Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, 1094, Budapest, Tűzoltó u. 37-47, Ungarn.
Chembiochem. 2020 Oct 15;21(20):2939-2944. doi: 10.1002/cbic.202000236. Epub 2020 Jul 14.
One of the pivotal steps in aptamer selection is the amplification of target-specific oligonucleotides by thermophilic DNA polymerases; it can be a challenging task if nucleic acids possessing modified nucleotides are to be amplified. Hence, the identification of compatible DNA polymerase and modified nucleotide pairs is necessary for effective selection of aptamers with unnatural nucleotides. We present an in-depth study of using 5-indolyl-AA-dUTP (TAdUTP) to generate oligonucleotide libraries for aptamer selection. We found that, among the eight studied DNA polymerases, only Vent(exo-) and KOD XL are capable of adapting TAdUTP, and that replacing dTTP did not have a significant effect on the productivity of KOD XL. We demonstrated that water-in-oil emulsion PCR is suitable for the generation of aptamer libraries of modified nucleotides. Finally, high-throughput sequence analysis showed that neither the error rate nor the PCR bias was significantly affected by using TAdUTP. In summary, we propose that KOD XL and TAdUTP could be effectively used for aptamer selection without distorting the sequence space of random oligonucleotide libraries.
在适体筛选中，至关重要的步骤之一是通过嗜热 DNA 聚合酶扩增靶特异性寡核苷酸；如果要扩增具有修饰核苷酸的核酸，则这可能是一项具有挑战性的任务。因此，为了有效地筛选具有非天然核苷酸的适体，有必要确定相容的 DNA 聚合酶和修饰核苷酸对。我们深入研究了使用 5-吲哚基-AA-dUTP（TAdUTP）生成适体选择的寡核苷酸文库。我们发现，在所研究的 8 种 DNA 聚合酶中，只有 Vent(exo-)和 KOD XL 能够适应 TAdUTP，并且用 TAdUTP 替换 dTTP 对 KOD XL 的生产力没有显著影响。我们证明油包水乳状液 PCR 适合于修饰核苷酸的适体文库的生成。最后，高通量序列分析表明，使用 TAdUTP 不会显著影响错误率或 PCR 偏倚。总之，我们提出，KOD XL 和 TAdUTP 可以有效地用于适体筛选，而不会扭曲随机寡核苷酸文库的序列空间。
