Toward the expansion of the genetic alphabet of DNA, several artificial third base pairs (unnatural base pairs) have been created. Organisms are defined by the information encoded in their genomes, and since the origin of life this information has been encoded using a two-base-pair genetic alphabet (A–T and G–C). In vitro, the alphabet have been expanded to include several unnatural base pairs (UBPs). A class of UBPs formed between nucleotides bearing hydrophobic nucleobases, exemplified by the pair formed between d5SICS and dNaM (d5SICS–dNaM) was developed, which is efficiently PCR-amplified and transcribed in vitro, and whose unique mechanism of replication has been characterized. However, expansion of an organism’s genetic alphabet presents new and unprecedented challenges: the unnatural nucleoside triphosphates must be available inside the cell; endogenous polymerases must be able to use the unnatural triphosphates to faithfully replicate DNA containing the UBP within the complex cellular milieu; and finally, the UBP must be stable in the presence of pathways that maintain the integrity of DNA. In a major breakthrough, it was reported that an exogenously expressed algal nucleotide triphosphate transporter efficiently imports the triphosphates of both d5SICS and dNaM (d5SICSTP and dNaMTP) into Escherichia coli, and that the endogenous replication machinery uses them to accurately replicate a plasmid containing d5SICS–dNaM was already reported. Neither the presence of the unnatural triphosphates nor the replication of the UBP introduces a notable growth burden. Thus, the resulting bacterium is the first semi-synthetic organism to propagate stably an expanded genetic alphabet. The unnatural base pair systems now have high potential to open the door to next generation biotechnology.

1. Is Colorful…….
Is Miracle…….
Is a Colorful Miracle……
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2. 2

3. ARIJIT SHOME
M-5490 (Veterinary Biochemistry)
Credit Seminar
DNA’S NEW ALPHABET
on
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4. Background
For billions of years, the history of life has been written with just four letters-
A, T, C and G
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5. A T
G C
“PEARL” OF SCIENCE-
The genetic code
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Scientific acumen of Scientists does not allow to be satisfied with tradition and
one always looks for “NEW” and beyond ………………
Is there anything
beyond A T G C... ??
Unnatural Base Pair

6. UNNATURAL BASE PAIR (UBP)
Designed subunit
(nucleobase) of
DNA
Created in a
laboratory
Does not occur
in nature.
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NEW, FUNCTIONAL DNA BASE PAIR
Code for proteins and
Organisms that have never existed before.
Requirements
Bind with an affinity comparable to that A, T, G
& C
Line up stably alongside the natural bases in a
zipper-like stretch of DNA.
Unzip and re-zip smoothly when worked on by
natural polymerase enzymes during DNA
replication and Transcription into RNA
Avoid being attacked and removed by natural
DNA-repair mechanisms
.
TARGET !!!!!!!
•Shape complementarity
• Polarity
• Electrical repulsion, and
• Hydrophobicity between
pairing bases, and
• Minor groove interactions with
polymerases,
Factors need to be
considered when creating
unnatural base pairs.

8. Steven A. Benner
- Biological Chemist
Steven Benner founded The Westheimer Institute of
Science and Technology (TWIST) and the Foundation
for Applied Molecular Evolution (FFAME),
Research summary
Initiated synthetic biology as a field. The Benner
group was the first to synthesize a gene for an
enzyme, and used organic synthesis to prepare
the first artificial genetic systems. These systems
have been used to direct the synthesis of artificial
proteins having unnatural amino acids.
PIONEER

9. Started a lab in 1986 to rebuild DNA’s backbone at Swiss Federal Institute
of Technology. Catalytic RNA – RNA catalysis
Putative Watson-Crick pairing interaction between iso-C and iso-G
BENNER’S GROUP IN 1989
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Isoguanine and isocytosine,
have their amine and
ketone inverted compared
to standard guanine and
cytosine
They showed Polymerase could read DNA containing unnatural bases and
insert the complimentary partners into a growing DNA or RNA strand.
Ribosome could also read an RNA snippet containing iso-C and use it to an
unnatural amino acid to a growing protein.
Bain et al. (1992) Nature.356:537-39
isoG:6-amino-2-ketopurine ,
isoC:2-amino-4-ketopyrimidine

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BENNER’S GROUP IN 1989
1. The UBP had a proton donor site as compared to natural
base pair which is unfavourable for polymerase recognition.
2. The isoC is chemically unstable under alkaline and acidic
conditions. Hence difficult to prepare in lab.
3. Hydrogen bonded UBP suffer from instability due to
tautomerism.
Although their efforts revealed the possibility of a genetic epansion system, they
also highlighted some shortcomings

11. Eric T. Kool
Department of Chemistry and
Synthetic Biology, University of
Stanford
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Kool's research interests lie in the interdisciplinary
fields of organic chemistry, chemical biology, and
biophysics.

