Supplementary information
HPLC gradients
On 4x 250 mm Nucleosil 120-5 C18 column products have been eluted using 1mL/min
gradient flow (Solvent A: 0.1 M NH4HCO3, Solvent B: 100% CH3CN). 0-5 min: 5% B; 5-12
min: 5-15% B; 12-15 min: 15-50% B; 15-20 min: 50-85 % B; 20-22 min: 85% B; 22-27 min:
85-5% B; 27-40 min: 5%B. On VP 250/10 Nucleodur 100-5 C18 ec column products were
eluted at 3 mL/min using the gradient: 0-5 min: 5% B; 5-20 min 5-20% B; 20-23 min: 2050% B; 23-25 min: 50-85% B; 25-28 min: 85% B; 28-33: 85-5% B; 33-45 min: 5%.
IE HPLC is done using column: Mini Q anion exchange Amersham Pharmacia; running
NaClO4 gradient from 0 to 1 M at pH 12. using the solvents: A: 2 mM TRIS*HClO4, pH 12;
B: 2 mM TRIS*HClO4/1 M NaClO4/20% acetonitrile, pH 12; gradient: 0-3 min 0-7% B; 3-11
min 7-11% B; 11-25 min 11-14% B; 25-55 min 14-20% B; 55-63 min 20-100% B; 63-65 min
100% B; 65-68 min 100-0% B; 68-90 min 0% B.
Matrix-Assisted-Laser-Desorption-Ionization-Time-Of-Flight (MALDI-TOF)
The measurements were made on Autoflex II-Mass Spectrometer from Bruker with N2 (337
nm) laser. 10µL of sample are treated with Dowex 50WX8-200 cation – exchange resin in
ammonium form, mixed with 5 µL of matrix solution (0.3M of 2,4,6-trihydroxyacetophenone
in EtOH mixed with 0.1M diammonium citrate in water in proportion 2:1), 1µL aliquot of the
mixture is taken and allowed to dry in a steel target plate.
Table S1. MALDI-TOF masses of synthesized products
Compound
nCAp
nCGp
nTAp
nTGp
nTGTGp
nCATGp
nTGCAp
nTACGp
nTGTACAp
nTGCGCAp
massteo
619.40
635.40
634.42
650.42
1284.9
1254.8
1254.8
1254.8
1871.3
1872.2
massexp
618.6
636.1
634.8
650.1
1283.8
1253.8
1254.2
1253.05
1870.9
1871.65
Table S2. MALDI-TOF masses of the products identified in the reaction mixture of 20 mM
nCAp, 0.4M EDC, 0.1M HEPES pH 7.55; after 24h at 2°C
Product
nCAp
nCAp(cycl)
EDCnCAp
(nCAp)2(cycl)
(nCAp)2
EDC(nCAp)2
(nCAp)3(cycl)
(nCAp)3
EDC(nCAp)3
(nCAp)4
EDC(nCAp)4
EDC(nCAp)5
Mtheo
619
601
774
1202
1220
1375
1806
1821
1971
2422
2577
3178
MMALDI
776
1204
1224
1377
1806
1823
1978
2424
2579
3182
Table S3. MALDI-TOF masses of the products identified in the reaction mixture of 20 mM
nTGp, 0.4M EDC, 0.1M HEPES pH 7.55; after 24h at 2°C
Product
nTGp
nTGp(cycl)
EDCnTGp
(nTGp)(cycl)
EDC
(nTGp)2(cycl)
(nTGp)2
EDC(nTGp)2cycl
EDC(nTGp)2
(nTGp)3(cycl)
(nTGp)3
(nTGp)
EDC
3(cycl)
EDC(nTGp)3
(nTGp)4(cycl)
Mtheo
651
633
806
788
1264
1282
1419
1437
1905
1923
2055
2078
2528
MMALDI
807
789
1266
1288 (cycl+23?)
