Background

Infectious diseases remain one of the biggest challenges for human health, with the current COVID-19 pandemic as a primary example. Accurate and early detection of viruses is crucial for transmission control through clinical diagnosis and therapy. To achieve this goal, DNA aptamers have emerged as a promising approach to develop sensors for on-site and real-time detection and quantitation. DNA aptamers are short single-strand DNA molecules with a specific sequence that allow them to form a specific three-dimensional conformation to recognize a certain target with high affinity and selectivity (Huizenga 1997; Cho et al., 2009; Liu et al., 2009; Xiang and Lu 2014; Xing et al., 2014; Li et al., 2019; Ma et al., 2021; Xie et al., 2021). They have been shown to be highly specific for binding viruses (Balogh et al., 2010; González et al., 2016; Zou et al., 2019; Kukushkin et al., 2019; Peinetti et al., 2021), rivaling antibodies. Furthermore, DNA aptamers have gained considerable attention because they are cost-effective, have easy chemical modifications, and have high chemical stability, binding affinity, repeatability, and reusability (Liu et al., 2009; Fang and Tan 2010; Dunn et al., 2017; Hong et al., 2020). All these features make them ideal candidates for developing affordable, rapid, sensitive methods able to identify new or emerging viruses, including virus variants (Xing et al., 2014; Zou et al., 2019; Li et al., 2021; Peinetti et al., 2021; Chakraborty et al., 2022). Aptamers are obtained from a random DNA library by an iterative procedure called Systematic Evolution of Ligands by EXponential enrichment (SELEX), which is carried out in test tubes with large sampling libraries (~10¹⁵) and is performed in a much shorter time with less cost than the generation of antibodies, peptides, and small molecule agents (Ellington and Szostak 1990; Tuerk and Gold 1990). More importantly, counter selection during SELEX to remove the DNA molecules that bind similar competing species can enhance the selectivity of aptamers and give them unique properties (Bruesehoff et al., 2002; Shen et al., 2016), such as differentiating an intact infectious virus from its non-infectious version (Peinetti et al., 2021).

Given the success of SELEX in obtaining aptamers for a wide variety of targets, including infectious viruses, it is important to measure their binding affinity to the targets and to similar competing species to understand the interactions of the aptamer with their targets (Sun et al., 2021; Zhang et al., 2021). In addition to validating the capability of the aptamers to bind their intended targets but not other targets, the results provide quantitative measures on how strong and how selective the binding is and thus play a vital role in optimizing their performance as sensors, imaging tools, or therapeutic agents.

Viruses generally present several proteins in high copy number on their surfaces that are potential targets for aptamer recognition (Kwon et al., 2020; Li et al., 2022). Considering the high copy number and possibility of higher-ordered structures and arrangements of the proteins on the virus surface, it is more reasonable to determine the binding affinity of aptamers using the whole virus, rather than using the viral protein or a domain of the protein in solution. There are several sources of discrepancies between using the whole virus or just the purified protein. One of the most critical differences is that surface proteins exist in their native state on the surface of the virus. For example, the spike protein of SARS-CoV-2 is in trimeric form on the virus surface, and thus it is very challenging to purify the protein in its native-like environments that preserve their conformation (Cai et al., 2020; Tai et al., 2020; Juraszek et al., 2021). Moreover, as there are multiple copies of the same protein in each virion, the avidity effect jointly with multivalent effects may significantly affect the binding capability (Kwon et al., 2020; Kim et al., 2022). An aptamer that would bind to a single protein may have a significantly different binding affinity or may not even bind at all to the protein when expressed on the surface of a virion, and vice versa. Therefore, to allow direct detection of the virus, it is best to determine the binding affinity between the aptamer and the intact virus.

To perform binding assays between aptamers and intact viruses, there are some aspects and challenges that need to be considered. First, the DNA aptamer structures depend on the experiment conditions more than other recognition macromolecules such as antibodies or other folded proteins (Liu et al., 2009; Xia et al., 2013). Therefore, determining the affinity constant of an aptamer requires rigorous control of the experimental conditions to obtain reproducible results. Second, the available techniques for binding assays that can be performed with the intact virus are limited compared to proteins. For instance, viruses are larger than proteins, and a common binding assay like Electrophoretic Mobility Shift Assay (EMSA) cannot be performed. Furthermore, handling the whole intact virus or intact pseudoviruses (when working with BSL3 viruses such as SARS-CoV-2) could require a biosafety level 2 (BSL2) facility or even more stringent safety considerations. As a result, the equipment for the measurement needs to be housed in a BSL2 lab and requires regular decontamination with solutions like bleach that can be damaging to instruments if there are not available disposable options to introduce the viral sample. Because of these requirements, it is difficult to use some techniques such as Surface Plasmon Resonance (SPR) or Isothermal Titration Calorimetry (ITC) that have been successfully applied to characterize other aptamer-target bindings (Ramakrishnan et al., 2012; Chen et al., 2015; Harazi et al., 2017).

Here, we describe two different techniques that can be used to obtain binding affinity of aptamers to viruses. First, Enzyme-Linked Oligonucleotide Assay (ELONA), which has been more extensively used for studying the binding between aptamers and viruses (Drolet et al., 1996; Balogh et al., 2010; Bai et al., 2018). This method immobilizes the virus on a surface and uses biotin-labeled aptamers and streptavidin-labeled horseradish peroxidase (HRP) to carry out colorimetric assays through oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate to 3,3',5,5'-tetramethylbenzidine diamine. By changing the concentration of the aptamer relative to the virus and measuring the amount of bound aptamer, it is possible to determine the binding affinity as a dissociation constant (K_D). Second, a more recent method, MicroScale Thermophoresis (MST), which monitors thermophoretic mobility change in a fluorescent DNA aptamer as a function of the concentration of virus to determine the binding affinity (Duhr et al., 2004; Plach et al., 2017). MST is a simpler assay to set up and offers the possibility to determine binding affinity, binding stoichiometry, and interaction thermodynamics (Baaske et al., 2010; Jerabek-Willemsen et al., 2011). In this work, we used both protocols to measure the binding of DNA aptamers and SARS-CoV-2 pseudoviruses (Peinetti et al., 2021). It is important to mention that pseudoviruses are used for SARS-CoV-2 to reduce the biosafety level required for SARS-CoV-2 viruses from BSL3 to BSL2. However, these methods can be used for other viruses, including different types of viruses, from enveloped RNA viruses like the one shown here, to non-enveloped DNA viruses, as demonstrated in our recent paper (Peinetti et al., 2021).