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.

Enhancing the management of long COVID in general practice: a scoping review.

BACKGROUND: Long COVID is a multifaceted condition, and it has impacted a considerable proportion of those with acute COVID-19. Affected patients often have complex care needs requiring holistic and multidisciplinary care, the kind routinely provided in general practice. However, there is limited evidence regarding GP interventions. AIM: This study aimed to identify key concepts and knowledge gaps around long COVID by conducting a scoping review of literature on the condition's management by GPs. DESIGN & SETTING: Arksey and O'Malley's six-stage scoping review framework, with recommendations by Levac et al, was used. METHOD: PubMed, Google Scholar, the Cochrane Library, Scopus, and Google searches were conducted to identify relevant peer reviewed and grey literature, and study selection process was conducted according to the PRISMA Extension for Scoping Reviews guidelines. Braun and Clarke's 'Thematic Analysis' approach was used to interpret data. RESULTS: Nineteen of 972 identified articles were selected for review. These included peer reviewed articles and grey literature spanning a wide range of countries. Six themes were identified regarding GP management of long COVID, these being: (1) GP uncertainty, (2) listening and empathy, (3) assessment and monitoring of symptoms, (4) coordinating access to appropriate services, (5) facilitating provision of continual and integrated multidisciplinary care and (6) need to provide or facilitate psychological support. CONCLUSION: The findings show that GPs can play and have played a key role in the management of long COVID, and that patient care can be improved through better understanding of patient experiences, standardised approaches for symptom identification and treatment, and facilitation of access to multidisciplinary specialist services when needed. Future research evaluating focused GP interventions is needed.

Cover Feature: Aptamers for SARS-CoV-2: Isolation, Characterization, and Diagnostic and Therapeutic Developments (Anal. Sens. 5/2022).

The cover feature image shows nucleic acid aptamers armed and ready for our battle against the monstrous SARS-CoV-2 virus. Often thought of as "chemical antibodies", these molecular recognition elements are equipped with several unique benefits and have thus been a popular research subject worldwide. Many aptamers for recognizing the spike and nucleocapsid proteins of SARS-CoV-2 have been developed and examined as diagnostic and therapeutic weaponry for the war against COVID-19 and future pandemics. More information can be found in the Review by J. D. Brennan, Y. Li, and co-workers.

A novel digital rectoscope for the triage of lower gastrointestinal symptoms in primary care: a prospective multicentre feasibility study.

BACKGROUND: Access to community rectoscopy might help to ease the burden on hospital services and reduce costs for the NHS. To assess this, a prospective multicentre observational phase I feasibility study of a novel digital rectoscope and telestration software for the triage of lower gastrointestinal (GI) symptoms was undertaken. AIM: To determine if digital rectoscopy is feasible, acceptable, and clinically safe. DESIGN & SETTING: Evaluation of clinician case reports and patient questionnaires from patients recruited from five primary care centres. METHOD: Adults meeting 2-week wait (2WW) criteria for suspected lower GI cancer, suspected new diagnosis, or flare-up of inflammatory bowel disease (IBD) were enrolled. Examinations were performed by primary care practitioners using the LumenEye rectoscope. The CHiP platform allowed immediate remote review by secondary care. A prospective analysis was performed of patient and clinician experiences, diagnostic accuracy, and cost. RESULTS: A total of 114 patients were recruited and 110 underwent the procedure (46 [42%] females and 64 [58%] males). No serious adverse events were reported. Eighty-two (74.5%) patients reported that examination was more comfortable than expected, while 104 (94.5%) felt the intervention was most convenient if delivered in the community. Clinicians were confident of their assessment in 100 (87.7%) examinations. Forty-eight (42.1%) patients subsequently underwent colonoscopy, flexible sigmoidoscopy, or computed tomography virtual colonoscopy (CTVC). The overall sensitivity and specificity of LumenEye in identifying rectal pathology was 90.0% and 88.9%. It was 100% and 100% for cancer, and 83.3% and 97.8% for polyps. Following LumenEye examination, 19 (17.3%) patients were discharged, with projected savings of 11 305 GBP. CONCLUSION: Digital rectoscopy in primary care is safe, acceptable, and can reduce referrals. A phase III randomised controlled trial is indicated to define its utility in reducing the burden on hospital diagnostic services.

Front Cover: A Universal DNA Aptamer that Recognizes Spike Proteins of Diverse SARS‐CoV‐2 Variants of Concern (Chem. Eur. J. 15/2022)

A unique DNA aptamer, denoted MSA52, displays universally high affinity for the spike proteins of the wild‐type SARS‐CoV‐2 as well as its Alpha, Beta, Gamma, Epsilon, Kappa, Delta and Omicron variants. This aptamer also recognizes pseudotyped lentiviruses expressing eight different spike proteins of SARS‐CoV‐2 with very high affinity, exhibiting dissociation constants (Kd) of 20–50 pM for these viruses. More information can be found in the Research Article by J. D. Brennan, Y. Li et al. (DOI: 10.1002/chem.202200078).

Aptamers for SARS-CoV-2: Isolation, Characterization, and Diagnostic and Therapeutic Developments.

The SARS-CoV-2 virus and COVID-19 pandemic continue to demand effective diagnostic and therapeutic solutions. Finding these solutions requires highly functional molecular recognition elements. Nucleic acid aptamers represent a possible solution. Characterized by their high affinity and specificity, aptamers can be rapidly identified from random-sequence nucleic acid libraries. Over the past two years, many labs around the world have rushed to create diverse aptamers that target two important structural proteins of SARS-CoV-2: the spike (S) protein and nucleocapsid (N) protein. These have led to the identification of many aptamers that show real promise for the development of diagnostic tests and therapeutic agents for SARS-CoV-2. Herein we review all these developments, with a special focus on the development of diverse aptasensors for detecting SARS-CoV-2. These include electrochemical and optical sensors, lateral flow devices, and aptamer-linked immobilized sorbent assays.

A Universal DNA Aptamer that Recognizes Spike Proteins of Diverse SARS-CoV-2 Variants of Concern.

Invited for the cover of this issue are John Brennan, Yingfu Li, and co-workers at McMaster University. The image depicts MSA52 as a universal DNA aptamer that recognizes spike proteins of diverse SARS-CoV-2 variants of concern. Read the full text of the article at 10.1002/chem.202200078.

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 Universal DNA Aptamer that Recognizes Spike Proteins of Diverse SARS-CoV-2 Variants of Concern.

We report on a unique DNA aptamer, denoted MSA52, that displays universally high affinity for the spike proteins of wildtype SARS-CoV-2 as well as the Alpha, Beta, Gamma, Epsilon, Kappa, Delta and Omicron variants. Using an aptamer pool produced from round 13 of selection against the S1 domain of the wildtype spike protein, we carried out one-round SELEX experiments using five different trimeric spike proteins from variants, followed by high-throughput sequencing and sequence alignment analysis of aptamers that formed complexes with all proteins. A previously unidentified aptamer, MSA52, showed Kd values ranging from 2 to 10 nM for all variant spike proteins, and also bound similarly to variants not present in the reselection experiments. This aptamer also recognized pseudotyped lentiviruses (PL) expressing eight different spike proteins of SARS-CoV-2 with Kd values between 20 and 50 pM, and was integrated into a simple colorimetric assay for detection of multiple PL variants. This discovery provides evidence that aptamers can be generated with high affinity to multiple variants of a single protein, including emerging variants, making it well-suited for molecular recognition of rapidly evolving targets such as those found in SARS-CoV-2.

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.