nagendra revised-supplementary material · 2005. 7. 6. · title: microsoft word - nagendra...

# Supplementary Material (ESI) for Chemical Communications # This journal is © The Royal Society of Chemistry 2005

1

PNA C-C+ i-motif: Superior stability of PNA TC8Tetraplexes compared to DNA TC8 tetraplexes Nagendra K. Sharma and Krishna N. Ganesh* Division of Organic Chemistry (Synthesis) National Chemical laboratory, Pune 411008, INDIA. Fax: 91 20 589 3153; Tel: 91 20 589 3153; E-mail: [email protected] CONTENTS

Experimental 2

HPLC of 1-4 3-5

UV-Tm experimental details 6

MALDI- TOF 7-8

UV-melting curves 9-10

UV-Tm table 11


2

Experimental Section:

General. Reagents for PNA monomers synthesis, were purchased from Lancaster, UK. DMF was

dried by vacuum distillation over P2O5, THF refluxed over sodium, pyridine refluxed over KOH

then CaH, CH2Cl2 refluxed over CaH and CH3CN refluxed over CaH then distilled under anhydrous

conditions. TLC were done using pre-coated silica gel plates 1.0554 DC-Alufolien 20 x 20 cm

Kieselgel 60 F254 from Merck. . 1H NMR (200 MHz) and 13C NMR were recorded in solvent

CDCl3 and values are quoted in δ ppm. Mass spectra were recorded using Finnigan MAT and

MALDI-TOF using ABI mass spectrometer.

General protocol for solid phase synthesis of PNA.

Synthesis of PNA was carried out using BOC-β-alanine derivatized Merrifield resin (Pharmecia)

(0.15 mmol/g substitution). The synthesis cycle was as follows: deprotection: 50% TFA in DCM

(15 min), wash with DCM, DMF and DCM , neutralize (5% DIEA in DCM), wash DCM, DMF and

DCM), coupling (4b/HOBT/HBTU/DIEA in DMF, 4 eq, 1.5 h) capping (10% Ac2O/Pyridine in

DCM), wash (DCM, DMF and DCM). Deprotection and amide coupling reactions were monitored

by Kaiser’s test. The purity of PNA was ascertained on an analytical RP C18 column.

Table 1. Oligomers for the study of i-motif of PNA PNA Sequences of PNA Molecular

Formula Molecular weight (Calculated)

1 H2N-T-C-C-βala-COOH C34H49N15O12 859.86 2 H2N-T-C-C-C-C-βala-COOH C44H62N20O15 1111.11 3 H2N-T-C-C-C-C-C-βala-COOH C54H75N25O18 1360.36 4 HN-Lys-T-C-C-C-C-C-C-C-C-CONH2 C99H137N47O30 2463.3

Table 2. DNA was synthesized using standard procedure on automated synthesizer.

5 d(TCCCC) 6 d(TCCCCCCCC)


3

PNA 1

Figure 1.HPLC of PNAs 1-4


4

PNA 3

PNA 4


5

PNA 2


6

UV-Tm experiments.

UV melting experiments were performed on Lambda-35 UV Spectrometer (Perkin-Elmer)

equipped with a thermal melt system, PTP-6 Peltier Temperature Programmer with water circulator

Thermohake K20. The sample for Tm measurement was prepared by mixing calculated amount of

stock oligonucleotide and PNA solutions together in 2 mL of sodium phosphate buffer (pH 7.1).

The samples 2 mL were transferred to quartz cell and sealed with Teflon stopper after degassing

with nitrogen gas for 15 min and equilibrated at the starting temperature for at least 30 min. The OD

at 260 nm was recorded in steps from 10-85 oC with temperature increment of 0.2 oC/min. The

results were normalized and analysis of data was performed on using Origin 5.0 (Microsoft Corp.).

