Lecture 3 Nucleic Acid Structure

Transcription

Lecture 3 Nucleic Acid Structure
Lecture 3 - Binding of small molecules to DNA
Binding of small molecules to DNA
- Electrostatics
- Intercalation
- Groove binding through Van der Waals and hydrogen
bonding
Distamycin
A-T
O
NH
N
R
O
O
H 2N
N
N
N
N
N3A
O2T
NH
Distamycin-Increasing selectivity
-Selectivity is not great i.e. (1/2) 5- one in every 32 sites is
an AT rich run of 5 bases
How do you increase selectivity?
-Simplest solution is to increase the length of polypyrrole
H
N
N
H
Distamycin = P3 (n=3)
H
N
O
N
O
n
This works for n≤ 5, but for n>5 the curvature of the polypyrrole backbone doesnt match the
curvature of DNA.
-To solve this add in an aminobuteric linker to reorient the pyrrole units
N
NH
O
K A~ 10 8 M -1
H
N
R
H
N
N
O
H
N
N
NH 2
NH 2
O
-Sequence specific - favors [A:T]n or [T:A]n, n=4,5.
-Binds and displaces the spine of hydration in the
minor groove.
-Bifurcated hydrogen bonding of one NH to two lone pairs
-Entropic driving force due to displacement of H2O.
-Enthalpic driving through Van der Waals and hydrogen
bonding.
-Can also have 2:1 binding mode (2 distamycin/site)
-No major change in DNA upon binding
O
H
N
P3
O
N
H
O
H
N
P3
NH
O
-One can also make a distamycin who's binding dependent on metal ion binding
-Add in a metal binding sequence i.e. a PEG spacer which will bind to a metal ion to form a crownether type structure
H
N
-Sr2+ and Ba 2+ show high binding affinity
O
O
H
-Unfortunately this doesn't work with Ca 2+
N
O
2+
M
O
HN
O
N
O
O
H
O
NH
O
HN
HN
N
O
Why not G:C?
NH 2
N
N
O
O
-Another solution would be to space distamycin with an interchelator
N
N
HN
N
H 2N
NH 2 group in minor groove sterically
inhibits binding of distamycin
P3
EtBr
P3
(A/T)n GC/CG (A/T) 2
Sequence Specific Binder
- How do you determine where small molecules bind?
-Take advantage of the following reaction:
DTT
H 2O 2
EDTA.Fe(II)
O2
OH
OR
O P O
O
H
OR
O P O
O
B
O
O
O P O
OR'
OH
Distamycin
OR
O P O
O
B
O
O
O P O
OR'
-If you bind a molecule (distamycin) to the DNA, it will provide either steric
blockage or quench the cleavage reaction
-The resulting gel will have a "foot-printed" binding site
B
HO O
O2
OR
O P O
O
O
H
small
O
O P O
OR'
To increase resolution add an Fe(II) binding motif to generate site specific
OH adjacent to ligand binding site.
H
O
O P O
OR'
B
labelled with
32P
treat with
EDTA.Fe(II),
O 2, DTT
"single hit"
kinetics
5'
N
O
O P O
OR'
Run on
polyacryamide gel
+
+
N
HO
O
O
O
O
O2
Fe(II)
DTT
FeII
H
N
OH
O
N
HN
N
O
n
-generates OH at the site of binding
-reacts at a diffusion controlled rate constant
-in water small molecules diffuse at 10 9-1010M -1s-1
-large molecules 10 6-107M -1s-1
-end up with local cleavage of distamycin binding site
large
5'
5'
HO
B
cleavage ladder
DNA backbone is cleaved
5'
O
HO
H
O
O
O P O
OR'
OR
O P O
O
O
Distamycin binding site:
Foot printing
O
HO
O
large
Distamycin binding site
small
-separate DNA to nucleotide resolution
-visualize fragments that are 32P labelled
-If you label both strands you can track cleavage on both strands
-but still limited to AT base pairs
3'-end labelled
NH 2
O
N
N
N
N
O
5'-end labelled
FeII
N
HN
H 2N
C
G
O2/N3
H-bond
Donor
H
N
H
N
N
5'
FeII
N
O
minor groove
3'
-TA vs. AT
O
C4H'
FeII
2 base
pairs
C4H'
-Fe(II) cleaves 2 base pairs away on the other
strand due to helicity of the strands
NH
N
N
O
closer
5'- T G G A C A
3'- A C C T G T
Py Py Py HP Im
5'
FeII
OH
N
N
HP Im Im Py Py
major groove
H
N
N
H 2N
further
-But helicity in the major groove is different, so Fe(II) cleavage of major groove
binders results in a shift in the 5' direction
-used to distinguish between major or minor groove binders
O
Im
Py
-this is due to the helicity of right-handed DNA
N
Modify to:
-observed that cleavage on the 5' strand was shifted in the 3' direction
3'
H-bond
Acceptor
N
O
HP
Im/Py
Py/Im
aminobutyric acid HP/Py
Py/HP
GC
CG
TA
AT
Typically bind
with KD<10 -9 M -1
Binding of longer sequences
N
major groove
O
N
NH
N
O
T
Chemical Modification of DNA
R
T
N
NH 2
N
O
N
O
A
N
HO
N
Cl
Mustards
N
H 2N
C
minor groove
O
N
HN
N
CH+
O
N
H pH 6.2
H 2N
N
N
Cl
G
X= NH, S
X
-Use Hoogsteen base pairing in the major groove
-Two hydrogen-bonds from protonated CH+ (pH 6.2) binding to GC
-Two hydrogen-bonds from T binding to AT
CH+- T - T -CH+- T
5' - G - A - A - G - A 3' - C - T - T - C - T -
5' A-A-(A)10
3' T- T-(T)10
O
Cl
5'- T-T- T-T- T-T- T
5'- A-A-A-A-A-A-A-A
3'- T-T- T-T- T-T- T-T
5'
3'
G N7 Alkylation leads to tautomerization and mispairing
O
O
H
O
O
N
N
R
N
NH
NH 2
N R
N
N
Cl
N7G
N7G
NH 2
N
O
O
H
O
OH
N
N
NH 2
N
HN
Cl
O
7
N
-Crosslinks the DNA
-Interstrand crosslinking negatively
impacts replication and transcription
-Hoogsteen is parallel Py/Pu versus antiparallel Py/Pu
R
N
7
N
anchimeric effect
-cleavage pattern shifted
in the 5' direction
-indicates major groove
binder
FeII
N
N
N
-This only works for -(Py)n- or -(Pu)n - sequences
(i.e. GATGA does not work).
NH 2
HN
Cl
Cl
T- T-(T)10
-Chlorambucil - anti-cancer drug
-Alkylates the N7 on G
Cl
3' TT(T)10
R
O
N
H
H 2N
O
5' AA(A)10
N R
N7G
N
N7G