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

Claudine Boiziau,Eric Dausse,Ludmila Yurchenko,Jean-Jacques Toulmé

doi:10.1074/jbc.274.18.12730

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 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.

Highlights

In the antisense strategy, a DNA oligonucleotide is designed to hybridize to an RNA sequence, in order to inhibit the reading of the encoded genetic information [1]
We previously demonstrated that in vitro selection of DNA ligands 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])
A DNA library comprising more than 1012 different sequences was screened on the basis of oligonucleotide ability to bind to TAR

Summary

Introduction

A DNA oligonucleotide is designed to hybridize to an RNA sequence, in order to inhibit the reading of the encoded genetic information [1]. 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) 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 upper part of the aptamer stem likely pre-organizes the loop to minimize the distortion required for loop-loop complex formation

Methods

Results

Conclusion