2.1 TetR DNA binding protein

The tetracycline repressor protein (tetR) is naturally present in gram-negative bacteria and is involved in the resistance mechanism against tetracycline (and derivatives). It does so by tightly controlling the gene expression of tetA, which encodes an efflux pump responsible for removing tetracycline from the cell. TetR binds selectively to two plaindromic recognition sequences (tetO>1,2) with high affinity. For DNA binding to occur tetR adopts a homodimeric structure and binds with two α-helix-turn- α-helix motifs (HTH) to two tandemly oriented tetO sequences. In the presence of tetracycline or its analogs, tetR undergoes a conformational change, which prevents it from binding to DNA, therby allowing gene expression(Orth et al. 2000; Kisker et al. 1995).
Due to its robust and highly regulatable DNA-binding properties, tetR has become a widely adopted tool in synthetic biology. Its ease of modification and ability to function in both prokaryotic and eukaryotic systems have made it an essential element in the development of gene regulation systems (Berens & Hillen, 2004).
In our project, tetR was integrated into the design of a modular DNA-stapling system because of its well-characterized behavior, ensuring reliable DNA interactions.

2.2mScarlet-I

mScarlet-I is a rapidly-maturing monomeric red fluorescent protein designed by Bindels et al. in 2016. It is a derivative of the original mScarlet, with a single amino acid substitution (T74I) that enhances its maturation speed and fluorescence properties (Bindels et al., 2016). mScarlet-I absorbs at 569 nm and emits at 594 nm.

mScarlet-I stands out among red fluorescent proteins for its high quantum yield (0.54), long fluorescence lifetime (3.1 ns), and rapid maturation time of approximately 36 minutes. These features ensure strong fluorescence intensity and make it an ideal candidate for Förster Resonance Energy Transfer assays (McCullock et al. 2020). When paired with mNeonGreen, mScarlet-I

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