Anti-parallel triplex DNA

advertisement
Non-Natural Nucleobases for the Triplex DNA Formation
as A New Strategy for Regulation of Gene Expression
Duplex DNA





TFO
(triplex-forming ODN)
Triplex DNA
Parallel triplex DNA
Anti-parallel triplex DNA
A pyrimidine-rich TFO binds parallel to the
homopurine strand of duplex DNA.
A purine-rich TFO binds anti-parallel to the
homopurine strand of duplex DNA.
Formation in the major groove of the duplex.
Inhibition of transcription (antigene)
Recruitment of biological systems
Site-specific modification of DNA
Initiation of DNA repair events
Probability in human genome
(15 bases length, >50% G with one pyrimidine)
 At least one exist in the promoter/transcribed
region of ~98% annotated human genes
 ~87% of them are the unique to one gene
Limitation of Duplex Sequence for Triplex Formation
TFO
(triplex-forming ODN)
Duplex DNA
Stable Triplexes
Form
with
HomopurineHomopyrimidine
Strand
TFO
A
A
T
G
G
C
A
A
T
A
A
T
G
G
C
G
G
C
G
G
C
Triplex DNA
Pyrimidine Inserts
Destabilize
Triplexes
Interrupting Site
TFO
A
A
T
G
G
C
A
A
T
T
A
A
G
G
C
G
G
C
G
G
C
Non-Natural Base Analogs Needed
for TA and CG Interrupting Sites
Interrupting Sites
Antiparallel Triplex
T FO
TFO
ReverseHoogsteen
H-bonds
WatsonCrick pair
Py
Pu
Py
Pu
T
A
A
T
T FO
TFO
ReverseHoogsteen
H-bonds
Pu
Py
Py
WatsonCrick pair
Pu
Pu
C
H
G
Non-Natural Base Analogs for TA and CG Interrupting Sites
In Parallel Triplex DNA
Ohkubo, K. Yamada, Y. Ito, K. Yoshimura, K. Miyauchi, T.
Kanamori, Y. Masaki, K. Seio, H. Yuasa, and M. Sekine,
Nucleic Acids Res., 2015, 1–12.
Y. Hari, M. Akabane, and S. Obika, Chem.
Commun., 2013, 49, 7421.
D. A. Rusling, Vicki E. C. Powers, R. T. Ranasinghe, Y.
Wang, S. D. Osborne, T. Brown, and K. R. Fox,
Nucleic Acids Res., 2005, 33, 3025–3032.
Li, J.-S., Chen, F.-X., Shikiya, R., Marky, L. A. & Gold,
B. J. Am. Chem. Soc. 127, 12657–12665 (2005).
Difference in Geometry between
Parallel and Antiparallel Triplexes
Antiparallel
Parallel
Natural T-A:T
TFO
Natural A-A:T
TFO
b
ReverseHoogsteen
a
Natural G-G:C
b
Design of Recognition Molecules for a TA and a CG Sites
Stacking part
•Hydrogen Bondings.
DNAO
O
DNAO
Recognition part
•Stacking Interactions.
•Shape complementarity.
O
W-Shaped Nucleic Analog (WNA)
1.A short spacer
O
O
O
O
5'
O
O
O
H N
N
H N
O
H
3'
N
N
N
N
3'
O
O
a
H
Aromatic ring
O
O
3'
N
Bicyclic ring
5'
5'
O
3. Stacking unit
2. Fixed conformation
O
H N
N
H N
O
3'
H
N
N
N
N
3'
O
H N
N
H N
O
H
N
N
N
3'
Diversity of WNA Structure
SubstitutedBenzene
Naphthalene
Thiophene
Furane
Pyrrole
etc
Aromatic part
D NA O
D NA O
O
O
Recognition part
H
guanine
thymine
adenine
cytosine
5-methylcytosine
pyridone
imidazole
etc
Synthesis of WNA
R=TB DPS
HO
1)Acetone, H+
O
HO
O H 2)Ac2O, Pyr
OH
3) piperidine
Ac O
O
O
OH 1) PCC
O
2) PhLi
RO
O
O
D-ribose
O H 1) TBDPSCl
O
RO
2) Allyltrimethylsilane
ZnBr2, CH3NO2
O
O
O
(α-Allyl: β-Allyl= 7 : 6)