12. The chemical structure of Kool’s base pairs
(a) The hydrophobic unnatural Z-F pair
(b) The natural A-T pair
(c) The hydrophobic unnatural Q-F pair.
Key point:
Emphasized the importance of shape complementarity of the pairing
bases rather than the hydrogen bonding interactions.
Non-hydrogen bonded hydrophobic base analogs could be potential
candidates for creating unnatural third base pair.
ERIC KOOL’S GROUP – Mid 1990s
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Z: 4-methylbenzimidazole
F: 2,4-difluorotoluene

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Ichiro Hirao
Team leader at the RIKEN System and Structural Biology Center, Yokohama,
Japan.
He is interested in the creation of unnatural base pairs towards the expansion
of the genetic code and re-engineering DNA/RNA materials

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ICHIRO HIRAO’S GROUP (1997)
UBP’s synthesized by Hirao and co –workers
x= 2-amino- 6-(N,N-dimethylamino)purine
y= pyridine-2-one
s= 2-amino-6-(2-thienyl)purine
y= pyridine-2-one
v= 2-amino-6-(2-thiazoyl)purine
y= pyridine-2-one
BP-1
BP-2
BP-3

15. Hydrogen-bonded, unnatural base pairs – Chemical structures of Hirao’s pairs – (a)
The unnatural x-y pair, (b) The unnatural s-y or v-y pair, (c) The non-cognate pairing of s
or v with T. (d) The cognate s-z pair.
(d)
ICHIRO HIRAO’S GROUP (1997)
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A two unnatural base pair system of the s-y and s-z pairs could be used as
a coupled transcription and translation system for the site specific
incorporation of nonstandard amino acids into proteins
In 2002, 3-chlorotyrosine incorporation at site 32 of Ras protein was
achieved and was the first evidence of direct synthesis of protein
containing a non-standard amino acid at a specific position by
transcription and translation system using its long DNA template.

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atoms of s, y, z attract natural hydrophilic bases in
replication so switched over to Hydrophobic unnatural
base pairs
Fig: Hydrogen bonded,
unnatural base pairs designed
by Hirao’s group
a) The cognate S-Y pair
b) The non cognate A-y pair
c) The cognate s-z pair
d) The non cognate A-z pair
Ichiro Hirao’s group

17. Successful UBP : – Ds-Diol-Px
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a
b
c
Fig. Hydrophobic, unatural base pairs.
a) The hypothetical self pair between 4-
methylpyridin-2-one (4MP) bases
b) The Q-F pair. The clash of the hydrogen atoms
in the centre of the Q-F pairing surface is
indicated in red
c) The Q-Pa pair
Ichiro Hirao’s group…2005

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Expansion of the Genetic Alphabet
Expansion of the genetic alphabet to include a third base pair would be
a fundamental accomplishment that would not only have immediate
utility for a number of biotechnology applications, such as site-specific
labeling of DNA and RNA, but would also lay the foundation for a semi-
synthetic organism with increased potential for information storage and
retrieval.
Floyd Romesberg, Biological
Chemist, leads the work at the
Scripps Research Institute in
California.

19. Selectivity of 5SICs-NaM is 99.8 % per replication in PCR
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Romesberg’s Group
The self pair between 6-propynylcarbostyril (PICS) bases
Romesberg’s Group 2009 reported the following modifying improvement of
(shape complementarity) their initial PICS-PICS pair

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Unnatural Base pair has
expanded
the genetic alphabet in the central dogma
FIGURE----The unnatural base pair (X–Y), which works together with
the natural A–T and G–C base pairs in the central dogma, allows the site-
specific incorporation of extra unnatural nucleotides (X and Y) into DNA
and RNA, and unnatural amino acids (unAA) into proteins.

21. Fig : Unnatural base pairs that can be used as a third base pair in
in vitro biology systems …until 2012
15 May 2014
MAJOR
BREAK THROUGH

22. Scientists create first living
organism that transmits added
letters in DNA 'alphabet'
A semi-synthetic organism with an expanded
GENETIC ALPHABET
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UBP’s in in-vivo system…
A major Breakthrough
Malyshev et al ., 2014- Nature 509: 385-388

23. An exogenously expressed algal Nucleotide Triphosphate
Transporter (NTT) efficiently imports the triphosphates of both
d5SICS and
dNaM (d5SICSTP and dNaMTP) into Escherichia coli,
and
The endogenous replication machinery uses them to accurately
replicate
a plasmid containing d5SICS–dNaM.
Found that the UBP is not efficiently excised by DNA repair pathways.
First Life with Alien DNA... Achieved!!
Thus, the resulting bacterium is the
first organism to propagate stably an expanded genetic alphabet.
Malyshev et al ., 2014- The study showed:
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A
Glimpse
http://synthorx.com/technology/

