supplementary information - the royal society of chemistry · 2013-04-03 · supplementary...

Supplementary Information

Polymerase Synthesis of Oligonucleotides Containing a Single

Chemically Modified Nucleobase for Site-Specific Redox

Labelling

Petra Ménová, Hana Cahová, Medard Plucnara, Luděk Havran, Miroslav Fojta, Michal Hocek

Electronic Supplementary Material (ESI) for Chemical CommunicationsThis journal is © The Royal Society of Chemistry 2013

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Contents:

Supplementary Information ........................................................................................................ 0

Contents: ................................................................................................................................. 1

Supplementary tables ............................................................................................................. 2

Table S1. Sequences of oligonucleotides used and synthesized in this study.[a]

............... 2

Table S2. MALDI-TOF analyses of ON products. ........................................................... 3

Table S3. Melting temperatures of DNA duplexes. .......................................................... 4

Supplementary figures ............................................................................................................ 5

Denaturing PAGE analyses of product of polymerase syntheses of ONs bearing a

single modification by SNI-PEX ..................................................................................... 5

Electrochemistry ............................................................................................................. 12

Synthesis of dGNO2

TP and dGNH2

TP .................................................................................... 13

Experimental ........................................................................................................................ 15

General remarks ............................................................................................................. 15

Synthesis of 2'-deoxy-5-(3-nitrophenyl)-7-deazaguanosine 5'-O-triphosphate (dG

NO2TP) ....................................................................................................................... 16

2'-deoxy-5-(3-nitrophenyl)-7-deazaguanosine (dGNO2

)................................................... 16

2'-deoxy-5-(3-nitrophenyl)-7-deazaguanosine 5'-O-triphosphate (dGNO2

TP) ................ 16

Biochemistry ................................................................................................................... 18

MALDI-TOF spectra of ON products .......................................................................... 21

References ............................................................................................................................ 27


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Supplementary tables

Table S1. Sequences of oligonucleotides used and synthesized in this study.[a]

Primers Sequence

prim 5‘-CATGGGCGGCATGGG-3‘

primC 5‘-CATGGGCGGCATGGGC-3‘

primC-1 5‘-CATGGGCGGCATGGC-3‘

primT 5‘-CATGGGCGGCATGGT-3‘

primA 5‘-CATGGGCGGCATGGA-3‘

Templates

MonoA 5‘-(bio)-CTAGCATGAGCTCAGTCCCATGCCGCCCATG-3‘

MonoC 5‘-(bio)-CTAGCATGAGCTCAGGCCCATGCCGCCCATG-3‘

MonoG 5‘-(bio)-CTAGCATGAGCTCAGCCCCATGCCGCCCATG-3‘

MonoT 5‘-(bio)-CTAGCATGAGCTCAGACCCATGCCGCCCATG-3‘

MonogAa 5‘-(bio)-CTAGCATGAGCTCATTCCCATGCCGCCCATG-3‘

MonogAc 5‘-(bio)-CTAGCATGAGCTCAGTCCCATGCCGCCCATG-3‘

MonogAt 5‘-(bio)-CTAGCATGAGCTCAATCCCATGCCGCCCATG-3‘

MonocAa 5‘-(bio)-CTAGCATGAGCTCATTGCCATGCCGCCCATG-3‘

MonocAt 5‘-(bio)-CTAGCATGAGCTCAATGCCATGCCGCCCATG-3‘

MonocAc 5‘-(bio)-CTAGCATGAGCTCAGTGCCATGCCGCCCATG-3‘

MonotAt 5‘-(bio)-CTAGCATGAGCTCAATACCATGCCGCCCATG-3‘

MonotAa 5‘-(bio)-CTAGCATGAGCTCATTACCATGCCGCCCATG-3‘

MonoaAa 5‘-(bio)-CTAGCATGAGCTCATTTCCATGCCGCCCATG-3‘

MonogGa 5‘-(bio)-CTAGCATGAGCTCATCCCCATGCCGCCCATG-3‘

MonogGc 5‘-(bio)-CTAGCATGAGCTCAGCCCCATGCCGCCCATG-3‘

MonotGc 5‘-(bio)-CTAGCATGAGCTCAGCACCATGCCGCCCATG-3‘

MonogCa 5‘-(bio)-CTAGCATGAGCTCATGCCCATGCCGCCCATG-3‘

MonogCt 5‘-(bio)-CTAGCATGAGCTCAAGCCCATGCCGCCCATG-3‘

MonotCt 5‘-(bio)-CTAGCATGAGCTCAAGACCATGCCGCCCATG-3‘

MonogTa 5‘-(bio)-CTAGCATGAGCTCATACCCATGCCGCCCATG-3‘

MonogTc 5‘-(bio)-CTAGCATGAGCTCAGACCCATGCCGCCCATG-3‘

MonotTc 5‘-(bio)-CTAGCATGAGCTCAGAACCATGCCGCCCATG-3‘

MonoC-short 5‘-(bio)-GCCCATGCCGCCCATG-3’

