1
Expanding the building bricks of life
Genetic code expansion, non-standard amino acids,
photocaging and optogenetics
Dr. Martin Marek
Loschmidt Laboratories
Faculty of Science, MUNI
Kamenice 5, bld. A13, room 332
martin.marek@recetox.muni.cz
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Questions
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Short recapitulation of previous lesson
• How to build multi-subunit protein nanomachines?
• How to reconstitute multi-subunit protein complexes?
• How to determine 3D structure of protein nanomachines?
• What are viruses? And why to engineer them?
• Which viruses are used in synthetic biology?
• What is immunotherapy and gene therapy? Approaches?
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Recapitulation: protein nanomachines
RNA polymerase
Ribosome
Membrane
protein
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Recapitulation: Design of self-assembling biomachines
Science 2012 336 (6085):1171-4. doi: 10.1126/science.1219364
(A) First, a target symmetric architecture is chosen. Octahedral point group symmetry is used in this example; the threefold
rotational axes are marked here by triangles and shown as black lines throughout. The dashed cube is shown to orient the
viewer. (B) Multiple copies of the building block are symmetrically arranged in the target architecture by aligning their shared
symmetry axes. The pre-existing organization of the oligomeric building block fixes several (in this case four) rigid body degrees
of freedom (DOFs). (C) Symmetrical docking is performed by systematically varying the two DOFs (moves are applied
symmetrically to all subunits) and computing the suitability of each configuration for interface design (red: more suitable; blue:
less suitable). Points corresponding to the docked configurations in panels (B), in which the building blocks are not in contact,
and (D), a highly complementary interface, are indicated (E, F).
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Recapitulation: multi-expression systems
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X-ray crystallography versus cryo-EM
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Constructing de novo synthetic viruses for biomedicine
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Expanding the building bricks of life
Concepts
Methods
Applications
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What will we talk about
• Genetic code expansion
• Goals and applications
• Non-standard amino acids
• Concepts, methodology, success stories
• Cell-free protein expression
• Strategies, methodology, disadvantages, success stories
• Optogenetics and photocaging in synthetic biology
• Concepts, methodology, success stories
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Building bricks: the LEGO system
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Genetic code expansion (GCE)
Concepts
Methods
Applications
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How DNA directs protein synthesis
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Standard building blocks of DNA and RNA
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Non-standard building blocks of DNA and RNA
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The eight nucleotides of hachimoji DNA and hachimoji RNA are
designed to form eight-letter genetic code
https://science.sciencemag.org/content/363/6429/884
Hydrogen bond donor atoms involved in pairing are blue; hydrogen bond acceptor atoms are
red. The left two pairs in each set are formed from the four standard nucleotides (note
missing hydrogen-bonding group in the A:T pair, a peculiarity of standard terran DNA and
RNA). The right two pairs in each set are formed from the four new nonstandard nucleotides.
Genetic system from eight (“hachi”) building block “letters” (“moji”)
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Crystal structures of PB, PC, and PP hachimoji DNA
(A)The host-guest complex with two N-terminal
fragments from Moloney murine leukemia virus reverse
transcriptase (in green and cyan) bound to a 16-mer PP
hachimoji DNA; in the duplex sphere model, Z:P pairs
are green and S:B pairs are magenta
(B) Hachimoji DNA structures PB (green), PC (red), and
PP (blue) are superimposed with GC DNA (gray)
(C) Structure of hachimoji DNA with self-complementary
duplex 5′-CTTATPBTASZATAAG (“PB”)
(D) Structure of hachimoji DNA with self-complementary
duplex 5′-CTTAPCBTASGZTAAG (“PC”)
(E) Structure of hachimoji DNA with self-complementary
duplex with six consecutive nonstandard 5′CTTATPPSBZZATAAG
(PP) components. DNA
structures are shown as stick models with P:Z pairs
(carbon atoms, green), B:S pairs (carbon atoms,
magenta), and natural pairs (carbon atoms, gray)
(F to I) Examples of largest differences in detailed
structures. The Z:P pair from the PB structure (F) is more
buckled than the corresponding G:C pair (G). The S:B
pair from the PB structure (H) exhibits a propeller angle
similar to that in the corresponding G:C pair (I).
https://science.sciencemag.org/content/363/6429/884
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Structure and fluorescent properties of hachimoji RNA molecules
(A) Schematic showing the full hachimoji spinach variant aptamer; additional nucleotide components of
the hachimoji system are shown as black letters at positions 8, 10, 76, and 78 (B, Z, P, and S,
respectively). The fluor binds in loop L12. Fluorescence of various species in equal amounts as
determined by UV. Fluorescence was visualized under a blue light (470 nm) with an amber (580 nm)
filter.
