Supporting Information

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1 Supporting Information Wiley-VCH Weinheim, Germany

2 High Thermal Stability of 5-5 Linked Alternate Hoogsteen Triplexes at Physiological ph Vyacheslav V. Filichev, Mads C. Nielsen, Niels Bomholt, Carsten H. Jessen, Erik B. Pedersen 1. General Remarks: NMR spectra were recorded on a Varian Gemini 2000 spectrometer at 300 MHz for 1 H and 75 MHz for 13 C. Internal standard used in 1 H NMR spectra was TMS (δ: 0.00) for CDCl 3 ; in 13 C NMR was CDCl 3 (δ: 77.0); in 31 P NMR was H 3 P 4 (δ: 0.00). Accurate ion mass determinations were performed using the 4.7 Tesla Ultima Fourier Transform (FT) mass spectrometer (Ion Spec, Irvine, CA). The [M + Na] + ions were peak matched using ions derived from the 2,5- dihydroxybenzoic acid matrix. EI-MS was performed on Finnigan SSQ 710. Thin layer chromatography (TLC) analyses were carried out with use of TLC plates 60 F 254 purchased from Merck and were visualized in an UV light (254 nm). The silica gel ( mm) used for column chromatography was purchased from Merck. Solvents used for column chromatography were distilled prior to use, while reagents were used as purchased. Unmodified Ns were purchased from DNA Technology A/S (Århus, Denmark) and from TAG Copenhagen A/S (Copenhagen, Denmark). 2. Molecular Modeling: The molecular modeling was performed with Maestro v7.5 from Schrödinger. All calculations were conducted with AMBER* force field and the GB/ SA water model. The dynamics simulations were preformed with stochastic dynamics, a SHAKE algorithm to constrain bonds to hydrogen, time step 1.5 fs and simulation temperature of 300 K. Simulation for 0.5 ns with an equilibration time of 150 ps generated 250 structures, which all were minimized using the PRCG method with convergence threshold of kj/mol. The minimized structures were examined with Xcluster from Schrödinger, and representative low-energy structures were selected. The starting structures were generated with Insight II v97.2 from MSI, followed by incorporation of the desired linkage between the two 5 -ends. Figures 1SI 3SI represent typical alternative positioning of linkers 2 and 3 in the core of the alternate strand triplex as determined by cluster analysis. Dr. V.V. Filichev, Mr. M. C. Nielsen, Mr. N. Bomholt, Dr. C. H. Jessen, Prof. E.B. Pedersen, Nucleic Acid Center, Department of Chemistry, University of Southern Denmark Campusvej 55, DK-5230 dense M, Denmark Fax: filichev@chem.sdu.dk

3 Figure 1SI. Typical alternative positioning of linker 2 in the alternate strand triplex. Picture to the right is top-view and picture to the left is side-view on the linker. Figure 2SI. Typical alternative positioning of linker 2 in the alternate starnd triplex. Picture to the right is top-view and picture to the left is side-view on the linker. Figure 3SI. Typical alternative positioning of linker 3 in the alternate strand triplex. Picture to the right is top-view and picture to the left is side-view on the linker.

