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Toward a Home Test for COVID‐19 Diagnosis: DNA Machine for Amplification‐Free SARS‐CoV‐2 Detection in Clinical Samples

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ChemMedChem
Authors:
Ahmed Eldeeb at ITMO University
Ahmed Eldeeb
Sofia S. Zablotskaya
Sofia S. Zablotskaya
Sofia S. Zablotskaya
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Maria S. Rubel at ITMO University
Maria S. Rubel
Moustapha Ahmed-Yassin Nour at ITMO University
Moustapha Ahmed-Yassin Nour
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Abstract and Figures

Nucleic acid‐based detection of RNA viruses requires an annealing procedure to obtain RNA/probe or RNA/primer complexes for unwinding stable structures of folded viral RNA. In this study, we designed a protein‐enzyme‐free nano‐construction, named four‐armed DNA machine (4DNM), that requires neither an amplification stage nor a high‐temperature annealing step for SARS‐CoV‐2 detection. It uses a binary deoxyribozyme (BiDz) sensor incorporated in a DNA nanostructure equipped with a total of four RNA‐binding arms. Additional arms were found to improve the limit of detection at least 10‐fold. The sensor distinguished SARS‐CoV‐2 from other respiratory viruses and correctly identified five positive and six negative clinical samples verified by quantitative polymerase chain reaction (RT‐qPCR). The strategy reported here can be used for the detection of long natural RNA and can become a basis for a point‐of‐care or home diagnostic test.
Design of the DNA constructs used in this study. A) BiDz probe: DNA strands Dza and Dzb, bind RNA analyte and form the catalytic core that cleaves fluorophore and quencher labelled F sub. B) The four‐armed DNA nanomachine (4DNM) has Dza and Dzb attached to a double stranded DNA scaffold (shaded grey). Arms 3 and 4 are designed to tightly bind natural RNA, thereby unwinding its secondary structure. F sub is not shown.
Design of the DNA constructs used in this study. A) BiDz probe: DNA strands Dza and Dzb, bind RNA analyte and form the catalytic core that cleaves fluorophore and quencher labelled F sub. B) The four‐armed DNA nanomachine (4DNM) has Dza and Dzb attached to a double stranded DNA scaffold (shaded grey). Arms 3 and 4 are designed to tightly bind natural RNA, thereby unwinding its secondary structure. F sub is not shown.
… 
4DNM selectively detects CoV2 RNA. A) Comparison of fluorescent response of 4DNM with BiDz in the absence or presence (dark grey and light grey bars, respectively) of 125 pM CoV2 RNA. F‐sub (200 nM) was incubated with either BiDz (20 nM Dza and 5 nM Dzb) or DNM (20 nM Dza and 5 nM of T1/T2 complex) in the presence 3.8×10⁹ of either CoV2 or OC43 RNA molecules in a reaction buffer 1 (50 mM HEPES, pH 7.5, 150 mM KCl, 15 mM NaCl, 200 mM MgCl2). Fluorescence at 517 nm (λex=485 nm) was registered after 1 or 3 h of incubation at 55 °C. The error bars represent one standard deviation from average values calculated after three independent experiments. B) Selectivity of 4DNM machine. Fluorescent response of 4DNM to 3.8×10⁹ molecules in 50 μL (125 pM) of CoV2 or OC43 RNA (See experimental section). The conditions were as described for panel A.
4DNM selectively detects CoV2 RNA. A) Comparison of fluorescent response of 4DNM with BiDz in the absence or presence (dark grey and light grey bars, respectively) of 125 pM CoV2 RNA. F‐sub (200 nM) was incubated with either BiDz (20 nM Dza and 5 nM Dzb) or DNM (20 nM Dza and 5 nM of T1/T2 complex) in the presence 3.8×10⁹ of either CoV2 or OC43 RNA molecules in a reaction buffer 1 (50 mM HEPES, pH 7.5, 150 mM KCl, 15 mM NaCl, 200 mM MgCl2). Fluorescence at 517 nm (λex=485 nm) was registered after 1 or 3 h of incubation at 55 °C. The error bars represent one standard deviation from average values calculated after three independent experiments. B) Selectivity of 4DNM machine. Fluorescent response of 4DNM to 3.8×10⁹ molecules in 50 μL (125 pM) of CoV2 or OC43 RNA (See experimental section). The conditions were as described for panel A.
