Coronavirus disease 2019 (COVID‑19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) . The virus was first identified in December 2019 in Wuhan, Hubei, China, and has resulted in an ongoing pandemic [2, 3]. The first confirmed case has been traced back to 17 November 2019 in Hubei . As of the 10th of August 2020, more than 19.9 million cases have been reported across 188 countries and territories, resulting in more than 732,000 deaths.
SARS-CoV-2 (COVID-19) is caused by inhalation of viral particles where epithelial cells are vulnerable to invasion by the virus. It is now well accepted that angiotensin converting enzyme 2 (ACE2) binds to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S1) and this ultimately leads to fusion of the viral particle with the cell membrane, viral endocytosis, and viral replication [6, 7].
Because of the well understood interaction between the S1 RBD and ACE2, inhibiting this interaction has been an attractive strategy for therapeutic development. Separately, however S1 is repeated some 50 to 200 times on the viral surface although the exact copy number is still a subject of research [8–11]. Nevertheless, repeated spikes make this surface target not only attractive for therapeutics but also for direct viral antigen detection in rapid diagnostics.
Base Pair’s contribution to rapid COVID-19 diagnostics development
Aptamers have unique attributes that are highly attractive for the development of field deployable or ‘point-of-care’ (POC) tests. Aptamers are DNA- or RNA-based ligands capable of binding practically any molecular target. Aptamers are uniquely suited to address the challenges associated with viral antigen detection. They are usually identified by an in vitro method of selection referred to as Systematic Evolution of Ligands by EXponential enrichment or “SELEX.” The process begins with a very large pool (~1015 unique sequences) of randomized polynnucleotides, which is narrowed to just a few aptamer binders per target [12, 13]. Figure 1 shows a simplified schematic for repeated rounds of SELEX of DNA aptamers. Once multiple typically 10-15) rounds of SELEX are completed, DNA sequences are identified by next generation sequencing.
Aptamers have been developed as ligands to important peptides and proteins, rivaling antibodies in both affinity and specificity [14–17]. Aptamers also have been developed to bind small organic molecules and cellular toxins [18–22], viruses [23, 24], and even targets as small as heavy metal ions [25–28]. In addition to the versatility of aptamers and their ability to be selected in vitro without the need for conventional immunization, aptamers have other advantages for POC tests. Specifically, they are heat stable and can be lyophilized on sensors, lateral flow strips, ELISA wells, etc. with almost indefinite shelf life. Aptamers are also synthetic and therefore, unlike antibodies, have zero lot-to-lot variability. With these considerations in mind, we sought to rapidly develop aptamers to the spike protein of SARS-CoV-2 to make high affinity aptamers available to the scientific community.
Target for Selection: SARS-CoV-2 (2019-nCoV) Spike S1+S2 ECD-His Recombinant Protein (Baculovirus Host expressed), Sino Biological Cat.# 40589-V08B1
Number of DNA Nucleotides: 70 or 32 for the truncated versions.
Aptamer was selected from a randomized Base Pair natural DNA library against the target molecule. At the end of the SELEX campaign, next generation sequencing and proprietary bioinformatic methods were used to choose the most promising aptamer candidates for binding characterization.
Affinity Determination Method: BioLayer Interferometry (BLI)
Biotinylated aptamers were loaded onto streptavidin-activated BLI sensors. The loaded sensors were then immersed into buffer containing the spike protein in free solution. Time 0-600s represents the association phase (aptamer binding to target), time 600-1200s represents the dissociation phase (release of target during a wash step). Background signal has been removed by subtracting the raw data from a (blank) zero target sample from the raw data for each other sample.
Buffer Used for Affinity Determination: 1x PBS, 1mM MgCl2, 10% human saliva
Figure 2 Average KD for CFA0688T: 3.52 nM (R2 = 0.9985)
Table 1 shows the dissociation constants of seven aptamer candidates measured by BLI.