MonogA-short 5‘-(bio)-TCCCATGCCGCCCATG-3‘

MonocA-short 5‘-(bio)-TGCCATGCCGCCCATG-3‘

MonotA-short 5‘-(bio)-TACCATGCCGCCCATG-3‘

MonoaA-short 5‘-(bio)-TTCCATGCCGCCCATG-3‘

PEX products

ON1 5‘-CATGGGCGGCATGGGACTGAGCTCATGCTAG-3‘

ON2 5‘-CATGGGCGGCATGGGCCTGAGCTCATGCTAG-3‘

ON3 5‘-CATGGGCGGCATGGGGCTGAGCTCATGCTAG-3‘

ON4 5‘-CATGGGCGGCATGGGTCTGAGCTCATGCTAG-3‘

ON5 5‘-CATGGGCGGCATGGGC-3‘

ON6 5‘-CATGGGCGGCATGGGAATGAGCTCATGCTAG-3‘

ON7 5‘-CATGGGCGGCATGGGACTGAGCTCATGCTAG-3‘

ON8 5‘-CATGGGCGGCATGGGATTGAGCTCATGCTAG-3‘

ON9 5‘-CATGGGCGGCATGGCAATGAGCTCATGCTAG-3‘

ON10 5‘-CATGGGCGGCATGGCATTGAGCTCATGCTAG-3‘

ON11 5‘-CATGGGCGGCATGGCACTGAGCTCATGCTAG-3‘

ON12 5‘-CATGGGCGGCATGGTATTGAGCTCATGCTAG-3‘

ON13 5‘-CATGGGCGGCATGGTAATGAGCTCATGCTAG-3‘


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ON14 5‘-CATGGGCGGCATGGAAATGAGCTCATGCTAG-3‘

ON15 5‘-CATGGGCGGCATGGGGATGAGCTCATGCTAG-3‘

ON13 5‘-CATGGGCGGCATGGGGCTGAGCTCATGCTAG-3‘

ON17 5‘-CATGGGCGGCATGGTGCTGAGCTCATGCTAG-3‘

ON18 5‘-CATGGGCGGCATGGGCATGAGCTCATGCTAG-3‘

ON19 5‘-CATGGGCGGCATGGGCTTGAGCTCATGCTAG-3‘

ON20 5‘-CATGGGCGGCATGGTCTTGAGCTCATGCTAG-3‘

ON21 5‘-CATGGGCGGCATGGGTATGAGCTCATGCTAG-3‘

ON22 5‘-CATGGGCGGCATGGGTCTGAGCTCATGCTAG-3‘

ON23 5‘-CATGGGCGGCATGGTTCTGAGCTCATGCTAG-3‘

[a] Italics: parts forming duplex with the primer; bold: position of the modification in the

product; bio = biotin.

Table S2. MALDI-TOF analyses of ON products.

ON calcd. [M+H]

+

found [M+H]

+

ON1 ANO2

9738.4 Da 9738.5 Da

ON1 ANH2

9708.4 Da 9708.8 Da

ON2 CNO2

9715.4 Da 9715.3 Da

ON2 CNH2

9685.4 Da 9685.8 Da

ON3 GNO2

9754.4 Da 9754.5 Da

ON3 GNH2

9724.4 Da 9724.6 Da

ON4 UNO2

9716.4 Da 9716.0 Da

ON4 UNH2

9686.4 Da 9686.8 Da


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Table S3. Melting temperatures of DNA duplexes.

Complementary strand

Base pair

Tm Tm Tm[a]

ON1 A ON1 ANO2

MonoT A 79.6(7) 79.1(5) - 0.5

MonoG C 78.8(4) 79.7(6) +0.9

MonoC G 81.1(5) 80.9(5) - 0.2

MonoA T 82.2(5) 80.9(4) - 1.3

ON2 C ON2 CNO2

MonoT A 82.8(7) 81.2(7) - 1.6

MonoG C 82.8(4) 80.6(5) - 2.2

MonoC G 85.1(5) 82.9(5) - 2.2

MonoA T 83.1(5) 82.3(4) - 0.8

ON3 G ON3 GNO2

MonoT A 80.8(5) 80.7(5) - 0.1

MonoG C 82.6(4) 82.5(5) - 0.1

MonoC G 80.9(6) 80.6(5) - 0.3

MonoA T 80.6(5) 80.1(5) - 0.5

ON4 T ON4 UNO2

MonoT A 82.1(5) 81.2(6) - 0.9

MonoG C 77.4(4) 79.2(5) +1.8

MonoC G 80.5(4) 80.1(5) - 0.4

MonoA T 77.2(5) 79.1(5) +1.9

[a] Tm = Tm(ON X

mod) – Tm(ON X).


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Supplementary figures

Denaturing PAGE analyses of product of polymerase syntheses of ONs bearing a single

modification by SNI-PEX

Figure S1. Denaturing PAGE analysis of products of polymerase synthesis of ONs bearing a

single modification (ANO2

(a), ANH2

(b), and AFc

(c)) by SNI-PEX. Diluted DNA polymerase

was necessary for successful SNI (0.02 U/µL for ANO2

, 0.004 U/µL for ANH2

, standard

conditions for AFc

). Experiments are supplemented with a primer (p). Lane 1: positive control

for SNI (only natural dATP present); lane 2: negative control for SNI (no dNTPs present);

lane 3: SNI of AX (only A

X present); lane 4: negative control for PEX (absence of natural

dATP); lane 5: positive control for PEX (all natural dNTPs present); lane 6: PEX with primer

extended with AX (all natural dNTPs present). For reaction conditions, see the Experimental.