(B) Control with fluor only, lacking RNA
(C) Hachimoji spinach with the sequence shown in (A)
(D) Native spinach aptamer with fluor
(E) Fluor and spinach aptamer containing Z at position 50, replacing the A:U pair at positions 53:29 with
G:C to restore the triple observed in the crystal structure. This places the quenching Z chromophore
near the fluor; CD spectra suggest that this variant had the same fold as native spinach
https://science.sciencemag.org/content/363/6429/884
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Hachimoji DNA- and RNA-like molecules: summary
• DNA- and RNA-like systems built from eight nucleotide “letters” (hence the
name “hachimoji”) that form four orthogonal pairs
• These synthetic systems meet the structural requirements needed to support
Darwinian evolution, including a polyelectrolyte backbone, predictable
thermodynamic stability, and stereoregular building blocks that fit a Schrödinger
aperiodic crystal
• Measured thermodynamic parameters predict the stability of hachimoji
duplexes, allowing hachimoji DNA to increase the information density of natural
terran DNA
• Three crystal structures show that the synthetic building blocks do not perturb
the aperiodic crystal seen in the DNA double helix
• Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a
functioning fluorescent hachimoji aptamer. These results expand the scope of
molecular structures that might support life, including life throughout the
cosmos.
https://science.sciencemag.org/content/363/6429/884
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Non-standard amino acids
Concepts
Methods
Applications
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The ribosome, a protein-RNA nanomachine for protein synthesis
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Redundancy of the genetic code
• Degeneracy of codons is the redundancy of the genetic code, exhibited as the
multiplicity of three-base pair codon combinations that specify an amino acid
• The genetic code is degenerate mainly at the third codon position
• The genetic code consists of 64 triplet codons specifying 20 canonical amino acids and
3 stop signals
Is it possible to expand genetic code for new amino acids?
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Expanded genetic code
https://en.wikipedia.org/wiki/Expanded_genetic_code
An expanded genetic code is an artificially modified genetic code in which one
or more codons have been re-allocated to encode an amino acid that is not
among the twenty naturally-encoded (canonical) amino acids
The key prerequisites to expand the
genetic code are:
• the non-standard amino acid to encode
• an unused codon to adopt
• a tRNA that recognises this codon
• a tRNA synthetase that recognises only
that tRNA and only the non-standard
amino acid
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Unnatural amino acids: examples
Unnatural amino acids are non-proteinogenic amino
acids that either occur naturally or are chemically
synthesized. Unnatural amino acids represent a
nearly infinite array of diverse structural elements
for the development of new synthetic proteins via the
expanded genetic code approach.
