DNA sequence selectivity of hairpin polyamide turn units

Article abstract of DOI:10.1016/j.bmcl.2009.03.072

A class of hairpin polyamides linked by 3,4-diaminobutyric acid, resulting in a β-amine residue at the turn unit, showed improved binding affinities relative to their α-amino-γ-turn analogs for particular sequences. We incorporated β-amino-γ-turns in six-

Full text of DOI:10.1016/j.bmcl.2009.03.072

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3919–3923
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
DNA sequence selectivity of hairpin polyamide turn units
*
Michelle E. Farkas, Benjamin C. Li, Christian Dose, Peter B. Dervan
California Institute of Technology, Division of Chemistry and Chemical Engineering, 1200 E California Blvd, Pasadena, CA 91125, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A class of hairpin polyamides linked by 3,4-diaminobutyric acid, resulting in a b-amine residue at the turn
Received 24 February 2009
Revised 13 March 2009
Accepted 18 March 2009
Available online 24 March 2009
unit, showed improved binding affinities relative to their
a-amino-c-turn analogs for particular
sequences. We incorporated b-amino- -turns in six-ring polyamides and determined whether there
c
are any sequence preferences under the turn unit by quantitative footprinting titrations. Although there
was an energetic penalty for GꢀC and CꢀG base pairs, we found little preference for TꢀA over AꢀT at the b-
amino-c-turn position. Fluorine and hydroxyl substituted a-amino-c-turns were synthesized for com-
Keywords:
parison. Their binding affinities and specificities in the context of six-ring polyamides demonstrated
overall diminished affinity and no additional specificity at the turn position. We anticipate that this study
will be a baseline for further investigation of the turn subunit as a recognition element for the DNA minor
groove.
Molecular recognition
DNA minor groove
Foldamer
Ó 2009 Elsevier Ltd. All rights reserved.
Hairpin pyrrole-imidazole (Py/Im) polyamides are a class of
programmable synthetic ligands able to bind a broad repertoire
of DNA sequences with affinities and specificities comparable to
those of DNA-binding proteins.1,2 They have been shown to localize
to the nuclei of living cells3,4 and regulate endogenous gene
expression by interfering with transcription factor/DNA inter-
faces.5–10 Discrimination of the four Watson–Crick base pairs is
dependent upon Py/Im ring pairings in the minor groove. Pairing
rules have been established whereby N-methyl imidazole/N-meth-
ylpyrrole (Im/Py) pairs target GꢀC, the reverse (Py/Im) target CꢀG,
and Py/Py pairs target AꢀT and TꢀA.11–13
The turn unit in the hairpin is a recognition element, favoring
TꢀA/AꢀT over GꢀC/CꢀG.14 There is an energetic penalty for unfavor-
able steric interaction with the exocyclic amine present at the edge
of the GꢀC base pair (Fig. 1). The question arises whether discrimi-
nation between TꢀA and AꢀT with the turn unit can be achieved.
Previous efforts toward the improvement of hairpin binding affin-
ity have involved modifications of the turn unit. Early studies
showed the optimal length of the turn element to be three methy-
lene units, resulting in the use of
Modification of the -position of the parent
diaminobutyric acid ( -amino- -turn) results in an approximately
15-fold increase in DNA-binding affinity.16 In contrast, hairpins
containing the opposite enantiomer, (S)- -amino- -turn, bind
c
-aminobutyric acid (
c
-turn).15
a
c-turn with (R)-2,4-
a
c
a
c
DNA with diminished affinities likely due to a steric clash of the
amine with the wall of the minor groove. Polyamides containing
a
-hydroxy-c a
-turns17 and -diaminobutyric acid18 turns have been
Figure 1. Schematic diagram of six-ring hairpin polyamide (ImImPy-turn-PyPyPy)
targeting the DNA sequence 50-WWGGWW-30. Dashed lines indicate hydrogen
bonds between the polyamide and DNA base pairs.
* Corresponding author. Tel.: +1 626 395 6002; fax: +1 626 683 8753.
E-mail address: dervan@caltech.edu (P.B. Dervan).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.072

