1. Sequence overview

2. Usage and Biology

2.3 Förster Resonance Energy Transfer (FRET)

Förster Resonance Energy Transfer (FRET) is a distance-dependent physical process where energy is transferred non-radiatively from an excited donor fluorophore to an acceptor fluorophore via dipole-dipole coupling. The efficiency of energy transfer is highly sensitive to the distance between the donor and acceptor, typically in the range of 1-10 nm, making FRET ideal for studying molecular proximity (Hochreiter et al., 2019). This proximity sensitivity is governed by the Förster radius (R₀), which is the distance at which 50% energy transfer occurs. Factors affecting FRET efficiency include the overlap of the donor's emission spectrum with the acceptor's absorption spectrum, the quantum yield of the donor, and the relative orientation of the fluorophores (Wu & Brand, 1994). These characteristics allow FRET to detect interactions such as protein-DNA binding or DNA proximity in real time. For our assay, we selected mNeonGreen and mScarlet-I as donor and acceptor, respectively, due to their strong fluorescence, spectral overlap, and minimal photobleaching, ensuring high FRET efficiency in our system (Bindels et al., 2017; Shaner et al., 2013). FRET's sensitivity to small changes in distance makes it especially powerful for analyzing molecular interactions in living cells (Okamoto & Sako, 2017).

Figure 2: Overview of excitation and emission spectrum of mNeonGreen and m-Scarlet and it's properties as a FRET pair

3. Assembly and part evolution

The amino acid sequence of mNeonGreen was taken from the fluorescent protein database (FPbase) and codon optimized for use in E. coli. It was fused to thhe N-terminus of Oct1-DBD (BBa_K52347004) for protein purification of Oct1-DBD and in vivo FRET measurements.

4. Results

The FRET assay was developed using a two-plasmid system in bacterial cells. The expression plasmid contains a tetR binding site (BBa_K5237019) and expresses three key proteins under the control of a single T7 promoter in a polycistronic operon: (1) tetR-Oct1, our simple staple fusion protein that acts as a bivalent DNA-binding protein, tethering two plasmids via tetR and Oct1 binding sites; (2) Oct1-mNeonGreen, serving as the FRET donor; and (3) tetR-mScarlet-I, the FRET acceptor. This ensures all three proteins are co-expressed in similar stoichiometry. The folding plasmid contains an Oct1 binding site (BBa_K5237018) for the staple and FRET donor binding.

When tetR-Oct1 binds its respective sites on both plasmids, it brings mNeonGreen and mScarlet-I into close proximity, facilitating FRET between the two fluorophores. Successful stapling of the plasmids results in increased energy transfer from mNeonGreen to mScarlet-I, which can be detected by exciting mNeonGreen and measuring enhanced emission from mScarlet-I. A positive control, consisting of a direct fusion of mNeonGreen and mScarlet-I, ensures maximal FRET efficiency and serves as a benchmark for the assay.

Fluorescence intensity, normalized to cell count, of mNeonGreen and mScarlet-I was measured 18 h after induction with varying IPTG concentration (Figure 3). An increasing expression strength is visible for decreasing IPTG concentrations. Fluorescence intensity of the positive control was significantly stronger compared to the negative control and staple. The negative control and staple, which both have the same expression plasmid construct, had similar fluorescence intensity for mNeonGreen and mScarlet-I down to approximately 0.05 mM. Lower concentrations resulted in strong discrepancies. To ensure comparability between the negative control and staple, further fluorescence intensity measurements were conducted after induction with 0.05 mM IPTG. Fluorescence measurement of the donor and acceptor showed similar intensities, with only a small significant difference for mNeonGreen. A large difference could be measured between the staple and negative control, indicating proximity induced FRET. (Figure 3)

Figure 3: Fluorescence of mNeonGreen, mScarlet-I and FRET. Fluorescence intensity of mNeonGreen (ex. 490 nm, em. 530 nm), mScarlet-I (ex. 560 nm, em. 600 nm), and FRET (ex. 490 nm, em. 600 nm) was measured 18 h after induction with IPTG and normalized to cell count (OD₆₀₀). A), B) Fluorescence intensity of mNeonGreen and mScarlet-I after induction with different IPTG concentrations. C) Fluorescence intensity of FRET pair induced with 0.025 mM IPTG. Statistical significance was tested for with Ordinary two-way ANOVA with Šidák's multiple comparison test, with a single pooled variance. *p < 0.05, ****p < 0.001. Only significant results, within groups are shown. Data is depcited as mean ± SD.

