Kincaid Kristi, Kuchta Robert D
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA.
Nucleic Acids Res. 2006;34(16):e109. doi: 10.1093/nar/gkl632. Epub 2006 Aug 31.
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.
长期以来，人们一直对发现可被聚合酶掺入DNA的非天然核苷酸感兴趣。然而，很难预测哪些核苷酸类似物将被证明具有生物学相关性。因此，我们开发了一种新的筛选方法来鉴定DNA聚合酶的新型底物。该技术利用聚合酶本身从潜在底物库中通过掺入到短寡核苷酸上来选择dNTP。然后通过高分辨率质谱鉴定非天然核苷酸。通过使用DNA聚合酶作为选择工具，仅一小部分核苷酸文库中具有生物学相关性的成员能够被快速确定。我们已经证明，该方法可用于发现掺入检测阈值≤10%的DNA中的非天然碱基对。
