, X-ray crystallographic analysis of nemanifuranone A, the nordammarane triterpenoid and 2,3-dihydro-2-hydroxy-2,4-dimethyl-5-trans-propenylfuran-3-one compounds, and comparison of NOE and vicinal 1H-1H coupling constants to literature data for relative stereochemical assignments. Nemanifuranone A possesses a rare C2 hemiacetal and was active against both Gram-negative and Gram-positive bacteria.

Carbon Monoxide-Releasing Molecule-401 Suppresses Polymorphonuclear Leukocyte Migratory Potential by Modulating F-Actin Dynamics.

Carbon monoxide-releasing molecules (CORMs) suppress inflammation by reducing polymorphonuclear leukocyte (PMN) recruitment to the affected organs. We investigated modulation of PMN-endothelial cell adhesive interactions by water-soluble CORM-401 using an experimental model of endotoxemia in vitro. Human umbilical vein endothelial cells (HUVEC) grown on laminar-flow perfusion channels were stimulated with 1 µg/mL lipopolysaccharide for 6 hours and perfused with 100 µmol/L CORM-401 (or inactive compound iCORM-401)-pretreated PMN for 5 minutes in the presence of 1.0 dyn/cm2 shear stress. HUVEC: PMN co-cultures were perfused for additional 15 minutes with PMN-free medium containing CORM-401/inactive CORM-401. The experiments were videorecorded (phase-contrast microscopy), and PMN adhesion/migration were assessed off-line. In parallel, CORM-401-dependent modulation of PMN chemotaxis, F-actin expression/distribution, and actin-regulating pathways [eg, p21-activated protein kinases (PAK1/2) and extracellular signal-regulated kinase (ERK)/C-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPK)] were assessed in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation. Pretreating PMN with CORM-401 did not suppress PMN adhesion to HUVEC, but significantly reduced PMN transendothelial migration (P < 0.0001) and fMLP-induced PMN chemotaxis (ie, migration directionality and velocity). These changes were associated with CORM-401-dependent suppression of F-actin levels/cellular distribution and fMLP-induced phosphorylation of PAK1/2 and ERK/JNK MAPK (P < 0.05). CORM-401 had no effect on p38 MAPK activation. In summary, this study demonstrates, for the first time, CORM-401-dependent suppression of neutrophil migratory potential associated with modulation of PAK1/2 and ERK/JNK MAPK signaling and F-actin dynamics.

Preparation of an 18 F-Labeled Hydrocyanine Dye as a Multimodal Probe for Reactive Oxygen Species.

Hydrocyanine dyes are sensitive "turn-on" type optical probes that can detect reactive oxygen species (ROS). We have developed a method to prepare an 18 F-labeled hydrocyanine dye as a multi-modal PET and optical "turn-on" probe. A commercially available near infrared (NIR) dye was modified with a fluorinated prosthetic group that did not alter its ROS sensing properties in the presence of superoxide and hydroxyl radicals. The 18 F-labeled analogue was produced using a single-step terminal fluorination procedure. Positron emission tomography (PET) imaging and quantitative in vivo biodistribution studies indicated this novel probe had remarkably different pharmacokinetics compared to the oxidized cyanine analogue. The chemistry reported enables the use of quantitative and dynamic PET imaging for the in vivo study of hydrocyanine dyes as ROS probes.

Total Synthesis of Aspergillomarasmine†A and Related Compounds: A Sulfamidate Approach Enables Exploration of Structure-Activity Relationships.

The fungal secondary metabolite aspergillomarasmine A (AMA) has recently been identified as an inhibitor of metallo-ß-lactamases NDM-1 and VIM-2. Described herein is an efficient and practical route to AMA and its related compounds by a sulfamidate approach. In addition, a series of derivatives has been prepared and tested for biological activity in an effort to explore preliminary structure activity relationships. While it was determined that natural LLL isomer of AMA remains the most effective inactivator of NDM-1 enzyme activity both in vitro and in cells, the structure is highly tolerant of the changes in the stereochemistry at positions 3, 6, and 9.

Total Synthesis and Activity of the Metallo-ß-lactamase Inhibitor Aspergillomarasmine†A.

Resistance to ß-lactam antibiotics is mediated primarily by enzymes that hydrolytically inactivate the drugs by one of two mechanisms: serine nucleophilic attack or metal-dependent activation of a water molecule. Serine ß-lactamases are countered in the clinic by several codrugs that inhibit these enzymes, thereby rescuing antibiotic action. There are no equivalent inhibitors of metallo-ß-lactamases in clinical use, but the fungal secondary metabolite aspergillomarasmine A has recently been identified as a potential candidate for such a codrug. Herein we report the synthesis of aspergillomarasmine A. The synthesis enabled confirmation of the stereochemical configuration of the compound and offers a route for the synthesis of derivatives in the future.