4 3. Synthesis of alternate linkers Synthesis of Linker 2: Br Br Br a b c d Br H H H e 8 9 H DMT NC P N DMT Scheme 1SI. Reagents and conditions: a) NBS, (PhC 2 ) 2, CCl 4, reflux, 24 h; b) ethylene glycol, NaH, 20 h; c) 1,3- diethynylbenzene, Pd(PPh 3 ) 2 Cl 2, CuI, NEt 3, DMF, 1,4-dioxane, 24 h; d) DMT-Cl, NEt 3, CH 2 Cl 2, 20 h; e) NC(CH 2 ) 2 P(NPr i 2) 2, diisopropylammonium tetrazolide, CH 2 Cl 2, 24 h Bromo-4-bromomethylnaphthalene (5). [1] A mixture of 1-bromo-4-methylnaphthalene (1; 20.9 g, 95.0 mmol), N- bromosuccinimide (20.2 g, mmol) and benzoyl peroxide (1.5 g, 6.3 mmol) in dry CCl 4 (300 ml) was refluxed for 24 h. The solvent was removed in vacuo. The residue was crystallized from hexane giving compound 2 as yellow crystals. Yield g (57%); mp ºC (lit ºC [1] ); 1 H NMR (CDCl 3 ) δ 4.89 (s, 2H, PhCH 2 Br), 7.36 and 7.69 (AB system, 2 2H, J = 7.8 Hz). 13 C NMR (CDCl 3 ) δ 30.9 (CH 2 Br); 124.2, 124.5, 127.4, 127.6, 127.9, 128.1, 129.5, 132.1, 132.4, (Ar) (4-Bromonaphthalen-1-ylmethoxy)ethanol (6). NaH (55% in mineral oil, 5.9 g, mmol) was stirred in cyclohexane (20 ml) for 30 min under N 2. Afterwards solution was decanted and ethylene glycol (50 ml) was added portionwise at 0ºC. The mixture was allowed to warm to rt. 1-Bromo-4-bromomethylnaphthalene (2; 8.0 g, 27.0 mmol) was added, and the mixture was heated with a heat gun until a clear orange solution was obtained. The mixture was stirred for 20 hours at rt, then poured into H 2 (300 ml) and extracted with Et 2 (3 100 ml). The combined organic phases were washed with brine (100 ml), dried (MgS 4 ), filtered evaporated in vacuo affording compound 3 as dark brown oil. Yield 7.42 g (99%). 1 H NMR (CDCl 3 ) δ 2.11 (br. s, 1H, H), 3.63 (m, 2H, CH 2 CH 2 H), 3.72 (m, 2H, CH 2 H), 4.94 (s, 2H, ArCH 2 ), 7.31 (d, J = 7.2, 1H, Ar), 7.58 (m, 2H, Ar), 7.72 (d, J = 7.8, 1H, Ar), 8.08 (m, 1H, Ar), 8.28 (m, 1H, Ar). 13 C NMR (CDCl 3 ) δ 61.8 (CH 2 H), 71.3 (CH 2 CH 2 H), 71.5 (PhCH 2 ), 123.5, 124.3, 126.8, 127.1, 127.2, 127.8, 129.2, 132.1, 132.8, (Ar). HR-MALDI-MS: m/z calcd for C 13 H 13 BrNa + 2 [M+Na] , found ,2 -[1,3-Phenylenebis(2,1-ethynediyl-4,1-naphthalenmethyleneoxy)bisethanol] (7). 1,3-Diethynylbenzene (1.6 g, 13.0 mmol), compound 3 (7.2 g, 25.6 mmol), Pd(PPh 3 ) 2 Cl 2 (360 mg, 0.5 mmol) and CuI (245 mg, 1.3 mmol) were suspended in the mixture of dry DMF (125 ml), 1,4-dioxane (50 ml) and Et 3 N (5.4 ml) under Ar. The mixture was refluxed for 24 hours. CH 2 Cl 2 (200 ml) was added and the mixture was washed with H 2 (200 ml), 0.3 M aq. EDTA (2 125 ml) and H 2

5 (3 200 ml). The organic phase was dried (Na 2 S 4 ), filtered and concentrated in vacuo. The residue was purified by dry column vacuum chromatography (silica gel; % EtAc in petroleum ether) to afford compound 4 as brown oil. Yield 1.42 g (21%). 1 H NMR (CDCl 3 ) δ 2.15 (br. s, 2H, 2 H), 3.67 (m, 4H, 2 CH 2 CH 2 H), 3.76 (m, 4H, 2 CH 2 H), 5.01 (s, 4H, 2 PhCH 2 ), 7.58 (m, 11H, Ar), 7.93 (s, 1H, Ar), 8.11 (m, 2H, Ar), 8.50 (m, 2H, Ar). 