S6


single modification (CNO2

(a), CNH2

(b), and CFc

(c)) by SNI-PEX using one-base-longer

primer (primC) in order to incorporate one redox label only. Experiments are supplemented

with a primer (p). Lane 1: positive control for SNI (only natural dCTP present); lane 2:

negative control for SNI (no dNTPs present); lane 3: SNI of CX (only C

X present); lane 4:

negative control for PEX (absence of natural dCTP); lane 5: positive control for PEX (all

natural dNTPs present); lane 6: PEX with primer extended with CX (all natural dNTPs

present). For reaction conditions, see the Experimental.


S7


single modification (GNO2

(a), GNH2

(b), and GFc

) by SNI-PEX. Experiments are

supplemented with a primer (p). Lane 1: positive control for SNI (only natural dGTP present);

lane 2: negative control for SNI (no dNTPs present); lane 3: SNI of GX (only G

X present);

lane 4: negative control for PEX (absence of natural dGTP); lane 5: positive control for PEX

(all natural dNTPs present); lane 6: PEX with primer extended with GX (all natural dNTPs



S8


single modification (UNO2

(a), UNH2

(b), and UFc

(c)) by SNI-PEX. Experiments are

supplemented with a primer (p). Lane 1: positive control for SNI (only natural dTTP present);

lane 2: negative control for SNI (no dNTPs present); lane 3: SNI of UX (only U

X present);

lane 4: negative control for PEX (absence of natural dTTP); lane 5: positive control for PEX

(all natural dNTPs present); lane 6: PEX with primer extended with UX (all natural dNTPs



S9


single modification (CNO2

(a), CNH2

(b), and CFc

(c)) by SNI-PEX into a sequence containing

two Cs in a row. Only one-base longer template is used for SNI. The SNI is followed by

magnetoseparation, a full-length template is added and the one-base extended primer is fully

extended with natural dNTPs. Experiments are supplemented with a primer (p). Lane 1:

positive control for SNI (only natural dCTP present, template MonoC-short); lane 2: SNI of

CX (only C

X present, template MonoC-short); lane 3: positive control for PEX (all natural

dNTPs present, template MonoC); lane 4: PEX with primer extended with CX (all natural

dNTPs present, template MonoC-short). For reaction conditions, see the Experimental.


S10


single modification (CNH2

) by SNI-PEX into a sequence containing two Cs in a row, using a

standard-length primer and 1 equiv. of CNH2

. The polymerase is unable to incorporate only

one base into a homo-C sequence and a mixture of primer, primer extended with one CNH2

and primer extended with two CNH2

s is obtained. Experiments are supplemented with a primer

(p). Lane 1: positive control for SNI (only natural dCTP present); lane 2: negative control for

SNI (no dNTPs present); lane 3: SNI of CX (only C

X present); lane 4: negative control for

PEX (absence of natural dCTP); lane 5: positive control for PEX (all natural dNTPs present);

lane 6: PEX with primer extended with CX (all natural dNTPs present). For reaction

conditions, see the Experimental.


S11


single modification (ANO2

(a) and ANH2

(b)) by SNI-PEX using the standard concentration of

DNA polymerase (0.1 U/µL). Modified AX are the best substrates for Vent(exo-) DNA

polymerase and the polymerase incorporates more AXs in a row regardless the template

sequence, thus forming mismatch pairs. Experiments are supplemented with a primer (p).

Lane 1: positive control for SNI (only natural dATP present); lane 2: negative control for SNI

(no dNTPs present); lane 3: SNI of AX (only A

X present); lane 4: negative control for PEX

(absence of natural dATP); lane 5: positive control for PEX (all natural dNTPs present); lane

6: PEX with primer extended with AX (all natural dNTPs present). For reaction conditions,

see the Experimental.


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Electrochemistry

Figure S8 Examples of electrochemical responses of ANO2

-modified ON products.

Left panel: ex situ cyclic voltammograms of the ONs with sequences shown in the legend; red

letters indicate the modified base. The CVs consist of three segments indicated by arrows: the

first from -0.2 to -0.64 V, the second from -0.64 to +0.1 V and the third one from +0.1 to -0.2

V. In the first catodic scan the nitro group is reduced irreversibly with four electrons to

hydroxylamino group, giving rise to the peak NO2red

. The hydroxylamine gives reversible

electrochemistry, being oxidized in the anodic scan (segment 2) with two electrons to nitroso

derivative (peak NHOHox

) and the latter in the cathodic segment 3 reduced back to

hydroxylamine (NOred

). Black and blue curves correspond to monoincorporation products

(full length ON1 after the two-step PEX and that with a single ANO2

incorporation only,

respectively). Dashed red curve corresponds to a single-step PEX reaction in the presence of

dANO2

TP, dCTP, TTP and dGTP (i.e. full length ON bearing five nitro groups, showing

proportionality between number of nitro tags incorporated and signal intensity), the green

curve is negative PEX control (the same dNTP mixture but with no polymerase added,

demonstrating that only incorporated NO2 groups, but not residual dANO2

TP from the reaction

mixture, give the signal).