… 
Analysis of clinical samples using 4DNM. All samples contained F‐sub (200 nM) and 4DNM (20 nM Dza and 5 nM of T1/T2 complex) in reaction buffer 1 in the absence (−) or presence of clinical samples. Positive control (last bar) contained 20 pM of the synthetic CoV2‐DNA1 analyte. Fluorescence at 517 nm (λex=485 nm) was registered after 3 h of incubation at 55 °C. Nine CoV‐2 positive and five negative samples were collected from 14 different individuals and confirmed by RT‐qPCR with indicated threshold cycle (Ct) values. The Ct values for positive samples were as follows: 6–20.4; 7–21.0; 8–21.8, 9–22.0; 10–22.9; 11–24.2; 12–25.5; 13–25.9; 14–27.7. Clinical samples 1–14 lack standard deviation since all available RNA from each sample was fully used only in one measurement to ensure the highest available concentration of viral RNA. Standard deviation for the negative (−) and the positive (+) controls were determined after three independent measurements. The threshold, line corresponds to 3 standard deviations above average (−) control.
Analysis of clinical samples using 4DNM. All samples contained F‐sub (200 nM) and 4DNM (20 nM Dza and 5 nM of T1/T2 complex) in reaction buffer 1 in the absence (−) or presence of clinical samples. Positive control (last bar) contained 20 pM of the synthetic CoV2‐DNA1 analyte. Fluorescence at 517 nm (λex=485 nm) was registered after 3 h of incubation at 55 °C. Nine CoV‐2 positive and five negative samples were collected from 14 different individuals and confirmed by RT‐qPCR with indicated threshold cycle (Ct) values. The Ct values for positive samples were as follows: 6–20.4; 7–21.0; 8–21.8, 9–22.0; 10–22.9; 11–24.2; 12–25.5; 13–25.9; 14–27.7. Clinical samples 1–14 lack standard deviation since all available RNA from each sample was fully used only in one measurement to ensure the highest available concentration of viral RNA. Standard deviation for the negative (−) and the positive (+) controls were determined after three independent measurements. The threshold, line corresponds to 3 standard deviations above average (−) control.
… 
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Toward a Home Test for COVID-19 Diagnosis: DNA Machine
for Amplification-Free SARS-CoV-2 Detection in Clinical
Samples
Ahmed A. El-Deeb,[a] Sofia S. Zablotskaya,[a] Maria S. Rubel,[a] Moustapha A. Y. Nour,[a]
Liubov I. Kozlovskaya,[b] Anna A. Shtro,[c] Andrey B. Komissarov,[c] and
Dmitry M. Kolpashchikov*[a, d, e]
Nucleic acid-based detection of RNA viruses requires an
annealing procedure to obtain RNA/probe or RNA/primer
complexes for unwinding stable structures of folded viral RNA.
In this study, we designed a protein-enzyme-free nano-
construction, named four-armed DNA machine (4DNM), that
requires neither an amplification stage nor a high-temperature
annealing step for SARS-CoV-2 detection. It uses a binary
deoxyribozyme (BiDz) sensor incorporated in a DNA nano-
structure equipped with a total of four RNA-binding arms.
Additional arms were found to improve the limit of detection at
least 10-fold. The sensor distinguished SARS-CoV-2 from other
respiratory viruses and correctly identified five positive and six
negative clinical samples verified by quantitative polymerase
chain reaction (RT-qPCR). The strategy reported here can be
used for the detection of long natural RNA and can become a
basis for a point-of-care or home diagnostic test.
Introduction
Current testing capacity of respiratory infections cannot meet the
unprecedented global demands for rapid, reliable, and accessible
nucleic acid-based diagnostics.[1–3] Reveres transcription quantita-
tive polymerase chain reaction (RT-PCR) is one of the most
common techniques used for the diagnosis of severe acute
respiratory syndrome coronavirus 2 (CoV2) infection. Several days
of the wait time for the test results during ongoing CoV2
outbreak revealed imperfection of the RT-qPCR and calls for new
inexpensive, accessible diagnostics affordable by general popula-
tion outside the specialized laboratories.[1] Towards this goal,
several RT-PCR-free CoV2 nucleic acid tests are under develop-
ment including those based on loop-mediated isothermal
amplification (LAMP),[4] recombinase polymerase amplification
(RPA),[5] and nucleic acid base amplification (NASBA)[6] among
others.[7] Majority of the tests, however, depend on nucleic acid
amplification procedures, which require expensive reagents
including perishable protein enzymes. The amplification step can
also add false positive results due to sample degradation or
contamination.[8] Amplification-free diagnostics would avoid the
need to use instruments that require specialized laboratories and/
or qualified personnel.[9] Visual[10] or tactile[11] signal outputs could
be the most user-friendly as they would not require equipment.
