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. 2006;34(16):e109.
doi: 10.1093/nar/gkl632. Epub 2006 Aug 31.

A mass spectrometry-based approach for identifying novel DNA polymerase substrates from a pool of dNTP analogues

Kristi Kincaid  1 , Robert D Kuchta
Affiliations

A mass spectrometry-based approach for identifying novel DNA polymerase substrates from a pool of dNTP analogues

Kristi Kincaid et al. Nucleic Acids Res. 2006.

Abstract

There has been a long-standing interest in the discovery of unnatural nucleotides that can be incorporated into DNA by polymerases. However, it is difficult to predict which nucleotide analogs will prove to have biological relevance. Therefore, we have developed a new screening method to identify novel substrates for DNA polymerases. This technique uses the polymerase itself to select a dNTP from a pool of potential substrates via incorporation onto a short oligonucleotide. The unnatural nucleotide(s) is then identified by high-resolution mass spectrometry. By using a DNA polymerase as a selection tool, only the biologically relevant members of a small nucleotide library can be quickly determined. We have demonstrated that this method can be used to discover unnatural base pairs in DNA with a detection threshold of < or =10% incorporation.

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Figures

Figure 1
Figure 1
Overview of nucleotide selection scheme. A template containing a nucleotide analog is incubated with a library of nucleoside triphosphate analogs and a DNA polymerase. The extended oligonucleotides are separated, enzymatically digested to nucleosides and analyzed by LC-MS.
Figure 2
Figure 2
The positive control unnatural base pair: 2,4-diaminopyrimidine (κ) and xanthine (X).
Figure 3
Figure 3
Sequences of oligonucleotides studied.
Figure 4
Figure 4
Testing the DNA sequences in Figure 3 for activity as substrates for pol α and KF. The polymerase used and the oligonucleotide length are indicated in the figure. For each length template, the 5 lanes are the: 1, no enzyme control; 2, no dNTP control; 3, +1 extension; 4, +2 extension; and 5, full extension.
Figure 5
Figure 5
Incorporation of dXTP opposite dκ by KF and pol α. Polymerase and amount of dXTP used are indicated. The lengths of the DNA bands are indicated with arrows.
Figure 6
Figure 6
Composition of the dNTP mixtures used in the full run-through of the selection scheme. The concentration of each nucleotide was 100 µM. dRTP, deoxyribose 5′triphosphate.
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