DNA aptamers selected against the HIV-1 trans-activation-responsive RNA element form RNA-DNA kissing complexes.
In vitro selection was performed in a DNA library, made of oligonucleotides with a 30-nucleotide random sequence, to identify ligands of the human immunodeficiency virus type-1 trans-activation-responsive (TAR) RNA element. Aptamers, extracted after 15 rounds of selection-amplification, either from a classical library of sequences or from virtual combinatorial libraries, displayed an imperfect stem-loop structure and presented a consensus motif 5'ACTCCCAT in the apical loop. The six central bases of the consensus were complementary to the TAR apical region, giving rise to the formation of RNA-DNA kissing complexes, without disrupting the secondary structure of TAR. The RNA-DNA kissing complex was a poor substrate for Escherichia coli RNase H, likely due to steric and conformational constraints of the DNA/RNA heteroduplex. 2'-O-Methyl derivatives of a selected aptamer were binders of lower efficiency than the parent aptamer in contrast to regular sense/antisense hybrids, indicating that the RNA/DNA loop-loop region adopted a non-canonical heteroduplex structure. These results, which allowed the identification of a new type of complex, DNA-RNA kissing complex, demonstrate the interest of in vitro selection for identifying non-antisense oligonucleotide ligands of RNA structures that are of potential value for artificially modulating gene expression.
Boiziau
C
C
INSERM U 386, IFR Pathologies Infectieuses, Université Victor Segalen, 146 rue Léo Saignat, F-33076 Bordeaux Cedex, France.
Dausse
E
E
Yurchenko
L
L
Toulmé
J J
JJ
D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D016914:Ribonuclease H / Q000378:metabolism
D017931:DNA Primers; D004279:DNA, Viral; D009692:Nucleic Acid Heteroduplexes; D012367:RNA, Viral; D016914:Ribonuclease H