Part:BBa_K5237009
Mini Staple: bGCN4
The bGCN4 Mini staple is a fusion of the basic leucine zipper GCN4 and rGCN4, offering a smaller, compact protein staple. With its well-characterized subunits and strong in silico and experimental validation, this Mini staple serves as a versatile foundation for expanding to similar staples.
Contents
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 175
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Small basic-region leucine zipper (bZip) proteins are characterized by their DNA-binding. The bZip motif
consists of a coiled-coil leucine zipper dimerization domain, and a highly charged basic region that binds to DNA
(Hollenbeck & Oakley, 2000). One well characterized example is the General Control
Protein 4 (GCN4), a well-characterized transcriptional activator from yeast (Arndt & Fink, 1986).
The amino acid sequences for GCN4 and rGCN4 were obtained from literature (Hollenbeck et al. 2001), and
codon-optimized for Escherichia coli.
The two leucine zippers were combined with a GSG linker harbouring a BamHI site to adapt the construct with different
linker designs, based on our dry lab DaVinci
model.
A FLAG-tag (DYKDDDDK) was added to the N-terminus for protein purification. The FLAG-tag can be cleaved off using an
Enterokinase, if necessary.
Expression of FLAG-GCN4 was done under an IPTG inducible T7 promoter in E. coli BL21 (DE3) cells.
The current version of the part with GCN4-rGCN4 fusion, linked by a GSG peptide linker has not demonstrated any DNA
binding in the tests conducted thus far.
Nevertheless, we believe the part to still be a valuable addition, as it can be further engineered with different
linker types to
create a functioning staple. Through dry lab modelling, various linker designs have already been simulated to
predict improved dimerization and DNA binding.
The bZip proteins GCN4, rGCN4 and the fusion thereof, bGCN4, were fused N-terminally to a FLAG-tag (DYKDDDDK).
All proteins could be readily
expressed under the T7 promoter in E. coli BL21 DE3 and purified with Anti-FLAG affinity
columns. The purity of the proteins was confirmed by SDS-PAGE (Fig. 2).
The Electrophoretic mobility shift assay (EMSA) is a widely adopted method used to study DNA-protein
interactions. EMSA functions on the basis that nucleic acids, bound to proteins, have reduced electrophoretic
mobility, compared to their counterpart. (Hellman & Fried, 2007). Mobility-shift
assays can both be used to qualitatively assess DNA binding capabilities or quantitatively to determine binding
stoichiometry and kinetics such as the apparent dissociation constant (Kd) (Fried, 1989).
To asses possible DNA binding, a qualitative EMSA was performed with the purified proteins. The same binding
buffer conditions were used, as previously described for GCN4 and rGCN4 (Hollenbeck et al. 2001).
DNA binding could only be shown for GCN4 and rGCN4, no visible band was observed for the bGCN4 fusion protein
(Fig. 4).
The EMSA is a widely adopted method used to study DNA-protein
interactions. EMSA functions on the basis that nucleic acids, bound to proteins, have reduced electrophoretic
mobility, compared to their counterpart. (Hellman & Fried, 2007). Mobility-shift
assays can be used both to qualitatively assess DNA binding capabilities or quantitatively to determine binding
stoichiometry and kinetics such as the apparent dissociation constant (Kd) (Fried, 1989).
To further analyze DNA binding of the staple subunits, quantitative shift assays were performed for GCN4 and rGCN4. Here
0.5 µM DNA were incubated with varying concentrations of protein until equilibration. After
electrophoresis, bands were stained with SYBR-Safe and quantified based on pixel intensity. The
obtained values were fitted to equation 1, describing formation of a 2:1 protein-DNA complex:
GCN4 binds to its optimal DNA binding motif with an apparent dissociation
constant KD of (0.293 ± 0.033) × 10-6 M, which is almost identical to the
rGCN4 dissociation constant
to INVii a KD of (0.298 ± 0.030) × 10-6 M.
Comparing them to literature values, our dissociation constants are approximately a factor 10 higher then those
described in literature ((9±6) × 10-8 M for
GCN4 and (2.9 ± 0.8) × 10-8 M for rGCN4) (Hollenbeck et al., 2001). The
differences could be due to the lower sensitivity of SYBR-Safe staining compared to radio-labeled oligos.
