Supporting Information- Part I

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1 Supporting Information- Part I Carba-LNA- 5Me C/A/G/T modified Oligos Show Nucleobase-specific Modulation of 3 -Exonuclease Activity, Thermodynamic Stability, RNA- Selectivity and RNase H Elicitation Synthesis and Biochemistry RamShankar Upadhayaya a, Sachin Gangadhar Deshpande a, Qing Li b, Ramakant Asaram Kardile a, Aftab Yusuf Sayyed a, Eknath Kamalakar Kshirsagar a, Rahul Vilas Salunke a, Shailesh Satish Dixit a, Chuanzheng Zhou b, András Földesi b and Jyoti Chattopadhyaya b * a Institute of Molecular Medicine, International Biotech Park, Genesis Campus, Phase II Opp Infosys, Tal Mulshi Hinjewadi, Pune , India. b Program of Bioorganic Chemistry, Institute of Cell and Molecular Biology, Biomedical Centre, Uppsala University, SE Uppsala, Sweden. * Corresponding Author: Bioorganic Chemistry, Institute of Cell and Molecular Biology. Biomedical Centre, Uppsala University, SE Uppsala, Sweden. Phone: , Fax: , jyoti@boc.uu.se Sachin Gangadhar Deshpande a, RamShankar Upadhayaya a and Qing Li b have contributed equally to this work 1

2 Table of Contents: General Methods:...3 Figure SI.1. PAGE pictures show the digestion pattern of AONs 9-16 by SVPDE...5 Fgiure SI.2. Degradation kinetics of AON 9 by SVPDE...6 Fgiure SI.3. Degradation kinetics of AON 10 by SVPDE...8 Figure SI.4. Degradation kinetics of AON 11 by SVPDE...10 Figure SI.5. Degradation kinetics of AON 12 by SVPDE...12 Figure SI.6. Degradation kinetics of AON 13 by SVPDE...14 Figure SI.7. Degradation kinetics of AON 14 by SVPDE...16 Figure SI 8. Degradation kinetics of AON 15 by SVPDE...18 Figure SI.9. Degradation kinetics of AON 16 by SVPDE...20 Figure SI.10. Autoradiograms of 20% denatured PAGE showing the stability of 5 -end 32P labeled AONs 1-8 in human blood serum Table 1. T m values of duplexes formed by modified AON 1-8 with complementary RNA and DNA a and MS characterization of AON Figure SI.11 MALDI-TOF spectrum of AON Figure SI.12 MALDI-TOF spectrum of AON Figure SI.13 MALDI-TOF spectrum of AON Figure SI.14 MALDI-TOF spectrum of AON Figure SI.15 MALDI-TOF spectrum of AON Figure SI.16 MALDI-TOF spectrum of AON Figure SI.17 MALDI-TOF spectrum of AON

3 General Methods: All chemicals and reagents used were of reagent grade. Purification and drying of reagents and solvents was carried out according to literature procedure. Thin layer chromatographic analyses were performed on E- Merck 60 F 254 precoated aluminum thin layer chromatographic plates. All air-sensitive reactions were carried out under nitrogen atmosphere. Melting points were determined on a Büchi melting point B-540 instrument and are uncorrected. 1 H and 13 C NMR spectra were recorded on Bruker Biospin 400 MHz, Avance DRX500 and DRX600 spectrometers in the indicated solvents (TMS as an internal standard). The values of chemical shifts are expressed in ppm and the coupling constants (J) in hertz (Hz). Mass spectra were recorded in API 2000 LC/MS/MS system spectrometer. HPLC separations: The mixture of compounds major isomer (7 R) 43a, minor isomer (7 S) 43b and endo product 43c was separated using high pressure preparative HPLC column CHIRALPAK-IC (4.6x250) 5 micron ARD/K/7461. An isocratic mobile phase was used consisting of hexane/ethanol (70:30) until the elution of all three products with the flow rate of 1.0 ml/minute. The minor isomer 43b elutes first with a retention time of min as peak 1, followed by major product 43a at minute as peak 2. The endo product 43c elutes at minute as peak 3. The chromatogram suggests the ratio of products 43a, 43b and 43c is 74.58:17.61:6.93, respectively. 3