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Incorporation of unnatural amino acid into proteins:
a basic concept
• The method involves the generation
of an orthogonal tRNA/synthetase
pair, which does not crosstalk with
endogenous tRNA/synthetase pairs
but is functionally compatible with the
protein translational machinery
• The orthogonal synthetase is
engineered to charge the desired
unnatural amino acid (Uaa) onto the
orthogonal tRNA, and the tRNA
decodes a unique codon (such as the
amber stop codon UAG) to
incorporate the Uaa into proteins
through translation
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Site-specific incorporation of NAAs into proteins
• (a) Initially, site-specific incorporation of NAA into a protein was based on nonsense suppression,
where the term “nonsense suppression” refers to the use of nonsense codons and suppressor tRNAs,
which recognize stop codons. The amber UAG stop codon has been most frequently used for the
incorporation of NAAs, but the use of the ochre (UAA) stop and the opal (UGA) stop codons have been
applied, especially for the incorporation of diverse NAAs into one recombinant protein. The major
advantage of the nonsense suppression technique lies in its simplicity. Nevertheless, even amber stop
codon terminates about 320 genes in the model laboratory strain of E. coli K-12, meaning that the NAA
would be incorporated not only in the protein of interest but also in response to TAG stop codons of the
genes present in the genome
• (b) Rarely used sense codons, such as arginine (AGG) or proline (CCC) codons of E. coli, can be used
for direct NAA incorporation in proteins
• (c) Finally, design of extended codons and frameshift suppression. In this approach, an mRNA
containing an extended codon consisting of four or five bases is read by a modified aa-tRNA containing
the corresponding extended anticodon, and a full-length protein containing NAA at the specific site is
obtained
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Site-specific incorporation of NAAs into proteins
(a) Genetic code expansion enables the site-specific
incorporation of an unnatural amino acid into a protein
via cellular translation
(b) The process of discovering orthogonal aminoacyltRNA
(transfer RNA) synthetases for unnatural amino
acids
(c) Orthogonality of synthetase/tRNA pairs in different
hosts. The solid lines indicate that a pair is orthogonal in
a host
(d) Sequential positive and negative
selections enable the discovery of synthetase/tRNA
pairs that direct the incorporation of unnatural amino
acids
Abbreviations: Ec TyrRS,Escherichia coli tyrosyl-tRNA
synthetase; Mj TyrRS, Methanococcus janaschii
tyrosyl-tRNA synthetase; mRNA, messenger
RNA; PylRS, pyrrolysyl-tRNA synthetase
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Unnatural acid incorporation into proteins
In a typical unnatural amino acid incorporation experiment, the unnatural amino acid is
added to the cell, recognized by the altered-specificity orthogonal synthetase, and used to
aminoacylate the orthogonal tRNA. The orthogonal tRNA is decoded by the ribosome in
response to a blank codon (which has been inserted into a gene of interest) on the
messenger RNA (mRNA), leading to unnatural amino acid incorporation at a genetically
defined position in the encoded polypeptide chain.
Genetic code expansion enables the site-specific incorporation of an unnatural
amino acid into a protein via cellular translation
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Orthogonal aminoacyl-tRNA synthetases
• For orthogonal synthetases that bind one of the 20
common amino acids (MjTyrRS, EcTyrRS, Ec
LeuRS), this process requires evolution of the
enzyme active site to destroy natural amino acid
binding and facilitate specific unnatural amino acid
binding
• PylRS naturally binds pyrrolysine but none of the
common amino acids, so it is not necessary to
mutate the active site to remove natural amino acid
binding. Moreover, while the substrate scope of
PylRS has been substantially expanded by
directed evolution, some useful unnatural amino
acids can also be incorporated using the wild-type
PylRS, thereby facilitating unnatural amino acid
incorporation
To use an orthogonal pair for genetic code expansion, the active site of the synthetase must
specifically bind an unnatural amino acid, and no natural amino acids, and must transfer the
unnatural amino acid onto its cognate orthogonal tRNA.
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Pyrrolysine
https://www.sciencedirect.com/science/article/pii/S2451945619301047
• Pyrrolysine is encoded by the 'amber' stop codon (UAG) is an amino acid
that is used in the biosynthesis of proteins in some methonogenic archeae
and bacteria.
• It is not present in eukaryotes.