Most likely, the protein concentration was miscalculated due to the presence of additional (lower intensity)
bands in
the SDS-PAGE analysis, indicating co-purification of small amounts of unspecific proteins.
We developed DaVinci, an in silico model, for rapid engineering and optimization of our PICasSO system. DaVinci
serves as a digital twin to PICasSO, analyzing the forces acting on the system, refining experimental parameters,
and identifying optimal interactions between protein staples and target DNA. The model was calibrated using
literature data and experimental affinity results from rGCN4 EMSA assays with purified proteins.
Akdel, M., Pires, D. E. V., Pardo, E. P., Janes, J., Zalevsky, A. O., Meszaros, B., Bryant, P., Good, L. L.,
Laskowski, R. A., Pozzati, G., Shenoy, A., Zhu, W., Kundrotas, P., Serra, V. R., Rodrigues, C. H. M., Dunham, A.
S., Burke, D., Borkakoti, N., Velankar, S., … Beltrao, P. (2022). A structural biology community assessment of
AlphaFold2 applications. Nat Struct Mol Biol, 29(11), 1056–1067. https://doi.org/10.1038/s41594-022-00849-w
Arai, R., Ueda, H., Kitayama, A., Kamiya, N., & Nagamune, T. (2001). Design of the linkers which effectively
separate domains of a bifunctional fusion protein. Protein Engineering, Design and Selection, 14(8),
529–532. https://doi.org/10.1093/protein/14.8.529 Arndt, K., & Fink, G. R. (1986). GCN4 protein, a positive transcription factor in yeast, binds general
control promoters at all 5’ TGACTC 3’ sequences. Proceedings of the National Academy of Sciences,
83(22),
8516–8520. https://doi.org/10.1073/pnas.83.22.8516 Chen, X., Zaro, J. L., & Shen, W.-C. (2013). Fusion protein linkers: Property, design and functionality.
Advanced Drug Delivery Reviews, 65(10), 1357–1369. https://doi.org/10.1016/j.addr.2012.09.039
Google DeepMind. (2024). AlphaFold Server. https://alphafoldserver.com/terms Guo, H.-B., Perminov, A., Bekele, S., Kedziora, G., Farajollahi, S., Varaljay, V., Hinkle, K., Molinero, V.,
Meister, K., Hung, C., Dennis, P., Kelley-Loughnane, N., & Berry, R. (2022). AlphaFold2 models indicate that
protein sequence determines both structure and dynamics. Scientific Reports, 12(1), 10696. https://doi.org/10.1038/s41598-022-14382-9
Ellenberger, T. E., Brandl, C. J., Struhl, K., & Harrison, S. C. (1992). The GCN4 basic region leucine
zipper
binds DNA as a dimer of uninterrupted alpha helices: Crystal structure of the protein-DNA complex. Cell,
71(7), 1223–1237. https://doi.org/10.1016/s0092-8674(05)80070-4 Hollenbeck, J. J., Gurnon, D. G., Fazio, G. C., Carlson, J. J., & Oakley, M. G. (2001). A GCN4 Variant with
a
C-Terminal Basic Region Binds to DNA with Wild-Type Affinity. Biochemistry, 40(46), 13833–13839. Hollenbeck, J. J., McClain, D. L., & Oakley, M. G. (2002). The role of helix stabilizing residues in GCN4
basic region folding and DNA binding. Protein Science, 11(11), 2740–2747. https://doi.org/10.1110/ps.0211102 Hollenbeck, J. J., & Oakley, M. G. (2000). GCN4 Binds with High Affinity to DNA Sequences Containing a
Single
Consensus Half-Site. Biochemistry, 39(21), 6380–6389. https://doi.org/10.1021/bi992705n Liu, J., Zheng, Q., Deng, Y., Cheng, C.-S., Kallenbach, N. R., & Lu, M. (2006). A seven-helix coiled coil.
