Half staple: Oct1-DBD

Oct1-DBD is the DNA-binding domain of the human Oct1 transcription factor. It can be readily fused with other DNA-binding proteins to form a functional staple to DNA-DNA proximity. We used this part as a component for our Simple staple (BBa_K5237006) and also fused to mNeonGreen, as part of a FRET readout system (BBa_K5237016).

While synthetic biology has in the past focused on engineering the genomic sequence of organisms, the 3D spatial organization of DNA is well-known to be an important layer of information encoding in particular in eukaryotes, playing a crucial role in gene regulation and hence cell fate, disease development, evolution, and more. However, tools to precisely manipulate and control the genomic spatial architecture are limited, hampering the exploration of 3D genome engineering in synthetic biology. We - the iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular toolbox for rationally engineering genome 3D architectures in living cells, based on various DNA-binding proteins.

The PICasSO part collection offers a comprehensive, modular platform for precise manipulation and re-programming of DNA-DNA interactions using engineered "protein staples" in living cells. This enables researchers to recreate naturally occurring alterations of 3D genomic interactions, such as enhancer hijacking in cancer, or to design entirely new spatial architectures for artificial gene regulation and cell function control. Specifically, the fusion of two DNA binding proteins enables to artificially bring otherwise distant genomic loci into spatial proximity. To unlock the system's full potential, we introduce versatile chimeric CRISPR/Cas complexes, connected either at the protein or - in the case of CRISPR/Cas-based DNA binding moieties - the guide RNA level. These complexes are referred to as protein- or Cas staples, respectively. Beyond its versatility with regard to the staple constructs themselves, PICasSO includes robust assay systems to support the engineering, optimization, and testing of new staples in vitro and in vivo. Notably, the PICasSO toolbox was developed in a design-build-test-learn engineering cycle closely intertwining wet lab experiments and computational modeling and iterated several times, yielding a collection of well-functioning and -characterized parts.

At its heart, the PICasSO part collection consists of three categories.
(i) Our DNA-binding proteins include our finalized Cas staple experimentally validated using an artificial "enhancer hijacking" system as well as "half staples" that can be combined by scientists to compose entirely new Cas staples in the future. We also include our Simple staples comprised of particularly small, simple and robust DNA binding domains well-known to the synthetic biology community, which serve as controls for successful stapling and can be further engineered to create alternative, simpler, and more compact staples.
(ii) As functional elements, we list additional parts that enhance and expand the functionality of our Cas and Basic staples. These consist of staples dependent on cleavable peptide linkers targeted by cancer-specific proteases or inteins that allow condition-specific, dynamic stapling in vivo. We also include several engineered parts that enable the efficient delivery of PICasSO's constructs into target cells, including mammalian cells, with our new interkingdom conjugation system.
(iii) As the final category of our collection, we provide parts that underlie our custom readout systems. These include components of our established FRET-based proximity assay system, enabling users to confirm accurate stapling. Additionally, we offer a complementary, application-oriented testing system based on a luciferase reporter, which allows for straightforward experimental assessment of functional enhancer hijacking events in mammalian cells.

The following table gives a comprehensive overview of all parts in our PICasSO toolbox. The highlighted parts showed exceptional performance as described on our iGEM wiki and can serve as a reference. The other parts in the collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their own custom Cas staples, enabling further optimization and innovation in the new field of 3D genome engineering.

Our part collection includes:

