Validating a NanoBiT protein complementation assay using an transfected NanoLuc-CyOFP HEK93T cell line?

Your concern is valid, as the components of the NanoBit optimized reporter are indeed structurally distinct from Nanoluc. Therefore, using an NanoLuc-CyOFP HEK293T cell line to validate the protein complementation assay may not necessarily prove that your compounds are not interacting with the NanoLuc fragments in the initial assay.

The issue arises because both NanoLuc and NanoBit are based on the luciferase enzyme, which has a certain degree of structural similarity between its different variants. This means that interactions between your compounds and the NanoLuc fragments in the initial assay could potentially also occur with the NanoLuc-CyOFP fusion protein used in the validation assay, even though they are structurally distinct.

To ensure that your compounds are truly specific to the target protein-protein interaction and not interacting with other luciferase variants, it would be best to use a different protein-protein interaction system in the validation assay. As you mentioned, using a well-known protein complex from a different pathway would be ideal, but this may not be feasible given your current constraints.

In light of this, here are some alternative strategies you could consider:

Use a different luciferase variant: Instead of using NanoLuc-CyOFP, you could try using a different luciferase variant, such as firefly luciferase or Renilla luciferase, to validate the protein complementation assay. Although these variants still share some structural similarities with NanoLuc, they are less likely to interact with your compounds in the same way.

Modify the NanoLuc-CyOFP fusion protein: You could modify the NanoLuc-CyOFP fusion protein to reduce its similarity to the luciferase variants used in the initial assay. For instance, you could introduce mutations or deletions in the luciferase domain that abolish its enzymatic activity or change its conformation, making it less likely to interact with your compounds.

Increase the stringency of the validation assay: To minimize false positives, you could optimize the conditions of the validation assay to make it more stringent. For example, you could use higher concentrations of your compounds, longer incubation times, or lower temperatures to reduce non-specific binding.

Use multiple validation assays: To further confirm the specificity of your compounds, you could perform multiple validation assays using different methods or targets. For instance, you could test your compounds against a different protein-protein interaction system or use a biochemical assay to measure their affinity for the target protein.

Use computational tools: Finally, you could use computational tools such as molecular dynamics simulations or docking algorithms to predict the binding mode of your compounds to the target protein and assess their potential interactions with other luciferase variants. While this approach cannot replace experimental validation, it can provide valuable insights into the potential mechanisms underlying your observations.

In summary, while using an NanoLuc-CyOFP HEK93T cell line for validation may not be perfect, there are ways to mitigate the limitations of this approach and increase the confidence in your results. By combining multiple lines of evidence and optimizing your validation assays, you can strengthen your conclusions about the specificity of your compounds.

All the best

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