Berens C, Thain A, Schroeder R
Institute of Microbiology and Genetics, Vienna Biocenter Dr. Bohrgasse 9/4, A-1030, Vienna, Austria.
Bioorg Med Chem. 2001 Oct;9(10):2549-56. doi: 10.1016/s0968-0896(01)00063-3.
Aptamers are perfect tools to study the interaction of small ligands with RNA. To study the mode of interaction of tetracycline with RNA, we isolated aptamers with high affinity to this antibiotic via in vitro selection. One of the selected aptamers, cb28, which has a comparable affinity to tetracycline as the small ribosomal subunit, was characterised in more detail. Cb28 binds only to typical tetracyclines, while atypical tetracyclines are not recognised. The hydroxyl group at position 6 is an essential determinant for recognition, while modifications at positions 4, 5 and 7 do not interfere with RNA binding. Binding of tetracycline to cb28 is magnesium dependent. The secondary structure of cb28 was determined by lead cleavage and DMS modification. Upon tetracycline binding, nucleotides in J2/3 and the P5 stem-loop are protected from cleavage by lead, indicating a conformational change in the RNA. This conformational change was confirmed by tetracycline dependent changes in the DMS modification pattern. Photo-induced affinity incorporation of tetracycline into cb28 resulted in a crosslink to position G76, a residue in L5. The mode of binding of tetracycline to the cb28 aptamer resembles its interaction with the primary binding site on the small ribosomal subunit.
适体是研究小分子配体与RNA相互作用的理想工具。为了研究四环素与RNA的相互作用模式，我们通过体外筛选分离出了对这种抗生素具有高亲和力的适体。其中一个被选中的适体cb28，它对四环素的亲和力与小核糖体亚基相当，对其进行了更详细的表征。Cb28仅与典型的四环素结合，而非典型四环素则不被识别。6位的羟基是识别的关键决定因素，而4、5和7位的修饰并不影响与RNA的结合。四环素与cb28的结合依赖于镁。Cb28的二级结构通过铅切割和DMS修饰来确定。四环素结合后，J2/3和P5茎环中的核苷酸受到保护，不被铅切割，这表明RNA发生了构象变化。这种构象变化通过DMS修饰模式中四环素依赖性变化得到了证实。四环素光诱导亲和掺入cb28导致与L5中的G76位点发生交联。四环素与cb28适体的结合模式类似于其与小核糖体亚基上主要结合位点的相互作用。