4 major isomer 43a minor isomer 43b endo product 43a 43b 43a 43c HPCL chromatogram showing the separation and composition of the product mixture 43a/b/c of Me C free radical cyclization. 4

5 ( s T (AON 9) ( s A (AON 11) ( s C (AON 10) ( s G (AON 12) mer 14mer ( s ru (AON 13) ( s ra (AON 15) ( s rc (AON 14) ( s rg (AON 16) mer 14mer Figure SI.1. PAGE pictures show the digestion pattern of AONs 9-16 by SVPDE. Because the present of thiophosphate linkage between T13 and T14, the SVPDE can only cleavage the last phosphate, giving the only product, 14mer oligonucleotides. Digestion conditions: AONs 1 μm, 100 mm Tris-HCl, ph 8.0, 100 mm NaCl and 15 mm MgCl 2, SVPDE unit for AONs 9-12, unit for AONs 13-16, 21 C, total reaction volume 30 μl. 5

6 Fgiure SI.2. Degradation kinetics of AON 9 by SVPDE. Digestion conditions: 100 mm Tris-HCl (ph 8.0), 100 mm NaCl, 15 mm MgCl 2, SVPDE unit, total reaction volume was 30 μl. The concentration of AON 9 varied from 1 μm to 100 μm. Reaction temperature is 21 C. [( s T (AON 9)] = 1 um [( s T (AON 9)] = 2 μm k = ± μm/min 1.90 k = ± μm/min [( s T (AON 9)] = 5 μm [( s T (AON 9)] = 10 μm k = ± μm/min 9.4 k = ± μm/min

7 [( s T (AON 9)] = 50 μm [( s T (AON 9)] = 100 μm C (mm) k = ± μm/min 95 k = ± μm/min T (MIN) Initial velocity of the digestion of AON 9 by SVPDE as a function of concentration ( s T (AON 9) V0 (μm/min) K M = 2.88 ± 0.65 μm V max = ± μm/min C 0 (μm) 7

8 Fgiure SI.3. Degradation kinetics of AON 10 by SVPDE. Digestion conditions: 100 mm Tris-HCl (ph 8.0), 100 mm NaCl, 15 mm MgCl 2, SVPDE unit, total reaction volume was 30 μl. The concentration of AON 10 varied from 1 μm to 100 μm. Reaction temperature is 21 C. [( s C (AON 10)] = 1 μm [( s C (AON 10)] = 2 μm C (mm) k = ± μm/min k = ± μm/min [( s C (AON 10)] = 5 μm [( s C (AON 10)] = 10 μm k = ± μm/min 9.3 k = ± μm/min

9 [( s C (AON 10)] = 50 μm [( s C (AON 10)] = 100 μm k = ± μm/min k = ± μm/min Initial velocity of the digestion of AON 10 by SVPDE as a function of concentration ( s C (AON 10) V o (μm/min) K M = 2.63 ± 0.59 μm V max = ± μm/min C0 (μm) 9

10 Figure SI.4. Degradation kinetics of AON 11 by SVPDE. Digestion conditions: 100 mm Tris-HCl (ph 8.0), 100 mm NaCl, 15 mm MgCl 2, SVPDE unit, total reaction volume was 30 μl. The concentration of AON 11 varied from 1 μm to 100 μm. Reaction temperature is 21 C [( s A (AON 11)] = 1 μm 1.96 [( s A (AON 11)] = 2 μm k = ± μm/min k = ± μm/min [( s A (AON 11)] = 5 μm [( s A (AON 11)] = 10 μm k = ± μm/min k = ± μm/min

11 [( s A (AON 11)] = 50 μm [( s A (AON 11)] = 100 μm k = ± μm/min 95 k = ± μm/min Initial velocity of the digestion of AON 11 by SVPDE as a function of concentration ( s A (AON 11) V0 (μm/min) K M = 2.63 ± 0.62 μm V max = ± μm/min C 0 (μm) 11