However, such assays have high limits of detection (LOD) and
thus doomed to be dependent on nucleic acid amplification.[10e,d]
We turned our attention to fluorescent binary RNA-cleaving
deoxyribozyme (BiDz) probe.[12] BiDz can reach LOD down to 1–
10 pM,[13] which is, to the best of our knowledge, the lowest
among protein enzyme-free testing systems that do not use
amplification cascades.[14] BiDz consists of two DNA strands, Dza
and Dzbin Figure 1A which hybridize to the analyzed nucleic acid
and form Dz catalytic core followed by the cleavage of
fluorophore and a quencher-labelled reporter substrate (F sub in
Figure 1).[12] One advantage of BiDz is its ability to amplify the
signal due to cleaving multiple F sub molecules. Another
advantage is its high selectivity towards single nucleotide
substitutions (SNS).[12,15] This is achieved by shortening Arm 2
complementary to the SNS site (Figure 1A). Short arm binds only
the fully complementary, but not single base mismatched
sequences.[15]
Results
Initially, optimization of the BiDz assay was performed using a
synthetic DNA analyte containing the sequence of 15398–15487
of CoV2 RdRp gene (CoV2-DNA1 in Table S1 and Figure S1). It was
[a] A. A. El-Deeb, S. S. Zablotskaya, M. S. Rubel, M. A. Y. Nour,
Prof. D. M. Kolpashchikov
Laboratory of Molecular Robotics and Biosensor Materials
SCAMT Institute, ITMO University
191002 Saint Petersburg (Russia)
E-mail: dmitry.kolpashchikov@ucf.edu
[b] Dr. L. I. Kozlovskaya
Chumakov Federal Scientific Center for Research and Development of
Immune-and-Biological Products
Russian Academy of Sciences
Institute of Poliomyelitis, Moscow (Russia)
[c] Dr. A. A. Shtro, Dr. A. B. Komissarov
Smorodintsev Research Institute of Influenza
197376 Saint Petersburg (Russia)
[d] Prof. D. M. Kolpashchikov
Chemistry Department
University of Central Florida
Orlando, FL 32816 (USA)
[e] Prof. D. M. Kolpashchikov
Burnett School of Biomedical Sciences
University of Central Florida
Orlando, FL 32827 (USA)
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202200382
ChemMedChem
www.chemmedchem.org
Research Article
doi.org/10.1002/cmdc.202200382
ChemMedChem 2022,17, e202200382 (1 of 5) © 2022 Wiley-VCH GmbH
Wiley VCH Mittwoch, 05.10.2022
2220 / 267194 [S. 72/76] 1
... [32] Expanding upon this approach, our study was based on earlier efforts by our team to develop the fourarmed DNA nanomachine (4DNM) (Figure 1A), as a selective DNA nanomachine for the detection of SARS-CoV-2 in clinical samples. [33] 4DNM features four target-binding arms, which play a crucial role in unwinding the secondary structure of SARS-CoV-2 RNA, thereby facilitating its detection. Building upon this foundation, we have developed a novel multicore DNA nanomachine (MDNM) ( Figure 1B, Figure S2), incorporating four BiDz, interconnected via a scaffold, which targets a sequence within the RdRp gene of SARS-CoV-2 ( Figure S1). ...
... Our group's prior work had shown that 4DNM demonstrated an LOD of 1 pM following a 3 h incubation time at 55°C with the synthetic DNA analyte. [33] On the other hand, under the same settings, MDNM detected the synthetic analyte with an LOD of only 0.2 pM (Figure 2) signifying a five-fold improvement in The noticed enhancement in LOD for MDNM proves our hypothesis, which shows that the Dz based multifunctional sensors operate greater when they have multiple catalytic cores. ...
... Following a 3 h incubation period at 55°C, MDNM demonstrated an LOD of 4 pM (Figure 3), which is over 5-fold lower than 4DNM's LOD for SARS-CoV-2 RNA detection after a 3 h assay (which was 26 pM). [33] Notably, MDNM exhibited the ability to detect viral RNA after just 1 h with an LOD of 28 pM (Figure 3), a result comparable to 4DNM's after 3 h. These findings underscore MDNM's potential to reduce the required time for efficient detection of SARS-CoV-2. ...
