Synthetic Polymers for DNA Binding

Synthetic Polymers for DNA Binding James E. Hanson, Cosimo Antonacci, Mohammed Elshaer, Sueyuan Li, Mira Yazigi Department of Chemistry and Biochemistry Seton Hall University South Orange, New Jersey

DNA Binding and Recognition • Small molecules • Proteins and peptides • Oligonucleotides All bind DNA and can recognize sequences • Synthetic Polymers? Can they bind and recognize DNA?

Synthetic Polymers that Bind DNA • Polyethylenimine Cationic polymer Often used in transfection • Other polycations Will bind nucleic acid polyanion – but what about recognition?

Molecular Imprinting • A mature technique Polymerize monomer and cross-linker in presence of template…. Grind cross-linked polymer… Extract template Porous material with affinity for template • Can we imprint Polyethyleneimine? (PEI)

PEI Cross-linking • Isophthalaldehyde as the cross-linker Imine formation in water

• Characterized by NMR, QELS, extraction

Can we imprint DNA? Add PEI to T4G4 quadruplex as template… CD tells us the quadruplex changes….

But what does it change into… and what would we imprint on?

NOESY • NOE of T4G4 quadruplex with and without PEI

Additional CD Data • CD with PEI (5:1) over time… a shift from parallel to antiparallel? PEI/Quadruplex DNA (T4G4) 5:1 Ratio Time Point Profile

Molar Ellipticity (mdeg)

1.1 0.6 PEI/DNA(5:1) Initial

0.1 200 -0.4

250

300

-0.9 -1.4 Wavelength (nm)

350

PEI/DNA(5:1) 2.5H PEI/DNA (5:1)4H

Other polymers • If PEI causes structural changes – are other polymers more gentle? • Initial screen with porphyrin binding:

Porphyrin_Quadruplex DNA_Poly(N-vinylpyrrolidone) 2.5 Porphyrin Q-T4G4 PNVP Porphyrin_DNA-PNVP

PEI – strong binding displaces porphyrin and lmax shifts back

Absorption

2 1.5 1 0.5 0 -0.5200

250

300

350

Wavelength(nm)

400

450

500

Porphyrin Screen Polymer C= Cationic N=Neutral

Poly(2-vinyl pyridine) (C)

Porphyrin (nm)

λmax

Porphyrin λmax after Added DNA (nm)

Porphyrin λmax after Added DNA and polymer (nm)

422

433

422

422

433

422

422

433

422

422

433

422

422

433

422

422

433

422

Poly(4-vinly-1methylpyridinium bromide) (C)

422

433

422

Poly(L-ornithine) HCL (C)

422

433

422

422

433

433

422

433

433

422

433

433

PEI (C) Poly(allylamine) 20%wt (C) Poly(L-lysine) (C) Metafectene Easy (C) Metafectene Pro (C)

Poly(N- vinylpyrrolidon) (N) Ploy(4-vinylpyridine N-oxide) (N) PEG (N)

Binding by DOESY PEI

PVP

PVPyNO

Black – DNA blue- 1:1 red-1:5 green-1:10

Porphyrin cross-linkers 0.15

MO_MTALPYP_1H_Methanol

0.14

N

0.13

0.12

N

NH

NH

N

N

N 9.03 9.01

0.10

9.47

9.45

0.11

+ 4Br-

0.09

5.69 5.68

N

0.07

5.93 5.93 5.90 5.82 5.82 5.80

0.06

0.05

6.60

0.03

0.02

6.54

6.57 6.56

0.04

9.35 9.34

Normalized Intensity

0.08

0.01

0

-0.01

-0.02 9.5

9.0

8.5

8.0

7.5 Chemical Shift (ppm)

7.0

6.5

6.0

5.5

5.0

Binding of mTAlPyP Δλ

424

1.0

Unbound Fully Bound

1.0

Increasing (TTAGGG)4

0.8

436

0.8

0.6

Absorbance

Absorbance

0.6

ΔH

0.4

0.4

0.2

0.2

0.0

0.0

380

400

420

440

460

480

Wavelength (nm)