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M. E. Farkas et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3919–3923
Table 1
Melting temperatures of DNA/polyamide complexes
DNA = 50-CTATGGTA GAC-30
Tm (°C)
Polyamides
—
D
Tm (°C)
45.1 ( 0.1)
—
1
54.3 ( 0.2)
59.8 ( 0.3)
60.0 ( 0.2)
59.8 ( 0.2)
54.9 ( 0.1)
57.0 ( 0.2)
9.2
2R
4R
4S
3R
5R
14.7
14.9
14.7
10.1
12.2
All values are derived from at least three melting temperature experiments, with
standard deviations indicated in parentheses.
DTm values are given as Tm (DNA/
poiyamide) ꢁ Tm (DNA).
both a shape element as well as a DNA recognition element, allow-
ing the molecule to fold in a U-conformation. The turn unit de-
serves more attention and this Letter represents an effort to
create a baseline for the field.
Recently, we introduced a new class of hairpin polyamides
linked by 3,4-diaminobutyric acid, resulting in a b-amino-
These molecules showed improved binding affinities relative to
their -amino- -turn analogs for A/T-rich sequences. Additionally,
polyamides containing the b-amino- -turn were found to have im-
c
-turn.22
a
c
c
proved tolerance for synthetic modification at the amine position
presumably due to their more central location on the floor of the
minor groove. Due to limitations of quantitative DNase I footprint-
ing titrations23–25 for compounds where Ka P 1010 Mꢁ1, relative
binding affinities for high affinity molecules are compared by using
their thermal stabilization of DNA duplexes. It has been shown pre-
viously that increases in melting temperatures (DTm) of DNA du-
plexes bound by hairpin polyamides correlate with DNA-binding
Figure 2. Chemical and ball-and-stick structures of polyamides containing free and
acetylated amines. Ball and stick symbols are defined as follows: pyrrole is denoted
by an open circle, imidazole is denoted by a filled circle, and b-alanine is denoted by
a diamond shape.
affinity.26,27
We report herein a comparison of the sequence specificities of
hairpin Py/Im polyamides containing the
b-amino- -turn. Additionally, we have synthesized both hydroxyl
and fluoro-substituted -turns and determined their affinities
a-amino-c-turn and the
c
c
reported to impart additional elements of specificity, but with the
cost of diminished binding affinities.
and specificities in polyamides with analogous core ring pairs. By
employing six-ring polyamides, which have lower binding affini-
ties compared to eight-ring polyamides,28 we are able to determine
reliable equilibrium association constants (Ka) via quantitative
DNase I footprinting titrations, and compare them with DNA du-
plex thermal stabilizations.
In a formal sense, a DNA minor groove binding hairpin Py/Im
polyamide is an early example of a class of oligomers encoded by
the order of monomer units that fold to a desired shape with a spe-
cific function, referred to as ‘foldamers.’ 19–21 The ring order of Py/
Im polyamides codes in a programmable manner for a specific,
contiguous sequence of Watson–Crick base pairs. The turn unit is
Polyamide synthesis. Six-ring hairpin polyamides (ImImPy-turn-
PyPyPy) targeting the DNA sequence 50-WWGGWW-30 were syn-
Figure 3. Illustration of the EcoRI/PvuII restriction fragment derived from plasmid pCDMF6, used to characterize polyamides. The designed polyamide binding sites are
indicated by boxes; single base-pair mismatches are indicated by shaded regions.

M. E. Farkas et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3919–3923
3921
Figure 4. Quantitative DNase I footprinting titration experiments for polyamides 1, 2R, and 4R (top, left to right) and 4S, 3R, and 5R (bottom, left to right) on the 50 end
labeled PCR product of plasmid pCDMF6: lanes 1–11, 100 nM, 30 nM, 10 nM, 3 nM, 1 nM, 300 pM, 100 pM, 30 pM, 10 pM, 3 pM, and 1 pM polyamide, respectively; lane 12,
DNase I standard; lane 13, intact DNA; lane 14, A reaction; lane 15, G reaction. Respective isotherms are shown below.