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... 30,35,36 We have been developing binary deoxyribozyme (BiDz) probes ( Figure 1A), which can achieve lower LOD than other fluorescent probes by at least 100 times. 15,35,37 BiDz is made up of two parts, Dza and Dzb, and is based on an RNAcleaving DNAzyme. When the complementary target analyte is present, the two subunits combine to form the Dz catalytic core, which cleaves a reporter substrate (F-sub) containing a fluorophore and a quencher on opposite sides of the cleavage site to generate detectable fluorescence. ...
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View
... Further development of the technique has shown that the suggested MDMR1 is excessively intricate. Thus, a simple DNA scaffold represented by a doublestranded DNA that combines a half of the catalytic core containing binary sensor with one or two additional analyte-binding elements into a rigid structure (Fig. 2c) allows detection of double-stranded DNAs [17], PCR amplification products, including very long ones (>1400 bp) [21,22], and intact viral RNAs [23], as well as identification of single-nucleotide substitutions in the ribosomal RNA that could not be detected by binary DNAzymes [24]. ...
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Full-text available
  • Apr 2022
Limiting transmission of SARS-CoV-2 from asymptomatic people assumes the paramount importance of keeping fragile subjects protected. We evaluated the utility of rapid SARS-CoV-2 antigen testing in asymptomatic subjects attending emergency departments in non-COVID-19 areas, using a single nasopharyngeal swab specimen collected in universal transport medium to perform both rapid antigen testing and rRT-PCR (used as reference standard) in a cohort of 899 patients. In the overall sample, the rapid antigen test had 43.9% sensitivity, 100% specificity, 100% positive predictive value, 93.6% negative predictive value. Considering subjects with rRT-PCR cycle threshold ≤30, the test had 80.4% sensitivity, 100% specificity, 100% positive predictive value, 98.8% negative predictive value. Considering subjects with rRT-PCR cycle threshold ≤25, the test had 94.7% sensitivity, 100% specificity, 100% positive predictive value and 99.7% negative predictive value. Despite low sensitivity, routine application of rapid antigen testing in the emergency department can lead to isolation in less than 30 min of about a half of asymptomatic COVID-19 subjects assigned to non-COVID-19 areas by clinical triage. The rapid test correctly identified 94.7% of asymptomatic patients with cycle threshold ≤ 25 that are supposed to be more infective; thus, it could be a useful measure to contain viral transmission in non-COVID-19 areas.
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DNA nano-machine for visual detection of structured RNA and double-stranded DNA
Article
Full-text available
  • Apr 2022
Modern medical diagnosis relies on identifying specific nucleic acids using hybridization probes, often failing to detect single-stranded (ss) RNA folded in stable secondary structures or double-stranded (ds) DNA. In this...
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Clinical validation of engineered CRISPR/Cas12a for rapid SARS-CoV-2 detection
Article
Full-text available
  • Jan 2022
Background The coronavirus disease (COVID-19) caused by SARS-CoV-2 has swept through the globe at an unprecedented rate. CRISPR-based detection technologies have emerged as a rapid and affordable platform that can shape the future of diagnostics. Methods We developed ENHANCEv2 that is composed of a chimeric guide RNA, a modified LbCas12a enzyme, and a dual reporter construct to improve the previously reported ENHANCE system. We validated both ENHANCE and ENHANCEv2 using 62 nasopharyngeal swabs and compared the results to RT-qPCR. We created a lyophilized version of ENHANCEv2 and characterized its detection capability and stability. Results Here we demonstrate that when coupled with an RT-LAMP step, ENHANCE detects COVID-19 samples down to a few copies with 95% accuracy while maintaining a high specificity towards various isolates of SARS-CoV-2 against 31 highly similar and common respiratory pathogens. ENHANCE works robustly in a wide range of magnesium concentrations (3 mM-13 mM), allowing for further assay optimization. Our clinical validation results for both ENHANCE and ENHANCEv2 show 60/62 (96.7%) sample agreement with RT-qPCR results while only using 5 µL of sample and 20 minutes of CRISPR reaction. We show that the lateral flow assay using paper-based strips displays 100% agreement with the fluorescence-based reporter assay during clinical validation. Finally, we demonstrate that a lyophilized version of ENHANCEv2 shows high sensitivity and specificity for SARS-CoV-2 detection while reducing the CRISPR reaction time to as low as 3 minutes while maintaining its detection capability for several weeks upon storage at room temperature. Conclusions CRISPR-based diagnostic platforms offer many advantages as compared to conventional qPCR-based detection methods. Our work here provides clinical validation of ENHANCE and its improved form ENHANCEv2 for the detection of COVID-19.
