Prepare Bioanalytical Aptamer Tools and Train Next-generation Scientists for SARS-CoV-3 - PROJECT SUMMARY/ABSTRACT In the past two decades, the world has witnessed the outbreaks of three Beta-coronaviruses: SARS-CoV-1 in 2002, MERS-CoV in 2012, and SARS-CoV-2 in 2019. As the potential for future outbreaks, including the possibility of SARS-CoV-3, remains high and unabated, the development of universal bioanalytical sensors in advance assumes paramount importance. Notably, while the spike proteins of different Beta-coronaviruses exhibit distinct characteristics, they also contain highly conserved domains. For instance, the S2 domains of SARS-CoV-1 and CoV-2 display significant conservation (approximately 93.1% conserved residues). Considering this scientific foundation, the anticipated emergence of the future Beta-coronavirus, SARS-CoV-3, from the same lineage B of SARS-CoV-1 and CoV-2, suggests the presence of the same highly conserved S2 domain. Consequently, it is imperative for scientists to develop probes targeting the S2 domain that can serve as universal tools for various Beta-coronaviruses. Recent studies have reported the efficient neutralization of diverse SARS-CoV-2 variants using anti-S2 antibodies, and we have successfully developed the first anti-S2 aptamer, which is a single-stranded deoxynucleic acid (DNA) molecule capable of specifically recognizing the S2 domain. Preliminary data support the following observations: 1) Our aptamers, S2A2C1 and A1C1, recognize conserved spike domains of various SARS-CoV-2 variants; 2) They also recognize the spike proteins of SARS- CoV-1; 3) Dimeric fusion aptamers demonstrate enhanced binding avidities compared to monovalent aptamers. Hence, we are confident in the feasibility of developing cost-effective DNA aptamers with the ability to universally recognize different spike proteins of diverse Beta-coronaviruses. Two Specific Aims include: 1) To determine the potential of our aptamers against various spike proteins and prepare aptamer based bioanalytical sensors; 2) To construct homo- and hetero-dimeric aptamer based biosensors. It is important to note that Aim 2 of our research is independent of Aim 1, with the exception of protein production considerations. Building upon the outcomes of the Specific Aims, we will assess these aptamer-based bioanalytical sensors against various Beta-coronavirus pseudoviruses following the construction of these tools. The proposed studies hold substantial potential for determining the efficacy of universal anti-Beta-coronavirus aptamers and lay the foundation for designing universal bioanalytical tools against diverse Beta-coronaviruses. Furthermore, our research on discovering universal aptamer tools targeting the highly conserved S2 domain can expand the scope of aptamer-based research, facilitate the development of bioanalytical sensors for molecular diagnostics of SARS-CoV-3, and provide valuable insights for the design of future anti-SARS-CoV-3 antibodies and vaccines that target conserved S2 domains.