RO
O
O
H
OsO4,
NaIO4, Pyr
O
O
5%H2SO4,
THF, 60
Ž
RO
O
HO
O HH
O H Ac O, Pyr
2
RO
O
H
AcO
O
OA c
Syntheses of WNA Analogs
Et3SiH
TMSOTf
CH2Cl2
O
RO
O
R'O
OA c
R : TBDPS
R' : Ac
RO
R'O
iBu-G
BSA
TMSOTf
CH3CN
Bicyclic intermediate
O
O
WNA-H: 72%
iBuNH
N
RO
O
R'O
N
O
WNA-bT: 42%
+ aT: 37%
Bz-C
BSA
SnCl4
CH3CN
Bz-mC
Thymine
HMDS
TMSCl
SnCl4
CH3CN
Bz-A
BSA
TMSOTf
CH3CN
BSA
SnCl4
CH3CN
RO
O
R'O
NH
O
N N
O
WNA-9bG: 38%
+9aG:15%, 7bG: 25%, 7aG: 10%
O
NHBz
NHBz
NH
N
N
RO
O
O
R'O
N
O
WNA-bmC: 53%
+ amC: 14%
O
RO
O
R'O
N
O
O
WNA-bC: 53%
+ aC: 40%
N
RO
O
R'O
O
N
N N
WNA-9bA: 53%
+ 9aA: 40%
NHBz
Formation of the Stable Triplex Having a TA Interrupting Site
3' GGA AGG A Z G GAG GGA GGA -32P
TFO(10nM)
5' GGG AGG GAG GGA AGG A X G GAG GGA GGA AGC (Pu)
duplex
3' CCC TCC CTC CCT TCC T Y C CTC CCT CCT TCG (Py)
Z=WNA-bT
DN A O
XY
GC
AT
0 4 8 10 40 100 0 4 8 10 40 100
O
O
D NA O
duplex
concentration
thymine
O
N
b
O
XY
nM
duplex
concentration 0
Triplex
Triplex
32P-TFO
32P-TFO
Ks (M-1x109)
0.082
< 0.001
NH
CG
4 8 10 40 100
0.015
TA
0 4 8 10 40 100
nM
0.30
Ks=
[triplex]
[TFO] x [duplex]
Gel shift assay for determination of triplex formation. Triplex formation was done for 12 hours at 22 °C
in the buffer containing 5 mM MgCl2, 20 mM Tris-HCl, 2.5 mM spermidine and 10 % sucrose at pH 7.5.
Electrophoresis was done at 10 °C with 20 % non-denatured polyacrylamide gel.
Recognition of all four base pairs by WNA analogs
3’ GGAAGG AZG GAGGAGGGA-32P 5’
Target 5’ GGGAGGGAGGGAAGG AXG GAGGAGGGAAGC 3’
Duplex 3’ CCCTCCCTCCCTTCC TYC CTCCTCCCTTCG 5’
TFO
NH2
O
DNA
DNA
NH
O
N
O
O b
O
O
N
O
(Thymine)
O b
O
WNA-bT
DNA
N
O
Selective to the TA pair
DNA
O
(Cytosine)
WNA-bC
Selective to the CG
pair
Stability constants (Ks, 109M-1)
XY
Z
TA
AT
CG
GC
dG
0.004
0.008
0.008
0.086
G / GC
dA
<0.001
0.074
<0.001
0.047
A / AT
WNA-bT
0.300
<0.001
0.015
0.082
WNA-bT / TA
WNA-bC
<0.001
0.025
0.115
0.047
WNA-bC / CG
*5mM MgCl2 , Ks =
[Triplex]
[TFO][Duplex]
S. Sasaki, et al., J. Am. Chem. Soc., 126, 516-528 (2004).
The selectivity of the WNA depends on the flanking bases
TFO
Target
Duplex
3’ GGAAGG NZN GAGGAGGGA-32P 5’
5’ GGGAGGGAGGGAAGG NXN GAGGAGGGAAGC 3’
3’ CCCTCCCTCCCTTCC NYN CTCCTCCCTTCG 5’
NH2
O
DNA
DNA
NH
O
N
O
O b
O
DNA
O
N
O
N
O
(Thymine)
O b
O
WNA-bT
DNA
O
(Cytosine)
WNA-bC
Stability constants (Ks, 109M-1)
Z /XY
3’-AZG-5’ 3’-GZG-5’ 3’-AZA-5’ 3’-GZA-5’
AXG
GXG
AXA
GXA
TYC
CYC
TYT
CYT
WNA-bT/TA
0.300
0.130
<0.001
<0.001
WNA-bC/CG
0.115
0.002
<0.001
0.004
Recognition ability of WNA depends of flanking base pairs
Suitable WNA Analogs for a TA Site
10 nM TFO 3’-GGAAGG
Target 5’-GGGAGGGAGGGAAGG
Duplex 3’-CCCTCCCTCCCTTCC
3’
Ks
(109
M-1)
0.3
AZG
AXG
TYC
NZN GAGGAGGGA
NXN GAGGAGGGAAGC
NYN CTCCTCCCTTCG
5’
Ks
(109 M-1)
0.25
0.12
Br
O
0.2
O
O
0.15
NH
O
O
0.05
GC
AT
CG
0
pCN-
pBr-
mBr-
oBr-
WNA-b T dG
M-1)
0.06
0.05
3’
AZA
AXA
TYT
0.06
0.04
0
O
5’
O
pCN-
pBr-
mBr-
oBr-
WNA-bT dG
TA
NH
O
