Is Colorful.

Is Miracle.

Is a Colorful Miracle.

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ARIJIT SHOME M-5490 (Veterinary Biochemistry)

Credit Seminar

DNAS 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

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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 DNAs backbone at Swiss Federal Institute of Technology.

Putative Watson-Crick pairing interaction between iso-C and iso-G

BENNERS 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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BENNERS GROUP IN 1989

1. The UBP had a proton donor site unlike 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 Kools 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 KOOLS 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 HIRAOS GROUP (1997)

UBPs 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 Hiraos 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 HIRAOS 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 Hiraos 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 Hiraos group

Successful UBP : Ds-Diol-Px 18

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 Hiraos group2005

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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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Romesbergs Group

The self pair between 6-propynylcarbostyril (PICS) bases

Romesbergs 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 (XY), which works together with the natural AT and GC 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 2014 MAJOR

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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UBPs 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 d5SICSdNaM.

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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Therefore,

Artificial rebuilding of central dogma by introducing third base pairmade possible

Any functional group of interest can be attached to UBPs 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.

Romesbergs 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

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-

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 Benners 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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UNBs application in in-vivo system would open up a new safer genetic expansion technology beyond the present genetic recombination techniques

In-vivo system using UBPs protein containing non standard amino acids can be found.

Future Prospects

We need to learn more about the ability and dynamics of UBPs 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.; Corra, 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) DNAs New Alphabet, Nature 491:516-518.

Schweitzer, B. A. & Kool, E. T. (1995). J. Am. Chem. Soc. 117, 18631872.

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 DNAs Alphabet, Raising Hope and Fear". New York Times.

REFERENCES

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ACKNOWLEDGEMENTS

I 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 wouldnt 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.