Figure 2. COVID-19 S Protein Binding The target used for affinity measurement was expressed in HEK293 cells (Amsbio Cat.# AMS.SPN-C52H4)
Table 1. Summary of Unique Aptamers Affinity
Figure 3. Spike protein Cross-reactivity The targets used for affinity measurements were; COV1-Spike-S1 Subunit: (Sino Biological, Cat# 40150-V05H). SARS-CoV-2 Spike: (Amsbio Cat.# AMS.SPNC52H4) The Buffer Used for Affinity Determination was; 1x PBS, 1mM MgCl2, 10% human saliva There was no binding observed between CFA0688T and the SARS-Cov-(1) spike protein.
Figure 3. Pseudovirus Binding The targets used for these measurements were two different types of SARS-CoV-2 S protein expressing pseudovirus; MLV = Maloneymurine Leukemia Virus HIV = Human Immunodeficiency Virus The number after the colon (;) indicates the volume of pseudovirus present in each sample (total volume = 200 μL). The Buffer Used for Affinity Determination was; 1x PBS, 1mM MgCl2, 10% human saliva
The aptamer generated very strong binding curves against both type of pseudovirus (PV). The HIV PV generated a stronger signal than the MLV PV, but there are a few very simple potential explanations for this; 1) the concentration of the HIV PV could be higher than that of the MLV PV, neither manufacturer was able to provide a particle concentration for the products, 2) even if at the same concentration the HIV particles (~120nm diameter) are larger than the MLV ones (80-100nm diameter), as BLI signal largely correlates to size the larger particles would generate a larger signal, furthermore, it is highly likely that the larger particles will have more S proteins/particle and could therefore benefit from increased binding due to increased avidity.
Available for Purchase
All of the aptamers in Table 1 are available for purchase from Base Pair for evaluation. Typical package sizes are 25 and 100 μgrams, however larger amounts can be readily synthesized. All of the most common fluorophores, immobilization moieties – thiol, biotin, primary amine, etc. – are also available. Please inquire on our website or at firstname.lastname@example.org for more information and quotes.
- Coronavirus disease 2019 (COVID-19) – Symptoms and causes – Mayo Clinic. https://www.mayoclinic.org/diseasesconditions/coronavirus/symptoms-causes/syc-20479963. Accessed 10 Aug 2020.
- Hui DS, I Azhar E, Madani TA, Ntoumi F, Kock R, Dar O, et al. The continuing 2019-nCoV epidemic threat of novel coronaviruses to global health — The latest 2019 novel coronavirus outbreak in Wuhan, China. Int J Infect Dis. 2020;91:264–6.
- WHO Director-General’s opening remarks at the media briefing on COVID-19 – 11 March 2020. https://www.who.int/dg/speeches/detail/who-director-general-s-opening-remarks-at-the-media-briefing-on-covid-19—11-march-2020. Accessed 10 Aug 2020.
- China’s first confirmed Covid-19 case traced back to November 17. South China Morning Post. 2020. https://www.scmp.com/news/china/society/article/3074991/coronavirus-chinas-first-confirmed-covid-19-case-tracedback. Accessed 10 Aug 2020.
- Coronavirus COVID-19 (2019-nCoV). https://gisanddata.maps.arcgis.com/apps/opsdashboard/index.html#/bda7594740fd40299423467b48e9ecf6. Accessed 10 Aug 2020.
- Walls AC, Park Y-J, Tortorici MA, Wall A, McGuire AT, Veesler D. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell. 2020;181:281-292.e6.
- Ou X, Liu Y, Lei X, Li P, Mi D, Ren L, et al. Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nat Commun. 2020;11:1620.
- Wrapp D, Wang N, Corbett KS, Goldsmith JA, Hsieh C-L, Abiona O, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science. 2020;367:1260–3.
- Robson B. COVID-19 Coronavirus spike protein analysis for synthetic vaccines, a peptidomimetic antagonist, and therapeutic drugs, and analysis of a proposed achilles’ heel conserved region to minimize probability of escape mutations and drug resistance. Comput Biol Med. 2020;121:103749.