12 Figure SI.5. Degradation kinetics of AON 12 by SVPDE. Digestion conditions: 100 mm Tris-HCl (ph 8.0), 100 mm NaCl, 15 mm MgCl 2, SVPDE unit, total reaction volume was 30 μl. The concentration of AON 12 varied from 1 μm to 100 μm. Reaction temperature is 21 C. [( s G (AON 12)] = 1 μm k = ± μm/min [( s G (AON 12)] = 2 μm k = ± μm/min [( s G (AON 12)] = 5 μm [( s G (AON 12)] = 10 μm k = ± μm/min 9.4 k = ± μm/min

13 [( s G (AON 12)] = 50 μm [( s G (AON 12)] = 100 μm k = ± μm/min k = ± μm/min Initial velocity of the digestion of AON 12 by SVPDE as a function of concentration ( s G (AON 12) V0 (μm/min) K M = 0.66 ± 0.19 μm V max = ± μm/min C 0 (μm) 13

14 Figure SI.6. Degradation kinetics of AON 13 by SVPDE. Digestion conditions: 100 mm Tris-HCl (ph 8.0), 100 mm NaCl, 15 mm MgCl 2, SVPDE unit, total reaction volume was 30 μl. The concentration of AON 13 varied from 1 μm to 100 μm. Reaction temperature is 21 C. [( s ru (AON 13)] = 1 μm [( s ru (AON 13)] = 2 μm k = ± μm/min k = ± μm/min [( s ru (AON 13)] = 5 μm [( s ru (AON 13)] = 5 μm k = ± μm/min k = ± μm/min

15 50 [( s ru (AON 13)] = 50 μm 100 [( s ru (AON 13)] = 100 μm k = ± μm/min 94 k = ± μm/min Initial velocity of the digestion of AON 13 by SVPDE as a function of concentration ( s ru (AON 13) V 0 (μm/min) K M = 1.80 ± 0.32 μm V max = ± μm/min C 0 (μm) 15

16 Figure SI.7. Degradation kinetics of AON 14 by SVPDE. Digestion conditions: 100 mm Tris-HCl (ph 8.0), 100 mm NaCl, 15 mm MgCl 2, SVPDE unit, total reaction volume was 30 μl. The concentration of AON 14 varied from 1 μm to 100 μm. Reaction temperature is 21 C. [( s rc (AON 14)] =1 μm [( s rc (AON 14)] = 2 μm C(μM) k = ± μm/min k = ± μm/min [( s rc (AON 14)] = 5 μm [( s rc (AON 14)] = 10 μm k = ± μm/min k = ± μm/min

17 50 [( s rc (AON 14)] = 50 μm 99 [( s rc (AON 14)] = 100 μm k = ± μm/min 93 k = ± μm/min Initial velocity of the digestion of AON 14 by SVPDE as a function of concentration 0.10 ( s rc (AON 14) 0.08 V0 (μm/min) K M = 3.19 ± 0.53 μm V max = ± μm/min C 0 (μm) 17

18 Figure SI 8. Degradation kinetics of AON 15 by SVPDE. Digestion conditions: 100 mm Tris-HCl (ph 8.0), 100 mm NaCl, 15 mm MgCl 2, SVPDE unit, total reaction volume was 30 μl. The concentration of AON 15 varied from 1 μm to 100 μm. Reaction temperature is 21 C. [( s ra (AON 15)] = 1 μm [( s ra (AON 15)] = 2 μm k = ± μm/min k = ± μm/min [( s ra (AON 15)] = 5 μm [( s ra (AON 15)] = 10 μm k = ± μm/min k = ± μm/min

19 [( s ra (AON 15)] = 50 μm [( s ra (AON 15)] = 100 μm k = ± μm/min k = ± μm/min Initial velocity of the digestion of AON 15 by SVPDE as a function of concentration 0.10 ( s ra (AON 15) 0.08 V0 (mm/min) K M = 3.97 ± 0.91 μm V max = ± μm/min [S0] (μm) 19