Right panel: details of NO2red

peaks obtained for full length products of ANO2

monoincorporation in different sequence contexts (see legend in the panel). For peak

potentials corresponding to these and other combinations see Table 1 in the article.

-0.6 -0.4 -0.2 0.0

-0.10

-0.05

0.00

0.05

I [

A]

E [V]

NO2red

NHOHox

NOred

-0.6 -0.5 -0.4 -0.3

-0.015

-0.010

-0.005

0.000

I [

A]

CAT

GAC

GAT

CAA

E [V]

NO2red

CATGGGCGGCATGGGACAGAGCTCATGCTAGCATGGGCGGCATGGGACATGGGCGGCATGGGACAGAGCTCATGCTAGCATGGGCGGCATGGG (negative PEX)

1 3

2


S13

Synthesis of dGNO2

TP and dGNH2

TP

The aqueous Suzuki cross-coupling reaction on guanosine derivatives is rather challenging

due to the fact that the guanine moiety has an acidic proton, which under reaction conditions

(high pH, high temperature) may be deprotonated to give an anion that can coordinate to

palladium.1 We have previously reported the synthesis of both guanosine derivatives

dGNO2

TP and dGNH2

TP, however, the reaction yields were very low and the products

contained a high proportion of the corresponding diphosphate.2 Here, we report on an

improved synthesis of both compounds, leading to higher yields and lower diphosphate

content.

Our standard synthetic approach to modified nucleoside triphosphates consists of

triphosphorylation of an iodo nucleoside and a following cross-coupling reaction. For

dGNO2

TP it turned out better to start with the cross-coupling reaction on the iodo nucleoside

dGI and then proceed with triphosporylation. The desired dG

NO2TP was obtained as a pure

compound in an 18% overall yield.

Scheme S1. Synthesis of dGNO2

TP.

However, this concept failed when applied to the synthesis of dGNH2

TP. While the Suzuki

cross-coupling of the 7-iodo-7-deaza-2´-deoxyguanosine dGI with 3-aminophenylboronic

acid proceeded well, affording the 3-aminophenyl nucleoside in a 57% yield, the following

triphosphorylation did not work under any conditions applied, presumably due to the presence


S14

of aromatic amino group. Therefore, the triphosphorylation had to be accomplished before the

cross-coupling reaction. Iodo triphosphate dGITP was obtained in a 51% yield according to a

known procedure.3 Lower reaction temperature (100 °C) and higher acetonitrile content (2:1

CH3CN:H2O) together with a higher excess of the boronic acid (5 equiv.) led to the desired

triphosphate dGNH2

TP in 41% yield. The triphosphate contained 25 % of diphosphate, which

could be separated by perfusion chromatography on POROS reverse-phase column. The

overall yield of dGNH2

TP was 16 %.

Scheme S2. Synthesis of dGNH2

TP.


S15

Experimental

General remarks

NMR spectra were recorded using a 500 MHz (1H at 500 MHz,

13C at 125.7 MHz,

19F at

470.3 MHz) spectrometer, in D2O or DMSO-d6. Chemical shifts are given in ppm (scale

and coupling constant (J) in Hz. Both low resolution and high resolution mass spectra were

performed using electrospray ionization. Semi-preparative separation of nucleosides and

nucleoside triphosphates was performed by HPLC on a column packed with 10 µM C18

reversed phase (Phenomenex, Luna C18(2)).

3-Amino- and 3-nitrophenylboronic acids were purchased from Sigma Aldrich, and 7-iodo-7-

deaza-2´-deoxyguanosine was purchased from Chembiotech. Synthesis and characterization

data for 2'-deoxy-5-(3-nitrophenyl)-7-deazaadenosine 5'-O-triphosphate (dANO2

TP)4 , 7-(3-

aminophenyl)-2'-deoxy-7-deazaadenosine 5'-O-triphosphate (dANH2

TP)4 , 2'-deoxy-5-(3-

nitrophenyl)cytidine 5'-O-triphosphate (dCNO2

TP)4 , 5-(3-aminophenyl)-2'-deoxycytidine 5'-

O-triphosphate (dCNH2

TP)4 , 2'-deoxy-5-(3-nitrophenyl)uridine 5'-O-triphosphate

(dUNO2

TP)4 , 5-(3-aminophenyl)-2'-deoxyuridine 5'-O-triphosphate (dU

NH2TP)

4 , and for 2'-

deoxy-7-iodo-7-deazaguanosine 5'-O-triphosphate (dGITP)

3 were reported previously.

