Liu Zhixia, Chen Tingjian, Romesberg Floyd E
Department of Chemistry , The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla , CA 92037 , USA . Email:
Chem Sci. 2017 Dec 1;8(12):8179-8182. doi: 10.1039/c7sc03747c. Epub 2017 Oct 16.
RNA or DNA aptamers with 2'-OMe-modifications have been pursued to increase resistance to nucleases, but have been difficult to identify because the OMe groups ablate polymerase recognition. We recently reported evolution of the thermostable DNA polymerases SFM4-6 and SFM4-9, which enable the efficient "transcription" and "reverse transcription", respectively, of 2'-OMe oligonucleotides. With these polymerases, we now report the first selection of fully 2'-OMe modified aptamers, specifically aptamers that bind human neutrophil elastase (HNE). Two aptamers, 2mHNE-1 and 2mHNE-2, were isolated after five rounds of selection, and four more, 2mHNE-3-6, after an additional five rounds that included selection pressure for binding in the presence of serum. All six aptamers bind with reasonable affinity, which requires the 2'-OMe substituents. Further characterization of one aptamer, 2mHNE-5, showed that unlike a previously reported natural anti-HNE aptamer, affinity for HNE is retained in the presence of high concentrations of salt or serum. The polymerases SFM4-6 and SFM4-9 should prove valuable for the production and further exploration of modified aptamers.
人们一直在探索具有2'-O-甲基修饰的RNA或DNA适配体，以提高其对核酸酶的抗性，但由于O-甲基基团会消除聚合酶识别能力，因此很难鉴定。我们最近报道了耐热DNA聚合酶SFM4-6和SFM4-9的进化，它们分别能够对2'-O-甲基寡核苷酸进行高效“转录”和“逆转录”。利用这些聚合酶，我们现在报道了首次筛选出完全2'-O-甲基修饰的适配体，特别是与人中性粒细胞弹性蛋白酶（HNE）结合的适配体。经过五轮筛选，分离出了两种适配体2mHNE-1和2mHNE-2，在另外五轮筛选（包括在血清存在下进行结合选择压力）后，又分离出了另外四种适配体2mHNE-3 - 6。所有六种适配体都具有合理的亲和力，这需要2'-O-甲基取代基。对其中一种适配体2mHNE-5的进一步表征表明，与先前报道的天然抗HNE适配体不同，在高浓度盐或血清存在下，它对HNE的亲和力得以保留。SFM4-6和SFM4-9聚合酶对于生产和进一步探索修饰后的适配体应该具有重要价值。
