OBJECTIVEIn vitro selection of specific RNA aptamers against microcystin-LR from a random RNA pool.

METHODSA 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.

RESULTSThe 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.

CONCLUSIONSubpopulations 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.

Aptamers, Nucleotide ; Bacterial Toxins ; chemistry ; Base Sequence ; Cyanobacteria ; chemistry ; Microcystins ; Molecular Sequence Data ; Peptides, Cyclic ; chemistry ; RNA