presentation on how to expand the genetic code; it's pre-requisites; xenobiology and xeno-nucleic acids; planet's first Semi-Synthetic Organism ( with 6 nucleotides) and Hachimoji DNA (8 Nucleotide DNA)

1. EXPANDING THE
CODE OF LIFE
Guide: Dr Radhakrishnan KB
Presented by Abin Nazar
SCT17BT004
704
1

2. How to explain LIFE?
L
I
F
E
2

3. Key prerequisites to expand the genetic code
An unused codon to adopt
Non Standard Amino Acids(NSAA) to encode
A tRNA synthetase that can recognise only that
tRNA(Orthogonal tRNA)
A tRNA synthetase that can aminoacylate the
orthogonal tRNA
71 NSAA’s with phenyl alanine core + tyrosine
variants
3 standard stop codons,4 base codons and
non standard codons
The orthogonal
pair
(tRNA+tRNA
synthetase)
activity 3

4. The 71 NSAA’s
and Tyrosine
variants
71 NSAA’s
• Have PHENYLALANINE core with substituents
• Added to E.coli , yeast or mammalian cells
• Availability of NSAA to organism either as it imports it
from medium or the organism is made to biosynthesise
it by bioengineering a biosynthetic pathway
• Functions as probes,labels and to produce translational
proteins in E.coli with eukaryotic post translational
modifications e.g.
phosphoserine,phosphothrionine,phosphotyrosine
4

5. Minimal codon availability
Limitation with genetic code expansion on standard 4 nucleotide
standard Pentose sugar Genetic Molecule (GM)
Non
standard
amino acids
can be
assigned to
The 3
stop
codons
4 base
codons
A need to
engineer
novel sugars
and bases into
the GM to
code
innumerable
aminoacid
substitueents
5

6. XENOBIOLOGY :
A branch of synthetic biology that describes
novel biological systems and biochemistries
that differ from the canonical DNA-RNA-
(20+3) aminoacid system
XENO NUCLEIC ACIDS (XNA)
Synthetic XNA’s created so far
1. 1,5-anHydroxitol Nucleic Acid (HNA)
2. Cyclohexane Nucleic acid(CeNa)
3. Threose Nucleic Acid (TNA)
4. Glycol Nucleic Acid (GNA)
5. Locked Nucleic Acid(LNA)
6. Peptide Nucleic Acid(PNA)
7. Fluoro Arabina Nucleic Acid (FANA)
• Have a non canonical sugar backbone
• Morethan 7 types of synthetic sugars are
shown to form nucleic acid backbone that can
store and retrieve information
• Should not compromise on the aperiodic
crystal structure of the double helix
• A DNAase that can read the XNA is required to
read and duplicate the information in the XNA
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7. Merits of XNA Life
• Resistant to viral and bacterial infections,hence the lifeform is
no longer a succeptible host
• Enables the creation of GENETIC FIREWALL
• Eliminate Horizontal Gene Transfer
• To create an orthogonal biological system that would be
incompatible with natural genetic system
• Act as template for DNA in E.coli in-vivo
• Catalyst: ability to cleave and ligate DNA,RNA and other XNA’s
• HNA: to build target sequences that target HIV
• CeNa: form stable duplexes with itself and RNA
Erwin Schrodinger’s What is life? (1944)
2 important concepts
1. Negentropy: the entropy that an organism
exports to its sorroundings to keep its
internal entropy low
2. Aperiodic Crystal: hereditary mechanism
should be housed in an aperiodic crystal.
• Lacks strict periodicity (exact
repetition of motif); because
periodicity equals poor information
and without some unpredictability
or novelty nothing new is learnt or
communicated
• Small and consistent molecule
• Allows to encode infinite possibilities
within a small number of atoms
• Genetic information stored in it’s
configuration of covalent chemical
bond
Aperiodic
crystal
Photo 51: X-ray diffraction
image of paracrystalline gel
composed of DNA fibre taken by
Rosalind Franklin and Raymond
Gosling in 1952
Fig: photo 51
7

8. Planet’s First SEMI-SYNTHETIC ORGANISM (SSO)
6 NUCLEOTIDE GENETIC CODE: A T G C + X Y
X: dNaM
Y:. dTPT3
• 152 novel aminoacids can be coded along with the standard
proteinogenic aminoacids
• XY is the first Unnatural Base Pair (UBP) successfully incorporated into
standard DNA to create a Semi-synthetic organism with positive in-vivo trails.
• NO H-bond formation between XY base pair where stability and replication is
mediated by unique mechanism that draws upon interbase hydrophobic and
packing interactions.
Created by a team of scientists from The Scripps Research Institute (TSRI),
USA lead by Dr. Floyd Romesberg.
SSO created by genetically engineering natural E.coli bacterium in 2017.
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9. Stages of Development of SSO
Stage 1: DM1 strain
• UBP used was dNaM – d5SICS
• PtNTT2 transporter from Plac UV5 promoter
transports the UBP compounds from medium into
the cell.
• Gave proof of expanded genetic alphabet.
• High force of hydrophobic interaction between
the base pairs kept the integrity of the double
helix intact when the UBP was engineered into the
standard DNA.
Drawbacks of DM1 which lead to stage 2
• Poor UBP retention
• UBP retention explored only in single locus of a single DNA sequence
• Reduced the growth of the organism, especially with high copy number
plasmids. 9