Synthetic oligonucleotides (primers Prim, and PrimC; PEX templates MonoA, MonoC-short,

MonoC, MonoG, and MonoT; biotinylated PEX templates MonoA-bio, MonoC-short-bio,

MonoC-bio, MonoG-bio, and MonoT-bio; for sequences see Table S1) were purchased from

Sigma-Aldrich. DNA polymerase Vent(exo-), as well as natural nucleoside triphosphates

(dATP, dCTP, dGTP, dTTP) were purchased from New England Biolabs. KOD XL DNA

polymerase was obtained from Merck and Pwo DNA polymerase from Peqlab. Streptavidin

magnetic particles were obtained from Roche. All solutions for the PEX experiments were

prepared in Milli-Q water. The primer was labeled using [γ32

P]-ATP according to standard

techniques.5

Mass spectra of the prepared ONs were measured by MALDI-TOF, on Reflex IV (Bruker

Daltonics, Germany) with nitrogen UV laser (337 nm). UV spectra were measured on Varian

CARY 100 Bio spectrophotometer and on NanoDrop1000 (ThermoScientific).


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Synthesis of 2'-deoxy-5-(3-nitrophenyl)-7-deazaguanosine 5'-O-triphosphate (dGNO2

TP)

2'-deoxy-5-(3-nitrophenyl)-7-deazaguanosine (dGNO2

)

A water-acetonitrile mixture (2:1, 4 mL) was added through a septum to an argon-purged vial

containing 7-iodo-7-deaza-2'-deoxyguanosine dGI (75 mg, 0.19 mmol), 3-nitrophenylboronic

acid (10 equiv., 317 mg, 1.90 mmol), and Cs2CO3 (5 equiv., 310 mg, 0.95 mmol). After the

solids had dissolved, a solution of Pd(OAc)2 (10 mol%, 4.3 mg, 0.019 mmol) and TPPTS

(5 equiv. to Pd, 54 mg, 0.095 mmol) in water-acetonitrile mixture (2:1, 0.5 mL) was added

and the resulting mixture was stirred at 120 °C for 30 min. The crude reaction mixture was

evaporated with silica gel (2 g) and subsequently separated by column chromatography on

silica gel using dichloromethane-chloroform 14:1 as a mobile phase. The fractions containing

the product were evaporated and dried under vacuum at 80 °C. Compound dGNO2

was

obtained as a yellow solid (58 mg, 78 %).

1H NMR (499.8 MHz, DMSO-d6): 2.12 (ddd, 1H, Jgem = 13.1, J2'b,1' = 5.8, J2'b,3' = 2.5, H-2'b);

2.47 (ddd, 1H, Jgem = 13.1, J2'a,1' = 8.4, J2'a,3' = 5.7, H-2'a); 3.50, 3.56 (2 × ddd, 2 × 1H,

Jgem = 11.6, J5',OH = 5.6, J5',4' = 4.9, H-5'); 3.78 (td, 1H, J4',5' = 4.9, J4',3' = 2.5, H-4'); 4.33 (m,

1H, J3',2' = 5.7, 2.5, J3',OH = 3.8, J3',4' = 2.5, H-3'); 4.92 (t, 1H, JOH,5' = 5.6, OH-5'); 5.23 (d, 1H,

JOH,3' = 3.8, OH-3'); 6.39 (dd, 1H, J1'2' = 8.4, 5.8, H-1'); 6.41 (bs, 2H, NH2); 7.593 (s, 1H,

H-8); 7.595 (ddd, 1H, J5,4 = 8.2, J5,6 = 7.8, J5,2 = 0.3, H-5-C6H4NO2); 8.01 (ddd, 1H, J4,5 = 8.2,

J4,2 = 2.4, J4,6 = 1.0, H-4-C6H4NO2); 8.39 (ddd, 1H, J6,5 = 7.8, J6,2 = 1.8, J6,4 = 1.0,

H-6-C6H4NO2); 9.01 (ddd, 1H, J2,4 = 2.4, J2,6 = 1.8, J2,5 = 0.3, H-2-C6H4NO2); 10.53 (bs, 1H,

NH). 13

C NMR (125.7 MHz, DMSO-d6): 39.20 (CH2-2'); 62.09 (CH2-5'); 71.07 (CH-3');

82.32 (CH-1'); 87.33 (CH-4'); 97.21 (C-4a); 116.90 (CH-8); 118.04 (C-5); 120.39

(CH-4-C6H4NO2); 122.11 (CH-2-C6H4NO2); 129.44 (CH-5-C6H4NO2); 133.62

(CH-6-C6H4NO2); 136.32 (C-1-C6H4NO2); 148.23 (C-3-C6H4NO2); 152.53 (C-7a); 153.06 (C-

2); 159.10 (C-4). MS (ESI+): m/z (%): 410.1 (100) [M+Na]

+. HRMS (ESI

+): m/z [M + H]

+

calculated for C17H18O6N5 388.12516; found 388.12519.