13 C NMR (CDCl 3 ) δ 61.7 (CH 2 H), 71.5 (CH 2 CH 2 H), 71.6 (PhCH 2 ), 88.2 (C CNaphth), 93.7 (C CNaphth), 121.2, 123.8, 124.2, 125.8, 126.8, 126.9, 128.4, 128.6, 129.9, 131.5, , , , 133.4, (Ar). HR-MALDI-MS: m/z calcd for C 36 H 30 Na + 4 [M+Na] , found {4-[3-(4-{2-[Bis-(4-methoxyphenyl)phenylmethoxy]ethoxymethyl}naphthalen-1- ylethynyl)phenylethynyl]naphthalen-1-ylmethoxy}ethanol (8). The diol 7 (1.16 g, 2.2 mmol) and dry Et 3 N (370 µl, 2.6 mmol) were dissolved in dry CH 2 Cl 2 (40 ml) under Ar. DMT-Cl (0.896 g, 2.6 mmol) in dry CH 2 Cl 2 (15 ml) was slowly added over 45 min. The mixture was stirred at rt for 20 hours, and the solvents were removed in vacuo. The residue was purified by dry column vacuum chromatography (silica gel; 1% Et 3 N v/v, 0-70% EtAc in petroleum ether) to afford compound 8 and diprotected compound as yellow oils. Yield of 8 is 700 mg (38%). 1 H NMR (CDCl 3 ) δ 2.03 (br. s, 1H, H), 3.30 (t, J = 4.8 Hz, 2H, CH 2 DMT), (m, 6H, CH 2 H, CH 2 CH 2 H and CH 2 CH 2 DMT), 3.76 (s, 6H, 2 Me), 5.02 (s, 2H, ArCH 2 ), 5.05 (s, 2H, ArCH 2 ), 6.79 (d, J = 8.7 Hz, 4H, DMT), 7.48 (m, 20H, Ar), 7.94 (s, 1H, Ar), 8.12 (m, 1H, Ar), 8.20 (d, J = 7.8, 1H, Ar), 8.51 (d, J = 7.8, 2H, Ar). 13 C NMR (CDCl 3 ) δ 55.2 (Me), 61.9 (CH 2 H), 63.3 (CH 2 DMT), 70.0 (CH 2 CH 2 DMT, CH 2 CH 2 H), 71.5, 71.6 (2 ArCH 2 ), 86.0 (Ar 3 C), 88.2, 88.4 (2 C CNaphth), 93.5, 93.7 (2 C CNaphth), 113.0, 126.6, 127.7, 128.2, 130.1, 136.3, 145.1, (DMT), 120.9, 121.3, 123.7, 123.9, 124.2, 125.4, 125.8, , , 126.8, 126.9, 128.4, , , 129.9, 130.0, 131.4, 131.5, 133.4, 134.6, (Ar). HR-MALDI-MS: m/z calcd for C 57 H 48 Na + 6 [M+Na] , found Yield of diprotected alcohol is 650 mg (26%). 1 H NMR (CDCl 3 ) δ 3.31 (t, J = 4.1 Hz, 4H, 2 CH 2 DMT), 3.76 (m, 12H, 4 Me), (m, 4H, 2 CH 2 CH 2 DMT), 5.10 (s, 4H,, 2 ArCH 2 ), (m, 12H, DMT), (m, 40H, Ar), 7.95 (s, 1H, Ar), 8.21 (d, J = 8.2, 2H, Ar), 8.51 (d, J = 8.2, 2H, Ar). 13 C NMR (CDCl 3 ) δ 55.2 (Me), 63.3 (CH 2 DMT), 70.0 (CH 2 CH 2 DMT), 71.5 (2 ArCH 2 ), 86.0 (Ar 3 C), 88.4 (C CNaphth) 93.5 (C CNaphth), (DMT, Ar). HR-MALDI-MS: m/z calcd for C 78 H 66 Na + 8 [M+Na] , found Phosphoramidite 9. The alcohol 8 (0.657 g, 0.79 mmol) was dissolved in dry CH 2 Cl 2 (20 ml). Diisopropylammonium tetrazolide (0.200 g, 1.20 mmol) and 2-cyanoethyl N,N,N,N -tetraisopropylphosphordiamidite (0.430 g, 1.40 mmol) were added under Ar. The mixture was stirred at rt for 24 hours. CH 2 Cl 2 (30 ml) was added and the reaction mixture was washed with H 2 (3 30 ml), dried (MgS 4 ), filtered and purified by dry column vacuum chromatography (silica gel; Et 3 N 1% v/v, 0-25% EtAc in petroleum ether) to afford compound 10 as a clear oil. Yield g (50%). 31 P NMR (CDCl 3 ) δ ESI- MS: m/z calcd for C 66 H 65 Na + N 2 7 P [M+Na] , found

6 3.2. Synthesis of Linker 3. I a b c H H H H d H 13 DMT N P CN 14 DMT Scheme 2SI. Reagents and conditions: a) Pd(PPh 3 ) 2 Cl 2, CuI, TMSA, NEt 3, THF, 2 days then NEt 3 3HF, 7 h; b) 1,3- diiodoazulene, Pd(PPh 3 ) 2 Cl 2, CuI, NEt 3, 3 days; c) DMT-Cl, NEt 3, CH 2 Cl 2, 2 days; d) NC(CH 2 ) 2 P(NPr i 2) 2, diisopropylammonium tetrazolide, CH 2 Cl 2, 2 days (4-Ethynylbenzyloxy)ethanol (11). 