- Li F. Structure, Function, and Evolution of Coronavirus Spike Proteins. Annu Rev Virol. 2016;3:237–61.
- Petrov D. Photopolarimetrical properties of coronavirus model particles: Spike proteins number influence. J Quant Spectrosc Radiat Transf. 2020;248:107005.
- Ellington AD, Szostak JW. In vitro selection of RNA molecules that bind specific ligands. Nature. 1990;346:818–22.
- Tuerk C, Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990;249:505–10.
- Lee JF, Hesselberth JR, Meyers LA, Ellington AD. Aptamer Database. Nucleic Acids Res. 2004;32 Database Issue:D95-100.
- Srisawat C, Engelke DR. Streptavidin aptamers: Affinity tags for the study of RNAs and ribonucleoproteins. RNA. 2001;7:632–41.
- Ruckman J, Green LS, Beeson J, Waugh S, Gillette WL, Henninger DD, et al. 2’-Fluoropyrimidine RNA-based Aptamers to the 165-Amino Acid Form of Vascular Endothelial Growth Factor (VEGF165), Inhibition Of Receptor Binding And Vegf-Induced Vascular Permeability Through Interactions Requiring The Exon 7-Encoded Domain. J Biol Chem.1998;273:20556–7.
- Jayasena SD. Aptamers: an emerging class of molecules that rival antibodies in diagnostics. Clin Chem. 1999;45:1628–50.
- Sazani PL, Larralde R, Szostak JW. A Small Aptamer with Strong and Specific Recognition of the Triphosphate of ATP. J Am Chem Soc. 2004;126:8371.
- Babendure JR, Adams SR, Tsien RY. Aptamers Switch on Fluorescence of Triphenylmethane Dyes. J Am Chem Soc. 2003;125:14716–7.
- Hirao I, Yoshinari S, Yokoyama S, Endo Y, Ellington AD. In vitro selection of aptamers that bind to ribosomeinactivating toxins. Nucleic Acids Symp Ser. 1997;37:283–4.
- Liu J, Lu Y. Fast Colorimetric Sensing of Adenosine and Cocaine Based on a General Sensor Design Involving Aptamers and Nanoparticles. Angew Chem Int Ed. 2006;117:90–4.
- Chu TC, Marks 3rd JW, Lavery LA, Faulkner S, Rosenblum MG, Ellington AD, et al. Aptamer:toxin conjugates that specifically target prostate tumor cells. Cancer Res. 2006;66 Jun 15:5989–92.
- Gopinath SCB, Sakamaki Y, Kawasaki K, Kumar PKR. An Efficient RNA Aptamer against Human Influenza B Virus Hemagglutinin. J Biochem 2006 1395837-846. 2006;139:837–46.
- Gopinath SCB, Misono TS, Kawasaki K, Mizuno T, Imai M, Odagiri T, et al. An RNA aptamer that distinguishes between closely related human influenza viruses and inhibits haemagglutinin-mediated membrane fusion. J Gen Virol. 2006;87:479–87.
- Wernette DP, Kim H-K, Liu J, Swearingen CB, Yue Z, Zavareh M, et al. New Catalytic DNA Biosensors for Trace Contaminants in Water.
- Swearingen CB, Wernette DP, Cropek DM, Lu Y, Sweedler JV, Bohn PW. Immobilization of a Catalytic DNA Molecular Beacon on Au for Pb(II) Detection. Anal Chem. 2005;77:442–8.
- Chang I-H, Tulock J, Liu J, Kim W-S, Cannon Jr. D, Lu Y, et al. Miniaturized Lead Sensor Based on Lead-Specific DNAzyme in a Nanocapillary Interconnected Microfluidic Device. Env Sci Technol. 2005;39:3756.
- Wrzesinski J, Ciesiolka J. Characterization of structure and metal ions specificity of Co2+-binding RNA aptamers. Biochemistry. 2005;44 Apr 26:6257–68.