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Monoterpene-Containing Substituted Coumarins as Inhibitors of Respiratory Syncytial Virus (RSV) Replication
Article
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Respiratory syncytial virus (RSV) is a critical cause of infant mortality. However, there are no vaccines and adequate drugs for its treatment. We showed, for the first time, that O-linked coumarin–monoterpene conjugates are effective RSV inhibitors. The most potent compounds are active against both RSV serotypes, A and B. According to the results of the time-of-addition experiment, the conjugates act at the early stages of virus cycle. Based on molecular modelling data, RSV F protein may be considered as a possible target.
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Regenerable and high-throughput surface plasmon resonance assay for rapid screening of anti-SARS-CoV-2 antibody in serum samples
Article
  • Apr 2022
  • ANAL CHIM ACTA
Current serological antibody tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) require enzyme or fluorescent labels, and the titer well plates cannot be reused. By immobilizing histidine (His)-tagged SARS-CoV-2 spike (S1) protein onto tris‒nitrilotriacetic acid (tris-NTA) sensor and using the early association phase for mass-transfer-controlled concentration determination, we developed a rapid and regenerable surface plasmon resonance (SPR) method for quantifying anti-SARS-CoV-2 antibody. On a five-channel SPR instrument and with optimized S1 protein immobilization density, each of the four analytical channels is sequentially used for multiple measurements, and all four channels can be simultaneously regenerated once they have reached a threshold value. Coupled with a programmable autosampler, each sensor can be regenerated at least 20 times, enabling uninterrupted assays of more than 800 serum samples. The accuracy and speed of our method compare well with those of the enzyme-linked immunosorbent assay (ELISA), and the detection limit (0.057 μg mL⁻¹) can easily meet the requirement for screening low antibody levels such as those in convalescent patients. In addition, our method exhibits excellent channel-to-channel (RSD = 1.9%) and sensor-to-sensor (RSD = 2.1%) reproducibility. Obviation of an enzyme label drastically reduced the assay cost, rending our method (<60 cents) much more cost effective than those of commercial ELISA kits ($4.4–11.4). Therefore, our method offers a cost-effective and high-throughput alternative to the existing methods for serological measurements of anti-SARS-CoV-2 antibody levels, holding great promise for rapid screening of clinical samples without elaborate sample pretreatments and special reagents.
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Rapid, multiplexed, and nucleic acid amplification-free detection of SARS-CoV-2 RNA using an electrochemical biosensor
Article
  • Sep 2021
  • BIOSENS BIOELECTRON
Considering the worldwide health crisis associated with highly contagious severe respiratory disease of COVID-19 outbreak, the development of multiplexed, simple and rapid diagnostic platforms to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is in high demand. Here, a nucleic acid amplification-free electrochemical biosensor based on four-way junction (4-WJ) hybridization is presented for the detection of SARS-CoV-2. To form a 4-WJ structure, a Universal DNA-Hairpin (UDH) probe is hybridized with two adaptor strands and a SARS-CoV-2 RNA target. One of the adaptor strands is functionalized with a redox mediator that can be detected using an electrochemical biosensor. The biosensor could simultaneously detect 5.0 and 6.8 ag/μL of S and Orf1ab genes, respectively, within 1 h. The biosensor was evaluated with 21 clinical samples (16 positive and 5 negative). The results revealed a satisfactory agreement with qRT-PCR. In conclusion, this biosensor has the potential to be used as an on-site, real-time diagnostic test for COVID-19.
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Recent progress on rapid SARS-CoV-2/COVID-19 detection by CRISPR-Cas13-based platforms
Article
  • Jun 2021
  • DRUG DISCOV TODAY
The limitations of conventional diagnostic procedures, such as real-time PCR-based methods and serological tests, have led the scientific community to innovate alternative nucleic acid detection approaches for SARS-CoV-2 RNA, thereby addressing the dire need for increased testing. Such approaches aim to provide rapid, accurate, cost-effective, sensitive, and high-throughput detection of SARS-CoV-2 RNA, on multiple specimen types, and without specialized equipment and expertise. The CRISPR-Cas13 system functions as a sequence-specific RNA-sensing tool that has recently been harnessed to develop simplified and flexible testing formats. This review recapitulates technical advances in the most recent CRISPR-Cas13-based methods for SARS-CoV-2/COVID-19 diagnosis. The challenges and opportunities for implementing mass testing using these novel CRISPR-Cas13 platforms are critically analyzed.
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