Supporting Information
for
DNA Strand Cleavage Near a CC Mismatch Directed by a Metalloinsertor
Mi Hee Lim, Irvin H. Lau and Jacqueline K. Barton
*
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 911
25
RECEIVED DATE (will be automatically inserted after manuscript is accepted)
Experimental
Materials and Procedures
All reagents and solvents were purchased from commercial suppliers and used
without
further
purification.
The
precursor
of
[Rh(chrysi)(phe
n)(bphen)]
3+
,
[Rh(chrysi)(phen)(bpy
#
)]
3+
(bpy
#
=
7-(4'
-methyl
-2,2'
-bipyridin
-4-
yl)heptan
-1-
amine
), and
5-(1,10
-phenanthrolin
-5-
ylamino)
-5-
oxopentanoic acid
(
phen’
) was prepared by
previously reported methods.
1,2
UV
-visible spectra were recorded on a Beckm
an DU
7400 UV
-visible spectrophotometer (Beckman Coulter). The samples for the
photocleavage experiments were irradiated by using a 1000 W Oriel Hg/Xe arc lamp
(Oriel, Stamford, CT) with 320
– 440 nm light. The oligonucleotides
5’-GAT GTC GGT
CCC
ACG ATG G
TG ACG GAT TAC C
-3’ and 3’
- CTA CAG CCA
GXG
TGC TAC CAC
TGC CTA ATG G
-5’ (X = G or C) were synthesized on an ABI 392 DNA/RNA
synthesizer (Applied Biosystems) and purified as previously described.
3
[Rh(chrysi)(phen)(bphen)]Cl
3
.
The Rh complex
was synthesize
d as described in
Scheme 1. The compounds
[Rh(chrysi)(phen)(bpy
#
)]
3+
(4.1 mg, 4.4
μ
mol),
phen’
(1.6 mg,
5.3
μ
mol), HOBt (2.1 mg, 16
μ
mol) and HBTU (6.0 mg, 16
μ
mol) were combined in a
S
2
round bottom flask and then dried in vacuo for 2 h. The reagents were di
ssolved in
DMF (1 mL) and
N
,
N
-diisopropylethylamine (DIEA, 6
μ
L, 32
μ
mol
) was added to the
solution. To the resultant reaction mixture another portion of DIEA (30
μ
L, 0.16 mmol)
was treated
after 1 h and allowed to stir under Ar at room temperature
. After
3 h, water
(4 mL) was combined with the reaction solution, which was loaded onto a Waters Sep
-
Pak (C18 column), washed with water, and then eluted with
1:1:0.001
(H
2
O:MeCN:TFA). The collected brown solution was lyophilized to dryness. The
complex
was purif
ied by preparative HPLC using a gradient of
H
2
O (with 0.1% TFA) to
CH
3
CN (with 0.1% TFA) over the course of 30 min and afterward converted to the
soluble Cl
–
salt by an anion exchange column on Sephadex QEA (yield 31%). Features in
UV/vis
of
[Rh(chrysi)(ph
en)(bphen)]Cl
3
are
identical
of
those
in
[Rh(chrysi)(phen)(bpy
#
)]Cl
3
.
1
ESI(+)MS (m/z) for (
M
–
3Cl
)
3+
calcd
: 371.3, found: 371.5;
for
(M -
3Cl
– H)
2+
calcd
: 556.2, found: 556.2; for (
M
– 3Cl
– 2H)
+
calcd: 1111.4, found:
1111.5.
References
(1)
Schatzschneide
r, U.; Barton, J. K.
J. Am. Chem. Soc.
2004
,
126
, 8630
-8631.
(2)
Sardesai, N. Y.; Lin, S. C.; Zimmermann, K.; Barton, J. K.
Bioconjugate Chem.
1995
,
6
, 302
-312.
(3)
Brunner, J.; Barton, J. K.
Biochemistry
2006
,
45
, 12295
-12302.
S
3
(a)
(b)
048
12
16
20
0
0.2
0.4
0.6
0.8
1
Fraction Cleaved
[Rh complex]
Figure S1.
(a) Autora
diogram of the denaturing 20% polyacrylamide gel displaying
DNA photocleavage by [Rh(bpy)
2
(chrysi)]
3+
and [Rh(chrysi)(phen)(bphen)]
3+
on DNA
with a CC mismatch. Conditions: Duplex (5
μ
M) was incubated with
[Rh(bpy)
2
(chrysi)]
3+
(variable concentrations) or
[Rh(chrysi)(phen)(bphen)]
3+
(variable
concentrations) in 10 mM Tris, pH 7.5, 50 mM NaCl for 10 min and irradiated for 30 min
by a solar simulator (325
– 450 nm). A+G and C+T, Maxam
-Gilbert sequencing
reactions. Lanes 1
-8, fragments with [Rh(bpy)
2
(chrysi)]
3+
(0, 1.25, 2.5, 5, 7.5, 10, 12.5, 15
and 20
μ
M); Lanes 9
-16, fragments with [Rh(chrysi)(phen)(bphen)]
3+
(0, 1.25, 2.5, 5, 7.5,
10, 12.5, 15 and 20
μ
M). The arrow marks the mismatched site. (b) A plot of fraction
cleaved
at
various
[Rh
complex]
([Rh(bpy)
2
(chrysi)]
3+
,
circle
in
red;
[Rh(chrysi)(phen)(bphen)]
3+
, square in blue). Data were analyzed using phosphorimager
and ImageQuant software (Molecular Dynamics).
S
4
Figure S2.
Sequences of 31mer oligonucleotides (top). Autoradiogram of a denaturing
20% polyacrylamide gel presenting DNA cleavage by
Cu
,
Rh+Cu
and
Rh
–Cu
with
matched and mismatched oligonucleotides at 37 ºC (bottom). A+G and C+T, Maxam
-
Gilbert sequencing reactions. * indicates 5’
-
32
P-end
-label of
the strand. Lanes 1
-2 and 7
-8,
fragments
with
Rh+Cu
; lanes 3
-4 and 9
-10, fragments with
Rh
–Cu
; lanes 5
-6 and 11
-12,
S
5
fragment with
Cu
. Time depicted in the figure shows incubation time after treatment of
sodium ascorbate. The arrow marks the mismatched site.
S
6
Figure S3.
Autoradiagram o
f a denaturing 20% polyacrylamide gel revealing DNA
cleavage by
Rh+Cu
with DNA containing a CC mismatch. A+G and C+T, Maxam
-
Gilbert sequencing reactions. Lanes 1 and 2, fragments from the reaction of
Rh+Cu
with
DNA for 5 or 10 min followed by treatment of
alkaline (10 mM) at 60
o
C for 30 min;
lanes 3 and 4, fragments from the reaction of
Rh+Cu
with DNA for 5 or 10 min without
addition of alkaline; lanes 5 and 6, fragments from the reaction of
Rh+Cu
with DNA for
5 or 10 min followed by addition of piperidine
(10% v/v) at 90
o
C for 30 min; lanes 7
-9,
fragments from the reaction of
Rh+Cu
with DNA for 5 or 10 min without piperidine
S
7
treatment. Time depicted in the figure presents incubation time after treatment of
sodium ascorbate. The arrow marks the mismatched
site.