DNA Aptamers Selected Against the HIV-1trans-Activation-responsive RNA Element Form RNA-DNA Kissing Complexes

Abstract

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 5ACTCCCAT 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.In the antisense strategy, a DNA oligonucleotide is designed to hybridize to an RNA sequence, in order to inhibit specifically the reading of the encoded genetic information (1). Although RNA is a single chain nucleic acid, it adopts secondary and tertiary structures, which can prevent the hybridization of the antisense sequence. This is one of the likely explanations of the poor inhibition efficiency, if any, induced by some antisense oligonucleotides. The aptamer strategy has been successfully used for the selection of ligands against a large range of targets, such as proteins and small molecules (nucleotides, amino acids, dyes) (see Ref. 2 for a review). This methodology offers an alternative way for designing nucleic acid ligands against an RNA structure. Indeed, we previously demonstrated that in vitro selection of DNA ligands (aptamers) against DNA secondary structures led to the identification of sequences able to recognize the DNA targets through base pair formation and additional unidentified interactions (3, 4). This might be of high potential interest, as numerous RNA structures display a regulatory function through interaction either with proteins (such as the iron-responsive element interacting with the ironresponsive element-binding protein (5), the HIV trans-activation-responsive (TAR) 1 element binding to the viral protein Tat (6), or the HIV Rev-responsive element promoting the export of retroviral RNA from the nucleus due to the binding with the viral protein Rev (6)) or with nucleic acids (like the dimerization-initiating sequence of HIV (7)). The binding of an oligonucleotide to such structures could prevent the interaction of the RNA with the regulator...

Liquid-crystal NMR structure of HIV TAR RNA bound to its SELEX RNA aptamer reveals the origins of the high stability of the complex

“…Several strategies have allowed for the identification of various ligands of TAR RNA (8)(9)(10)(11). A large field of investigation concerns RNA aptamers that specifically bind TAR, which potentially offer a good selectivity and affinity for the RNA target.…”

NMR characterization of a kissing complex formed between the TAR RNA element of HIV-1 and a DNA aptamer

“…The TAR-DNA aptamer: a loop-loop interaction Footprint studies performed on TAR bound to different DNA aptamers confirmed the loop-loop interaction suggested by the complementarity of their loop sequences (7). Preliminary 1D NMR studies were performed on a 29 nt RNA (TAR-29) corresponding to the apical region of the wild-type TAR sequence known to retain complete biological activity in vivo (1).…”

“…All the DNA molecules identified at the end of this SELEX process displayed an imperfect stem-loop with a consensus sequence 5′-ACTCCCAT-3′ containing five bases complementary to the TAR loop sequence, suggesting the possible formation of a loop-loop interaction. In addition, D-04, the aptamer displaying the highest affinity among the selected candidate, exhibited a thymine residue in the upper part of the stem, which was suggested to form a single base bulge in a previous study (7). Such anti-TAR aptamers might be of interest for the control of HIV development.…”

“…This TAR loop sequence is critical for in vivo transactivation and has been shown to be the binding site of viral and cellular proteins (5,6). DNA molecules were previously selected in vitro for their specific binding to the TAR sequence (7). All the DNA molecules identified at the end of this SELEX process displayed an imperfect stem-loop with a consensus sequence 5′-ACTCCCAT-3′ containing five bases complementary to the TAR loop sequence, suggesting the possible formation of a loop-loop interaction.…”

Modulating viral gene expression by aptamers to RNA structures

“…This is probably due to the different geometry adopted by the RNA-RNA helices in the TAR-R06 complex and the DNA-RNA (looploop hybrid) and DNA-DNA (the aptamer stem) helices in the TAR-aptamer complexes. Interestingly, in vitro selection using an oligodeoxynucleotide pool against TAR RNA led to aptamers that also form kissing complexes with substantial differences compared to the selected RNAs (Boiziau et al, 1999). At high (10 mM) magnesium concentration, the loop of the best candidate DI-04 forms five base pairs with the TAR loop.…”