N
O
O
Ks
(109 M-1)
0.12
mBr-WNA-bT
3’
GZA
GXA
CYT
5’
0.1
CN
0.08
O
0.03
0.02
O
0.01
GC
AT
CG
0
pBr-
GC
AT
CG
0.02
TA
0.04
pCN-
5’
0.08
Br
O
Ks
GZG
GXG
CYC
0.1
N
oBr-WNA-bT
0.1
(109
0.14
O
3’
mBr-
oBr- WNA-b T
dG
TA
N H 0.04
O
O
0.06
N
O
pCN-WNA-bT
O
0.02
GC
AT
CG
0
pCN- pBr-
mBr- oBr- WNA-b T
dG
TA
1) Y. Taniguchi, et al. Tetrahedron, 64, 7164–7170 (2008), 2) Y. Taniguchi, et al. J. Org. Chem., 71, 2115–2122 (2006), 3) Y. Taniguchi, et al. Nucleosides,
Nucleotides and Nucleic Acids, 24, 823–827 (2005)
An Application of TFO for Antiproliferative Effects to A549 Cells
Blc2 TFO(bT)
Survivin TFO(nat)
Survivin TFO(bT)
Random
Cell viability (%)
Bcl2 TFO(nat)
(mM)
The A549 cell was treated with the complex of TFOs and OligofectamineTM and PLUS Reagent in
DMEM medium containing 10%FBS, incubated for 72 hr under 5% CO2 at 37 oC. The cell
proliferation was checked by CellTiter 96® (PROMEGA). (n = 3)
Bcl2
Survivin
3’ ---------AGGGG GTGGTGGAGGAAGAGGGGTG GGGAG- ------5’
.
3’ ---------GTGAC GGAAGAAGGAGGGAGTGAAGAG
TGGAC- --5’
5’ ---------TCCCC CACCACCTCCTTCTCCCCAC CCCTC-------3’
5’ ---------CACTG CCTTCTTCCTCCCTCACTTCTC ACCTG- --3’
Bcl2 TFO(bT) 5’ GbTGGbTGGUGGUUGUGGGGbTG-amino 3’
Survivin TFO(bT) 5’ GGUUGUUGGUGGGUGbTGUUGUG-amino 3’
Bcl2 TFO(nat) 5’ GTGGTGGUGGUUGUGGGGTG-amino 3’
Survivin TFO(nat) 5’ GGUUGUUGGUGGGUGTGUUGUG-amino 3’
Random 5’ UUGTGGUGGGUGGUGGUGUGUU-amino 3’
O
HO
O
HO
N
O
NH
O
WNA-bT
Taniguchi Y and Sasaki S.,,Org. Biomol. Chem., 10(41),8336-8341 (2012).
Desigen of N 2-modified Isocytidine Derivatives for
Selective CG Base Pair Recognition
Dr. Hidenori Okamura,
Associate Prof. Yosuke Okamura
Non-natural
nucleoside
TFO
Duplex
DNA
Triplex
DNA
N = A, G,T, C
GuanidinoisodC
Thymidine
C
G
Weak and low-selective
interaction
C
G
AP-isodC
C
G
H. Okamura, Y. Taniguchi and S. Sasaki, Org. Biomol. Chem., 2013
H. Okamura, Y. Taniguchi and S. Sasaki, ChemBioChem, 2014
Confirmaiton of Isocytidine Derivatives for Selective
CG Base Pair Recognition
TFO
Duplex DNA
Z=
3’-GGAAGGG Z AGAGGAGGGA
5’-GAGGGAAGGG X AGAGGAGGGAAGC
3’-CTCCCTTCCC Y TCTCCTCCCTTCG-FAM
XY =
TFO (nM)
Triplex DNA
Duplex DNA
(100 nM)
GC
0 10 50 100 500 1000
Conditions : 20 mM Tris-HCl buffer,
20 mM MgCl2, 5 mM spermidine, pH 7.5
Ks (10-6 M-1) = [Triplex] / ([TFO][Duplex])
n.d.: not determined
CG
0
10 50 100 500 1000
AT
0 10 50 100 500 1000
TA
0 10 50 100 500 1000
Ks = 6.9
Ks = 6.7
Ks = 39.2
Ks = 0.5
Ks = 0.2
Ks = 0.6
n.d.
n.d.
n.d.
Ks = 12.09
n.d.
n.d.
Triplex DNA
Duplex DNA
(100 nM)
Triplex DNA
Duplex DNA
(100 nM)
Iso-dC derivatives selectively recognize CG base pair
But affinity needs to be improved
Challenge to In Vivo Application of Triplex DNA and
Selective Chemical Modification of RNA
 Triplex DNA
 Covalent
Modification
Delivery System
Release
Antisense
Delivery
Penetration
Chemical
Modification
Nucleus
Antigene
Efficient Inhibition
Editing
mRNA
mRNA
Triplex
DNA
DNA
17
Download