The UV study of the i-motif formation by PNA was done in acidic pH by monitoring at 295

nm. 100mM sodium acetate buffer was used for pH 3.0-5.0, while 10mM potassium phosphate

buffer with KCl for pH 5.9-7.0. Figures 3 and 4 show the first derivative UV-melting curves for

different PNAs at different pH. Tables 3 and 4 are the Tms computed from the UV data. The ITC

results are shown in Figure 5.

240 245 250 255 260 265 270 275 280 285 290 295 3000.000.050.100.150.200.250.300.350.400.450.500.550.60

4.05

3.64

3.473.23

3.07 2.88 2.78 neat

Abso

rban

ce

Wavelength (nm)

UV-spectra of TC8 PNA 4 at different pHs


7

Figure 2. Mass spectra of PNAs 1-

810.0 841.8 873.6 905.4 937.2 969.0Mass (m/z)

0

1.3E+4

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

Voyager Spec #1 MC=>AdvBC(32,0.5,0.1)=>NR(2.00)=>AdvBC(32,0.5,0.1)=>AdvBC(32,0.5,0.1)=>NR(2.00)[BP = 868.5, 12614]868.57

867.58

869.57

870.56919.53893.52845.51 871.51

PNA1 M+= 859 (M+8H)+=868.57

1072.0 1119.4 1166.8 1214.2 1261.6 1309.0Mass (m/z)

0

1.3E+4

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

Voyager Spec #1[BP = 691.8, 50647]

1112.15

1111.151113.12

1102.20

1134.021103.17

PNA 2 M+= 1111.11 (M+H)+=1112.5

1168.0 1331.8 1495.6 1659.4 1823.2 1987.0Mass (m/z)

0

4.1E+4

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

Voyager Spec #1=>AdvBC(32,0.5,0.1)=>NR(2.00)[BP = 1113.1, 58357]

1322.90

1344.92

1345.90

1388.93

1360.90 M+= 1360

PNA 3


8

PNA 4


9

10 20 30 40 50 60 70 80 90-0.10.00.10.20.30.40.50.60.70.80.91.0

6 4pH 5.9

Norm

alis

ed A

bsob

ance

at 2

95nm

Temperature (oC)

A

10 20 30 40 50 60 70 80 90

0.0

0.2

0.4

0.6

0.8

1.0

4

6 pH 7.0

Norm

aliz

ed A

borb

ance

at 2

95nm

Temperature (00C)

C

Figure 3. UV-melting profiles of PNA (4,)/DNA (6) at wavelength 295nm in 100mM sodium acetate buffer in acidic pH. Plots D, E and F arerepresented in pH 5.9, 6..5 and 7.0 respectively.

UV-Tm curves


10

10 20 30 40 50 60 70 80 90

-0.030

-0.025

-0.020

-0.015

-0.010

-0.005

0.000 pH 3.03 4 5 6

d(Ab

sorb

ance

295)/

dT

Temperature (0C)

H

20 40 60 80-0.020

-0.018

-0.016

-0.014

-0.012

-0.010

-0.008

-0.006

-0.004

-0.002

0.000pH 4.5

5

3

4

d(Ab

sorb

ance

295)/

dT

Temperature (0C)

I

20 30 40 50 60 70 80-0.05

-0.04

-0.03

-0.02

-0.01

0.00 6pH 4.5

35

4

d(Ab

sorb

ance

295 nm

)/dT

Temperature (0C)

Figure 4. Derivative UV absorbance (295)-temperature profiles, A. PNA/DNA13 hybrids and B. PNA:poly rA hybrids a. 8 b.9 c.10 d.11


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Table 3. Length dependent Tm of TCn, in PNA/ DNA

PNA/DNA Tm at different pH 5.0 (0C) *

1 Not formed 2 Not formed 3 40.5 4 46.0 6 55.7

Table 2. Tm of TCn in PNA and DNA Tm at different

pH (0C) * PNA/DNA

3.0 4.5 3 67.3 40.0 4 67.4 55.0 5 47.5 43.0 6 58.4 58.7


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nagendra revised-supplementary material · 2005. 7. 6. · title: microsoft word - nagendra...

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