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NanoLuc-mNeonGreen

NanoLuc is a deep-sea shrimp-derived luciferase, which is smaller compared to the Firefly and Renilla luciferases and therefore offers certain advantages over the traditional methods. NanoLuc has increased stability, smaller size and a >150-fold increase in luminescence (England, 2016). Furthermore, NanoLuc displays high physical stability, maintains its activity during incubation up to 55 ^oC or in culture medium for >15 h at 37 ^oC and shows no evidence of posttranslational modifications or subcellular partitioning in mammalian cells (Hall, 2012). Furimazine, NanoLuc’s substrate, shows increased stability and lower background activity which enhances the possibilities for bioluminescence imaging (England, 2016). Furimazine reacts with NanoLuc in the presence of oxygen. Furimazine is converted to Furimamide and normally a blue luminescent output occurs. This part consists of NanoLuc which is merged with mNeonGreen, a tetrameric fluorescent protein derived from the cephalochordate Branchiostoma lanceolatum. mNeonGreen is three to five times brighter than GFP and is more stable and less vulnerable to laser induced bleaching (Shaner, 2013). Because the emission spectrum of NanoLuc and the excitation spectrum of mNeonGreen overlap, bioluminescence resonance energy transfer (BRET) occurs and the green emission from mNeonGreen is observed instead, without further external illumination (figure 1). The closer the donor and acceptor are, the more efficient the BRET is. Therefore, the C terminus of mNeonGreen overlaps with the N terminus of NanoLuc, no linker is used. A His-tag in the front and a Strep-tag at the end are included for protein purification.

Figure 1. Schematic representation BRET between the donor NanoLuc and the acceptor mNeonGreen.

Usage and Biology

NanoLuc-mNeonGreen allows ratiometric measurements where NanoLuc-mNeonGreen acts as a calibrator luciferase to allow for time and concentration independent measurements. This is due to the fact that NanoLuc and NanoLuc-mNeonGreen both convert the same substrate and have a similar decay in intensity over time. This then allows for measurements that are more reliable than non-ratiometric measurements due to the independence of time and concentration. This can be implemented easily when performing measurements with NanoLuc and wishing to do ratiometric calibration measurements.

Characterization

Expression

NanoLuc-mNeonGreen was cloned into a pET28a (+) vector, subsequently expressed in BL21 (DE3) E. coli and purified using Ni-NTA affinity chromatography and Strep-Tactin purification. Expression was subsequently analyzed on SDS-PAGE (figure 2). This shows that after purification there is a clear blob just above 37 kDa for NanoLuc-mNeonGreen, which corresponds with the molecular weight of 6xHis-tagged and Strep-tagged NanoLuc-mNeonGreen. This indicates that protein expression and purification were successful for both proteins.

Figure 2. SDS-PAGE of NanoLuc-mNeonGreen (29 µM) after purification.

Luminescence measurement

The functionality was examined by measuring the luminescence upon addition of the substrate (Furimazine). The figure below clearly shows the energy transfer from NanoLuc (460 nm) to mNeonGreen (517 nm).

Figure 3. BRET spectrum NanoLuc-mNeonGreen.

To prove that time and concentration independent ratiometric measurements could be performed with NanoLuc-mNeonGreen as calibrator luciferase, measurements with different concentrations of NanoLuc and NanoLuc-mNeonGreen were executed as well as measurements over time (Figure 4 & 5). The measurements with varying concentrations of NanoLuc and the calibrator NanoLuc-mNeonGreen indicate that the ratio (being ±4.5) between these two is constant for different concentrations, showing that concentration independent measurements can be performed with use of the calibrator luciferase. The measurements over time indicate that the ratio (being ±3.5) between NanoLuc and the calibrator NanoLuc-mNeonGreen stays constant over time, enabling time independent measurements.

Figure 4. Luminescence intensity at emission maximum for NanoLuc (460 nm) and NanoLuc-mNeonGreen (517 nm) for increasing concentrations of both proteins.

Figure 5. Luminescence intensity at emission maximum for NanoLuc (460 nm) and NanoLuc-mNeonGreen (517 nm) over time.

NanoLuc-mNeonGreen as a calibrator luciferase

For these measurements, the intensity of the calibrator luciferase should be around 10% of the intensity of the luciferase, facilitating measurements of concentrations that are ten times as high as well as ten times as low. For our paired dCas9-Split-NanoLuc system, the concentration of NanoLuc-mNeonGreen needs to be around a thousandfold lower than the concentration of dCas9-Split-NanoLuc to achieve this 10% intensity as can be seen in Figure 6, where the intensity of 2 pM NanoLuc-mNeonGreen (at its maximum emission wavelength of 517 nm) corresponds with 10% of the intensity of 2 nM dCas9-Split-NanoLuc (at 460 nm).

Figure 6. Relative bioluminescence for dCas9-Split-NanoLuc and different concentrations of NanoLuc-mNeonGreen.

References

Arts, R., Aper, S. J., & Merkx, M. (2017). Engineering BRET-sensor proteins. In Methods in enzymology (Vol. 589, pp. 87-114). Academic Press.

England, C. G., Ehlerding, E. B., & Cai, W. (2016). NanoLuc: a small luciferase is brightening up the field of bioluminescence. Bioconjugate chemistry, 27(5), 1175-1187.

Hall, M. P., Unch, J., Binkowski, B. F., Valley, M. P., Butler, B. L., Wood, M. G., ... & Robers, M. B. (2012). Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS chemical biology, 7(11), 1848-1857.

Shaner, N.C., et al. (2013). A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods.  10(5), 407-409.

Sequence and Features