2-(4-Iodobenzyloxy)ethanol [2] (10; 6.0 g, 21.6 mmol) was dissolved in dry THF (100 ml) and dry Et 3 N (6 ml). The solution was bubbled by Ar for 5min before Pd(PPh 3 ) 2 Cl 2 (300 mg, 0.43 mmol), CuI (209 mg, 1,10 mmol) and trimethylsilylacetylene (2.6 g, 26.8 mmol) were added. The mixture was stirred under Ar at rt for 42 hours in a flask covered by aluminium foil. Afterwards, NEt 3 3HF (3.0 g, 18.5 mmol) was added and the mixture was stirred for 7 hours. Solvents were removed in vacuo. The residue were dissolved in CH 2 Cl 2 (100 ml) and washed with H 2 (2 100 ml), 0.2 M aq. EDTA (2 100 ml) and brine (100 ml). The organic phase was dried (MgS 4 ), filtrated and concentrated under reduced pressure. The residue was purified by dry column vacuum chromatography (silica gel; 0-35 %, EtAc in cyclohexane, v/v) to afford compound 11 as a ginger oil. Yield 1.9 g (50%). 1 H NMR (CDCl 3 ) δ 2.49 (br. s, 1H, H), 3.08 (s, 1H, C CH), 3.56 (m, 2H, 2 CH 2 CH 2 H), 3.73 (br. s, 2H, 2 CH 2 H), 4.53 (s, 1H, 2 PhCH 2 ), 7.28 (d, J = 8.4 Hz, 2H, Ph), 7.47 (d, J = 8.4 Hz, 2H, Ph). 13 C NMR (CDCl 3 ) δ 61.7 (CH 2 H), 71.5 (CH 2 CH 2 H), 72.6 (PhCH 2 ), 77.3 (C CH), 83.4 (C CH), 121.3, 127.4, 132.1, (Ph). EI-MS: 176 (C 11 H 12 2 ; calc ). MS (EI): m/z 176 (20%) [C 11 H 12 2 ] +, 131 (30%) [M CH 2 CH 2 H] +, 115 (100%) [M CH 2 CH 2 H] (4-{3-[4-(2-Hydroxyethoxymethyl)phenylethynyl]azulen-1-ylethynyl}benzyloxy)ethanol (12). 2-(4- Ethynylbenzyloxy)ethanol (11; 650 mg, 3.7 mmol) was dissolved in dry Et 3 N (50 ml) and flushed with Ar for 10 min before CuI (32 mg, 0.17 mmol), Pd(PPh 3 ) 2 Cl 2 (60 mg, 0.08 mmol) and 1,3-diiodoazulene [3] (640 mg, 1.7 mmol) were added. The mixture was stirred under Ar at rt for 3 days, in a flask covered by aluminium foil. Solvent was removed under diminished pressure. The residue was dissolved in CH 2 Cl 2 (80 ml) and washed with 0.2 M aq. EDTA (2 100 ml), H 2 (100 ml) and brine (100 ml). The organic phase was dried (MgS 4 ), filtrated and concentrated under reduced pressure. The residue was purified by dry column vacuum chromatography (silica gel; % EtAc in cyclohexane, v/v) to afford compound 12 as green crystals. Yield 845 mg (100%). 1 H NMR (CDCl 3 ) δ 2.21 (br. s, 2H, 2 H), 3.60 (m, 4H, 2 CH 2 CH 2 H), 3.77 (m, 4H, 2 CH 2 H), 4.58 (s, 4H, 2 PhCH 2 ), 7.41 (m, 11H, Ar), 8.11 (s, 1H, Ar), 8.57 (d, J = 9,6 Hz, 2H, Ar). 13 C NMR (CDCl 3 ) δ 61.9 (CH 2 H), 71.5 (CH 2 CH 2 H), 72.9 (PhCH 2 ), 85.0 (PhC C), 93.9 (PhC C), 110.5, 123.2, 125.7, 127.5, , 131.4, 132.6, 137.1, 137.8, 140.0, 141.4, (Ar). HR-MALDI-MS: m/z calcd for C 32 H 28 4 [M] , found

7 {4-[3-(4-{2-[Bis-(4-methoxyphenyl)phenylmethoxy]ethoxymethyl}phenylethynyl)azulen-1- ylethynyl]benzyloxy}ethanol (13). The diol 12 (800 mg, 1.68 mmol) and dry Et 3 N (187 mg, 1.85 mmol) were dissolved in dry CH 2 Cl 2 (50 ml) and flushed with Ar for 5 min. DMT-Cl (625 mg, 1.85 mmol) in dry CH 2 Cl 2 (15 ml) was slowly added over 1 hour. The mixture was stirred under Ar at rt for 20 hours in a flask covered by aluminium foil. An additional amount of DMT-Cl (176 mg, 0.52 mmol) in dry CH 2 Cl 2 (5 ml) was slowly added over 30 min and reaction mixture was stirred for 24 hours. The mixture was washed with H 2 (3 50 ml) and the organic phase was dried (MgS 4 ), filtered and concentrated under reduced pressure. The residue was purified by dry column vacuum chromatography (silica gel; 0.5% Et 3 N v/v, % EtAc in cyclohexane) to afford compound 13 and diprotected diol as green oils. Yield of 13 is 400 mg (30%). 