DNA - BVL

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
N
N
dR
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
CF 3
O P
OO
Base
O
O
O
O
O
O P
O
O
SP
O
Base
BH 3-
O-
O
Base
O
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
N
P
O
O
Alternative sugar backbone
Hexitol nucleic acids
‐
O
O
P
‐
O
O
O Base
O
P
O
O
O
HNA
DNA
O
O
– Still susceptible to oxidative and UV damage
tr
Serum
No
• Low toxicity
• Poorly incorporated by natural polymerases
O
ea
DN tme
nt
as
e RN I
as
e BA I
L‐
31
• Can base pair with natural nucleic acids • Not naturally synthesised
• Increased chemical and biological resistance
DNA
h : 0 2 12 24 48
HNA
h : 0 24 48
DNA
HNA
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
X
X
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
•
RNA
Notrepis
Proteins
Proteins
Metabolism
Metabolism
These can be introduced in parallel
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