In vitro selection of aptamers that act with Zn2+.
An in vitro selection was carried out with Zn2+ to isolate novel RNA molecules, 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 random RNAs and regenerated to the next generation of the RNA library by subsequent reverse transcription, polymerase chain reaction, and transcription. Sequences of the selected RNAs were determined after 6 and 12 rounds of the selection. The control clones after normal selection procedure with Mg2+ had a consensus UUG that resembled essential sequences of TAR or Arg aptamers. On the other hand, many unique sequences were revealed from a library selected 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 RNA was predicted by using a computational prediction with our thermodynamic parameters and enzymatic scission of the RNA. Several model RNAs were prepared and the binding property of these RNAs to Tat were investigated. As a result, all the model RNAs did not reproduce the binding property of clone 31. Therefore, the Tat aptamer that acts with Zn2+ should require a relatively longer region of the sequence which is able to offer tertiary cooperation of several motifs for the binding.
Kawakami
J
J
Department of Chemistry, Faculty of Science, Konan University, Kobe, Japan.
Imanaka
H
H
Yokota
Y
Y
Sugimoto
N
N
D001483:Base Sequence; D015696:Gene Products, tat / Q000138:chemical synthesis / Q000378:metabolism*; D015497:HIV-1 / Q000737:chemistry; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D009843:Oligoribonucleotides; D054322:tat Gene Products, Human Immunodeficiency Virus; D012313:RNA; D015032:Zinc