2'-deoxy-5-(3-nitrophenyl)-7-deazaguanosine 5'-O-triphosphate (dGNO2

TP)

Dry POCl3 (2 equiv., 7.2 µL, 0.077 mmol) was added to a solution of dGNO2

(15 mg,

0.039 mmol) in dry trimethyl phosphate (750 µL) at 0 °C under argon atmosphere. The

reaction mixture was stirred at 0 °C for 7 hours. Next, an ice-cold solution of

(NHBu3)2H2P2O7 (5 equiv., 106 mg, 0.193 mmol) and tributylamine (75 µL, 0.420 mmol) in

anhydrous DMF (750 µL) was added. The reaction mixture was stirred at -4 °C for 1 hour.

The reaction was quenched with aqueous solution of TEAB (2M, 1 mL) and the mixture was


S17

concentrated under reduced pressure. The residue was five times co-evaporated with water.

The product was purified on semi-preparative HPLC on a C18 column with a linear gradient

of 0.1 M TEAB in H2O to 0.1 M TEAB in H2O/MeOH 1:1 as eluent. The obtained

triphosphate was converted to sodium salt using ionex (Dowex 50WX8 in Na+ cycle) and

freeze-dried from water. Compound dGNO2

TP was obtained as yellow solid (6.4 mg, 23 %).

NMR data are in accord with those in the literature.2

1H NMR (499.8 MHz, D2O, pD = 7.1, refdioxane = 3.75 ppm): 2.43 (ddd, 1H, Jgem = 14.0,

J2'b,1' = 6.2, J2'b,3' = 3.0, H-2'b); 2.71 (ddd, 1H, Jgem = 14.0, J2'a,1' = 7.8, J2'a,3' = 6.5, H-2'a); 4.14,

4.18 (2 × m, 2 × 1H, H-5'); 4.21 (m, 1H, H-4'); 4.76 (overlapped with HDO signal, H-3'); 6.47

(dd, 1H, J1'2' = 7.8, 6.2, H-1'); 7.40 (s, 1H, H-8); 7.59 (t, 1H, J5,4 = J5,6 = 8.1, H-5-C6H4NO2);

8.08 (bd, 1H, J6,5 = 8.1, H-6-C6H4NO2); 8.11 (dd, 1H, J4,5 = 8.1, J4,2 = 2.0, H-4-C6H4NO2);

8.70 (t, 1H, J2,4 = J2,6 = 2.0, H-2-C6H4NO2). 13

C NMR (125.7 MHz, D2O, pD = 7.1,

refdioxane = 69.3 ppm): 40.85 (CH2-2'); 68.35 (d, JC,P = 7.0, CH2-5'); 73.96 (CH-3'); 85.67

(CH-1'); 87.78 (d, JC,P = 9.7, CH-4'); 100.53 (C-5); 120.04 (CH-8); 121.72 (C-7); 124.07

(CH-4-C6H4NO2); 125.82 (CH-2-C6H4NO2); 132.07 (CH-5-C6H4NO2); 136.82

(CH-6-C6H4NO2); 137.60 (C-1- C6H4NO2); 150.48 (C-3-C6H4NO2); 155.08 (C-4); 155.67

(CH-2); 163.76 (C-6). 31

P {1H} NMR (202.3 MHz, D2O, pD = 7.1, refphosphate buffer =

2.35 ppm): -21.27 (b, P); -10.29 (d, J = 18.8, P); -6.62 (b, P).

Synthesis of 7-(3-aminophenyl)-2'-deoxy-7-deazaguanosine 5'-O-triphosphate (dGNH2

TP)

7-(3-aminophenyl)-2'-deoxy-7-deazaguanosine 5'-O-triphosphate (dGNH2

TP)

A water-acetonitrile mixture (1:2, 500 µL) was added through a septum to an argon-purged

vial containing 7-iodo-7-deaza-2'-deoxyguanosine 5'-O-triphosphate dGITP (36 mg,

0.05 mmol), 3-aminophenylboronic acid (5 equiv., 46 mg, 0.26 mmol), and Cs2CO3 (5 equiv.,

85 mg, 0.26 mmol). After the solids had dissolved, a solution of Pd(OAc)2 (10 mol%, 1.1 mg,

0.005 mmol) and TPPTS (5 equiv. to Pd, 14.2 mg, 0.025 mmol) in water-acetonitrile mixture

(1:2, 300 µL) was added and the resulting mixture was stirred at 100 °C for 30 min. The crude

reaction mixture was several times co-evaporated with water. The product was purified on

semi-preparative HPLC on a C18 column with a linear gradient of 0.1 M TEAB in H2O to

0.1 M TEAB in H2O/MeOH 1:1 as eluent. The obtained triphosphate was converted to

sodium salt using ionex (Dowex 50WX8 in Na+ cycle) and freeze-dried from water.