DNA-Binding Proteins: Modular building blocks for engineering of custom staples to mediate defined DNA-DNA interactions in vivo
BBa_K5237000	Fusion Guide RNA Entry Vector MbCas12a-SpCas9	Entry vector for simple fgRNA cloning via SapI
BBa_K5237001	Staple Subunit: dMbCas12a-Nucleoplasmin NLS	Staple subunit that can be combined with crRNA or fgRNA and dSpCas9 to form a functional staple
BBa_K5237002	Staple Subunit: SV40 NLS-dSpCas9-SV40 NLS	Staple subunit that can be combined with a sgRNA or fgRNA and dMbCas12a to form a functional staple
BBa_K5237003	Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS	Functional Cas staple that can be combined with sgRNA and crRNA or fgRNA to bring two DNA strands into close proximity
BBa_K5237004	Staple Subunit: Oct1-DBD	Staple subunit that can be combined to form a functional staple, for example with TetR. Can also be combined with a fluorescent protein as part of the FRET proximity assay
BBa_K5237005	Staple Subunit: TetR	Staple subunit that can be combined to form a functional staple, for example with Oct1. Can also be combined with a fluorescent protein as part of the FRET proximity assay
BBa_K5237006	Simple Staple: TetR-Oct1	Functional staple that can be used to bring two DNA strands in close proximity
BBa_K5237007	Staple Subunit: GCN4	Staple subunit that can be combined to form a functional staple, for example with rGCN4
BBa_K5237008	Staple Subunit: rGCN4	Staple subunit that can be combined to form a functional staple, for example with rGCN4
BBa_K5237009	Mini Staple: bGCN4	Assembled staple with minimal size that can be further engineered
Functional Elements: Protease-cleavable peptide linkers and inteins are used to control and modify staples for further optimization for custom applications
BBa_K5237010	Cathepsin B-cleavable Linker: GFLG	Cathepsin B-cleavable peptide linker that can be used to combine two staple subunits to make responsive staples
BBa_K5237011	Cathepsin B Expression Cassette	Expression cassette for the overexpression of cathepsin B
BBa_K5237012	Caged NpuN Intein	A caged NpuN split intein fragment that undergoes protein trans-splicing after protease activation, which can be used to create functionalized staple subunits
BBa_K5237013	Caged NpuC Intein	A caged NpuC split intein fragment that undergoes protein trans-splicing after protease activation, which can be used to create functionalized staple subunits
BBa_K5237014	Fusion Guide RNA Processing Casette	Processing cassette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D genome reprogramming
BBa_K5237015	Intimin anti-EGFR Nanobody	Interkingdom conjugation between bacteria and mammalian cells, as an alternative delivery tool for large constructs
BBa_K4643003	IncP Origin of Transfer	Origin of transfer that can be cloned into the plasmid vector and used for conjugation as a means of delivery
Readout Systems: FRET and enhancer recruitment readout systems to rapidly assess successful DNA stapling in bacterial and mammalian cells
BBa_K5237016	FRET-Donor: mNeonGreen-Oct1	FRET donor-fluorophore fused to Oct1-DBD that binds to the Oct1 binding cassette, which can be used to visualize DNA-DNA proximity
BBa_K5237017	FRET-Acceptor: TetR-mScarlet-I	Acceptor part for the FRET assay binding the TetR binding cassette, which can be used to visualize DNA-DNA proximity
BBa_K5237018	Oct1 Binding Casette	DNA sequence containing 12 Oct1 binding motifs, compatible with various assays such as the FRET proximity assay
BBa_K5237019	TetR Binding Cassette	DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET proximity assay
BBa_K5237020	Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64	Readout system that responds to protease activity, which was used to test cathepsin B-cleavable linker
BBa_K5237021	NLS-Gal4-VP64	Trans-activating enhancer, that can be used to simulate enhancer hijacking
BBa_K5237022	mCherry Expression Cassette: UAS, minimal Promoter, mCherry	Readout system for enhancer binding, which was used to test cathepsin B-cleavable linker
BBa_K5237023	Oct1 - 5x UAS Binding Casette	Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay
BBa_K5237024	TRE-minimal Promoter- Firefly Luciferase	Contains firefly luciferase controlled by a minimal promoter, which was used as a luminescence readout for simulated enhancer hijacking

1. Sequence overview

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

2. Usage and Biology

Oct1-DBD is the DNA-binding domain of the human transcription factor Oct1 (POU2F1), which plays a key role in gene regulation, immune response, and stress adaptation in eukaryotic cells. This domain specifically binds to the octamer motif (5'-ATGCAAAT-3') within promoter and enhancer regions, influencing transcriptional activity (Lundbäck et al., 2000). The Oct1-DBD consists of both a POU-specific domain and a POU homeodomain, which work together to form a stable complex with DNA (Park et al., 2013, Stepchenko et al. 2021).