5. Conclusion

Our FRET assay successfully demonstrated the proximity of two DNA strands in living cells using mNeonGreen and mScarlet-I as a donor-acceptor pair. The assay was optimized for maximal FRET efficiency and validated with a positive control. The results showed a significant difference in fluorescence intensity between the staple and negative control, indicating successful DNA stapling and FRET. This assay provides a powerful tool to engineer and test out novel Staples. Future work will focus on further optimizing the assay in mammalian cells and quantifying interactions.

6. References

Bindels, D. S., Haarbosch, L., van Weeren, L., Postma, M., Wiese, K. E., Mastop, M., Aumonier, S., Gotthard, G., Royant, A., Hink, M. A., & Gadella, T. W. J. (2017). mScarlet: A bright monomeric red fluorescent protein for cellular imaging. Nature Methods, 14(1), 53–56. https://doi.org/10.1038/nmeth.4074

Hochreiter, B., Kunze, M., Moser, B., & Schmid, J. A. (2019). Advanced FRET normalization allows quantitative analysis of protein interactions including stoichiometries and relative affinities in living cells. Scientific Reports, 9(1), 8233. https://doi.org/10.1038/s41598-019-44650-0

Lundbäck, T., Chang, J.-F., Phillips, K., Luisi, B., & Ladbury, J. E. (2000). Characterization of Sequence-Specific DNA Binding by the Transcription Factor Oct-1. Biochemistry, 39(25), 7570–7579. https://doi.org/10.1021/bi000377h

Okamoto, K., & Sako, Y. (2017). Recent advances in FRET for the study of protein interactions and dynamics. Current Opinion in Structural Biology, 46, 16–23. https://doi.org/10.1016/j.sbi.2017.03.010

Park, J. H., Kwon, H. W., & Jeong, K. J. (2013). Development of a plasmid display system with an Oct-1 DNA-binding domain suitable for in vitro screening of engineered proteins. Journal of Bioscience and Bioengineering, 116(2), 246–252. https://doi.org/10.1016/j.jbiosc.2013.02.005

Park, Y., Shin, J., Yang, J., Kim, H., Jung, Y., Oh, H., Kim, Y., Hwang, J., Park, M., Ban, C., Jeong, K. J., Kim, S.-K., & Kweon, D.-H. (2020). Plasmid Display for Stabilization of Enzymes Inside the Cell to Improve Whole-Cell Biotransformation Efficiency. Frontiers in Bioengineering and Biotechnology, 7. https://doi.org/10.3389/fbioe.2019.00444

Shaner, N. C., Lambert, G. G., Chammas, A., Ni, Y., Cranfill, P. J., Baird, M. A., Sell, B. R., Allen, J. R., Day, R. N., Israelsson, M., Davidson, M. W., & Wang, J. (2013). A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nature Methods, 10(5), 407–409. https://doi.org/10.1038/nmeth.2413

Stepchenko, A. G., Portseva, T. N., Glukhov, I. A., Kotnova, A. P., Lyanova, B. M., Georgieva, S. G., & Pankratova, E. V. (2021). Primate-specific stress-induced transcription factor POU2F1Z protects human neuronal cells from stress. Scientific Reports, 11(1), 18808. https://doi.org/10.1038/s41598-021-98323-y

Wu, P. G., & Brand, L. (1994). Resonance Energy Transfer: Methods and Applications. Analytical Biochemistry, 218(1), 1–13. https://doi.org/10.1006/abio.1994.1134