In vitro selection of DNA aptamers against the HIV-1 TAR RNA hairpin.
In vitro selection was performed to identify DNA aptamers against the TAR RNA stem-loop structure of HIV-1. A counterselection step allowed the elimination of kissing complex-forming aptamers previously selected (Boiziau et al. J. Biol. Chem. 1999; 274:12730). This led to the emergence of oligonucleotides, most of which contained two consensus sequences, one targeted to the stem 3'-strand (5'-CCCTAGTTA) and the other complementary to the TAR apical loop (5'-CTCCC). The best aptamer could be shortened to a 19-mer oligonucleotide, characterized by a dissociation constant of 50 nM. A 16-mer oligonucleotide complementary to the TAR stem 3'-strand could also be derived from the identified aptamers, with an equal affinity (Kd = 50 nM). Experiments performed to elucidate the interaction between TAR and the aptamers (UV melting measures, enzymatic and chemical footprints) demonstrated that the TAR stem 5'-strand was not simply displaced as a result of the complex formation but unexpectedly remained associated on contact with the antisense oligonucleotide. We suggest that a multistranded structure could be formed.
Sekkai
Dalila
D
INSERM U 386, Université Victor Segalen Bordeaux 2, France.
Dausse
Eric
E
Di Primo
Carmelo
C
Darfeuille
Fabien
F
Boiziau
Claudine
C
Toulmé
Jean-Jacques
JJ
D016373:DNA, Antisense / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008024:Ligands; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism
D016373:DNA, Antisense; D008024:Ligands; D012367:RNA, Viral