Proceedings of the National Academy of Sciences, 103(42), 15457–15462. https://doi.org/10.1073/pnas.0604871103
Lupas, A. N., Bassler, J., & Dunin-Horkawicz, S. (2017). The Structure and Topology of α-Helical Coiled
Coils. Fibrous Proteins: Structures and Mechanisms, 82, 95–129. https://doi.org/10.1007/978-3-319-49674-0_4 Woolfson, D. N. (2023). Understanding a protein fold: The physics, chemistry, and biology of α-helical coiled
coils. Journal of Biological Chemistry, 299(4), 104579. https://doi.org/10.1016/j.jbc.2023.104579
2. Usage and Biology
At its N-terminus, GCN4 contains basic residues, the so-called bZip domain, through which it binds specifically to
the CRE (cyclic AMP
response element) DNA sequence (5' ATGACGTCAT 3') (Hollenbeck et al., 2002). A variant of GCN4 with the DNA
binding bZip-domain at the C-terminus (rGCN4) has been engineered to bind to the inverted CRE sequence, INV2 (5'
GTCAtaTGAC 3', upper
case letters indicate direct interaction between protein and DNA) with similar affinity
(Hollenbeck et al., 2001). By genetically fusing GCN4 to rGCN4, we created a small, bivalent DNA binding
staple with less than 150 amino acids.3. Assembly and Part Evolution
4. Results
4.1 Protein Expression and Purification
4.2 Electrophoretic Mobility Shift Assay
4.2.1 Qualitative DNA Binding Analysis
To analyze the binding DNA affinity an EMSA was performed, in which
bGCN4 was incubated in binding buffer with a 20 bp DNA probe containing the either the CRE (GCN4 binding)
sequence (5' ATGACGTCAT 3') or the INVii (rGCN4 binding) sequence (5' GTCAtaTGAC 3') until equilibration.
Subsequently the formed protein-DNA complexes were loaded on a native PAGE. Afterwards the DNA bands were
stained with SYBR-safe.
The bGCN4 fusion protein did not show any DNA binding for both target sites.
4.2.2 Quantitative DNA Binding Analysis
Θapp = Θmin + (Θmax - Θmin) ×
(Ka2 [L]tot2) / (1 + Ka2
[L]tot2)
Equation 1
Here [L]tot describes the total protein monomer concentration, Ka
corresponds
to the apparent monomeric equilibration constant. The Θmin/max values are the
experimentally
determined site saturation values (For this experiment 0 and 1 were chosen for min and max
respectively).
The FLAG-tag fusion to the N-terminus of proteins could potentially decrease binding affinity, likely due
to steric hindrance affecting the interaction with DNA. Interestingly, the differences in binding affinity
between GCN4 and rGCN4 appear negligible. Since GCN4 binds to DNA via its N-terminus and rGCN4 binds
C-terminally, the FLAG-tag likely does not directly influence DNA binding. However, it may influence the
dimerization of the proteins, which is necessary for DNA binding. To further investigate this, the
FLAG-tag can be cleaved using an enterokinase and potential changes in binding affinity analyzed.
Furthermore, coiled coil formation, and the amount of dimeric and monomeric proteins could be further analyzed
with circular dichroism spectroscopy (Greenfield, 2006).
4.3 In Silico Characterization Using DaVinci
DaVinci operates in three phases: static structure prediction, all-atom dynamics simulation, and long-range DNA
dynamics simulation. We applied the first two phases to our components, allowing us to characterize the structure
and dynamics of the DNA-binding interactions.
For our bivalent DNA-binding Mini staple (BBa_K5237009),
consisting of GCN4 fused via a GSG-linker to rGCN4
(BBa_K5237008), we predicted the structure and binding
affinity and tested various linker options. We evaluated
the flexibility and rigidity of the constructs using pLDDT values from the predictions. Flexible linkers, like
('GGGGS')n, and rigid linkers, like ('EAAAK')n, were assessed (Arai et al., 2001). Predictions were colored by
pLDDT scores, providing insights into chain rigidity (Akdel et al., 2022; Guo et al., 2022). Construct C (Fig. 5)
was tested in the wet lab as part of BBa_K5237009, but it failed to bind DNA due to excessive rigidity, which
inhibited subunit dimerization.
5. References
| None |