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Pyrrolysyl-tRNA synthetase (PylRS) and tRNAPyl
https://www.sciencedirect.com/science/article/pii/S2451945619301047
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Incorporation of the non-natural lysine derivatives
https://www.sciencedirect.com/science/article/pii/S2451945619301047
• A mutant pyrrolysyl-tRNA synthetase, PylRS(Y306A/Y384),
acts on diverse amino acids
• The PylRS mutant and tRNAPyl incorporated 17 non-natural
amino acids into proteins
• Crystal structures of the PylRS mutant bound with 14 of the
amino acids were solved
• This information will facilitate the structure-based design of
novel amino acids
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Mechanism of genetic code expansion for incorporation of
an unnatural amino acid by amber suppression
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610526/
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Use of amber suppression to switch on protein production
(A) Absence of the unnatural amino acid leads to recognition of the UAG codon for
translation termination. (B) Addition of the unnatural amino acid leads to amber
suppression and successful production of the full-length and functional protein.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610526/
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Allowed and not allowed reactivities between the
orthogonal and endogenous aaRS/tRNA pairs
(A) Matching amino acid and aaRS/tRNA pairs; (B) mismatched amino acids;
(C) mismatched aaRS/tRNA pairs.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610526/
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Example of the small-molecule approach to protein inhibition
• Use of unnatural amino acid incorporation for selective inhibition of protein function by
bioorthogonal tethering
• In comparison with light-induced activation or inhibition, small molecules can be used
to activate or inhibit the target protein in deep animal tissue or intact animals which are
not easily accessible by light
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610526/
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Site-specific incorporation of NAAs into proteins
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Cell-free protein synthesis (CFPS)
Concepts
Methods
Applications
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Cell-Free Protein Synthesis (CFPS)
• Cell-free protein synthesis
(CFPS) is a platform technology
that provides new opportunities
for protein expression
• The advantages of CFPS over in
vivo protein expression include
its open system, the elimination
of reliance on living cells, and the
ability to focus all system energy
on production of the protein of
interest
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Cell-free platforms and their applications
(A) Web of the applications enabled by low adoption cell-free platforms. Connections shown
are based on applications that have been published or that have been proposed in
publications.
(B) Cumulative number of peer-reviewed publications over the last 60 years for cell-free
platforms.
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Preparation of cell-free extract and set-up of CFPS reactions
General workflow for preparation of cell-free extract and set up of CFPS reactions.
A visualization from cell growth to the CFPS reaction is depicted above for a new
user, highlighting the main steps involved.
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CFPS reaction incorporating nonstandard amino acids
• Scheme of cell-free protein
synthesis reaction
incorporating nonstandard
amino acid to investigate
the effect of RF1
• Cell extracts containing
transcription and translation
machinery are prepared
from rEc.E13 or
rEc.E13.ΔprfA strains
• Plasmid DNA template of
sfGFP containing single or
multiple amber codon
sites, orthogonal
tRNA/aaRS, NSAA, T7
RNA polymerase, and other
cofactors are added as
necessary to activate the
cell-free protein synthesis
(CFPS) reaction
p-acetyl-L-phenylalanine
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CFPS from extracts of a genomically recoded organism
• Schematic of the production and utilization of crude extract from genomically recoded organisms with
plasmid overexpression of orthogonal translation components for cell-free protein synthesis
• CFPS reactions are supplemented with the necessary substrates (e.g., amino acids, NTPs, etc.) required
for in vitro transcription and translation as well as purified orthogonal translation system (OTS) components
to help increase the ncAA incorporation efficiency
• aaRS, aminoacyl tRNA synthetase; ncAA, non-canonical amino acid; T7P, T7 RNA polymerase; UAG,
amber codon
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Genetic code expansion in eukaryotes
• Over last decades, discovery of macromolecular protein structures and complexes has
played a major role in advancing a more systems-oriented view of protein interaction and
signalling networks
• The design of biological systems often employs structural information or structure-based
protein design to successfully implement synthetic signalling circuits or for rewiring
signalling flows
Membraneless OT organelles enable mRNA-specific GCE in eukaryotes. OT organelles are designed organelles enriched in
a suppressor tRNA/tRNA synthetase pair and a specific mRNA binding domain (MCP) by means of using an assembler
protein (such as FUS and/or KIFs). A spatially distinct set of ribosomes associated with the OT organelle preferentially
translates recruited mRNAs tagged with ms2 loops to yield the selected protein with the targeted site-specific noncanonical
functionality.
