DNase I Footprinting and Alkylation Assays of Linear β-linked

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Appendix B

DNase I Footprinting and Alkylation Assays of Linear β-linked

Compounds and Hairpin Polyamides Targeted to GAA Repeats

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and a single hairpin polyamide (overview in Figure B.1) targeted to (AAG)₃·(CTT)₃ (Figures B.2-B.5). A single thermal cleavage gel of chlorambucil alkylator Im-Py-β-Im-Py-β-Im- β-Dp-Chl (polyamide 7) alkylating (GAA)₃₃·(TTC)₃₃ is shown (Figure B.6). Table B.1 summarizes the fi ndings in Figures B.2–B.5. Table B.2 summarizes MALDI-TOF Mass Spectrometric data collected for the seven compounds used in these studies.

The design of plasmid pJWP12 (Figure B.1B) was based on initial CSI motif data for polyamide 6, in which 5’-AAGATGTTC-3’ was the preferred motif, 5’-AAGAAGTTC-3’

an acceptable similar site for the primary motif, and 5’-AAGAAGAAG-3’ the binding site for which the polyamide was originally designed. Data from Figure B.2 shows that the affi nity of 1 for the principal motif, 5’-AAGATGTTC-3’ is less than that for the designed site, 5’-AAGAAGAAG-3.’ Chapter 3 probes this discrepancy more in depth. The CSI microarray intensities correlate well with polyamide binding affi nities but do not necessarily correlate with the sequence motif generated from the 300 highest affi nity binding sites.

Polyamide alkylation studies were undertaken with polyamide 7 due to a collaboration with the Robert D. Wells laboratories at Texas A&M. Preliminary data suggested that polyamide 7 may be able to upregulate frataxin expression in GM15850 cells.

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Figure B.1. Overview of Polyamides Studied on pJWP12 and pJWP11 and plasmid sequences of pJWP12 and pJWP11. a) Polyamides studied. b) Plasmid insert sequences

N N

HN

O N

HN O

N N

NH O

H N

O NH 3 N NH

N O NH

O NH

O N H

5'-A A G A A G A A G-3'

3'-T T C T T C T T C-5'⁺ ⁺

N N N H N O N H O

N H O

N N N H N O N H O

N H O

N N O N H N H O

N N

H O H N

Cl Cl

5'-A A G A A G A A G-3' 3'-T T C T T C T T C-5' Chl (+)

N N NH N O N H O

N H O N

N NH N O NH

O

N H O N O N N H N H O

N H

NH R

5'-A A G A A G A A G-3' 3'-T T C T T C T T C-5'⁺ *

N N N

H N O N H O

N H O

N N N H N O NH

O

NH O

N O N NH NH

O

RHN

5'-A A G A A G A A G-3' 3'-T T C T T C T T C-5' Cy3-(+)

5'-A A G A A G A A G-3'

3'-T T C T T C T T C-5'⁺ ^N

N NH

O N NH

O

NH O

N N NH

O N N H O

N H O

N O N N N H H O

N H

5'-GATC CGGCC AAGAAGAAG CCGGGGCC AAGATGTTC CCGGGGCC AAGAAGTTC CCGGAT AGCT-3' 3'-CTAG GCCGG TTCTTCTTC GGCCCCGG TTCTACAAG CCGGGGCC TTCTTCAAG GGCCTA TCGA-5'

32P pJWP-12 270 bp

I II III

Eco RI Pvu II

N N NH N O N H O O

NH N

N NH

O N N H O

NH O

N N N H O

NH O

N H

5'-A A G A A G A A G-3' 3'-T T C T T C T T C-5'⁺

N N NH

O N N H O N NH

O

N N N H O N NH

O

NH O

N N N H O

N H O

N H

5'-A A G A A G A A G-3' 3'-T T C T T C T T C-5'