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M. E. Farkas et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3919–3923
Table 2
tolerance for modifications at the turn. Acetylated turn units have
Binding affinities (Mꢁ1) for polyamides
been shown to improve nuclear uptake of polyamides.4
50-ATGGTT-30
50-ATGGTA-30
50-ATGGTC-30
50-ATGGTG-30
Thermal stabilization of DNA duplexes. Spectroscopic analyses
were performed on the 11mer DNA duplex shown in Table 1.22
All hairpins analyzed provided an increase in melting temperature,
1
2.1 ( 0.6)x 108
1.6 ( 0.7) ꢃ 109
3.3 ( 0.1} ꢃ 109
1.1 ( 0.1) ꢃ 109
3.2 ( 0.4) ꢃ 108
2.0 ( 0.4) ꢃ 109
2.0 ( 0.5) ꢃ 108
1.7 ( 0.9) ꢃ 109
1.8 ( 0.2) ꢃ 109
1.3 ( 0.2) ꢃ 109
2.1 { 0.3) ꢃ 108
5.2 ( 1.6) ꢃ 108
<107
<107
<107
<107
<107
<107
<107
2R
4R
4S
3R
5R
3.9 ( 2.0) ꢃ 107
5.0 ( 1.1) ꢃ 107
6.0 ( 0,4) ꢃ 107
<107
confirming the formation of DNA/polyamide complexes. The
values obtained for polyamides containing a free amine were with-
in error of each other. However, acetylated polyamide 3R ( -amino
turn) showed a greater decrease in affinity than 5R (b-amino-
turn). As had been demonstrated with eight-ring hairpin mole-
cules,22 improvements in binding affinities for b-over
-amino-
DTm
a
8.9 ( 2.8) ꢃ 107
c-
Equilibrium association constants are reported as mean values from three DNase I
footprinting titration experiments. Standard deviations are shown in parentheses.
a
c-
turn six-ring polyamides are more pronounced with decreasing
imidazole content (Supplementary Fig. 1, Table 1).
DNA binding affinity and sequence selectivity. The plasmid
pCDMF6 was prepared to characterize polyamides targeting the se-
quence 50-WWGGWW-30 (Fig. 3). The designed insert contains four
binding sites, varying the nucleotide base pair present under the
turn unit. Quantitative DNase I footprinting titrations were per-
formed with the polyamides in order to measure their binding site
affinities and specificities, as previously described.25 As expected,
Table 3
Relative binding affinities for polyamides
R1,R2,R3
TꢀA
AꢀT
CꢀG
GꢀC
H, H, H
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
0.5
1.2
0.7
0.3
<0.01
0.02
0.02
0.05
<0.01
0.04
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
NH3þ, H, H
þ
the parent hairpin containing the
ing affinity, while experiments for 2R, 4R, and 4S (Fig. 4, Table 2)
corroborated the similar Tm values obtained for duplex stabiliza-
c-turn retained the lowest bind-
H, H, NH3
H, NH3þ, H
D
NH-Ac, H, H
H, H, NH-Ac
tion. None of these molecules bound the GꢀC base pair, and binding
to CꢀG was greatly diminished. Polyamide 4R revealed only a ꢂ2-
fold specificity for TꢀA over AꢀT (Table 3). Study of an additional
polyamide series revealed similar trends (Supplementary Fig. 2, Ta-
ble 2). Analysis of polyamides 3R and 5R (Fig. 4, Table 2) revealed a
Relative binding affinities are reported as ratios of binding affinities (Ka) as deter-
mined by DNase I footprinting titration experiments for polyamides targeting 50-
WGGWW-30.
greater decrease in binding affinities for the
ecule than the b. Between 2R and 3R there is a five-fold decrease
-turn
a-amino-c-turn mol-
over TꢀA, and an eight-fold decrease over AꢀT. For b-amino-
c
thesized by solid phase methods on Pam resin (Fig. 2, Supplemen-
tary data 1).22,29 In addition to the parent molecule containing an
unsubstituted
ing an amine moiety in the
two polyamides with acetylated amines (3R, 5R) were also synthe-
polyamides 4R and 5R, there are 1.7 and 3.2-fold decreases for
TꢀA and AꢀT, respectively. 5R shows ꢂ3-fold preference for binding
TꢀA versus AꢀT (Table 3).
c
-aminobutyric acid hairpin (1), oligomers contain-
and b turn positions (2R, 4R, 4S) and
a
Fluoro and hydroxyl substituted turn units. Fluorine and hydroxyl
substituted hairpin turns were synthesized (Supplementary Figs. 3
and 4, Supplementary data 130,31) and incorporated in six-ring
sized. The acetylated, or capped, molecules were used to determine
Figure 5. Chemical and ball-and-stick structures of polyamides containing fluoro and hydroxyl substituted hairpin turns. Ball and stick symbols are defined as follows:
pyrrole is denoted by an open circle, imidazole is denoted by a filled circle, and b-alanine is denoted by a diamond shape.

M. E. Farkas et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3919–3923
3923
Table 4
all four Watson–Crick base pairs and add an additional element for
DNA recognition.
Melting temperatures of DNA/ polyamide complexes
DNA = 50-CTATGGTA GAC-30
Acknowledgments
Polyamide
—
Tm (°C)
D
Tm (°C)
45.1 ( 0.1)
55.1 ( 0.2)
—
We are grateful to the National Institutes of Health for research
support. We thank David M. Chenoweth for helpful discussions.
Mass spectrometry analyses were performed in the Mass Spec-
trometry Facility of the Division of Chemistry and Chemical Engi-
neering at the California Institute of Technology.
6R
7R
6S
7S
10.0
8.3
5.8
7.9
53.4 ( 0.2)
50.9 (+0.2)
53.0 ( 0.2)
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.03.072.
All values are derived from at least three melting temperature experiments, with
standard deviations indicated in parentheses.
DTm values are given as Tm (DNA/
polyamide) ꢁ Tm (DNA).
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hairpin polyamides targeting the DNA sequence 50-WWGGWW-30
(Fig. 5). Polyamides were synthesized on Pam resin using standard
solid phase methods.
Thermal stabilization analysis of the polyamides on an 11mer
DNA duplex revealed that fluoro and hydroxyl substituted hairpins
resulted in lower stabilizations than the corresponding amine-
substituted and acetylated polyamides (Table 4, Supplementary
Table 3). DNase I footprinting titrations on the plasmid pCDMF6
(Fig. 3, Supplementary Fig. 5) showed that both enantiomers of
the hydroxyl and fluoro-hairpin turns resulted in decreased poly-
amide binding affinities relative to their amine substituted coun-
terparts (Supplementary Table 4). Additionally, none of these
subunits resulted in increased elements of specificity at the turn
position of the molecule (Table 5).
By utilizing six-ring hairpin polyamides, we were able to com-
bine DNase I footprinting titration and duplex stabilization analy-
ses in order to fully characterize the binding preferences of various
hairpin turn subunits. Although the hairpin turns investigated
herein show modest DNA binding specificities, we anticipate that
further study will yield moieties enabling discrimination amongst