Supporting Information

Supporting Information Wiley-VCH 2007 69451 Weinheim, Germany

DNA minicircles with gaps for versatile functionalization** Goran Rasched, Damian Ackermann, Thorsten L. Schmidt, Peter Broekmann, Alexander Heckel and Michael Famulok * SUPPORTING INFORMATION Synthesis of the anthracene-functionalized 2 -desoxyuridine phosphoramidite (6) Modified oligonucleotides DNA minicircle synthesis by phosphorylation and ligation of oligonucleotides Analysis and purification of DNA minicircles by native gel electrophoresis Nuclease digestion AFM Imaging Calculation of ring size Instrumentation: NMR spectra were recorded on DPX 400 spectrometer from Bruker (Bremen, Germany) operated at 400.1 Mhz. Chemical shifts were recorded by calibrating the signal of the used solvent to literature values. Couplings were measured with 1D-WinNMR (Bruker) and MestReC 4.9.9 (Mestrelab Research). EI-MS spectra were recorded on a Finnigan MAT 95XL (Thermo Finnigan, Germany), FAB-MS spectra were recorded on a Concept 1H (Kratos, Germany), using meta-nitrobenzoicalcohol (mnba) as matrix. HRMS-ESI spectra were measured on a Bruker APEX IV Fourier-Transform Ion-Cyclotron-Resonance (FT-ICR) and LC-MS measurements were performed on an esquire6000 (Bruker) coupled to an Agilent 1100 HPLC System (Agilent Technologies, Böblingen, Germany). TLC analysis was performed on silica Gel F254 plates (Merck, Darmstadt, Germany), spots were visualized by UV light or staining with anisaldehyde/sulfuric acid/acetic acid/ethanol and subsequent heating. If necessary, all reactions were performed under an argon atmosphere and all solvents were dried using standard procedures. 5-Iodo-2 -deoxyuridine was obtained from Pharma-Waldhof, Düsseldorf, Germany. Automated solid-phase oligonucleotide synthesis was performed on an ABI 394 DNA-Synthesizer (Applied Biosystems). Atomic force microscopy (AFM) was conducted in situ (liquid cell) with an PicoScan AFM (former Molecular Imaging Inc. now Agilent Series 4500 SPM, PicoSPM I) using magnetic actuated mode (MAC- Mode). Silicon cantilevers with a thin magnetic coating on the back side were used (MAC Levers Type II, Agilent) purchased from Lot-Oriel GmbH (Darmstad, Germany) with nominal spring constant of 2.8 N/m.

Synthesis of the anthracene-functionalized 2 -desoxyuridine phosphoramidite (1) Supplementary Scheme 1. Synthesis of the functionalized desoxuridine phosphoramidite 1 as a substrate for automated DNA solid phase synthesis. a) Pd(Ph 3 ) 4, CuI, Et 3 N, DMF, b) DMTCl, Pyridine, c) aq. NH 3 (33%), MeOH, d) DIPEA, TSTU, Anthracene-9-carbamido-ε-aminocaproic acid, DMF/Dioxan/H 2 O, e) DIPEA, ClP(OCH 2 CH 2 CN)(i- Pr) 2 N, THF. Synthesis of 5-[3-(Trifluoracetyl amino)-prop-1-ynyl]-2 -desoxyuridin 5: 5-Iodo-2 - deoxyuridine (2, 5 g, 14.12 mmol) was dissolved in DMF (20 ml) and CuI (268.5 mg, 1.41 mmol) was added. The solution was degassed with argon and triethylamine (4 ml, 28.3 mmol), Trifluoro- N-prop-2-ynyl-acetamide (7.6 ml, 70.6 mmol) and Pd(PPh 3 ) 4 (808.9 mg, 0.7 mmol) were added successively. The yellow solution was stirred for 1.5 h at room temperature. Complete conversion of the starting material was detected by RP-TLC (acetonitrile:water, 1:3). The reaction was concentrated to dryness. The residue was dissolved in CH 2 Cl 2 :MeOH (1:1, 25 ml) and anion exchange resin (Amberlite IRA-400, HCO - 3 -form, 1.5 g) was added. The mixture was stirred for 30 min at room temperature, the resin was removed by filtration and the solvent was evaporated in vacuo. The crude mixture was purified by column chromatography on SiO 2 (CH 2 Cl 2 :MeOH, 9:1) to afford 2 (3.37 g, 8.9 mmol, 63% yield) as a pale yellow solid. R f =0.45. 1 H-NMR (400.1 MHz, DMSO, d6): δ = 11.59 (s, 1H, N3-H) 10.02 (t, 3 J(H,H) = 5.34 Hz, 1H, NHCH 2 ), 8.18 (s, 1H, H-6), 6.09 (t, 3 J(H,H) = 6.64 Hz, 1H, H-1 ), 4.95 (br, 2H, 3 -OH, 5 -OH), 4.23-4.21 (m, 3H, NHCH 2, H- 4 ), 3.78 (m, 1H, H-3 ), 3.63-3.52 (s, 2H, H-5 ), 2.13-2.10 (m, 1H, H-2 ). 13 C-NMR (100.6 MHz, DMSO, d6): δ = 161.97 (C4), 156.49 (q, 2 J(C,F) = 36.67 Hz, CF 3 C=O), 149.82 (C5), 144.53 (C6), 116.21 (q, 1 J(C,F) = 288.21 Hz, CF 3 ), 98.10 (C5), 88.09 (C C), 85.25 (C4 ), 85.99 (C1 ), 75.80 (C C), 70.61 (C3 ), 61.42 (C5 ), 48.99 (C2 ), 29.86 (NHCH 2 ). MS (FAB): calcd C 14 H 15 F 3 N 3 O 6 [M+H] + = 378.09, found 378.1 (32%).

