Molecular Sciences II.
Bálint Kiss1, Dominik Sziklai1, Luca Elizabet Kosik1, Zoltán Kis2, Hedvig Tordai1, Miklós Kellermayer1
1 Semmelweis University, Department of Biophysics and Radiation Biology, Budapest
2 National Biosafety Laboratory, National Public Health Center, Budapest
Viruses have put major pressure on our healthcare system during the COVID-19 pandemic. Even though vaccines are highly effective in prevention, certain “escape” variants can still pose a threat. By utilizing single particle approaches combined with the development of a molecular handle, our goal is to gain better understanding of the nanomechanics of the SARS-CoV-2 infection process. In our approach we have combined fluorescent imaging with either atomic force microscopy (AFM) or optical tweezers. Using immunofluorescence, we have effectively identified SARS-CoV-2 infected cell cultures and determined the infection efficiency. Successfully infected cell monolayers were loaded into the AFM, which can provide high resolution images of individual cells. In these images we have identified cell adhered SARS-CoV-2 virions in the midst of infection. Since our long-term goal is to uncover the nanomechanics of the infection process we have developed a multipurpose molecular handle, which can enable the manipulation of individual virions. First a short (30 bp ~ 10 nm), sticky ended DNA oligomer was designed, its opposing end was activated using a heterobifunctional crosslinker. Through this crosslinker, individual proteins of choice, in our case antibodies were covalently bound to the activated end of the DNA. We have optimized the production of these molecular handles, which can later be used to functionalize AFM tips allowing us to study virus-host interactions. Such DNA handles, although practical for AFM tip functionalization are rather short; thus, they cannot be effectively utilized in optical tweezer experiments. To overcome this issue, we have created longer (9500 bp ~ 3.2 μm) biotinylated DNA fragments by polymerase chain reaction (PCR), which can be hybridized and ligated to the short, functionalized oligomers via their compatible sticky ends. The strength of these long-range DNA handles was tested with optical tweezers, combined with fluorescent microscopy imaging. The development of variable length molecular handles provides a tool for single virion-host interaction assays enabling the characterization of SARS-CoV-2 infection either by AFM or by optical tweezers.