Is Colorful…….

Is Miracle…….

Is a Colorful Miracle…….

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ARIJIT SHOME

M-5490 (Veterinary Biochemistry)

Credit Seminar

DNA’S NEW ALPHABET

on

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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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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

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

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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.

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

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

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.

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

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

Successful UBP : – Ds-Diol-Px

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a

bc

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.

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.

Fig : Unnatural base pairs that can be used as a third base pair in

in vitro biology systems …until 2012

15 May 2014MAJOR

BREAK THROUGH

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

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/

25http://synthorx.com/technology/

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

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’steam 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

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.

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

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.