+

5'-GATC CGGCC AAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAG CCGGAT AGCT-3' 3'-CTAG GCCGG TTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTC GGCCTA TCGA-5'

pJWP-11 269 bp

32PEco RI PvuII

1

2

3

4

5

6

7

Polyamide Ball-and-stick Structure Chemical Structure

A

B

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Polyamide 1

5'-GATC CGGCC AAGAAGAAG CCGGGGCC AAGATGTTC CCGGGGCC AAGAAGTTC CCGGATAGCT-3' 3'-CTAG GCCGG TTCTTCTTC GGCCCCGG TTCTACAAG CCGGGGCC TTCTTCAAG GGCCTA TCGA-5'

32 PpJWP-12270 bp IIIIII

Eco RIPvu II

norm Θ

Ka (M-1)Binding Site 5’-AAGAAGTTC-3’ 5’-AAGATGTTC-3’ 5’-AAGAAGAAG-3’ Hill equation (n=1)

3.9 (± 0.5) • 109 7.1 (± 0.7) • 109

1.6 (± 0.1) • 109

5’-32 P label 123456789101112131415

Intact GA DNase I 100 nM

1 pΜ

III

[polyamide]

5'-A A G A A G A A G-3' 3'-T T C T T C T T C-5'+

NN

N H

ON

N H

O

N H

O N

N

N H

ON

N H

O

N H

O N

N

O

N H

O NH -0.20

0.2

0.4

0.6

0.8

11.2 10-1310-1210-1110-1010-910-810-710-6

III II I

Figure B.2. Quantitative DNase I footprint of a linear β-linked polyamide targeting GAA repeats. CSI Motif preferred binding site III does not correlate with polyamide preferred binding site I.

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5’-AAGAAGTTC-3’ 5’-AAGATGTTC-3’ 5’-AAGAAGAAG-3’

7.3 (± 3.5) • 107 ≤ 1 • 107

≤ 1 • 107 III

III

Polyamide 2

Eco RIPvu II Polyamide 3

norm Θ

Ka (M-1)Binding Site 5’-AAGAAGTTC-3’ 5’-AAGATGTTC-3’ 5’-AAGAAGAAG-3’ Hill equation (n=1)3.3 (± 0.5) • 107 1.1 (± 0.1) • 107

2.3 (± 0.1) • 107

5’-32 P label 123456789101112131415

Intact G ADNase I 1 μM

10 pΜ

III

III Hill equation (n=1)

norm Θ

[polyamide]

5’-32 P label Ka (M-1)Binding Site

123456789101112131415

Intact GA DNase I 1 μM

10 pΜ

[polyamide]

5'-A A G A A G A A G-3' 3'-T T C T T C T T C-5'

NN

N H

ON

N H

OO

N H

N

N H

ON

N H

O

N H

O N

N

N H

O

N H

O NH NN

N H

ON

N H

ON

N H

O N

N

N H

ON

N H

O

N H

O N

N

N H

O

N H

O NH ++ -0.20

0.2

0.4

0.6

0.8

11.2 10-1210-1110-1010-910-810-710-6

III II I -0.20

0.2

0.4

0.6

0.8

11.2 10-1210-1110-1010-910-810-710-6

III

Figure B.3. Quantitative DNase I footprints for two linear β-linked polyamides, one containing a β → Gly point mutation and the other containing a β → Py point mutation

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Figure B.4. Quantitative DNase I footprint of a hairpin polyamide targeting GAA repeats

Polyamide 4

Eco RIPvu II

norm Θ

1.6 (± 0.4) • 109 1.2 (± 0.1) • 109

6.1 (± 0.8) • 109

5’-32 P label 123456789101112131415

Intact G ADNase I 1 μM

10 pΜ

III

[polyamide]

N N O N H

N N H O

N H O

N N O NH

H N O

NH 3

N

H N O

N

H N O

N

H N O

N

H N O

N H

5'-A A G A A G A A G-3' 3'-T T C T T C T T C-5'++ -0.20

0.2

0.4

0.6

0.8

11.2 10-1210-1110-1010-910-810-710-6

III II I

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Eco RIPvu II 5’-AAGAAGTTC-3’ 5’-AAGATGTTC-3’ 5’-AAGAAGAAG-3’