https://science.sciencemag.org/content/363/6434/eaaw2644
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Spatial separation of the key components for orthogonal translation to
decode a specific stop codon in a specifically tagged mRNA
(A) Expression of the synthetase PylRS leads to aminoacylation of its cognate stop codon suppressor tRNAPyl with a custom
designed ncAA. This leads to site-specific ncAA incorporation whenever the respective stop codon occurs in the mRNA of the
POI. Given that many endogenous mRNAs terminate on the same stop codon, using this approach in the cytoplasm potentially
leads to misincorporation of the ncAA into unwanted proteins. (B) To avoid this, we propose to spatially enrich all components
to an OT organelle, including the mRNA of the POI, the aminoacyl-tRNA synthetase, the tRNA, and ribosomes through the use
of “assemblers.” Aminoacylated tRNAPyl should only be available in direct proximity of the OT organelle, so that only here stop
codon suppression can occur. The corresponding stop codon in mRNAs that are not targeted to the OT organelle should not
get translated. Whereas in (A) GCE is stop codon specific, in (B), it is stop codon– and mRNA-specific.
https://science.sciencemag.org/content/363/6434/eaaw2644
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A designed metalloprotein using an unnatural amino acid
An example of computational protein design II.
• Genetically encoded unnatural amino acids could facilitate the design of
proteins and enzymes of novel function
• The Rosetta design was used to design metalloproteins in which the amino acid (2,2′bipyridin-5yl)alanine
(Bpy-Ala) is a primary ligand of a bound metal ion
• A buried metal binding site with octahedral coordination geometry consisting of Bpy-Ala,
two protein-based metal ligands, and two metal-bound water molecules
• Experimental characterization revealed a Bpy-Ala-mediated metalloprotein with the ability
to bind divalent cations including Co2+, Zn2+, Fe2+, and Ni2+, with a Kd for Zn2+ of ∼40 pM
Mills et al., J. Am. Chem. Soc., 135: 13393-13399 (2013)
(2,2′-bipyridin-5yl)alanine
Theozyme for secondround
design
calculations
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• X-ray crystal structures of the designed protein bound to Co2+ and Ni2+ have RMSDs to the
design model of 0.9 and 1.0 Å respectively over all atoms in the binding site.
Mills et al., J. Am. Chem. Soc., 135: 13393-13399 (2013)
A designed metalloprotein using an unnatural amino acid
An example of computational protein design II.
X-ray crystallographic analysis of MB_07 bound to
Co2+ and Ni2+. Electron density from a 2Fo–Fc map
in the vicinity of the Bpy-Ala bound to Co2+ (a) and
Ni2+ (c) contoured at 1.0 σ. Density for Bpy-Ala, Co2+
or Ni2+ (pink and green spheres, respectively), D184,
E159, and a metal-bound water molecule (red
spheres) is visible.
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Optogenetics
Concepts
Methods
Applications
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What is optogenetics?
• Optogenetics provides the ability to gain light-control over genetically encoded cell
components, and thereby ultimately over signalling pathways or biological processes
• Contrary to chemically inducible systems, where spatiotemporal capabilities are limited
by the diffusion and the half-life of inducing molecules, light provides an exquisite
precision of control and thus opens unique possibilities to disentangle the principles
that govern nature
• By delivering optical control at the speed (millisecond-scale) and with the precision (cell
type–specific) required for biological processing, optogenetic approaches have opened
new landscapes for the study and engineering of biological systems
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Optogenetic approaches to control gene regulation,
protein function and subcellular compartments
https://doi.org/10.1016/j.copbio.2017.06.010
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Toolbox of light-responsive modules
Optogenetic switch modules. AsLOV2, Avena sativa light-oxygen-voltage domain 2; BphP1, bacterial bathy
phytochrome RpBphP1; CIB1, cryptochrome-interacting basic helix-loop-helix 1; COP1, constitutive
photomorphogenesis protein 1; CPH1, cyanobacterial phytochrome 1; CRY2, cryptochrome 2; FKF1, Kelch
repeat, F-box protein; GI, GIGANTEA; PHYB, phytochrome B; PIF, phytochrome interacting factor; POI,
protein of interest; PpsR2, oxygen-responsive transcription regulator; UVR8, ultraviolet B receptor; VVD,
vivid.