(125)I-Tetrazines and Inverse-Electron-Demand Diels-Alder Chemistry: A Convenient Radioiodination Strategy for Biomolecule Labeling, Screening, and Biodistribution Studies.

A convenient method to prepare radioiodinated tetrazines was developed, such that a bioorthogonal inverse electron demand Diels-Alder reaction can be used to label biomolecules with iodine-125 for in vitro screening and in vivo biodistribution studies. The tetrazine was prepared by employing a high-yielding oxidative halo destannylation reaction that concomitantly oxidized the dihydrotetrazine precursor. The product reacts quickly and efficiently with trans-cyclooctene derivatives. Utility was demonstrated through antibody and hormone labeling experiments and by evaluating products using standard analytical methods, in vitro assays, and quantitative biodistribution studies where the latter was performed in direct comparison to Bolton-Hunter and direct iodination methods. The approach described provides a convenient and advantageous alternative to conventional protein iodination methods that can expedite preclinical development and evaluation of biotherapeutics.

Fluorous Analogue of Chloramine-T: Preparation, X-ray Structure Determination, and Use as an Oxidant for Radioiodination and s-Tetrazine Synthesis.

A fluorous oxidant that can be used to introduce radioiodine into small molecules and proteins and generate iodinated tetrazines for bioorthogonal chemistry has been developed. The oxidant was prepared in 87% overall yield by combining a fluorous amine with tosyl chloride, followed by chlorination using aqueous sodium hypochlorite. A crystal structure of the oxidant, which is a fluorous analogue of chloramine-T, was obtained. The compound was shown to be stable for 7 days in EtOH and for longer than three months as a solid. The oxidant was effective at promoting the labeling of arylstannanes using [(125)I]NaI, where products were isolated in high specific activity in yields ranging from 46% to 86%. Similarly, iodinated biologically active proteins (e.g., thrombin) were successfully produced, as well as a radioiodinated tetrazine, through a concomitant oxidation-halodemetalation reaction. Because of its fluorous nature, unreacted oxidant and associated reaction byproducts can be removed quantitatively from reaction mixtures by passing solutions through fluorous solid phase extraction cartridges. This feature enables rapid and facile purification, which is critical when working with radionuclides and is similarly beneficial for general synthetic applications.

Carbon monoxide-releasing molecule 3 inhibits myeloperoxidase (MPO) and protects against MPO-induced vascular endothelial cell activation/dysfunction.

Polymorphonuclear leukocyte (PMN)-derived myeloperoxidase (MPO) contributes to the pathophysiology of numerous systemic inflammatory disorders through: (1) direct peroxidation of targets and (2) production of strong oxidizing compounds, e.g., hypohalous acids, particularly hypochlorous acid, which furthers oxidant damage and contributes to the propagation of inflammation and tissue injury/dysfunction. Carbon monoxide-releasing molecules (CORMs) offer potent anti-inflammatory effects; however, the mechanism(s) of action is not fully understood. This study assessed the potential of MPO activity inhibition by a water-soluble CORM, CORM-3. To this end, we used in vitro assays to study CORM-3-dependent modulation of MPO activity with respect to: (1) the inhibition of MPO's catalytic activity generally and (2) the specific inhibition of MPO's peroxidation and halogenation (i.e., production of hypochlorous acid) reactions. Further, we employed primary human umbilical vein endothelial cells (HUVECs) to investigate MPO-dependent cellular activation and dysfunction by measuring intracellular oxidant stress (DHR-123 oxidation) and HUVEC permeability (flux of Texas red-dextran), respectively. The results indicate that CORM-3 significantly inhibits MPO activity as well as MPO's peroxidation and hypohalous acid cycles specifically (p<0.05 vs uninhibited MPO). In addition, CORM-3 significantly decreases PMN homogenate- or rhMPO-induced intracellular DHR-123 oxidation in HUVECs and rhMPO-induced HUVEC monolayer permeability (p<0.05 vs untreated). In all assays the inactivated CORM-3 was significantly less effective than CORM-3 (p<0.05). Taken together our findings indicate that CORM-3 is a novel MPO inhibitor and mitigates inflammatory damage at least in part through a mechanism involving the inhibition of neutrophilic MPO activity.