DNA Templated Synthesis (DTS) -Nature’s effective molarity based approach-

Organic Seminar 27th May 2013

Bioorganic Chemistry Laboratory

D2 Naohiro Terasaka

1

Effective-molarity-based control of bond formation

2 Xiaoyu Li and David R. Liu, Angew. Chem. Int. Ed., 2004, 43, 4848-4870

nM-μM concentrations of many

reactants in one solution

macromolecule-

templated synthesis

selective product

formation

one possible

product

mM-M concentrations

isolated in one vessel

Chemists’ approach

Nature’s approach

The reactivities of these molecules are directed by modulating the effective molarity

of reactive groups and by providing catalytic functionality.

Nucleic acid templated synthesis –Translation–

3

Transcription Translation

DNA RNA

Protein

Replication

mRNA

tRNA

Nucleic acid templated synthesis plays a

important role in “central dogma”.

Nucleic acids have amplifiability,

inheritability, and the ability to be

diversified.

Central dogma

There are many applications of

nucleic acid templated synthesis to

non-biological reactants.

LG=

Chemical DNA/RNA ligation

pH7.0, 4 °C

Non-enzymatically backbone formation

4

Tan Inoue and Leslie E. Orgel, J. Am. Chem. Soc., 1981, 103, 7666-7667

Xiaoyu Li and David R. Liu, Angew. Chem. Int. Ed., 2004, 43, 4848-4870

John M. Pascal et al., Nature., 2004, 432, 473-478

Ligation by Human DNA ligase

There are several reports about the templated synthesis of nucleic acid

backbones and their analogs like phophonamide and amide bonds.

DTS unrelated to the DNA backbone

5

X

-ACCTGACAA-

Y

-GACGGCACC-

X

-CTGCCGTGG-

Y

-GACGGCACC-

No reaction

-CTGCCGTGG-

-GACGGCACC-

X

|

Y

Effective molarity

~60 nM

Effective molarity

~1 M

Zev J. Gartner, David R. Liu, J. Am. Chem. Soc., 2001, 123, 6961-6963

-CTGCC TGG

-GACGGCACC A T

C A

X Y

-CTGCC TGG

-GACGGCACC A T

C A

X-Y

DNA-templated synthesis (DTS) have the ability to direct the creation of

structures unrelated to the nucleic acid back-bone

Contents

Characteristics of DNA-templated synthesis (DTS)

• Template architecture

• Design of linker

Recent Applications

• Nucleic acid sensing

• Reaction discovery

• In vitro selection of kinase inhibitor

6

Template architecture (end, hairpin, Ω)

7 Zev J. Gartner et al., Angew. Chem. Int. Ed., 2003, 42, 1370-1375

end-of-helix (E)

n=1

hairpin (H)

n=1 omega, 3-base

constant region (Ω-3)

n=10

end-of-helix (E)

n=10

DNA duplex rigidity can inhibit

3-component reactions that

use template architectures

with terminal reactive groups

Template architecture (three substates)

8

amino modified dT

Y-architecture

The modified dT does not inhibit

the hybridization and PCR reaction.

Zev J. Gartner et al., Angew. Chem. Int. Ed., 2003, 42, 1370-1375

Lars H. Eckardt et al., Nature, 2002, 420, 286

T-architecture

Design of linker

9 Zev J. Gartner et al., J. Am. Chem. Soc., 2002, 124, 10304-10306

Cleavage of “scarless linker” generates a functional group that serves as a

substrate in subsequent steps.

A “scarless linker” is cleaved without introducing additional unwanted

functionality.

An “autocleaving linker” is cleaved as a natural consequence of the reaction.

Enzyme-free translation of DNA by DTS

10 Jia Niu, et al., Nat. Chem., 2013, 5, 282-292

Polymers were synthesized like

translation system by ribosome.

Translation by ribosome and tRNAs

miRNA imaging in human cell by DTS

11 Gorska Katarzyna, et al., Chemical Science., 2011, 2, 1969-1975

tmTCEP = transmembrane TCEP

PM = perfect match

MM = mismatch

Azidrhodamine was reducted to rhodamine by Staudinger reaction with phosphine-

PNA.

This method enables to quantified the amount of RNAs in living cells.

RNA-templated molecule release in bacterial cells

12 Aya Shibata, et al., Chem. comm., 2013, 49, 270-272

This system enabled to release a active molecule in response to the

sequence of a target gene.

This system could be applied to specific drug release to target cells.

no probe

Reaction discovery by DTS and in vitro selection

13 Matthew W. Kanan, et al., Nature, 2004, 431, 545-549

Post-selected DNAs

Pre-selected DNAs

No reaction

Reaction

Reaction discovery by DTS and in vitro selection

14 Matthew W. Kanan, et al., Nature, 2004, 431, 545-549

This system was available in

the condition where DNA can

make duplex.

Reaction discovery by non-hybridized DTS

15 Mary M Rozenman, et al., J. Am. Chem. Soc., 2007, 129, 14933-14938

Reaction discovery by non-hybridized DTS

16 Yiyun Chen, et al., Nat. Chem., 2011, 3, 146-153

A biomolecule-compatible visible-light-induced azide reduction was

discovered by this non-hybridized DTS system.

Translation of DNA into a small molecule library

17 Ralph E. Kleiner, et al., J. Am. Chem. Soc., 2010, 132, 11779-117791

In Vitro selection of a DTS small-molecule library

18 Ralph E. Kleiner, et al., J. Am. Chem. Soc., 2010, 132, 11779-117791

12×12×12×8 = 13824 compounds

Highly specific Src inhibitors from DTS library

19 Ralph E. Kleiner, et al., J. Am. Chem. Soc., 2010, 132, 11779-117791

George Georghiou, et al., Nat. Chem. Biol., 2012, 8, 366-374

IC50 = 15 μM

IC50 = 6.8 μM

IC50 = 0.13 μM

IC50 = 0.099 μM

Src is one of tyrosine kinase and src gene is oncogene.

2

Improve

9

4b

25b

The highly specific inhibitors

against Src kinase were

obtained by DTS selection

and further optimization.

These mutation positions are

different between Src and Hck.

Hck is Src-family kinase.

Summary

• DTS enables the reactivity of synthetic molecules to

be controlled by modulated effective molarities.

• DTS can translate amplifiable information into

synthetic structures and it was easily detected by

PCR, microarray and sequencing.

• There are some applications including nucleic acid

detection, synthetic small-molecule and polymer

discovery and reaction discovery.

20