Driving in vitro selection of anti-HIV-1 TAR aptamers by magnesium concentration and temperature.
In vitro selection with either DNA or RNA libraries was performed against the TAR RNA element of HIV-1. The role of the selection conditions on the outcome of the selection was evaluated by varying the magnesium concentration and the temperature. The selection stringency was demonstrated to determine i) the affinity of the best identified aptamers for the TAR target, and ii) the type of interaction between the two partners. Selections performed with a DNA library under low (4 degrees C, 10 mM magnesium) and high stringency (23 degrees C, 3 mM magnesium) led to the emergence of "kissing aptamers"; but even if the motif interacting directly with the TAR loop were identical in the two kinds of aptamers, the consensus was extended from eight to thirteen nucleotides when the Mg(2+) concentration was decreased from 10 to 3 mM. Similar kissing aptamers were selected at 23 degrees C and 37 degrees C starting with two different RNA libraries under identical ionic conditions. In addition, selection performed at 37 degrees C yielded a significant proportion of antisense sequences. Only antisense RNAs complementary to the TAR loop competitively inhibited the association of a Tat peptide with TAR.
Darfeuille
F
F
INSERM U 386, Université Victor Segalen Bordeaux 2, 146 rue Léo-Saignat, F-33076 Bordeaux cédex, France.
Sekkai
D
D
Dausse
E
E
Kolb
G
G
Yurchenko
L
L
Boiziau
C
C
Toulmé
J-J
JJ
D001483:Base Sequence; D004279:DNA, Viral / Q000737:chemistry* / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D015497:HIV-1 / Q000235:genetics*; D008274:Magnesium / Q000737:chemistry*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics; D013696:Temperature
D004279:DNA, Viral; D009841:Oligonucleotides; D012367:RNA, Viral; D008274:Magnesium