## 3 Conclusions
Even though two mutations of C11 are not on the CDRs, they provide structural modification such that more salt bridges can form between BA.5-C11. A previous study reported that salt bridges and hydrogen bonding interactions significantly enhanced the stability of the binding between synthetic Nbs and the flexible RBD loop [37]. As for K9 Nb, various point mutations as well as large differences in CDR1 and CDR3, facilitated interactions with more contact residues, enabling specific residues to interact with key amino acids of the RBD of BA.5 variant. Furthermore, prediction using molecular docking revealed that the K9 Nb binds to the RBD of JN.1 variant with the highest affinity. As SARS-CoV-2 is still rapidly evolving, as of August 20, 2024, the SARS-CoV-2 Omicron variants KP.2, KP.2.3, KP.3, KP.3.1.1, and LB.1, all descendants of the JN.1 variant, have a high prevalence in the United States. [38]  
It should be noted that the binding of Nb to RBD is complex. It is mediated by a large number of interactions that remodel the way protein complexes react to molecular forces in cellular environments. [PERSON] et al. [39]. demonstrated the mechanical stability of mutated nanobodies that interact with the RBD of the Delta and XBB.1.5 variants under simulated biophysical conditions, using molecular dynamics simulations. They reported that the transition from the bound to unbound state occurs without unfolding, driven by a balance of forces that both strengthen and weaken the protein interface. Many of these forces were nonpolar, highlighting the importance of hydrophobic interactions in maintaining the stability of the complex. Their finding strongly supports our results, which also show that nonpolar and aromatic amino acids at the contact surface between the RBD and the nanobody play a key role in stabilizing the interaction. Further study should be performed to investigate interactions between the obtained Nbs and RBD variants under biophysical conditions, similar to the study performed by [PERSON] et al. [40], using spike protein expressed on a mammalian cell line. In this study, we demonstrated the use of directed evolution by error-prone PCR to create mutants and the FLI-TRAP method to facilitate the isolation of Nbs that bind to a closely related specific target by addressing a viral variant with several mutations. The FLI-TRAP selection method offers a dual advantage, as the selection is based on the Nb's function, thus assessing its solubility and binding affinity simultaneously, guaranteeing that both characteristics are preserved. Recently, another clever method called 'autonomous hypermutation yeast surface display' (AHEAD) was developed for the molecular evolution of Nbs [41]. It utilizes yeast cells to mimic vertebrate somatic hypermutation by combining orthogonal DNA replication (OrthoRep) with yeast surface display (YSD) for rapid antibody evolution. The process involves an orthogonal error-prone DNA polymerase that replicates a special cytosolic plasmid (p1) encoding antibody fragments, thus allowing yeast cells to autonomously diversify YSD Nbs as they are cultured. High-affinity, high-quality Nbs were isolated through several cycles of yeast growth and fluorescence-activated cell sorting (FACS)-based selection. The main advantage of the AHEAD method is that the yeast cells can self-diversify Nb sequences. Since in vitro library construction is not required, several Nbs can be easily evolved at the same time. However, the selection method requires a flow cytometer with sorting capabilities, which is expensive and requires skilled operators [42]. Although our method requires in vitro mutated library construction, the in vivo selection method is straight forward, low-cost, and can be performed in most molecular biology laboratories. We expect that our strategy will be valuable in developing Nbs for various applications. Unfortunately, the SARS-CoV-2 virus has proven to be difficult to combat due to its very fast mutation rate.