How far can biology be
redesigned?
Orthogonality as a biosafety tool
Dr. Vitor B. Pinheiro
Lecturer in Synthetic Biology
Institute of Structural Molecular Biology
BVL 2015 Challenges Symposium – 05.11.14
UK Parliamentary copyright:
Reproduced with the permission of
Parliament
Synthetic Biology – more than genetic engineering
• Traditional scientific approach:
– Real systems are complex
– Minimise complexity to make system tractable – top-down approach
• Synthetic Biology
– Biology as engineering – bottom-up approach
– Can complex systems be assembled from parts?
– Can we redesign biological systems?
“What I cannot create, I cannot understand.”
Richard Feynman
Synthetic
Natural
Custom genomes
Unnatural
substrates
Transgenic
pathways
Gene circuits
Engineered
enzymes
Transgenics
Targeted mutants
Random mutants
Natural isolates
Synthetic Biology – more than genetic engineering
Synthetic Biology
Design
Genetic engineering
adapted from de Lorenzo (2010) Bioessays
10.1002/bies.201000099
Limitations of our existing tools
• Biological systems are the result of an evolutionary process
–
–
–
–
Probabilistic nature
Extensively but not thoroughly explored
Not conscious, not targeted, not designed
N = 1 problem
• So, why the systems we can observe have settled to their
current arrangement?
• Is the natural setup an intrinsic limitation or a frozen
accident?
• Can we try something different?
Synthetic
Synthetic
Natural
Redesigned
biological processes
Non-canonical
chemistries
Custom genomes
Unnatural
substrates
Transgenic
pathways
Gene circuits
Engineered
enzymes
Transgenics
Targeted mutants
Random mutants
Natural isolates
Xenobiology – unnatural biological systems
Xenobiology
Orthogonality
Synthetic Biology
Design
Genetic engineering
adapted from de Lorenzo (2010) Bioessays
10.1002/bies.201000099
Information transfer in biology
DNA
• Information storage and propagation
are essential for life
• Information only accessible from DNA
and RNA in biological systems
– The Central Dogma
RNA
• There is a change in information media
between RNA and proteins
– The Genetic code
Proteins
Information transfer in biology – The central dogma
DNA
• Information storage and propagation
are essential for life
• Information only accessible from DNA
and RNA in biological systems
– The Central Dogma
RNA
• Propagation is viable because of the
efficient and unambiguous base pairing
O
H2N
Proteins
NH 2
O
N
N
N
NH
dR
N
HN
N
O
O
N
dR
N
NH 2
G•C
N
dR
N
N
A•T
dR
Nucleic acid structure and function
Phosphate
Nucleobase
-O
O
P
O
O
Base
O
DNA
O
Ribofuranose sugar
Nucleic acid structure and function
Phosphate
Nucleobase
-O
O
P
O
O
Base
O
DNA
O
Ribofuranose sugar
• All three chemical moieties
contribute to nucleic acid
chemical properties, structure
and function
• Modification in any of the
moieties generates a synthetic
nucleic acid (XNA)
– Range of compatibility with natural
systems
– Range of chemical and biological
stability
Xenobiotic nucleic acids
Nucleobase substitutions
-O
Alternative base pairings
3S
N+
CF 3
OH
NH 2
N
N
O
N
HN
OH
S
O
N
H
N
H
N
N
H
O
SO 3-
NH 2
R
S
N
N
N
O 2N
NH
O
O P
N
O-
O
N
O
S
O
O
O
O
P
H 2N
O
O
H
O
N
N
N
H
NH
N
NH
O
CF3
O P
OO
Base
O
O
O
O
O
O P
O
O
SP
O
Base
BH 3-
O-
O
Base
O
NH
O
P
O
O
O
O
P
O-
OO
Pinheiro and Holliger (2014) Trends in Biotechnology
10.1016/j.tibtech.2014.03.010
Base
O
O
Base
F
O
Alternative internucleotide linkages
Base
O
O
O
O-
O
O
O
N
P
O
O
Alternative sugar backbone
Hexitol nucleic acids
• Can base pair with natural nucleic
acids
• Not naturally synthesised
• Increased chemical and biological
resistance
-
O
O
P
-
O
O
O Base
O
O
P
O
O
O
HNA
DNA
O
O
– Still susceptible to oxidative and UV
damage
• Low toxicity
• Poorly incorporated by natural
polymerases
Serum
DNA
h:
DNA
0
2
12 24 48
HNA
HNA
h:
0
24
48
Base
Expanding the central dogma
• Establish an XNA as a genetic
material
DNA
RNA
Proteins
XNA
– Code (DNA  XNA) and decode (XNA
 DNA) information from a synthetic
backbone using biocompatible routes
• Engineering DNA polymerases for
XNA synthesis and reverse
transcription
From XNA to Xenobiology
• XNA genetic material is the first step
towards an XNA episome
DNA
RNA
Proteins
XNA
– XNA genetic element stored
independently and maintained stably
within an organism
• Make information in XNA
inaccessible to general biology
• XNA maintenance in vivo depends
on XNA information being required
for cell survival
– Link to metabolism
• Many viable topologies integrating
XNA information to the cellular
function
From XNA to Xenobiology
XNA as a biosafety tool
• XNA as a dead man’s switch
DNA
RNA
XNA
– Synthetic precursors
– XNA traits are isolated from biology
– Cell’s dependence on XNA limits its
ecological impact
• Containment failure depends on
shortest evolutionary distance
– Archaeal XNA RT was a single mutation
Proteins
Metabolism
• Xenobiology systems are additive and
can be systematically integrated to
increase containment likelihood
Information transfer in biology – The genetic code
DNA
• Information storage and propagation
are essential for life
• Information only accessible from DNA
and RNA in biological systems
– The Central Dogma
RNA
• There is a change in information media
between RNA and proteins
– The Genetic code
Proteins
The genetic code
• Information in RNA is stored in nucleotides while information
in proteins is stored in amino acids
The genetic code
• Genetic code emerged early
in evolution
• Universal bar a handful of
exceptions
• Can it be modified?