Synthesis of DMT-protected nucleosid 6: Nucleoside 5 (500 mg, 1.33 mmol) was coevaporated with pyridine (3 x 3mL) and thoroughly dried before adding 4,4 -dimethoxytrityl chloride (496 mg, 1.46 mmol) in pyridine (3 ml). After stirring for 15 h at room temperature MeOH is added (10 ml) and the solvents evaporated in vacuo. After additional coevaporation with MeOH (10 ml) the crude product was purified by column chromatography on SiO 2 (CH 2 Cl 2 :MeOH:Et 3 N, 95:4:1) to afford 3 (677 mg, 1 mmol, 75% yield) as a yellowish solid. R f =0.46 (CH 2 Cl 2 :MeOH, 9:1). 1 H-NMR (400.1 MHz, CDCl 3 ): δ = 8.12 (s, 1H, H-6), 7.34 (d, 3 J(H,H) = 7.16 Hz, 2H, Ar), 7.26-7.23 (m, 7H, Ar), 7.13 (t, 3 J(H,H) = 7.14 Hz, 1H, NH), 6.76 (d, 3 J(H,H) = 8.94 Hz, 4H, Ar), 6.29-6.25 (m, 1H, H-1 ), 4.51 (m, 1H, H-3 ), 4.07 (m, 1H, H-4 ), 3.86 (d, 3 J(H,H) = 5.30 Hz, 2H, CH 2 C C), 3.70 (s, 6H, OCH 3 ), 3.29 (s, br, 2H, H-5 ), 2.50-2.47 (m, 1H, H-2 ), 2.24 (m, 1H, H-2 ). 13 C-NMR (100.6 MHz, CD 3 OD): δ = 162.73 (C4), 158.69 (C=O), 158.66 (Ar, q), 149.54 (C5), 143.55 (C6), 135.52 (Ar, q), 130.04, 129.97, 128.05, 127.92, 127.02, 113.39 (br, CF 3 ), 98.99 (C5), 87.33 (C C), 87.10 (Ar 3 C-O- 5 ), 86.93 (C4 ), 85.99 (C1 ), 75.49 (C C), 72.23 (C3 ), 63.58 (C5 ), 55.28 (OCH 3, 2C), 41.64 (C2 ), 30.41 (NHCH 2 ). MS (FAB): calcd C 35 H 32 F 3 N 3 O 8 [M ] + = 679.21, found 679.3 (32%). Synthesis of anthracene modified nucleosid 4: Nucleoside 6 (977.59 mg, 1.43 mmol) was desolved in MeOH (10 ml) and aq. Ammonia solution (33%, 35 ml) was slowly added while stirring. The solvents were removed in vacuo followed by coevaporation of the residue with MeOH (2 x 20 ml). Intermediate 3 was used in the next reaction step without further purification. Anthracene-9-carbamido-ε-aminocaproic acid (479.62 mg, 1.43 mmol) was dissolved in DMF (3 ml) and DIPEA (0.735 ml, 4.29 mmol). TSTU was added under stirring and after 15 min. a solution of nucleoside 3 (834.6 mg, 1.43 mmol) in DMF (5mL) was added. After stirring for 1 h at room temperature the reaction mixture was evaporated to dryness and the residue purified by column chromatography on SiO 2 (EE:MeOH:Et 3 N, 98:1:1) to afford nucleoside 4 (470 mg, 0.52 mmol, 37% yield) as a yellow foam. R f =0.57. 