微囊藻毒素-LR特异性RNA配基体外筛选探讨

目的利用大容量寡核苷酸库筛选富集具有分子识别、能与微囊藻毒素LR特异结合的RNA配基。方法将一段中间为40个随机核苷酸排列、两端为恒定区域的DNA库转录成RNA随机库,然后将RNA随机库与共价结合在琼脂糖上的微囊藻毒素LR反应、洗脱、收集能与靶分子特异结合的微量RNA,反转录为cDNA,PCR扩增。如此重复,经13轮筛选富集,再对筛选核酸配基克隆、测序,进一步比较各克隆RNA配基的亲和性。结果能与靶分子特异结合的微量RNA经过每轮筛选逐步增加,到第11～13轮进入平台期;60个克隆产物中有38个成功测定序列和分析,将其分成3组5对,所有克隆序列中未发现组间共有保守序列。选择有代表性的克隆RNA配基,比较其对靶分子的亲和力,克隆RNA配基MC1、MC25、MC17和MC32对靶分子微囊藻毒素LR具有较高的亲和力;他们与不同浓度系列的靶分子反应,经数学模型外推克隆MC1、MC25、MC17和MC32RNA配基以及RNA随机库对微囊藻毒素LR的解离常数值分别为84、46、57、91和>256μmol/L,其中MC25检测微囊藻毒素LR最低下限至少可达05μmol/L。结论从大容量RNA随机库中分离富集到一类能够识别并特异性结合微囊藻毒素LR的配基,为研究与检测环境中藻类毒素提供了一种新的手段。

In vitro selection of specific aptamers against microcystin-LR

OBJECTIVE: In vitro selection of specific RNA aptamers against microcystin-LR from a random RNA pool. METHODS: A RNA library with 40 randomized nucleotide positions was applied to select for specific aptamers to microcystin-LR covalently linked to Sepharose by using a standard in vitro selection protocol. RESULTS: The specific enriched RNA aptamer for microcystin-LR increased step by step from initial round to 11th round after which a plateau of the aptamer quantity was observed between 11th and 13th round. The enriched RNAs from last round were reverse transcribed, PCR amplified and cloned into E. coli DH10 b competent cells. Sixty colonies were sequenced from which 38 sequences were aligned and classified into 3 families and 5 duplicates and no conserved sequences were found among them. Eight representative clones from the groups were selected for further binding experiments comparing with original pool RNA. Four clone RNAs were identified with relatively high affinity to microcystin-LR, of which MC25 clone RNA could combine with microcystin-LR as lower as 0.5 micromol/L. CONCLUSION: Subpopulations of RNA molecules that bind specifically to microcystin-LR have been isolated from a population of random sequence RNA molecules, which might provide a new way for future application in environmental monitoring of microcystin.