1 H NMR (CDCl 3 ) δ 2.74 (br. s, 1H, H), 3.29 (m, 2H, CH 2 DMT), (m, 6H, CH 2 H, CH 2 CH 2 H and CH 2 CH 2 DMT), 3.78, 3.79 (s, 6H, 2 Me), 4.60, 4.60 (s, 4H, 2 PhCH 2 ), 6.82 (d, J = 8.7 Hz, 4H, DMT), (m, 27H, Ph). 13 C NMR (CDCl 3 ) δ 55.2 (2 Me), 61.9 (CH 2 H), 63.3 (CH 2 DMT), 69.9 (CH 2 CH 2 DMT, CH 2 CH 2 H), 72.8, 72.9 (2 PhCH 2 ), 84.8, 85.0 (2 PhC C), 94.1, 94.5 (2 PhC C), 113.0, 113.1, 126.7, 127.7, 127.8, 129.1, 136.3, 147.3, (DMT), 110.2, 125.7, 127.1, 127.3, 127.5, 127.6, 127.7, 128.1, 128.2, 130.1, 131.3, 131.4, 131.5, 132.5, 132.6, (Ar). + HR-MALDI-MS: HR-MALDI-MS: m/z calcd for C 53 H 47 6 [M+H] , found Yield of diprotected diol is 660 mg (36 %). 1 H NMR (CDCl 3 ) δ 3.29 (m, 4H, 2 CH 2 DMT), (m, 4H, 2 CH 2 CH 2 DMT), 3.78 (s, 12H, 4 Me), 4.63 (s, 4H, 2 PhCH 2 ), (m, 16H, DMT), (m, 24H, Ar). 13 C NMR (CDCl 3 ) δ 55.2 (Me), 63.3 (CH 2 DMT), 69.9 (CH 2 CH 2 DMT), 72.7 (PhCH 2 ), 84.6 (C=CPh), 85.9 (CPh 3 ) 94.0 (C=CPh), 113.0, 113.0, 126.6, 126.6, 127.7, 127.7, 128.1, 128.2, 130.0, 130.1, 136.3, 136.3, 145.0, 145.1, 158.3, (DMT), 110.6, 123.0, 125.6, 127.3, 127.4, 131.3, 131.3, 132.4, 137.1, (Ar). To obtain additional amount of compound 13, the diprotected diol (600 mg, 0.55 mmol) was dissolved in dry MeCN (20 ml) and 80% aq. AcH (250 µl, mmol) was added. After stirring at rt for 3 days, 1M aq. KH (4.5 ml) was added. The mixture was diluted by MeCN (30 ml) and Et 2 (50 ml), washed with H 2 (3 50 ml). The organic phase was dried (MgS 4 ), filtrated and concentrated under reduced pressure. The residue was purified by dry column vacuum chromatography (silica gel; EtAc:cyclohexane:Et 3 N; 49.5:49.5:1 %, v/v/v) to afford compound 13 as a green oil. Yield 210 mg (47%) Phosphoramidite 14. The alcohol 13 (200 mg, 0.26 mmol) was dissolved under Ar in dry MeCN (10 ml). Diisopropylammonium tetrazolide (107 mg, 0.62 mmol) and 2-cyanoethyl N,N,N,N -tetraisopropylphosphordiamidite (192 mg, 0.64 mmol) were added, and the mixture was stirred at rt for 2 days in a flask covered by aluminium foil. CH 2 Cl 2 (25 ml) was added and the mixture was washed with H 2 (3 50 ml). The organic phase was dried (MgS 4 ), filtrated and concentrated under reduced pressure. The residue was purified by dry column vacuum chromatography (silica gel; Et 3 N 0.05% v/v, 0-100% EtAc in cyclohexane) to afford compound 14 as a green oil. Yield 267 mg (100%). 31 P NMR (CDCl 3 ) δ HR-MALDI-MS: m/z calcd for C 62 H 63 N 2 7 PNa + [M+Na] , found Synthesis and Purification of ligonucleotides: Ns were synthesized on an Expedite Nucleic Acid Synthesis System Model 8909 from Applied Biosystems using 4,5-dicyanoimidazole as an activator for commercial phosphoramidites and TINA phosphoramidite. Solutions of phosphoramidites 9 and 14, and phosphoramidite of the linker 1 [2] were prepared in a 1 ml plastic syringes in dry CH 2 Cl 2 (0.075 M, 350 µl) followed by addition of 0.1 M solution of 1H-tetrazol in MeCN (350 µl) and used in a coupling step on a DNA synthesizer (2 min reaction time). An increased deprotection time (100 sec) and coupling time (2 min) for M solution of the TINA phosphoramidite (P) was applied. The DMT-on Ns were cleaved off from the solid support (rt, 2h) and deprotected (55 C, overnight) using 32 % aqueous ammonia. Purification of DMT-on Ns was accomplished using a reverse-phase semipreparative HPLC on Waters Xterra MS C 18 column. Buffer A [0.05 M triethylammonium acetate in H 2 (ph = 7.0)] and buffer B (75% CH 3 CN in H 2 ). Flow 2.5 ml/min. Gradients: 2 min 100 %