In synthetic biology, Oct1-DBD has been previously used for plasmid display technology due to its strong binding affinity (K_D = 9 × 10^-11 M), additionally proteins fused with Oct1-DBD showed increased expression and protein solubility (Parker et al. 2020).

This part was further used in BBa_K5237002 as a fusion with tetR, resulting in a bivalent DNA binding staple, and also fused to mNeonGreen, as part of a FRET readout system (BBa_K5237016).

3. Assembly and Part Evolution

The Oct1-DBD amino acid sequence was obtained from UniProt (P14859, POU domain, class 2, transcription factor 1) and the DNA binding domain was extracted based on information given from Park et al. 2013 & 2020. An E. coli codon optimized DNA sequence was obtained through gene synthesis and used to clone further constructs.

4. Results

4.1 Protein Expression and EMSA

Oct1 was N-terminally fused to His6-mNeonGreen. This fusion protein was expressed under a T7 promoter and subsequently purified using metal affinity chromatography with Ni-NTA beads (Fig. 2, left). DNA binding affinity was estimated with an electrophoretic mobility shift assay (EMSA). For this, three different buffer conditions were tested (Binding buffer 1: 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂HPO₄, 0.1 % (v/v) IGEPAL® CA-360, 1 mM EDTA; Binding buffer 2: 10 mM Tris, 50 mM KCl; NaP250: Na₂HPO₄, 150 mM NaCl, 250 mM Imidazol). DNA binding could only be detected for Binding buffer 1. (Fig. 2, right)

Figure 2: Expression and DNA Binding Analysis of His₆-mNeonGreen-Oct1-DBD.
Left image:Lane 1: raw lysate of E. coli expression culture after sterile filtration; Lane 2: Flow through of first wash (10 bed volumes of NaP10 (Na₂HPO₄, 150 mM NaCl, 10 mM Imidazol)); Lane 3: Flow through of second wash (10 bed volumes of NaP20 (Na₂HPO₄, 150 mM NaCl, 20 mM Imidazol)); Lane 4: Elution of purified protein.
1 µL of each fraction was loaded after mixing and heating with 4x Laemmli buffer, on a 4-15% TGX-Gel. The expected band size of the protein is 56 840.23 Da, highlighted in red on the gel.
Right image Purified mNeonGreen-Oct1 fusion-protein (1000 nM, 100 nM or 10 nM) were equilibrated with 0.5 µM DNA, containing three Oct1 binding sites, in different buffer compositions. (Binding buffer 1: 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2HPO4, 1.8 mM KH2HPO4, 0.1 % (v/v) IGEPAL® CA-360, 1 mM EDTA; Binding buffer 2: 10 mM Tris, 50 mM KCl; NaP250: 50 mM NaH2PO4, 150 mM NaCl, 250 mM Imidazol) Bands were visualized with SYBR-Safe staining.

4.2 In Silico Characterization using DaVinci

We developed the in silico model DaVinci for rapid engineering and development of our PICasSO system. DaVinci acts as a digital twin to PICasSO, designed to understand the forces acting on our system, refine experimental parameters, and find optimal connections between protein staples and target DNA. We calibrated DaVinci with literature and our own experimental affinity data calculated from EMSA assays with purified proteins DaVinci is divided into three phases: static structure prediction, all-atom dynamics simulation, and long-ranged DNA dynamics simulation. We applied the first two to our parts, characterizing structure and dynamics of the DNA-binding interaction.
The structures shown in Figure 4 were predicted using the AlphaFold server and the protein-DNA interaction further analyzed with all atom dynamics simulations. The depicted structures show favorable DNA binding, and no apparent problems with the fusion protein and DNA binding were detected.

Figure 4: Representations of the Simple Staple Constructs Proteins are shown in full color (top row) and by their predicted structural accuracy during DNA interaction. The linkers were selected based on their structural property providing maximal flexibility. All structures were predicted using the AlphaFold server (Google DeepMind, 2024).

5. References

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

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

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

Part:BBa_K5237004