20 Figure SI.9. Degradation kinetics of AON 16 by SVPDE. Digestion conditions: 100 mm Tris-HCl (ph 8.0), 100 mm NaCl, 15 mm MgCl 2, SVPDE unit, total reaction volume was 30 μl. The concentration of AON 16 varied from 1 μm to 100 μm. Reaction temperature is 21 C. [( s rg (AON 16)] = 1 μm k = ± μm/min [( s rg (AON 16)] = 2 μm k = ± μm/min [( s rg (AON 16)] = 5 μm [( s rg (AON 16)] = 10 μm k = ± μm/min k = ± μm/min

21 50 [( s rg (AON 16)] = 50 μm 100 [( s rg (AON 16)] = 100 μm C(μM) k = ± μm/min 94 k = ± μm/min Initial velocity of the digestion of AON 16 by SVPDE as a function of concentration 0.10 ( s rg (AON 16) 0.08 V 0 (μm/min) K M = 3.47 ± 0.81 μm V max = ± μm/min C 0 (μm) 21

22 Figure SI.10. Autoradiograms of 20% denatured PAGE showing the stability of 5 -end 32P labeled AONs 1-8 in human blood serum h Native AON h h AON 3 AON h 19mer 17mer 18mer AON 5 AON 6 AON 7 AON h h h h 19mer 17mer 22 18mer 19mer

23 Table 1. Tm values of duplexes formed by modified AON 1-8 with complementary RNA and DNA a and MS characterization of AON 2-8. Oligo name Sequence of AONs Tm of AON/RN A ΔTm of AON/RN A b Tm of AON/DN A ΔTm of AON/DN ΔΔTm A c d (ΜΗ) + e Cacl. Found AON 1 5 TCC CGC CTG TGA CAT GCA TT 74.0 C 0 o C 71.0 o C 0 o C +3.0 o C - - AON 2 5 TCC CGC CTG TGA CAT GCA TT 77.5 C +3.5 C 74.7 o C +3.7 o C +2.8 o C AON 3 5 TCC CGC CTG TGA CAT GCA TT 80.0 C +6 C 71.2 o C +0.2 o C +8.8 o C AON 4 5 TCC CGC CTG TGA CAT GCA TT 82.0 C +8 C 76.7 o C +5.7 o C +5.3 o C AON 5 5 TCC CGC CTG TGA CAT GCA TT 83.0 C +9 C 76.4 o C +5.4 o C +6.6 o C AON 6 5 TCC CGC CTG TGA CAT GCA TT 78.0 C +4 C 72.4 o C +1.4 o C +5.6 o C AON 7 5 TCC CGC CTG TGA CAT GCA TT 79.0 C +5 C 72.8 o C +1.8 o C +6.2 o C AON 8 5 TCC CGC CTG TGA CAT GCA TT 87.5 C C 76.3 o C +5.3 o C o C a T m Values measured at the maximum of the first derivative of the melting curve (A260nm Vs temperature) in medium salt buffer (60 mm tris-hcl at ph 7.5, 60 mm KCl, 0.8 mm MgCl2) with temperature 20 C 90 C using 1μM concentrations of two complementary strands. The values of Tm given are averages of three independent measurements (the error of the three consecutive measurements is within C). A = clna-a; G = clna-g; C = clna- Me C; T = clna-t. b ΔTm of AON/RNA was obtained by comparing the Tm of AON/RNA with that of the native AON 1/RNA. c ΔTm of AON/DNA was obtained by comparing the Tm of AON/DNA with that of the native AON 1/DNA. d RNA-selectivity: ΔTm = (Tm of AON/RNA) - (Tm of AON/DNA). e Mass spectra of AON2-8 were recorded by MALDI-TOF mass spectroscopy. 23

24 Figure SI.11 MALDI-TOF spectrum of AON2 24

25 Figure SI.12 MALDI-TOF spectrum of AON3 25

26 Figure SI.13 MALDI-TOF spectrum of AON4 26

27 Figure SI.14 MALDI-TOF spectrum of AON5 27

28 Figure SI.15 MALDI-TOF spectrum of AON6 28

29 Figure SI.16 MALDI-TOF spectrum of AON7 29

30 Figure SI.17 MALDI-TOF spectrum of AON8 30