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. 2015 Jan 1;6(1):575-587.
doi: 10.1039/c4sc01906g. Epub 2014 Sep 16.

Azidophenyl as a click-transformable redox label of DNA suitable for electrochemical detection of DNA-protein interactions

Jana Balintová  1 , Jan Špaček  2 , Radek Pohl  1 , Marie Brázdová  2 , Luděk Havran  2   3 , Miroslav Fojta  2   3 , Michal Hocek  1   4
Affiliations

Azidophenyl as a click-transformable redox label of DNA suitable for electrochemical detection of DNA-protein interactions

Jana Balintová et al. Chem Sci. .

Abstract

New redox labelling of DNA by an azido group which can be chemically transformed to nitrophenyltriazole or silenced to phenyltriazole was developed and applied to the electrochemical detection of DNA-protein interactions. 5-(4-Azidophenyl)-2'-deoxycytidine and 7-(4-azidophenyl)-7-deaza-2'-deoxyadenosine nucleosides were prepared by aqueous-phase Suzuki cross-coupling and converted to nucleoside triphosphates (dNTPs) which served as substrates for incorporation into DNA by DNA polymerase. The azidophenyl-modified nucleotides and azidophenyl-modified DNA gave a strong signal in voltammetric studies, at -0.9 V, due to reduction of the azido function. The Cu-catalyzed click reaction of azidophenyl-modified nucleosides or azidophenyl-modified DNA with 4-nitrophenylacetylene gave nitrophenyl-substituted triazoles, exerting a reduction peak at -0.4 V under voltammetry, whereas the click reaction with phenylacetylene gave electrochemically silent phenyltriazoles. The transformation of the azidophenyl label to nitrophenyltriazole was used for electrochemical detection of DNA-protein interactions (p53 protein) since only those azidophenyl groups in the parts of the DNA not shielded by the bound p53 protein were transformed to nitrophenyltriazoles, whereas those covered by the protein were not.

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Figures

Scheme 1
Scheme 1. Synthesis of modified nucleosides: (i) Suzuki–Miyaura cross-coupling: 1, PdCl2(dppf), Cs2CO3, MeOH, 2 h, 80 °C; (ii) CuAAC: 2 (or 3), sodium ascorbate, CuSO4·5H2O, tBuOH–H2O (1 : 1), 12 h, rt; (iii) triphosphorylation of modified nucleosides: (1) PO(OMe)3, POCl3, 0 °C; (2) (NHBu3)2H2P2O7, Bu3N, DMF, 0 °C; (3) TEAB; (iv) PEX experiment; (v) azide–alkyne Huisgen cycloaddition: 2 (or 3), sodium ascorbate, CuBr, TBTA ligand, tBuOH–DMSO (1 : 3), 2 h, 37 °C.
Fig. 1
Fig. 1. CV responses of dNATPs, dNTPTPs and dNTNOTPs at HMDE.
Fig. 2
Fig. 2. PAGE analysis of PEX single-incorporations into 19 nt DNA using dNATP, tempA and tempC template and KOD XL polymerase.
Fig. 3
Fig. 3. PAGE analysis of PEX incorporations into 31 nt DNA using dNATP, template temprnd16 and KOD XL polymerase, followed by click reaction with 1-ethynyl-4-nitrobenzene and phenylacetylene.
Fig. 4
Fig. 4. PAGE analysis of PEX single-incorporations into 19 nt DNA using dNTNO2TP or dNTPTP, tempA or tempC template and KOD XL polymerase.
Fig. 5
Fig. 5. PAGE analysis of PEX reactions with dNTPTP or dNTNO2TP using template temprnd16 and KOD XL polymerase leading to 31 nt DNA.
Fig. 6
Fig. 6. CV responses at a HMDE of PEX products synthesized with a temprnd16 template and dNTP mixes containing a dNATP conjugate (as specified in the legend) complemented with three respective unmodified dNTPs and PEX products after click reaction with (nitro)phenyltriazole. Peak G corresponds to oxidation of a reduction product of guanine generated at the electrode. For full CV scans and other details see Fig. S14–S15 and Experimental section.
Fig. 7
Fig. 7. CV responses at a HMDE of PEX products synthesized with a temprnd16 template and dNTP mixes containing a dNTxTP conjugate (as specified in the legend) complemented with three respective unmodified dNTPs. For full CV scans and other details see Fig. S16–S17 and Experimental section.
Scheme 2
Scheme 2. The principle of electrochemical detection of protein–DNA interactions.
Fig. 8
Fig. 8. PAGE analysis of PEX reactions with dNATP using template tempp53-1a2G and KOD XL DNA polymerase, giving 50 nt DNA products.
Fig. 9
Fig. 9. (a) Native PAGE analysis of the 50-mer DNA1a2G_p53CD_GST complex. Lane 1: natural DNA; 2 : 0.4 equiv.; 3 : 0.7 equiv.; 4 : 1.2 equiv.; 5 : 1.8 equiv. of protein p53CD_GST to DNA; lane 6: DNAA; 7 : 0.4 equiv.; 8 : 0.7 equiv.; 9 : 1.2 equiv.; 10 : 1.8 equiv. of protein p53CD_GST to DNA. (b) Native PAGE analysis of the stability of the DNA_p53CD_GST complex after click reaction of the DNA. Templatep53_1a2G : lane 11: DNAA; lane 12: protein–DNA complex; lane 13: protein–DNA complex, 0.5 mM 4-nitrophenylacetylene, 5 μM CuBr; 25 μM TBTA ligand, 65 μM Na ascorbate, 20 °C, 1 h.
Fig. 10
Fig. 10. CV responses at a HMDE of PEX products synthesized with tempp53_1a2G template and a dAATP conjugate complemented with three respective unmodified dNTPs (red curve); PEX products after click reaction with nitrophenylacetylene (green curve); DNA–p53 complex after click reaction followed by denaturation (violet curve); and the control with BSA (black curve). For full CV scans and other details see Fig. S18 and Experimental section.
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