Selection of High-Affinity RNA Aptamers That Distinguish between Doxycycline and Tetracycline.,Biochemistry

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Selection of High-Affinity RNA Aptamers That Distinguish between Doxycycline and Tetracycline.
Biochemistry ( IF 2.9 ) Pub Date : 2020-08-28 , DOI: 10.1021/acs.biochem.0c00586
Zachary J Tickner ₁ , Guocai Zhong ₁ , Kelly R Sheptack ₁ , Michael Farzan ₁

Affiliation

Oligonucleotide aptamers are found in prokaryotes and eukaryotes, and they can be selected from large synthetic libraries to bind protein or small-molecule ligands with high affinities and specificities. Aptamers can function as biosensors, as protein recognition elements, and as components of riboswitches allowing ligand-dependent control of gene expression. One of the best studied laboratory-selected aptamers binds the antibiotic tetracycline, but it binds with a much lower affinity to the closely related but more bioavailable antibiotic doxycycline. Here we report enrichment of doxycycline binding aptamers from a selectively randomized library of tetracycline aptamer variants over four selection rounds. Selected aptamers distinguish between doxycycline, which they bind with dissociation constants of approximately 7 nM, and tetracycline, which they bind undetectably. They thus function as orthogonal complements to the original tetracycline aptamer. Unexpectedly, doxycycline aptamers adopt a conformation distinct from that of the tetracycline aptamer and depend on constant regions originally installed as primer binding sites. We show that the fluorescence emission intensity of doxycycline increases upon aptamer binding, permitting their use as biosensors. This new class of aptamers can be used in multiple contexts where doxycycline detection, or doxycycline-mediated regulation, is necessary.

中文翻译：

选择区分多西环素和四环素的高亲和力 RNA 适体。

寡核苷酸适体存在于原核生物和真核生物中，它们可以从大型合成文库中选择，以高亲和力和特异性结合蛋白质或小分子配体。适体可用作生物传感器、蛋白质识别元件和核糖开关的组件，允许依赖配体控制基因表达。研究得最好的实验室选择的适体之一与抗生素四环素结合，但它与密切相关但生物利用度更高的抗生素强力霉素的结合亲和力要低得多。在这里，我们报告了在四轮选择中从四环素适体变体的选择性随机库中富集强力霉素结合适体。选定的适体区分强力霉素，它们以大约 7 nM 的解离常数结合，和四环素，它们无法察觉地结合在一起。因此，它们作为原始四环素适体的正交互补物起作用。出乎意料的是，强力霉素适体采用与四环素适体不同的构象，并依赖于最初作为引物结合位点安装的恒定区。我们表明多西环素的荧光发射强度在适体结合后增加，允许它们用作生物传感器。这种新型适体可用于需要强力霉素检测或强力霉素介导调节的多种环境。我们表明多西环素的荧光发射强度在适体结合后增加，允许它们用作生物传感器。这种新型适体可用于需要强力霉素检测或强力霉素介导调节的多种环境。我们表明多西环素的荧光发射强度在适体结合后增加，允许它们用作生物传感器。这种新型适体可用于需要强力霉素检测或强力霉素介导调节的多种环境。

更新日期：2020-09-22

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