External Binding: < 8 nm & < 10% Intercalation: > 15nm & > 35%

380

400

420

440

460

480

Wavelength (nm)

%H =

ε𝐹 −ε𝐵 ε𝐹

× 100

Δλ = λ𝐵 − λ𝐹

%H = 34

Δλ = 12 𝑛𝑛

Tetra-pyridyl-porphyrin-tetra-hexanoic acid HO O

N

N

H

O

O

O +

NH

OH Reflux, 1.5h

NH

N

Br

OH

N

N HN

N

DMF, 72h , N2

NH HO

N

O

OH

N

N

O

HN

N

N + 4Br-

N

N

mTPyP

O OH

mTCAPyP Chemical Formula: C64H70N8O84+ Molecular Weight: 1079.29

Characterization 0.35

HO O

N

ME_CAP72_1H_Methanol-D6

NH HO 0.30

N O

HN

N

N 0.25

O OH

Normalized Intensity

0.20

0.15

0.10

0.05

0 23.88 9.5

8.00 9.0

8.5

8.0

7.5

7.0

6.5

6.0 5.5 5.0 Chemical Shift (ppm)

16.12 4.5

4.0

x10

3.5

5

3.0

2.5

16.43 2.0

1.5

Cpd 1: C64 H66 N8 O8: +ESI Scan (0.133-0.316 min, 23 Scans) Frag=365.0V SHJHJH20A.d Subtract

481.2240 2 1

359.1750 (M+3H)+3

1 0 0

100 200 300 400 Counts vs. Mass-to-Charge (m/z)

500

600

700

800

900

1000 101100044 ( 1200

OH

N

N O

) 1300

1400

1500

1600

But it doesn’t bind DNA! Unbound Fully Bound

0.4

Absorbance

Absorbance

0.4

0.2

0.0

380

0.2

0.0

400

420

440

460

480

380

Wavelength (nm)

400

420

440

Wavelength (nm)

No Bathochromic Shift! Small Hypochromicity!

460

480

Cross-linking PEI with mTHexPyP 50 mg of 10,000 MW PEI (5 x 10-6 moles) was dissolved in 1 mL of pH 7.2 KPBS 4x and .25x moles of mTCAPyP were added (2.65 x 10-9 M stock) 4 x Sulfo-NHS with respect to functionality was added Mixture was shaken vigorously for 15 min 10x EDC with respect to functionality was added Mixture was vortexed intermittently for 2 hr and allowed to react overnight Solution was dialyzed using a 1000 MWCO vs ddH2O (2x in 48hr) Water removed by lyophilization

Cross-linking ESI-MS PEI

4:1 Excess Porphyrin

Excess (4:1) Porphyrin

Excess PEI (1:4) 1:4 Excess PEI

mTCAPyP

Porphyrin cross-linked PEI does not bind DNA at porphyrin •

No bathochromic shift of soret band when x-linked PEI is added to Quadruplex

•

Electrostatic interaction between PEI and DNA dominate the binding interactions

•

Porphyrin assay demonstrated this effect PEI disrupts the porphyrin-DNA binding event 0.5

Free mTOPrPyP Fully Bound mTOPrPyP PEI-Porphyrin Adduct

0.4

O.D

0.3

0.2

0.1

0.0 380

400

420

440

Wavelength (nm)

460

480

Conclusions • Synthetic polymers bind DNA cationic bind strongest but perturb structure • Effective cross-liking strategies for PEI isophthalaldehyde, porphyrins • Analytical methods NMR, light scattering, MS, CD • PEI may cause quadruplex structure transition evidence is complex – not confirmed.

Acknowledgements

Seton Hall University Research Council Pfizer PURE Program Hunter College MS facility