In vitro selection of aptamers that act with Zn2+

An in vitro selection was carried out with Zn2+ to isolate novel RNA molecu les, zinc-dependent aptamers, that bind to HIV-1 Tat protein. RNAs bound to Tat were collected by using a nitrocellulose filter from a library of rand om RNAs and regenerated to the next generation of the RNA library by subseq uent reverse transcription, polymerase chain reaction, and transcription. S equences of the selected RNAs were determined after 6 and 12 rounds of the selection. The control clones after normal selection procedure with Mg2+ ha d a consensus UUG that resembled essential sequences of TAR or Arg aptamers . On the other hand, many unique sequences were revealed from a library sel ected with Zn2+ and the RNA with most abundant sequence (clone 31) bound to Tat tightly only when Zn2+ existed. The secondary structure of clone 31 RN A was predicted by using a computational prediction with our thermodynamic parameters and enzymatic scission of the RNA. Several model RNAs were prepa red and the binding property of these RNAs to Tat were investigated. As a r esult, all the model RNAs did not reproduce the binding property of clone 3 1. Therefore, the Tat aptamer that acts with Zn2+ should require a relative ly longer region of the sequence which, is able to offer tertiary cooperati on of several motifs for the binding. (C) 2000 Elsevier Science B.V. All ri ghts reserved.