Protein biosynthesis
Nascent
protein
tRNA synthetase
tRNA
RNA
Ribosome
Synthetase orthogonality
• For the genetic code to be
specific, aaRS must be
orthogonal
– Charge only its cognate amino
acid
– Charge only its cognate tRNAs
• Multiple interactions ensure
aaRS specificity
– Amino acid and enzyme
– tRNA and enzyme
– Downstream interactions also
reported
Expanding the genetic code
• Make use of unused rare codons
to introduce unnatural amino
acids
– E. coli only uses TAG as a stop
codon in 8% of its genes
– It can be modified without
greatly affecting the bacteria
– It can be removed by systematic
genomic editing
Jackson et al. (2006) JACS
10.1021/ja061099y
Rewriting the genetic code
S
S
TCC
TCC
• In vivo and in vitro approaches currently
being developed
– Multiple reassignments need to be carried out
to regenerate a viable code
S
ATG
Rewriting the genetic code
… TCC ATG ATT CTG GAG TAG …
S
… ATG TCC ATT CTG GAG TAG …
… ATG TCC ATT CTG GAG TAG …
TCC
E
L
GAG
CTG
I
M
ATT
ATG
… SMILE
… MSILE
… SMILE
Rewriting the genetic code as a biosafety tool
• A genetically recoded organism
(GRO) would not be able to
exchange information with natural
organisms
DNA
– GRO  Nature will not generate
viable proteins
– Nature  GRO will not generate
viable proteins
RNA
Notrepis
Proteins
Proteins
• A GRO auxotroph would be
contained
– Semantic firewall
Metabolism
Metabolism
Xenobiology as a biosafety tool
DNA
XNA
• Biosafety can be introduced in
multiple layers
– Genetic firewall
– Semantic firewall
– Metabolic firewall
• These can be introduced in
parallel
RNA
Notrepis
Proteins
Proteins
Metabolism
Metabolism
Human risk of Xenobiology
• ‘Xeno’-organisms are still biological systems
– As a class, broadly similar risks and hazards as posed by GMOs
• Additional considerations required depending on
modification, its implementation and purpose:
– Input compounds – e.g. XNA precursors – chemical toxicity of
precursors, contaminants from precursor synthesis, abiotic precursor
breakdown
– Intermediates and side reactions – e.g. unnatural amino acids –
biological modification or misuse of input compounds, pathway
intermediates, truncation products, biologically accessible bypass
alternatives
– Output compounds – e.g. XNAs – biological activity or toxicity of
intended products or molecules, and of their breakdown products by
natural metabolic or environmental routes, co-option by cellular
mechanisms
Acknowledgements
•
•
•
•
Leticia Torres
Antje Krüger
Eszter Csibra
Pinheiro Group
•
•
•
•
Phil Holliger
Piet Herdewijn
Philippe Marliere
Chris Cozens
•
•
•
•
John Ward
Helen Hailes
Gary Lye
Jack Stilgoe
“The farther [from biology], the safer.”
Philippe Marliere
Environmental risk of GMOs
• GMOs pose two potential risks:
– Ecological – the direct risk of a GMO interfering with other organisms
and altering ecological niches
– Informational – the risk that at least part of the genetic information of
a GMO can spread to natural organisms, conferring an ecological
advantage
• Reports of controlled release of GMOs for bioremediation
suggest GMOs pose negligible risk to the environment
– GMOs could not establish themselves in the tested niches
Environmental risk of GMOs
• Risk is only one
element in looking at
potential hazards
Sterling (2010) Nature
10.1038/4681029a
Routes to safe bioprocessing
Bioprocessing
focus
2nd containment
(facility design)
Natural
organisms
1st containment
(equipment design)
Genome
ablation
Cell-free
chassis
Component
focus
Genetic firewall
Auxotrophy
Inducible expression
systems
Semantic firewall
Metabolic
containment
Chassis
focus