Morris K N, Tarasow T M, Julin C M, Simons S L, Hilvert D, Gold L
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder 80309.
Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):13028-32. doi: 10.1073/pnas.91.26.13028.
RNA molecules that bind a transition state analog for a Diels-Alder reaction (Kd = 0.35 +/- 0.05 mM) were isolated from a starting pool of approximately 10(14) sequences by affinity chromatography. After the initial rise and plateau of the amount of RNA that eluted with soluble analog, a step gradient elution was used to further enrich the pool for sequences with higher affinities for the target. To our knowledge, the isolation of RNA molecules that bind either a nonplanar or a hydrophobic ligand has not been reported previously. A conserved nucleotide sequence and secondary structure present in many of the RNA molecules are necessary but not sufficient for binding the analog. No catalysts of the targeted Diels-Alder reaction were found among the binders. The absence of catalysis contrasts with previous successful experiments with antibodies and suggests that other strategies may be needed to identify oligonucleotides with diverse catalytic activities.
通过亲和色谱法从大约10¹⁴个序列的起始文库中分离出与狄尔斯-阿尔德反应过渡态类似物结合的RNA分子（解离常数Kd = 0.35 ± 0.05 mM）。在用可溶性类似物洗脱的RNA量最初上升并达到平稳后，采用分步梯度洗脱进一步富集文库中对靶标具有更高亲和力的序列。据我们所知，此前尚未报道过与非平面或疏水性配体结合的RNA分子的分离情况。许多RNA分子中存在的保守核苷酸序列和二级结构对于结合类似物是必要的，但并不充分。在这些结合分子中未发现靶向狄尔斯-阿尔德反应的催化剂。催化作用的缺失与之前抗体的成功实验形成对比，这表明可能需要其他策略来鉴定具有多种催化活性的寡核苷酸。