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Examples of optogenetic applications
(a) Light-controlled activation of a receptor tyrosine kinase (RTK) signaling pathway as a platform for
optogenetics-assisted small-molecule screening
(b) Light-controlled CNK1 clustering reveals the protein's role as a molecular platform for cellular decision-
making
(c) Light-controlled localization of Rho guanine exchange factor ECT2 as an optogenetic system to show
that the protein's plasma membrane association during anaphase is necessary and sufficient for
cytokinesis
(d) Light-controlled localization of 5-phosphatase OCRL as an optogenetic tool to control cell contractility
during tissue morphogenesis
Legend: AKT, protein kinase B; CNK1, connector enhancer of KSR 1; ERK1/2, extracellular signalregulated
kinase; GFP, green fluorescent protein; RAF, rapidly accelerated fibrosarcoma kinase; SRE,
serum response element
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Optogenetics: conclusions
• Optogenetics uses light to control genetically encoded cell
components
• It is a precise tool for systematically addressing complex
biological questions
• It has a potential for applications in synthetic biology,
medicine, pharmacology and elsewhere
• The latest optogenetic systems move from single cells to
in vivo applications
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Photocaging in synthetic biology
Concepts
Methods
Applications
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Photochemical control of protein function
• Biological processes are regulated with a high level of spatial and temporal resolution
• To understand and manipulate these processes, we need to be able to regulate them
with Nature's level of precision
• In this context, light is a unique regulatory element because it can be precisely
controlled in terms of location, timing and amplitude
• Moreover, most biological laboratories have a wide range of light sources as standard
equipment
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Photochemical regulation of gene expression
(a) Light-activatable small-molecule inhibitors of gene expression repressor proteins. Caged IPTG 1
(b) Bacterial lithography experiment with 1 showing light-controlled induction and spatial control of βgalactosidase
(left) and GFP (right) in a bacterial lawn (∅ 10 cm)
(c) Caged doxycycline 2
(d) Spatial control of GFP expression using 2, with irradiation through a 344-μm photomask (scale bar
250 μm); irradiated (blue) and non-irradiated (red) cells were quantified and fluorescence intensity is
shown as a function of time. The light-removable caging groups are shown in red
https://doi.org/10.1016/j.tibtech.2010.06.001
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Photochemical control over enzyme activity
(a) Photo-control over the activity of histone deacetylase 2 (HDAC2). Based on known
HDAC2 inhibitor vorinostat, compound 1 was designed. Upon irradiation, compound 1
switches from trans to cis form, becoming 39-fold more active as an HDAC2 inhibitor.
(b) Dose–response curve for compound 1 in trans (blue) and cis (orange) form on cell
viability of HeLa cells.
https://doi.org/10.1016/j.tibs.2018.05.004
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Reversible photochemical activation of K+ ion channel
(a) Schematic of PAL-gated K+ channel: The sphere labelled with a plus sign represents a quaternary
ammonium group, which blocks the channel when the diazobenzene is in the trans conformation.
Irradiation at 380 nm switches the diazobenzene from trans to cis, thus enabling K+ flow. Irradiation
with 500 nm light reverses the switching event, thus blocking the ion channel.
(b) Light-controlled membrane potential of a PAL-gated K+ channel measured in dependence of light
irradiation.
https://doi.org/10.1016/j.tibtech.2010.06.001
Three distinct features:
• a quaternary ammonium group
• a photo-isomerizable azobenzene group
• an electrophilic cysteine-reactive group (e.g. acrylamide,
chloroacetamide, epoxide or maleimide)
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Photochemical protein localization in cells
(a) Genetically encoded caged lysine (lightremovable
caging group shown in red)
(b) Gene diagram of p53–egfp with the
crucial lysine K305 in bold
(c) Nuclear import of EGFP in cells after
introduction and photolysis of the caged
lysine 3 introduced into position 305 in
NLSp53. A mutation of K305 to tyrosine
(K305→Y) blocks transport into the
nucleus, regardless of light irradiation
(left). Introduction of the caged lysine 3
at position 305 (K305→3) blocks
transport of p53–EGFP, but facilitates
translocation into the nucleus after brief
light irradiation (365 nm, 5 s) (right).
