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. 2015 Jul 24;20(8):13591-602.
doi: 10.3390/molecules200813591.

Synthesis and Enzymatic Incorporation of Modified Deoxyuridine Triphosphates

Erkai Liu  1 , Curtis H Lam  2 , David M Perrin  3
Affiliations

Synthesis and Enzymatic Incorporation of Modified Deoxyuridine Triphosphates

Erkai Liu et al. Molecules. .

Abstract

To expand the chemical functionality of DNAzymes and aptamers, several new modified deoxyuridine triphosphates have been synthesized. An important precursor that enables this aim is 5-aminomethyl dUTP, whereby the pendent amine serves as a handle for further synthetic functionalization. Five functional groups were conjugated to 5-aminomethyl dUTP. Incorporation assays were performed on several templates that demand 2-5 sequential incorporation events using several commercially available DNA polymerases. It was found that Vent (exo-) DNA polymerase efficiently incorporates all five modified dUTPs. In addition, all nucleoside triphosphates were capable of supporting a double-stranded exponential PCR amplification. Modified PCR amplicons were PCR amplified into unmodified DNA and sequenced to verify that genetic information was conserved through incorporation, amplification, and reamplification. Overall these modified dUTPs represent new candidate substrates for use in selections using modified nucleotide libraries.

Keywords: Vent (exo-) DNA polymerase; incorporation; modified nucleoside triphosphates.

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Conflict of interest statement

The authors declare no conflict of interest in this work.

Figures

Figure 1
Figure 1
Modified dUTPs.
Figure 2
Figure 2
Screening various DNA polymerases with compound 1A and T1. Lane 1, primer only; lane 2, ddTTP (control); lane 3, dTTP (control); lane 4, Tth; lane 5, Tfl; lane 6, Thermosequenase; lane 7, Vent(exo-); lane 8, Therminator; lane 9, KOD DASH; lane 10, Klenow; lane 11, Sequenase v.2.0.
Figure 3
Figure 3
Full length incorporation with T2 and Vent(exo-) DNA polymerase. Lane 1: primer only; lane 2: dATP, dGTP, dCTP + dTTP; lane 3: dATP, dGTP, dCTP + compound 1A; lane 4: dATP, dGTP, dCTP + compound 1B; lane 5: dATP, dGTP, dCTP + compound 1C; lane 6: dATP, dGTP, dCTP + compound 1D; lane 7: dATP, dGTP, dCTP + compound 1E.
Figure 4
Figure 4
Agarose gel analysis of PCR products obtained with Vent (exo-). Total reaction mixture (20 μL) consisted of 1 pmole T3, 150 pmole of each primer, 0.2 mM of each dNTP and 2 unit of enzyme. Reactions were performed at 1X commercial thermopol buffer with a final Mg+2 concentration of 7 mM. Lane 1: 67mer DNA template; lane 2: no dNTP’s; lane 3: dATP, dGTP, dCTP + dTTP; lane 4: dATP, dGTP, dCTP + compound 1A; lane 5: dATP, dGTP, dCTP + compound 1B; lane 6: dATP, dGTP, dCTP + compound 1C; lane 7: dATP, dGTP, dCTP + compound 1D; lane 8: dATP, dGTP, dCTP + compound 1E.
Scheme 1
Scheme 1
Synthesis of modified dUTPs.
See this image and copyright information in PMC

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