dna templated synthesis (dts) - university of...
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DNA Templated Synthesis (DTS) -Nature’s effective molarity based approach-
Organic Seminar 27th May 2013
Bioorganic Chemistry Laboratory
D2 Naohiro Terasaka
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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.
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Nucleic acid templated synthesis –Translation–
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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.
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LG=
Chemical DNA/RNA ligation
pH7.0, 4 °C
Non-enzymatically backbone formation
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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.
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DTS unrelated to the DNA backbone
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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
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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
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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
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DNA duplex rigidity can inhibit
3-component reactions that
use template architectures
with terminal reactive groups
Template architecture (three substates)
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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
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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.
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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
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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.
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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
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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
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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.
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Reaction discovery by non-hybridized DTS
15 Mary M Rozenman, et al., J. Am. Chem. Soc., 2007, 129, 14933-14938
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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.
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Translation of DNA into a small molecule library
17 Ralph E. Kleiner, et al., J. Am. Chem. Soc., 2010, 132, 11779-117791
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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
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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.
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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.
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