1 H-NMR (400.1 MHz, CDCl 3 ): δ = 8.25 (s, 1H, Anthracene H-10), 7.88 (d, 3 J(H,H) = 8.60 Hz, 2H, Ar Anthracene), 7.82 (s, 1H, H-6), 7.80-7.78 (d, 3 J(H,H) = 8.37 Hz, 2H, Ar Anthracene), 7.36-7.28 (m, 6H, Ar Anthracene/DMT), 7.19-7.17 (m, 6H, Ar Anthracene/DMT), 7.09 (t, 3 J(H,H) = 7.26 Hz, 1H, Phenyl H-6), 6.71 (d, 3 J(H,H) = 8.73 Hz, 4H, Ar DMT), 6.65 (br, 1H, NH), 6.04 (m, 1H, NH, 1H, H-1 ), 4.24 (s, 1H, H-3 ), 3.89 (m, 1H, H-4 ), 3.68 (d, J(H,H) = 10.10 Hz, 2H, CH 2 C C), 3.63 (s, 6H, OCH 3 ), 3.48-3.46 (m, 2H, CH 2 CH 2 NH), 3.19-3.11 (m, 2H, H-5 ), 2.26 (m, 1H, H-2 ), 1.95 (m, 1H, H-2 ), 1.87 (t, 3 J(H,H) = 7.06 Hz, 1H, NHCOCH 2 ), 1.57-1.54 (m, 2H, CH 2 CH 2 NH), 1.49-1.45 (m, 2H, CH 2 CH 2 CH 2 CH 2 NH), 1.30-1.27 (m, 2H, CH 2 CH 2 CH 2 CH 2 NH). 13 C-NMR (100.6 MHz, CD 3 OD): δ = 172.4 (C=O), 169.7 (C=O), 162.3 (C4), 158.6 (Ar, q), 149.2 (C5), 144.7 (Ar, q), 143.0 (C6), 135.67 (Ar, q), 132.1 (Ar, q), 131.0 (Ar, q), 130.0 (Ar, 4C, CH), 128.5 (Ar, 2C, CH), 128.0 (Ar, 4C, CH), 128.0 (Ar, CH), 127.9 (Ar, CH), 127.0 (Ar, CH), 126.7 (Ar, 2C, CH), 125.6 (Ar, 2C, CH), 125.00 (Ar, 2C, CH), 113.37 (Ar, 4C, CH), 99.1 (C5), 89.4 (C C), 86.9 (Ar 3 C-O-5 ), 86.6 (C4 ), 85.8 (C1 ), 74.0 (C C), 71.8 (C3 ), 63,6 (C5 ), 55.3 (OCH 3, 2C), 41.6 (C2 ), 40.0 (CH 2 ), 35.7 (CH 2 ), 29.7 (CH 2 ), 29.2 (CH 2 ), 26.5 (CH 2 ), 25.0 (CH 2 ). MS (FAB): calcd C 54 H 52 N 4 O 9 [M ] + = 900.37 found 900.4 (10%).

Synthesis of nucleoside phosphoramidite 1: To a solution of nucleoside 4 (470 mg, 0.52 mmol) in dry THF (4 ml) DIPEA was added (0.45 ml, 2.63 mmol) and the reaction mixture was stirred for 10 min at room temperature before dropwise adding of 2-cyanoethyl-N,Ndiisopropylchlorophosphoramidite (0.23 ml, 1.03 mmol). Stirring continued for 1h at room temperature. The reaction was quenched by addinging 5% aq. NaHCO 3 -solution and extracting the organic phase with EE. The organic phase is washed with sat. aq. NaCl-solution and dried over MgSO 4. After removal of the solvents in vacuo the crude mixture was purified by column chromatography on SiO 2 (EE:MeOH:Et 3 N, 94:5:1) to afford the nucleoside phosphoramidite 1 (480 mg, 0.44 mmol, 84% yield) as a yellowish foam. R f =0.53. 1 H-NMR (400.1 MHz, CD 3 CN): δ = 8.52 (s, 1H, Anthracene H-10), 8.05-7.99 (m, 4H, Ar Anthracen), 7.86 (d, 3 J(H,H) = 11.73 Hz, 1H, H-6), 7.55-7.44 (m, 6H, arom. Anthracene/DMT), 7.36-7.29 (m, 6H, Ar Anthracene/DMT), 7.24 (t, 3 J(H,H) = 7.25 Hz, 1H, Phenyl H-6), 7.15 (br, 1H, NH), 6.89-6.86 (m, 4H, Ar DMT), 6.28 (br, 1H, NH), 6.13-6.06 (m, 1H, H-1 ), 4.65-4.56 (m, 1H, H-3 ), 4.15-4.10 (m, 1H, H-4 ), 3.79 (m, 2H, CH 2 C C), 3.76 (s, 3H, OCH 3 ), 3.75 (s, 3H, OCH 3 ), 3.74-3.57 (m, 2H, CH 2 P, 2H, NCH(CH 3 ) 2 ), 3.57-3.52 (m, 2H, CH 2 CH 2 NH), 3.37-3.24 (m, 2H, H-5 ), 2.64 (t, 3 J(H,H) = 5.96 Hz, 1H, NCCH 2 CH 2 OP), 2.53 (t, 3 J(H,H) = 5.96 Hz, 1H, NCCH 2 CH 2 OP), 2.50-2.29 (m, 2H, H-2 ), 2.08-2.04 (m, 2H, NHCOCH 2 ), 1.72-1.65 (m, 2H, CH 2 CH 2 NH), 1.62-1.56 (m, 2H, CH 2 CH 2 CH 2 CH 2 NH), 1.46-1.39 (m, 2H, CH 2 CH 2 CH 2 CH 2 NH), 1.24-1.06 (m, 12H, CH(CH 3 ) 2 ). 