Masse J E, Bortmann P, Dieckmann T, Feigon J
Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, CA 90095-1569, USA.
Nucleic Acids Res. 1998 Jun 1;26(11):2618-24. doi: 10.1093/nar/26.11.2618.
The use of uniformly 13C,15N-labeled RNA has greatly facilitated structural studies of RNA oligonucleotides by NMR. Application of similar methodologies for the study of DNA has been limited, primarily due to the lack of adequate methods for sample preparation. Methods for both chemical and enzymatic synthesis of DNA oligonucleotides uniformly labeled with 13C and/or 15N have been published, but have not yet been widely used. We have developed a modified procedure for preparing uniformly 13C,15N-labeled DNA based on enzymatic synthesis using Taq DNA polymerase. The highly efficient protocol results in quantitative polymerization of the template and approximately 80% incorporation of the labeled dNTPs. Procedures for avoiding non-templated addition of nucleotides or for their removal are given. The method has been used to synthesize several DNA oligonucleotides, including two complementary 15 base strands, a 32 base DNA oligonucleotide that folds to form an intramolecular triplex and a 12 base oligonucleotide that dimerizes and folds to form a quadruplex. Heteronuclear NMR spectra of the samples illustrate the quality of the labeled DNA obtained by these procedures.
使用均匀 13C、15N 标记的 RNA 极大地促进了通过核磁共振对 RNA 寡核苷酸的结构研究。将类似方法应用于 DNA 研究受到限制，主要是由于缺乏合适的样品制备方法。关于化学合成和酶促合成均匀标记有 13C 和/或 15N 的 DNA 寡核苷酸的方法已经发表，但尚未得到广泛应用。我们基于使用 Taq DNA 聚合酶的酶促合成开发了一种制备均匀 13C、15N 标记 DNA 的改进方法。该高效方案可实现模板的定量聚合以及标记 dNTP 的约 80%掺入。给出了避免非模板核苷酸添加或去除它们的方法。该方法已用于合成几种 DNA 寡核苷酸，包括两条互补的 15 碱基链、一条折叠形成分子内三链体的 32 碱基 DNA 寡核苷酸以及一条二聚化并折叠形成四链体的 12 碱基寡核苷酸。样品的异核核磁共振谱说明了通过这些方法获得的标记 DNA 的质量。
