SPR to identify enzymes for COVID RNA

The proposed experiment centers around isolating an ideal phosphatase enzyme that would prevent phosphorylation of the nucleocapsid of COVID-19. The reasoning behind targeting this component of the virus is that phosphorylation of the capsid protein is thought to regulate the proper folding of the viral RNA and therefore has significant impact on viral gene expression. Surface plasmon resonance (SPR) is utilized to assess a library of phosphatase enzymes that would be able to dephosphorylate the capsid protein and test the kinetics of the molecular response of viral RNA to the protein after it has been exposed to the library. As a control, this experiment also includes a native phosphorylated RNA binding protein (pRBP) which is tested for its sensitivity to the library. The goal is to isolate a biomolecule that will only target the viral capsid protein while leaving everything else untouched.

Documented outbreaks of coronaviruses demonstrate that this family encapsulates a range of respiratory viruses including SARS and MERS. COVID-19 shows significant overlap with SARS and MERS structures which serves the source of inspiration for a miracle drug. The problem is how to identify a drug that would target the essential nucleocapsid proteins of the coronavirus. This solution is an investigation into the role of phosphorylation of N protein as it is recognized by the viral RNA of COVID-19.

Recognizing viral genomic RNA by the virus-encoded N protein for RNA synthesis and proper RNA folding is fundamental to the virus life cycle. Phosphorylation of N protein is thought to regulate many of these events. Thus, this solution aims to create a drug using an ideal phosphatase that would undo phosphorylation of N protein which would disrupt synthesis and proper folding of the molecule, ideally rendering the virus inactive. Phosphorylation affects binding affinity by altering charge. Using SPR, this experiment relies on differences in affinity between phosphorylated and unphosphorylated nucleocapsid (N protein) of COVID-19 to alter viral RNA regulation. We test various phosphatase enzymes designed specifically for viral RNA that would not disrupt normal phosphorylation metabolism already present in the body.

The aim of this experiment is to assess a library of phosphatases via SPR to determine which of those best dephosphorylates and therefore lowers the binding affinity of nucleocapsid (N protein) when it binds to viral RNA. The first step in the procedure is to obtain a source of phosphorylated N protein and a native phosphorylated RNA binding protein (pRBP). The capsid protein is purified from a mucous sample of coronavirus. Ammonium sulfate and anion exchange chromatography is used to separate the protein from the rest of the contaminant proteins. This method relies on exploiting the capsids innate ability to polymerize and depolymerize in vitro. Similarly, pRBP is obtained by affinity chromatography. Affinity chromatography under denaturing conditions is combined with subsequent on-column refolding, to prevent self-association of pRBP while removing any contaminants from the end product.

 It is important to keep in mind that although two different RNA’s are being tested (viral and native human) we are looking for a change in binding affinity for each respective RNA binding protein. The goal is to find the right phosphatase enzyme, whether natural or synthetic, that will dephosphorylate the viral nucleocapsid protein but not the native pRBP.

This is one of the numerous ways in which laboratories around the world are working to find magic bullets for the COVID-19 pandemic. This solution in particular is a creative experimental approach to isolating the fickle nature of this virus. This pitch serves as another contribution to the brain storm currently sweeping over every biochemistry lab in the world to stop the pandemic. 

The data that will be collected consists of the molecular binding interactions of the nucleocapsid of COVID-19 and the immobilized viral RNA. This solution collects the kinetics of molecular binding events between RNA and (dephosphorylated) protein. It can be observed by monitoring the change in SPR response over time. For each combination of protein and enzyme (or the null without any added enzyme) the ratio of Ka and Kd is determined and this information explains whether or not the enzyme alters the binding affinity for the RNA on the sensor chip.

Surface plasmon resonance (SPR) has been around for a long time in many biochemical labs. SPR technology is frequently used to study molecular binding interactions of free-floating analytes in solution and probe molecules (usually probe molecules either linked to or immobilized onto the sensor surface). Using this pre-existing technique, the proposition aims to identify the best phosphatase that will affect viral RNA enzymes while leaving all other enzymes alone. 

Both N protein and pRBP are introduced to a library of 20 different phosphatases for 2 hours, at physiological temperature and pH. Then, one by one, each solution of protein (viral and native) and each variation of phosphatase enzyme are prepped for immobilization for their respective RNA sample. The library of phosphatase enzymes comes from the available list at Sigma Aldrich: wheatgerm, cow intestine, potato, shrimp, human placenta, e. Coli, mouse, and goat. 

Surface plasmon resonance (SPR) is a powerful optical detection technique for studying label free biomolecular interactions in real time. Its simplicity, every SPR instrument uses a Kretschmann Configuration- a setup whereby a thin metal film is evaporated onto a glass block. Then, the ligand-capture molecule (in this case, the RNA sample) is immobilized onto the metal. Based on the majority of SPR publications, the most common strategy for immobilization is chemical coupling with amine which creates a stable covalent bond between the ligand and surface. The analyte (phosphorylated or dephosphorylated RNA binding protein) is introduced in the liquid phase and will potentially bind to the immobilized ligand. When light hits the block, an ephemeral wave penetrates the thin metal film and the plasmons (resonating electrons) are subsequently excited on the outer side of the film.[1] When the light reflects off the backside of the sensor chip surface (the metal film) it hits a detector. At a certain incident resonance angle, light is absorbed by the electrons in the metal film, causing them to resonate. The surface plasmons are sensitive to the environment. An intensity loss due to the plasmons appears as a dark band which translates to a dip in the SPR intensity curve (where the x axis is the change in resonance angle and the y axis is % SPR reflection intensity). The shape and location of the dip translates to new info obtained about the sensor’s surface.[2]

Chen, H., Gill, A., Dove, B. K., Emmett, S. R., Kemp, C. F., Ritchie, M. A., … Hiscox, J. A. (2004). Mass Spectroscopic Characterization of the Coronavirus Infectious Bronchitis Virus Nucleoprotein and Elucidation of the Role of Phosphorylation in RNA Binding by Using Surface Plasmon Resonance. Journal of Virology, 79(2), 1164–1179. doi: 10.1128/jvi.79.2.1164-1179.2005

https://www.ncbi.nlm.nih.gov/pubmed/15613344

This is a source of inspiration from an older outbreak and a separate family of coronaviruses in 2005.

This solution, if given the chance, can serve everyone affected by COVID-19. So, the whole world.

1. This solution could theoretically reach everyone around the globe in need. 

2. The amount of time necessary would likely be a year or two.

3. In five years, the number of people served could be in the millions. 

Using this approach, I hope to contribute to the litany of research currently underway in the era of the coronavirus pandemic. 

I am a graduate from the University of Texas at Austin. I have a BA in Plan II Honors and a BSA in Biochemistry. This is the brainchild of my time spent in several biochemistry labs using SPR as a way to target proteins from different strains of viruses.

I am trying to find a phosphatase or a set of phosphatases that will target capsid proteins from viral RNA from coronavirus but will leave all other human RNA proteins untouched. The goal is to render viral N proteins unphosphorylated in order to interrupt proper regulation and folding which inactivates the virus as a whole. This is achieved using sensory technology like SPR.

I hope this idea can make it into biolabs across the world. Perhaps capsid proteins need more testing!