Compound dGNH2

TP was obtained as white solid (11.4 mg, 32 %). NMR data are in accord

with those in the literature.2


S18

1H NMR (499.8 MHz, D2O, pD = 7.1, refdioxane = 3.75 ppm): 2.39 (ddd, 1H, Jgem = 14.0, J2'b,1'

= 6.3, J2'b,3' = 3.2, H-2'b); 2.69 (ddd, 1H, Jgem = 14.0, J2'a,1' = 7.7, J2'a,3' = 6.3, H-2'a); 4.14, 4.18

(2 × m, 2 × 1H, H-5'); 4.22 (m, 1H, H-4'); 4.74 (dt, 1H, J3',2' = 6.3, 3.2, J3',4' = 3.2, H-3'); 6.47

(dd, 1H, J1'2' = 7.7, 6.3, H-1'); 6.80 (bd, 1H, J4,5 = 7.5, H-4-C6H4NH2); 7.21 (m, 2H,

H-2,6-C6H4NH2); 7.24 (s, 1H, H-8); 7.26 (bt, 1H, J5,4 = J5,6 = 7.5, H-5-C6H4NH2). 13

C NMR

(125.7 MHz, D2O, pD = 7.1, refdioxane = 69.3 ppm): 40.81 (CH2-2'); 68.34 (d, JC,P = 5.7,

CH2-5'); 73.98 (CH-3'); 85.61 (CH-1'); 87.72 (d, JC,P = 8.8, CH-4'); 100.65 (C-5); 117.97

(CH-4-C6H4NH2); 119.02 (CH-8); 119.11 (CH-2-C6H4NH2); 122.64 (CH-6-C6H4NH2);

123.69 (C-7); 132.13 (CH-5-C6H4NH2); 137.19 (C-1-C6H4NH2); 148.21 (C-3-C6H4NH2);

154.80 (C-4); 155.53 (CH-2); 163.75 (C-6). 31

P {1H dec.} NMR (202.3 MHz, D2O, pD = 7.1,

refphosphate buffer = 2.35 ppm): -21.40 (t, J = 19.3, P); -10.35 (d, J = 19.3, P); -6.68 (d, J = 19.3,

P).

Biochemistry

Single nucleotide incorporation and primer extension (SNI-PEX) for analysis by

polyacrylamide gel electrophoresis

The reaction mixture (10 µL) contained 5'-32

P-labelled primer (3 µM, 0.5 µL), template

(3 µM, 0.75 µL), Vent(exo-) DNA polymerase (2 U/µL, amount specified in Table S4), and

modified dNTP (4 µM, 0.5 µL) in ThermoPol buffer (10×, 1 µL) supplied by the

manufacturer with the enzyme. The reaction mixture was incubated at 60 °C for 5 min. For

the subsequent extension, a mixture of natural dNTPs (4 mM, 0.5 µL) was added and the

reaction mixture was incubated for further 20 min. The reaction was stopped by the addition

of PAGE stop solution (20 µL, 80% [v/v] formamide, 20mM EDTA, 0.025% [w/v]

bromophenol blue, 0.025% [w/v] xylene cyanol) and heated at 95 °C for 5 min. Aliquots

(2 µL) were subjected to vertical electrophoresis in 12.5% denaturing polyacrylamide gel

containing 1x TBE buffer (pH 8) and 7M urea at 45 mA for 50 min. The gels were dried

(85 °C, 70 min), audioradiographed and visualized by phosphorimager (Typhoon 9410,

Amersham Biosciences).


S19

Table S4. Amount of Vent(exo-) DNA polymerase for single nucleotide incorporation

experiments.

dNTP Vent(exo-)

ANO2

0.020 U/µL

ANH2

0.004 U/µL

CNO2

, CNH2

G

NO2, G

NH2

UNO2

, UNH2

0.100 U/µL

Preparative single nucleotide incorporation and primer extension (SNI-PEX) for

electrochemical and thermal studies

The reaction mixture (200 µL) contained primer (10 µM, 27 µL), 5´-biotinylated template

(10 µM, 27 µL), Vent(exo-) DNA polymerase (2 U/µL, 10 µL for all modified dNTPs), and

modified dNTP (40 µM, 8.8 µL) in ThermoPol buffer (10×, 20 µL) supplied by the

manufacturer with the enzyme. The reaction mixture was incubated at 60 °C and 450 rpm for

8 min. For the subsequent extension, a mixture of natural dNTPs (10 mM, 27 µL) was added

and the reaction mixture was incubated for further 20 min. The reaction was stopped by

cooling to 4 °C.

Streptavidin magnetic particles (Roche, 100 μL) were washed with Binding buffer TEN100

(10 mM Tris, 1 mM EDTA, 100 mM NaCl, pH 7.5) (3 × 500 μL). The reaction mixture after

the extension was diluted with the Binding buffer TEN100 (200 μL), the solution was added to

the pre-washed magnetic beads and the resulting mixture was incubated for 20 min at 18 °C

and 1200 rpm. After the incubation, the magnetic beads were collected on a magnet

(PureProteome Magnetic Stand, Merck) and the solution was discarded. The beads were

washed successively with Wash buffer TEN1000 (10 mM Tris, 1 mM EDTA, 1 M NaCl,

pH 7.5) (2 × 500 μL), and water (3 × 500 μL). Then water (50 μL) was added and the sample

was denatured for 2 min at 65 °C and 900 rpm. The beads were collected on a magnet and the

solution was transferred into a clean vial.