8 A, linear gradient to 70 % B in 38 min, linear gradient to 100 % B in 7 min, 100 % B in 3 min and then 100 % A in 10 min. Corresponding fractions with Ns were evaporated, and DMT-group was cleaved by 80% aq. AcH (100 µl) in 20 min followed by dilution with 1 M aq. NaAc (150 µl) and precipitation of Ns from chilled EtH (550 µl). The modified Ns were confirmed by MALDI-TF analysis on a Voyager Elite Biospectrometry Research Station from PerSeptive Biosystems. The purity of the final Ns was found to be more than 85% by analytical ion-exchange chromatography using LaChrom system from Merck Hitachi on GenPak-Fax column (Waters). ligonucleotide concentrations were determined by absorbance at 260 nm and the calculated single-strand extinction coefficients were based on a nearest neighbor model (the extinction coefficient at 260 nm for monomer P is 22400, for the linker 2 is 22830, for the linker 3 is 13030). Table 1SI. MALDI-TF MS and ion-exchange HPLC analysis of Ns synthesized ligonucleotides m/z [M+H] +, calcd (Da) m/z [M+H] +, found (Da) N N N N N N N N N N N N Melting Temperature Measurements. Melting temperature measurements were performed on a Perkin-Elmer UV/VIS spectrometer Lambda 35 fitted with a PTP-6 temperature programmer. The triplexes were formed by mixing the two strands of the Watson-Crick duplex, each at a concentration of 1.0 µm, followed by addition of the third (TF) strand (1.5 µm) in the corresponding buffer solution. The solution was heated to 80 C for 5 min, cooled to room temperature and then kept at 5 C for 1 hour. Solutions of triplexes containing N4 were kept at 4 C for 24 hours. The melting temperature (T m, C) was determined as the maximum of the first derivative plots of the melting curves obtained by measuring absorbance at 260 nm against increasing temperature (1.0 C per 1 min). Lower speed of increasing the temperature (0.5 C per 1 min) resulted in the same curves. Experiments were also done at 373 nm for Ns possessing monomer P. All melting temperatures are within the uncertainty ± 0.5 C as determined by repetitive experiments.

9 N1/D1 N3/D1 N5/D1 70 d(abs 260 )/dt T ( C) Figure 1SI. Representative curves of the first derivatives obtained from melting curves (260 nm) recorded for N1, N3 and N5 toward D1 in the buffer (20 mm sodium cacodylate, 100 mm NaCl, 10 mm MgCl 2 ) at ph 7.2. Apparent melting temperatures are reported without mathematical curve resolution of triplex and duplex melting curves. Reference: [1] M. C. Carreño, R. Hernández-Sánchez, J. Mahugo, A. Urbano A. J. rg. Chem. 1999, 64, [2] C. H. Jessen, E. B. Pedersen, Helv. Chim. Acta 2004, 87, [3] K. H. H. Fabian, A. H. M. Elwahy, K. Hafner, Tetrahedron Lett. 2000, 41,