1.4 (± 0.7) • 108 2.6 (± 1.1) • 108

1.0 (± 0.5) • 107 III

III

Polyamide 5 Hill equation (n=1)

norm Θ

[polyamide]

5’-32 P label Ka (M-1)Binding Site

123456789101112131415

Intact GA DNase I 1 μM

10 pΜ

5'-A A G A A G A A G-3' 3'-T T C T T C T T C-5' -0.20

0.2

0.4

0.6

0.8

11.2 10-1210-1110-1010-910-810-710-6

III II I

NN

N H

ON

N H

O

N H

O N

N

N H

ON

N H

O

N H

O N

NO

N H

O NRH Cy3-(+)

Polyamide 6

norm Θ

1.1 (± 0.1) • 108 1.6 (± 0.3) • 108

8.5 (± 1.5) • 107

5’-32 P label 123456789101112131415

Intact GA DNase I 100 nM

1 pΜ

III

[polyamide]

N

N H

ON

N H

O

N H

O N

N

N H

ON

N H

O

N H

O N

NO

N H

O NH NHR 5'-A A G A A G A A G-3' 3'-T T C T T C T T C-5'+* -0.20

0.2

0.4

0.6

0.8

11.2 10-1210-1110-1010-910-810-710-6

III II I

Figure B.5. Quantitative DNase I footprint of two Cy3-labeled, linear β-linked polyamides targeting GAA repeats

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Figure B.6. A Chlorambucil-labeled GAA-targeting β-linked polyamide alkylates (GAA)₃₃ repeat sequences

5'-GATC CGGCC AAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAG CCGGAT AGCT-3' 3'-CTAG GCCGG TTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTC GGCCTA TCGA-5' pJWP-11 269 bp 32 P Eco RI Pvu II

5'-AAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAG CCGGAT AGCT-3' 3'-TTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTC GGCCTA TCGA-5'

1 2 3 4 5 6 7 8 9 10 11 12 13 14

t c a t n I G A

M n 0 0 1

p 1

Μ

N N

N H

O N

N H

O

N H

O N

N

N H

O N

N H

O

N H

O N

N

O

N H

O N

N H H O N C l

C l 5'-A A G A A G A A G-3' 3'-T T C T T C T T C-5'Chl (+) Polyamide 7

p 300

Μ

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Table B.1. Summary data from Figures B.2–B.5. All values are listed as K_a (M^-1) of the polyamide for the specifi ed binding site. Bolded entries represent the highest affi nity within a row

+ +

+ *

Cy3-(+) + + + 1

2 3 4 5 6

Polyamide Ball-and-stick Structure AAGAAGAAG AAGATGTTC AAGAAGTTC 7.1 (± 0.7) · 10⁹

1.1 (± 0.1) · 10⁷

≤ 1 · 10⁷

1.2 (± 0.1) · 10⁹ 2.6 (± 1.1) · 10⁸ 1.6 (± 0.3) · 10⁸

2.3 (± 0.1) · 10⁷

≤ 1 · 10⁷

6.1 (± 0.8) · 10⁹ 1.0 (± 0.5) · 10⁷ 8.5 (± 1.5) · 10⁷

3.3 (± 0.5) · 10⁷ 7.3 (± 3.5) · 10⁷ 1.6 (± 0.4) · 10⁹

1.4 (± 0.7) · 10⁸ 1.1 (± 0.1) · 10⁸ 1.6 (± 0.1) · 10⁹ 3.9 (± 0.5) · 10⁹

Table B.2. MALDI-TOF Mass Spectral Data for polyamides 1–7

+ +

+ *

Cy3-(+)

Chl-(+) + + + 1

2 3 4 5 6

Polyamide Ball-and-stick Structure [M+H]⁺ calc’d [M+H]⁺ obsv’d 914.4

900.4 965.5 1187.6 1569.7 1569.7

900.5 965.4 1187.6 1569.6 1569.7

7 1242.6 1242.5

914.4