1421
1440
1898
1920
2053
2075
2528
Table S4. MALDI-TOF masses of the products identified in the reaction mixture of 20 mM
nTAp, 0.4M EDC, 0.1M HEPES pH 7.55; after 24h at 2°C
Product
nTAp
(nTAp)2
(nTAp)3
(nTAp)4
(nTAp)5
(nTAp)5
Mtheo
634
1250
1866
2482
3098
3714
MMALDI
1251
1868
2503 (M+22)
3120 (M+22)
3734 (M+22)
Table S5. MALDI-TOF masses of the products identified in the reaction mixture of 20 mM
nCGp, 0.4M EDC, 0.1M HEPES pH 7.55; after 24h at 2°C
Product
nCGp
(nCGp)2
(nCGp)3
(nCGp)4
n
CG
Mtheo
635
1252
1869
2486
MMALDI
635
1252
1868
2485
p
10mer
20mer
30mer
40mer
50mer
60mer
10
20
30
40
Retentionszeit [min]
A
B
Figure SI1. nCGp polymerization. (A) 50 mM nCGp after 6h of reaction at 2°C. (B) 20 mM
nCGp after 2h, 4h, 8h, 24h and 48h of reaction. Marker line is a ladder with 10 nt step.
1285
2490
1222
1887
758
1824
3092
1000
2000
3000
3689
3758
4000
5000
m/z
Figure SI 2. MALDI TOF spectrum of 10 mM nTGTGp + 10 mM nCAp polymerization in
presence of 0.4M EDC, 0.1M HEPES buffer, pH 7.55; after 2h of the reaction.
Table S6. MALDI TOF spectrum of 10 mM nTGTGp + 10 mM nCAp polymerization in
presence of 0.4M EDC, 0.1M HEPES buffer, pH 7.55; after 2h of the reaction.
Product
EDCnCAp(cycl)
nCACAp
nTGTGp
nCACACAp
nTGTGCAp
nTGTGCACAp
nTGTGCACACAp
nTGTGCACACACAp
nTGTGTGTGCACAp
Mtheo
759
1220
1284
1822
1885
2487
3088
3690
1254
MMALDI
759
1222
1285
1824
1887
2490
3092
3689
3758
nCGp
(nCGp)cycl.
360 min
240 min
180 min
120 min
60 min
30 min
10 min
0
5
10
15
20
25
retention time (min)
Figure SI3. HPLC plot of nCGp cyclization. Reaction of 0.85 mM nCGp, 0.4M EDC, 0.1M
HEPES pH 7.55 at 30°C.
Figure SI 4. Effect of the ionic strength on polycondensation efficiency. nTGp with nCAp
in 1:1 ratio. Total nucleotide concentration is 20 mM. 20% polyacrylamide denaturating gel
(7M Urea) is stained with SybrGold® gel stain for 20 min. To estimate the length of the
product Ultra Low Range DNA ladder by Fermentas® was used with a fragments length
equal to 300, 200, 150, 100, 75, 50 (the most intense band), 35, 25, 20 and 15nt.
Figure SI 5. The crystal growth during the nCAp with nTGp polycondensation. nCAp+
nTGp 20 mM, 0.04M EDC, 0.1M HEPES pH 7.5 (Na+ salt), 8°C. Above: a crystal formed
after 24h of reaction. Center: the same crystal after 48h of reaction. Below: crystal after 48h
under polarized light. Scale bar corresponds to 50 μm. Compare the pictures with the increase
in product length in the figure 3C.
Figure SI 6. Crystals stained with Hoechst 33258 stacking dye sensitive to double
stranded DNA. (Above) For fluorescent microscopy a Zeiss Microscope LSM 510 was used
with excitation at 450-480nm and emission at 500nm. The stock solution of the Hoechst
33258 stacking dye was added to the reaction mixture to achieve a 0.5 µg/mL final
concentration. (Below) The same crystals under white light.
14
2°C
8°C
14°C
20°C
26°C
32°C
38°C
42°C
12
10
8
CD, mdeg
6
4
2
0
-2
-4
-6
-8
-10
220
240
260
280
300
320
340
wavelength, nm
CD276nm
10
9
CD, mdeg
8
7
6
5
4
3
0
10
20
30
40
50
Temperature, °C
Figure SI 7. Melting curve of unreacted dinucleotides in solution. 10 mM nCAp + 10 mM
nTGp in HEPES buffer pH 7.5.; 0.1 cm path length cell (above)
2
5°C
10°C
20°C
30°C
40°C
50°C
60°C
70°C
80°C
85°C
CD, mdeg
1
0
-1
-2
260
280
300
320
340
Wavelength, nm
0.8
CD, mdeg
0.4
0.0
-0.4
-0.8
0
20
40
60
80
100
Temperature, °C
Figure SI 8. Melting curves of the diluted reaction products. 10 mM nCAp + 10 mM
nTGp after 72h of reaction in 0.4M EDC, 0.1M HEPES, pH 7.55, at 2°C; diluted 100 times
and transferred to 1 cm cell.