Scale bar, 10 μm.
https://doi.org/10.1016/j.tibtech.2010.06.001
61
Overview of photocaging applications in biology
Representation of the photoprotecting groups, caged molecules, photochemical reactions behind the
use of photocaging approaches in different biomedical applications.
6262
Questions
6363
Supplementary materials
64
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4090895/
https://pubs.acs.org/doi/10.1021/sb300094q
65
Structure-based engineering and design of parts
• Until now, structural biology has been
mainly used in synthetic biology
approaches to design new
parts/components and tools
• Many of the characterized components
can be found in the Registry of Standard
Biological Parts (http://partsregistry.org/)
• Of special importance is the recently
developed “SYNZIP protein toolbox”
because it contains a complete biophysical
quantitative description (i.e., affinities) of
synthetic domains
66
Genetic circuits for signal processing
Genetic circuits are commonly built by synthetic biologists to enable cells to take
some input and compute what to do. Beyond a cell being activated by a single
input, cells could be engineered to respond to multiple signals and to make
decisions based on both its neighbouring cells and environment.
One published example of mammalian genetic circuits is ‘Boolean logic and
arithmetic through DNA excision’ (BLADE) and it was able to build 113 different
circuits in embryonic kidney and Jurkat T cells. The BLADE system
uses recombinases to create logic gates but can also interface with CRISPR–
Cas9 to regulate host cell genes. Large circuits like the ones demonstrated with
BLADE could be combined with synthetic receptors so cells can make
decisions based on what combination of signals they see.
67
Receptor engineering
New types of receptors can be engineered to detect different types of molecules
and turn on some cellular output function. The more receptors that can be
readily engineered the more molecules that can be used as inputs to a
therapeutic cell. It has been shown that synthetic notch receptors can be made
for use with different recognition domains as well as different transcription
factors as activation domains. That means that a researcher can decide both
what the receptor responds to and what it turns on after it binds its target.
Typical CAR-Ts only change the input of the receptor, but this kind of receptor
can swap out either the input or output or both. Having several independent
receptors would allow much more complex programming of therapeutic cells to
respond to signals within the human body.
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Safety kill switch
Since toxicity can be an issue, it would be good to have an off switch in the event
of a problem. One form of toxicity that can happen with CAR-T is called
“Cytokine Release Syndrome” or “Cytokine Storm” for especially severe cases.
This can happen when the immune system has such a strong response that
many inflammatory cytokines are released triggering mild to severe symptoms
including fever, headache, rash, rapid heartbeat, low blood pressure, and
trouble breathing.
Synthetic biologists have already worked on a number of “kill switches” in
different organisms. A kill switch could be applied to CAR-T by having a druginducible
kill-switch in the the T-cells that would give the medical team a way to
kill off the modified cells as soon as a bad side effect starts to show up.
Researchers and companies are also seeking multiple ways to either switch off
or kill the T-cells when they’re not needed. These variations on a kill switch can
help to make sure CAR-T cell therapy has extra safety measures in place.
There are many types of cancer and many ways in which a cancer can avoid
successful treatment. Hopefully mammalian synthetic biology can add
something to the immunotherapy toolkit by improving CAR-T or other
approaches to enlist the immune against cancer.
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(a) Recombinases can perform
simple BUF logic
operations, either by
tyrosine-recombinasemediated
excision (left) or
serine-integrase-mediated
inversion (right).
(b) Recombinases are tested
for their recombination
efficiency and orthogonality
on all BUF logic reporters.
(c) A 6-input AND-gate that
produces GFP when all
inputs are present. MFI,
mean fluorescence intensity
from n = 3 transfected cell
cultures; a.u., arbitrary units.
Error bars, s.e.m.
