Primer (3 µM) was mixed with template DNA (3 µM), dNTPs (200 µM each) and KOD XL polymerase. The reaction mixtures were incubated
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Doctoral thesis – Olena Mayboroda 143
for 40 minutes at 60 °C and 2 minutes at 95 °C. The PEX products were mixed with loading buffer (80% formamide, 10 mm ethylenediaminetetraacetic acid (EDTA), 1 mgmL-1 xylene cyanol, 1
mgmL-1 bromophenol blue) and subjected to electrophoresis in 12% w/v
denaturing polyacrylamide gel containing 1xTBE buffer (pH 8) and 7M urea at 25W for 50 min. Gels were dried, autoradiographed and visualised using Phosphorimager Storm.
5.4. Results and discussion
The modified dNXTPs were prepared using two different
approaches: A. modification of the corresponding nucleoside followed by triphosphorylation and B. direct cross-coupling reaction (Figure 5.1) in analogy to previously developed procedures 20,21.
Directly linked NH2 derivatives of nucleosides dANH2 and dCNH2
were prepared, achieving good yields of 71 – 89 % in one step by Suzuki– Miyaura cross-coupling using dAI or dCI as initial substrates. Suzuki–
Miyaura cross-coupling of halogenated dNITPs (dAITP and dCITP) under
the same aqueous conditions did not give the desired NH2-modified
dNTPs (dANH2TP and dCNH2TP), so we applied an alternative strategy of
triphosphorylation 22 of the corresponding nucleosides (dNNH2) achieving
a yield of 25%.
Directly linked NO2 derivatives of nucleosides dANO2 and dCNO2
were prepared, achieving good yields of 71.5 – 91 % in one step by Suzuki– Miyaura cross-coupling using dAI or dCI as initial substrates.
Suzuki–Miyaura cross-coupling of halogenated dNITPs (dAITP and dCITP)
under the same aqueous conditions did not give the desired NO2-modified
dNTPs (dANO2TP and dCNO2TP), so we applied an alternative strategy of Olena Mayboroda
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144 Doctoral thesis – Olena Mayboroda
triphosphorylation of the corresponding nucleosides (dNNO2) achieving a
yield of 30%.
Directly linked BF derivatives of nucleosides dABF and dCBF were
prepared, with good yields of 70 – 90 % in one step by Suzuki– Miyaura cross-coupling using dAI or dCI as initial substrates. Suzuki–Miyaura
cross-coupling of halogenated dNITPs (dAITP and dCITP) under the same
aqueous conditions gave the desired BF-modified dNTPs (dABFTP and
dCBFTP), with a yield of 71%. We then applied an alternative strategy for
triphosphorylation of the corresponding nucleosides (dNBF) achieving a
comparable yield of 75 %.
These straightforward and efficient approaches gave the desired functionalised dNXTPs, which are suitable as substrates for polymerase
incorporation.
A)
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Doctoral thesis – Olena Mayboroda 145
B)
Figure 5.1 General strategy for the synthesis of modified dANH2TP by (A)
modification of the corresponding nucleoside followed by triphosphorylation and (B) direct cross-coupling reaction
All conjugates were successfully incorporated into
oligonucleotides through a primer extension (PEX) reaction catalysed by KOD XL DNA polymerase (Figure 5.2).
Figure 5.2 PEX incorporation of labelled dNTPs into one ON by KOD XL. P: primer; +: natural dNTPs; A-: dCTP, dGTP, dTTP; C-: dATP, dGTP, dTTP;
ANH2: dANH2TP, dCTP, dGTP, dTTP; CNH2: dCNH2TP, dATP, dGTP, dTTP; ANO2:
dANO2TP, dCTP, dGTP, dTTP; CNO2: dCNO2TP, dATP, dGTP, dTTP; ABF: dABFTP,
dCTP, dGTP, dTTP; CBF: dCBFTP, dATP, dGTP, dTTP; CNO2n: new batch of
dCNO2TP, dATP, dGTP, dTTP. Olena Mayboroda
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146 Doctoral thesis – Olena Mayboroda
5.5. Conclusions
In conclusion, we have applied two of the most common synthesis strategies to achieve aminophenyl-, nitrophenyl- and benzofurazane- containing dNXTPs. In all cases, the best performing method was the
Suzuki–Miyaura cross-coupling of the corresponding nucleosides followed by triphosphorylation, resulting in the desired products with relatively good yields. The modified dNXTPs were further efficiently
incorporated by KOD XL DNA polymerase to form NH2, NO2 or BF-
modified oligonucleotides. This gives us the possibility to further use these dNXTPs in DNA amplification and electrochemical detection.
5.6. Acknowledgements
This work has been carried out with partial financial support from Spanish Ministerio de Economía y Competitividad (SEASENSING BIO2014-56024-C2-1). OM thanks the Generalitat de Catalunya for a FI pre-doctoral scholarship.
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