COVID19 aptamers

Background

Coronavirus disease 2019 (COVID‑19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1]. 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 [4]. 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.

Selection Information

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

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

Cross-reactivity

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.

Pseudovirus Binding

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 info@basepairbio.com for more information and quotes.  

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