10. Stage 2: YZ3 strain
• dNaM-d5SICS UBP got replaced by dNaM-dTPT3
UBP ; scientists tried this new pair as the dNaM-
d5SICS pair had too high hydrophobic forces of
interactions which hindered the replication process
and hence wanted a pair with weaker hydrophobic
interaction.
• Codon optimised, chromosomally integrated N-
terminal truncated PtNTT2(66-575) transporter
whose 1-65 aminoacids were cleaved, was used in
YZ3 strain.
• YZ3 strain bearing 3 plasmid study conducted: to
determine whether the optimised transporter
system would facilitate high UBP retention
• pUCX2 showed good UBP retention
PLASMID pUCX1 pUCX2 pBRX2
Copy
Number
Specifica
tion
High High Low
Position
of UBP
with
respect
to Ori
Proximal Distal Distal
10

11. • pUCX2 selected to explore the effect of local sequence context
on UBP retention: 16 pUCX2 variants in which the UBP was
flanked by each possible combination of natural base pairs
within a fragment of GFP gene, results were satisfactory.
SSO growth and retention was poor in solid media: CRISPR Cas 9
type II system used to overcome this in stage 3
Stage 3: YZ4 strain
• Sequences with UBP were found to have reduced Cas
9(endonuclease) activity compared to sequences fully
complimentary to sg (single guide) RNA
• Immunity to UBP loss methodology: Cells with Cas 9
programmed with sgRNA(s) complimentary to natural
sequences that arise from UBP loss would enforce retention
ina population of plasmids
UBP loss was undetectable when a pCas 9 plasmid which
expressed 2 sgRNA(s) was used;
one targeted most common substitution mutation
. Other targeted most common deletion mutation 11

12. Finally a pAIO2X plasmid which contains 2
UBP’s was used,
• 1 UBP in Green Fluorescent Protein
(GFP) gene and
• 1 UBP in Ser T gene
It also contains sg RNA with most
common substitution mutation in both
UBP’S with Cas 9 endonuclease.
Here 17% increase in doubling time and
100% UBP retention was accomplished
The SSO went on to express both the
genes in vivo and lived healthy until it ran
out of raw materials for UBP from its
sorroundings.
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13. HACHIMOJI DNA: 8 letter DNA
• A Genetic System with 8 nucleotides: 4 natural + 4
synthetic.
• Devoloped by a team of scientists from Harvard University
lead by Dr. Steven Benner.
• The system meets with the structural requirements needed
to support: Darwinian Evolution
Predictable thermodynamic stability
Stereo regular building blocks that fit a
Schrodinger Aperiodic crystal
• Increased information storage density
• The four synthetic bases are P, B (purines) Z,S
(pyramidines).
• PZ and BS are the 2 UBP’s with 3 H-bonds between the
complimentary bases.
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14. Experiments conducted to test stability, mutability and transcription of the Genetic System
1. Evaluation on Energy of Duplex formation to
predict Duplex stability
• Tests done on 94 HACHIMOJI duplexes
• Nearest neighbour thermodynamic
parameter of all base pair Dimers were
evalauated
• 40 parameters were required
• Showed stability readings matching Standard
DNA
• Gibbs free energy at different temperatures
were read to draw conclusions
“S” is3-methyl-6-amino-5(1′-b-D-2′-
deoxyribofuranosyl)-pyrimidin-2-one
“B” is 6-amino-9[(1′-b-D-2′-
deoxyribofuranosyl)4-hydroxy-5-
(hydroxymethyl)-oxolan-2-yl]1H-
purin-2-one
“Z” is 6-amino-3-(1′-b-D-
2′deoxyribofuranosyl)-5-nitro-1H-
pyridin-2-one
“P” is 2-amino-8-(1′-b-D-2′-
deoxyribofuranosyl)imidazo-[1,2a]-
1,3,5-triazin-[8H]-4-one.
14

15. 2. To test Mutation ability without damaging Schrodinger’s Aperiodic crystal structure
• 3 Self complimentary HACHIMOJI duplexes;PB,PC and PP were crystallised by Moloney murine leukemia
virus reverse transcriptase
• Host- guest complex formed: 2 protein molecules (of host) bound to each of 16 mer duplex guest
sequence
• The intervening 10 BP’s were free to adopt a sequence dependent structure.
• All 3 duplexes formed B-DNA structure with 10.2 -10.4 BP’s/turn.
• The UBP’s were compared to natural base pairs and was shown to keep the Aperiodic crystal structure
needed for evolution.
15

16. 3. Transcription test
• Natural T7 polymerase transcribed all other 3
base pairs but failed to incorporate S opposite to
template dB.
• This was due to the absence of electron density
in S
• Polymerases recognise such electron densities
which is present in all other triphosphate
substrates
• Finally FAL RNA Polymerase successfully
transcribed all the four base pairs Hence HACHIMOJI DNA is a stable mutable
GM that can transcribe into HACHIMOJI RNA
with the largest information density among
GM’s upto date.
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17. Lets conclude with the facts that;
1. Our GENETIC SYSTEM is not unique.
2. Life could have evolved differently and
terran biology is simply one of the many
paths to life.
3. The Age of Semi Synthetic life have
begun.
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18. References
1. Y Zhang et al(2017); A semi synthetic Organism engineered for the stable expansion of the genetic alphabet
2. Hoshika et al (2019); HACHIMOJI DNA and RNA: a genetic system with eight building blocks
3. Malyshev DA , Romesberg FE(2015): the expanded genetic alphabet
4. Kimoto M, Hirai(2014); creation of Unnatural base pair systems towards new DNA/RNA technologies
5. Leduc S (1911); the mechanisms of life
6. Erwin Schrodinger (1944); What is life?
7. Varn DP, Crutchfield JP. 2016 What did Erwin mean? The physics of information from the materials genomics of
aperiodic crystals and water to molecular information catalysts and life. Phil. Trans. R.
Soc. A 374: 20150067.
http://dx.doi.org/10.1098/rsta.2015.0067
8. Adding new chemistries to the genetic code; Chang C. Liu and Peter Schultz, doi:
10.1146/annurev.biochem.052308.105824
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