Implementation of multi-input gates in mammalian cells
Weinberg et al., Nature Biotechnology 35: 453-462 (2017)
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(a)2-input BLADE template on one plasmid with a single
transcriptional unit. This template contains four distinct regions of
DNA (addresses) downstream of a promoter. Each address
corresponds to an output function and is accessed or deleted via
site-specific DNA recombination. Each address can be
programmed from different configurations ranging from zeroinputs
to Boolean functions. The first address (Z00), which is the
closest to the promoter, corresponds to a state where no
recombinase is expressed (A = 0, B = 0). If the Z00 address
contains a protein coding sequence, then that gene will be
expressed. Gene expression from the other addresses
downstream of Z00 will be blocked by the presence of the Z00
protein coding region. In the presence of recombinase A, which
corresponds to state (A = 1, B = 0), addresses Z00 and Z01 will
be removed, thus moving address Z10 directly downstream of
the promoter and allowing gene expression of address Z10 only
to occur. Similarly, when only recombinase B is present (A = 0, B
= 1), addresses Z00 and Z10 are excised, allowing Z01 to be
moved directly downstream of the promoter. Finally, when both
recombinases are expressed (A = 1, B = 1), addresses Z00, Z01,
Z10 are all excised, thus placing Z11 downstream of the
promoter unobstructed by the other addresses.
(a)Integrated 2-input BLADE decoder with tagBFP, EGFP,
iRFP720, and mRuby2 as addresses Z00, Z10, Z01, and Z11
respectively. Plasmids constitutively expressing Cre and/or Flp
are then stably integrated. Three days of doxycycline (DOX)
treatment is used to permit the rtTA-VP48 protein to bind to the
tetracycline response elements promoter (pTRE) to activate
gene expression. Mean fluorescence intensity (MFI) is plotted of
either n = 1 or n = 2 independent integrations. a.u., arbitrary
units.
Four distinct output functions based on two inputs
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• Koukni na toto
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Light-regulated protein-protein interactions
(a) Light-controlled expression of lacZ via PhyB–PIF protein interaction. Schematic of the lightcontrolled
transcriptional activator. Phytochrome (Phy), which is fused to the DNA-binding region of
GAL4 (GBD), reversibly binds to PIF. PIF is fused to the transcriptional activating domain of GAL4
(GAD), thus activating gene expression on exposure to red light and deactivating it by dissociation
on exposure to IR light.
(b) LacZ (β-galactosidase) activity induced with a pulse of red light (Rp, black line), and arrested with
pulses of far-red light (FRp, red line), thus showing that the system is reversible and can be used to
control lacZ expression.
https://doi.org/10.1016/j.tibtech.2010.06.001
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Genetically encoded photoresponsive protein domains
(a) Mechanism of LovTAP, a reversibly
switchable DNA-binding protein. Before
exposure to blue light, Jα (dark blue) is
associated with the LOV domain (light blue),
which renders the Trp repressor region (light
orange) inactive. After irradiation with blue
light (470 nm) a conformational change
occurs and Jα (now dark orange) dissociates
from the LOV domain, in turn activating the
Trp repressor. The active protein binds to
DNA at a lac operator region. The original
conformation of the protein is slowly (tens of
seconds) reassumed after incubation in the
dark and the Trp repressor region dissociates
from the DNA, thus making the activity of
LovTAP reversible.
(b) LovTAP protects DNA against RsaI digestion
at the lac operator site. An increase in the
concentration of LovTAP decreases
digestion, as does irradiation with blue light
(dashed lines, irradiated; solid lines, non-
irradiated).
https://doi.org/10.1016/j.tibtech.2010.06.001
75
Light-induced activation or inhibition (photocaging)
• Use of light to unmask or modify unnatural amino acids and subsequently regulate protein
function
• Depending on the nature of the light-responsive group, it is possible to either activate,
inhibit, or reversibly switch on/off protein function
• Unnatural amino acids containing a photocage (i.e. a photolabile protecting group) have
been widely used for protein activation
• When replacing a functionally critical amino acid residue with the corresponding
photocaged amino acid, the target protein becomes inactive; upon light irradiation, the
photocage is removed, thereby restoring the protein’s function
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610526/
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Dr. Martin Marek
Loschmidt Laboratories
Faculty of Science, MUNI
Kamenice 5, bld. A13, room 332
martin.marek@recetox.muni.cz
77
Dr. Martin Marek
Loschmidt Laboratories
Faculty of Science, MUNI
Kamenice 5, bld. A13, room 332
martin.marek@recetox.muni.cz