MALDI-TOF characterization of oligonucleotides

Oligonucleotides were characterized by MALDI-TOF mass spectrometry. A mixture of

3-hydroxypicolinic acid (HPA)/picolinic acid (PA)/ammonium tartrate in the ratio 8/1/1 in

50% acetonitrile was used as matrix for MALDI-TOF measurement. Then 2 μL of the matrix

and 1 μL of the sample were mixed on MTP 384 polished steel target by use of anchor-chip


S20

desk. The acceleration tension in reflectron mode was 19.5 kV and range of measurement

3−13 kDa.

Measuring of melting temperatures of DNA duplexes

Single-stranded oligonucleotides were prepared according to the large-scale SNI-PEX

protocol (vide infra). The samples for Tm measurement were prepared by mixing a

functionalized ssON (0.2 nmol), commercial complementary or partially complementary

strand (MonoA, MonoC, MonoG or MonoT; 0.2 nmol) in phosphate buffer (pH 6.7, 50 mM)

to the final volume of 120 µL. The concentrations of ssONs were determined from

absorbance at 260 nm and estimated extinction coefficients that were calculated using online

calculator by IDT Biophysics.6 Melting experiments were conducted on a Varian CARY 100

Bio spectrophotometer using Thermal application software. The absorbance values at 260 nm

were monitored in the temperature range of 95–25 °C. Both heating (denaturation) and

cooling (renaturation) transition curves were recorded at a controlled rate of temperature

change of 0.5 °C/min. Melting profile of the buffer alone was subtracted from the raw

absorbance versus temperature curves of DNA samples. Four melting curves were collected

for each DNA duplex and evaluated separately. The reported melting temperatures were

obtained as means of the four measurements.

Electrochemical analysis

Single-stranded monoincorporation and strand extension products isolated by

magnetoseparation protocol (vide supra) were analysed by ex situ (adsorptive transfer

stripping) cyclic voltammetry (CV) at hanging mercury drop electrode (HMDE). The ONs

were accumulated at the HMDE from 5 L aliquots containing 0.2 M NaCl for 60 s. The

electrode was then rinsed with deionized water and placed in the electrochemical cell. CV

settings: scan rate 0.5 V/s, initial potential -0.2 V, negative switching potential -0.64 V,

positive switching potential +0.1 V, final potential -0.2 V. Background electrolyte: 0.5 M

ammonium formate, 0.05 M sodium phosphate, pH 6.8. All measurements were performed at

ambient temperature in deaerated solution using an Autolab analyzer (Eco Chemie, The

Netherlands) in connection with VA-stand 663 (Metrohm, Herisau, Switzerland). The three-

electrode system was used with Ag/AgCl/3M KCl electrode as a reference and platinum wire

as an auxiliary electrode.


S21

MALDI-TOF spectra of ON products

General remarks:

Peaks at [M – 125] can be assigned to dethymination.

Peaks at [M + 313.2] can be assigned to product extended with additional adenosine (which is

the best substrate for Vent(exo-) DNA polymerase).

Fragmentation of XNO2

modified products corresponds with literature data for mass spectra of

nitrocompounds to contain peaks at M – 16.7

Figure S8. MALDI-TOF spectrum of ON1 ANH2

.

[M+H]+ (calc.) = 9708.4 Da, M (found) = 9708.8 Da.


S22

Figure S9. MALDI-TOF spectrum of ON1 ANO2

.


Figure S10. MALDI-TOF spectrum of ON2 CNH2

.



S23

Figure S11. MALDI-TOF spectrum of ON2 CNO2

.


Figure S12. MALDI-TOF spectrum of ON3 GNH2

.



S24

Figure S13. MALDI-TOF spectrum of ON3 GNO2

.


Figure S14. MALDI-TOF spectrum of ON4 UNH2

.



S25

Figure S15. MALDI-TOF spectrum of ON4 UNO2

.



S26

NMR spectra of compound dGNO2

Figure S16. 1H NMR spectrum of compound dG

NO2.

Figure S17. 13

C NMR spectrum of compound dGNO2

.


S27

References

1. E. C. Western, K. H. Shaughnessy, J. Org. Chem., 2005, 70, 6378–88.

2. P. Horáková, H. Macíčková-Cahová, H. Pivoňková, J. Špaček, L. Havran, M. Hocek, M.

Fojta, Org. Biomol. Chem., 2011, 9, 1366–1371.

3. J. Ludwig, Acta Biochim. Biophys. Acad. Sci. Hung., 1981, 16, 131–133.

4. H. Cahová, L. Havran, P. Brázdilová, H. Pivoňková, R. Pohl, M. Fojta, M. Hocek, Angew.

Chem. Int. Ed. 2008, 47, 2059–2062.

5. H. Macíčková-Cahová, M. Vrábel, M. Hocek, Curr. Protoc. Chem. Biol., 2010, 2, 1–14.

6. A. V. Tataurov, Y. You, R. Owczarzy, Biophys. Chem., 2008, 133, 66–70.

7. a) T. Ramdahl, K. Urdal, Anal. Chem., 1982, 54, 2256–2260; b) J. B. Kyzioł, Z.

Daszkiewicz, J. Hetper, J. Mass Spectrom., 2005, 22, 1096–9888.


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