DNA Templated Synthesis (DTS) - University of Tokyocms.chem.s.u-tokyo.ac.jp/SysChem/files/public/... · Chem. Int. Ed., 2004, 43, 4848-4870 nM-μM concentrations of many reactants
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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
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
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