13 C-NMR (100.6 MHz, CD 3 CN): δ = 173.29, 173.27, (C=O), 171.73, 169.81 (C=O), 162.62 (C4), 159.78 (Ar, q), 150.20 (C5), 146.00 (Ar, q), 144.02 (C6), 136.85, 136.63 134.24, 132.13, 131.17, 131.14, 131.11, 131.08, 129.45, 129.43, 129.05, 128.98, 128.94, 128.77, 128.56, 127.94, 127.56, 126.60, 126.24, 118.32 (N C), 114.28 (Ar, 4C, CH), 99.79, 99.70 (C5), 90.39, 90.36 (C C), 87.70, 87.68 (Ar 3 C-O-5 ), 86.65, 86.61 (C4 ), 86.41, 86.35 (C1 ), 74.72, 74.69 (C C), 74.39 (d, 2 J(C,P) = 17.24 Hz, C3 ), 73.91 (d, 2 J(C,P) = 16.35 Hz, C3 ), 64.23, 63.98 (C5 ), 59.65, 59.45 (CH 2 OP), 56.03(OCH 3 ), 55.98 (OCH 3 ), 44.17, 44.05 (NCH(CH 3 ) 2 ), 40.78, 40.73 (C2 ), 40.45 (CH 2 ), 36.46 (CH 2 ), 30.00, 29.97 (CH 2 ), 27.37 (CH 2 ), 25.97 (CH 2 ), 24.99, 24.92, 23.29-22.79 (m, 2C, CH 3 ), 23.29-22.79 (m, 2C, CH 3 ).21.19-20.99 (m, 2C, CH 3 ). 31 P-NMR (162.0 MHz, CD 3 CN): δ = 151.02 (s). MS (FAB): calcd C 63 H 69 N 6 O 10 P [M ] + = 1100.48 found 1100.5 (12%); calcd C 63 H 69 N 6 O 10 PNa [M+Na] + = 1123.47 found 1123.5 (17%).

Modified oligonucleotides: Supplementary Figure 1: Sequence of the oligonucleotide modified with two anthracene moieties (functionalized du derivatives are framed black) Anthracene modified oligonucleotides were prepared by standard phosphoramidite chemistry on low volume polystyrene columns (0.2 µmol scale) using trityl-on mode. The oligomers were cleaved from the solid support and deprotected by addition of 1 ml aq. NH 3 -solution (33%, DNAgrade) and heating at 55 C for 5 h. After removal of the resin and evaporation of the solvents the crude product was dissolved in TEAA-buffer (100 µl, 0.1 M) and purified by RP-HPLC (Agilent 1100 HPLC System, Nucleosil 100-5 C18 column, CS-Chromatographie Service GmbH, Germany). Flow rate 1.0 ml/min at room temperature was applied. 0.1 M TEAA-buffer was used as mobile phase A and 100% MeCN was used as mobile phase B. The DMT-group was removed by adding 75 µl of aq. acetic acid (80%) to the dry, purified oligomers followed by 25 µl TEAA-buffer (1M) after 20 min. The final oligonucleotide products were obtained after additional purification by RP- HPLC. ESI-MS: calculated [M-H] 7103.4, found 7104.4; 7127.6.