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http://synthorx.com/technology/

26. Therefore,
Artificial rebuilding of central dogma by introducing third base pair—made possible
 Any functional group of interest can be attached to UBP’s to get nucleic acids
with desired functionalities.
KEY OBSERVATION:
The new bases can only get into the cell if--- turn on the 'base transporter' protein.
Without this transporter or when new bases are not provided, the cell will revert
back to A, T, G, C, and the d5SICS and dNaM will disappear from the genome.”
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Expanded Genetic Code
63 = 216 codons.
216-44=172 amino
acids

28. Incorporation of a
toxic amino acid
into a protein to
ensure that it kills
only cancer cells
Development of glowing
amino acids that could
help scientists to track
biological reactions
under the microscope.
Romesberg’s
team has founded
a company called
Synthorx in San
Diego, California,
to commercialize
the work.
Malyshev, D. A. et al. Nature /10.1038/(2014).
APPLICATIONS
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Ichiro Hirao founded company ‘TagCyx Biotechnologies’ with Dr. Shigeyuki
Yokoyama, to provide unnatural base pair technologies toward the expansion of
the genetic alphabet of DNA

29. Inserting unnatural
bases into some of the
sequences in the
sample makes the HIV-
1 RNA easier to detect
at low levels
Unique unnatural
base pairs function
as a molecular
beacon and in real-
time quantitative
PCR to detect a
target nucleic acid
sequence
Ds base binds much
better than natural
sequences to IFN-γ
and vascular
endothelial growth
factor(VEGF) a
therapeutic target in
cancer and eye
disease.
Malyshev, D. A. et al. Nature /10.1038/(2014).
APPLICATIONS
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Siemens Health Care Diagnostics, New York & Luminex Texas
Use Benner’s isoC and isoG to improve detection and monitoring of viral
infections.

30. FUTURE Applications
Regulation of
lymphocyte
activation
Transfer of
peptide-MHC
complexes from
APCs to
lymphocytes
Direct secretion of
cytokines or lytic
granules
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UNB’s application in in-vivo system would open up a new safer
genetic expansion technology beyond the present genetic
recombination techniques
In-vivo system using UBP’s protein containing non standard
amino acids can be found.
Future Prospects
We need to learn more about the ability and dynamics of UBP’s in
the cell which in turn will provide NEW SYNTHETIC BIOLOGY
TOOL to elucidate molecular mechanisms in living organisms.
Future Directions

32. Malyshev, D.A.; Dhami, K.; Lavergne, T; Chen, T; Dai, N; Foster, J, M.; Corrêa, I,
R.; Romesberg, F, E. (2014). "A semi-synthetic organism with an expanded genetic
alphabet". Nature 509:385-388 .
Hirao, I,; Kimoto,M.(2012) Unnatural base pair systems towards the expansion of
the genetic alphabet in the central dogma. Proc. Jpn. Acad., Ser B 88: 345-367.
Kwok, R,; (2012) DNA’s New Alphabet, Nature 491:516-518.
 Schweitzer, B. A. & Kool, E. T. (1995). J. Am. Chem. Soc. 117, 1863–1872.
 Callaway, Ewan (2014). "Scientists Create First Living Organism With 'Artificial'
DNA". Nature News (Huffington Post).
 Fikes, Bradley J. (2014). "Life engineered with expanded genetic code". San
Diego Union Tribune.
 Pollack, Andrew (2014). "Scientists Add Letters to DNA’s Alphabet, Raising
Hope and Fear". New York Times.
REFERENCES
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ACKNOWLEDGEMENT
SI would never have been able to finish my seminar without the guidance of
my teachers , help from friends, and support from my father .
I would like to express my deepest gratitude to my teacher, Dr. C. Madan Mohan,
for his excellent guidance, infusing the scientific acumen in me.
I would like to thank Dr. Sohini Dey, who patiently corrected my writing.
I would also like to thank my advisor Dr. Mohini Saini for providing me with the
articles needed for the presentation. Without you madam this wouldn’t be possible.
I would like to thank Dr. Sunil Kumar S .P. , who as a good friend, was always
willing to help and give his best suggestions in improving my presentation.
Many thanks to Dr. Susweta Das Mitra , Research Associate at the National
Institute of Veterinary Epidemiology and Disease Informatics ( NIVEDI),
Bengaluru for bringing presentation to present shape.
Lastly but not the least I thank my dear father Dr. Bibek R. Shome who guided
me step by step in the entire journey of my seminar and helped in understanding the
subject in detail.