Supplementary Figure 2. DNA minicircle synthesis by phosphorylation and ligation of oligonucleotides. The oligonucleotides (200 pmol each, Eurogentec) of the DNA minicircle subunits α and β were phosphorylated together with 20 U of T4 polynucleotide kinase (NEB, New England Biolabs) for 45 min at 37 C in a 50 µl reaction volume containing ATP (200 nmol) and ligase buffer as recommended by the manufacturer. The oligonucleotides (200 pmol each) of subunit γ were phosphorylated separately in a 50 µl reaction volume as described above. The phosphorylated subunits α and β were ligated by adding 6 µl ligase buffer (NEB) and 2 µl T4 DNA ligase (NEB) and incubating at 16 C. After 6 h subunit γ, 6 µl ligase buffer and 2 µl T4 DNA ligase were added to the ligation mixture of α and β and incubation continued at 16 C for another 6 h. Analysis and purification of DNA minicircles by native gel electrophoresis: Separation in the first dimension was conducted with a nondenaturing 5% polyacrylamide gel in TBE (89 mm Tris/89 mm boric acid/2 mm EDTA, ph 8.3). Separation in the second (perpendicular) dimension was in a nondenaturing 8% polyacrylamide gel in TBE with chloroquine phosphate (50 µg/ml) in the gel and buffer. The ratio of acrylamide to bisacrylamide was 19:1 (wt/wt). The gels were run at 4 C in TBE buffer at 8 V/cm. The gels were exposed to storage phosphor screens and visualized by phosphorimaging (Scanner: Fujifilm, FLA 3000, Raytest, Straubenherdt, Germany). The product bands were excised, and the gel slices soaked over night at 30 C under mild shaking. After the removal of gel residues by filtration (Ultrafree-MC Centrifugal Devices, Millipore) the oligonucleotides were precipitated with ethanol and the pellets dissolved in water. The oligonucleotide solution was finally desalted by centrifugation through G25 columns (illustra MicroSpin G-25 Column, GE Healthcare). The concentration was determined by UV absorbtion (ε DNA-minicircle = 2. 10 6 M 1 cm 1 )

Nuclease Digestion: An analytical amount (5 µl, ca 2 pmol) of a DNA sample was treated with 0.2 µl BAL-31 Nuclease (1000 U/mL, New England Biolabs, Frankfurt, Germany) in 25 µl BAL-31 Nuclease Buffer (as recommended by the manufacturer) and 19.8 µl water for 1.5 h at 30 C. AFM Imaging: The PicoSPM AFM has a sample plate magnetically suspended below its top-down scanner. The liquid cell is a one-piece teflon well which is clipped onto the freshly cleaved mica on the sample plate. 10 µl (10-50 nm) of a solution of DNA minicircles were placed into the liquid cell on the mica, followed by 10 µl (100 mm) of an aq. NiCl 2 solution and 10 µl (100 mm, ph 7.5) TrisHCl-Buffer. Water was added to reach a volume of 100 µl and the sample was incubated at RT for 20 min. Before scanning another 300 µl (10 mm) TrisHCl-Buffer were added to the liquid cell. The images were processed with WSxM 3.0 beta 9.0 software (Nanotech Electronica, Spain). Sequences of oligonucleotides (Eurogentec, Metabion) subunit name sequence α β γ γ gap α-f α-r β-f β-r γ-f γ-r γf 20 -γf 15 γ-r 5 -TCTCTAAAAAATATATAAAAATCTCTAAAAAATATATAAAAATCTCTAAAAAATAT-3 3 -TTTTTTATATATTTTTAGAGATTTTTTATATATTTTTAGAGATTTTTTATATATTT-5 5 -ATTTTTTAGAGATTTTTATATATTTTTTAGAGATTTTTATATATTTTTTAGAGATT-3 3 -TTAGAGATTTTTTATATATTTTTAGAGATTTTTTATATATTTTTAGAGATTTTTTA-5 5 -AAAATATATAAAAATCTCTAATAATTACTATCATGTCTGCGAAAAATATATAAAAA-3 3 -TATATTTTTAGAGATTATTAATGATAGTACAGACGCTTTTTATATATTTTTAGAGA-5 5 -AAAATATATAAAAATCTCTA-3 5 -AAAAATATATAAAAA-3 3 -TATATTTTTAGAGATTATTAATGATAGTACAGACGCTTTTTATATATTTTTAGAGA-5 Calculation of ring size