Jean-Jacques Toulmé

Specific regulation of gene expression by antisense, sense and antigene nucleic acids

The regula t ion of gene express ion in l iving organisms rests main ly upon the recogni t ion of nucleic acid base sequences by pro te ins [1,2]. However , nucleic acids can also p lay a role. Several examples of regula t ion b y complemen ta ry R N A so cal led ant isense R N A have been descr ibed in p roka ryo tes [3,4]. N a t u r a l regu la tory nucleic acids exert their effect th rough several d is t inct mechanisms. They can hybr id ize to a messenger R N A leading to t rans la t ion arrest [3]. They m a y also induce degrada t ion of the target R N A , e.g., th rough an R N a s e I I I -dependen t mechan i sm [5]. Hybr id i za t i on to a nascent messenger R N A m a y lead to p rema tu re t ranscr ip t ion t e rmina t ion [6]. A n ant isense R N A m a y also induce an i rreversible modi f i ca t ion of its target messenger R N A

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.

Antimessenger oligodeoxyribonucleotides: an alternative to antisense RNA for artificial regulation of gene expression — a review

Synthetic oligodeoxyribonucleotides (oligos) are now widely used as artificial regulators for gene expression both in cell-free media and in cultured cells. We describe the biological consequence of the various chemical modifications that have been introduced into the molecules to improve their resistance against nuclease attack, their affinity for the target mRNA and their uptake by cells. We also describe the rising generation of antimessenger oligos. Covalently linked to reactive groups these molecules direct irreversible modifications of the complementary nucleic acids. We anticipate that these oligos will be targeted to double-stranded nucleic acids to interfere with gene expression at the DNA level.

New candidates for true antisense

cleotides have been widely used for artificially regulating gene expression through RNA targeting. One class of these, the phosphorothioate oligodeoxynucleotides1, were originally developed as nuclease-resistant alternatives to phosphodiester oligomers, which are rapidly degraded and thus unsuitable for in vivo use. Some phosphorothioates have proven therapeutic efficacy; in fact, in 1998 the Food and Drug Administration (FDA, Rockville, MD) approved a phosphorothioate oligomer against cytomegalovirus retinitis for clinical use. Unfortunately, despite becoming by far the most frequently used derivatives, the mechanism of action of these molecules is complex and may well have little to do with the translation inhibition for which they are intended. In this issue, Giovannangeli and coworkers3 develop a system for examining the mode of action of a new type of derivative, the N3 ́–P5 ́ phosphoramidate (NP), which is notable for both its extremely high nuclease resistance and its tight binding to singlestranded RNA (Fig. 1A). Several studies have already demonstrated sequence-specific effects in cell culture and in vivo for these derivatives, but evidence for a true antisense mechanism is poorly documented. In the present paper, the authors establish a reporter system to verify whether NP oligonucleotides act by selective binding to RNA or through nonspecific interactions with other cellular components. As a test, they designed an NP 18-mer to target a sequence, designated PPT, that lies immediately upstream of the firefly luciferase initiation codon. When the plasmid encoding the reporter gene and the antisense oligomer were co-transfected into HeLa cells by cationic lipids, a dose-dependent reduction of luciferase activity (50% inhibitory concentration (IC50) ≈ 100 nM) was observed. Luciferase expression was also strongly inhibited when this oligomer was administered to streptolysin O-permeabilized cells. The issue of sequence specificity, the Achilles heel of phosphorothioates, was also carefully addressed. When cotransfected with the reporter construct in HeLa cells, the NP oligomer did not reduce luciferase expression either when the target sequence was absent/ mutated or when the NP oligomer was designed with inverted polarity. This is consistent with what one expects with true antisense oligonucleotides: decreased expression of the target gene is strictly correlated to the formation of a RNA–oligonucleotide duplex. To test the mechanism of the NP 18-mer in vivo, the reporter construct was introduced, together with the oligonucleotide, into mouse skeletal muscle fiber by electricpulse-mediated transfer. As in cell culture, luciferase expression in muscle lysate was decreased in animals treated with the antisense sequence, but not with the inverted sequence, or when the target sequence was absent or mutated. In vivo effects of NP oligonucleotides were previously reported by Skorski et al2. Inoculation of HL60 acute leukemia cells in severe combined immunodeficient (SCID) mice induces a process reminiscent of the human disease, leading to the death of the animal five to seven weeks later. Intravenous injection of a NP 15-mer targeted to the c-myc oncogene resulted in a prolonged survival, up to 32 weeks. No such an effect was observed with a mismatched NP oligomer. In both studies2,3 phosphoramidates were about 10-

Aptamers: a new class of oligonucleotides in the drug discovery pipeline?

Aptamers are oligonucleotides identified in a randomly synthesized library containing up to 10(15) different molecules that fold into defined three-dimensional structures. Following their selection for predetermined properties at the end of an iterative process known as SELEX (Systematic Evolution of Ligands by Exponential enrichment) they can be chemically modified in order to provide them with additional properties. These molecules display both high affinity and specificity for their target. Aptamers constitute promising molecules for therapeutic applications as exemplified by pegaptanib, an aptamer-derived anti-VEGF compound shown to be effective in treating age-related macular degeneration.

Loop–loop interaction of HIV-1 TAR RNA with N3′ → P5′ deoxyphosphoramidate aptamers inhibits in vitro Tat-mediated transcription

A hairpin RNA aptamer has been identified by in vitro selection against the transactivation-responsive element (TAR) of HIV-1. A nuclease-resistant N3′ → P5′ phosphoramidate isosequential analog of this aptamer also folds as a hairpin and forms with TAR a loop–loop “kissing” complex with a binding constant in the low nanomolar range as demonstrated by electrophoretic mobility-shift assays and surface plasmon resonance experiments. The key structural determinants, which contribute to the stability of the RNA aptamer–TAR complex, loop complementarity and the GA residues closing the aptamer loop, remain crucial for the N3′ → P5′ aptamer–TAR complex. Moreover, the N3′ → P5′ phosphoramidate aptamer specifically interferes with the binding of a peptide derived from the transactivator protein (Tat) peptide to TAR and selectively inhibits the Tat-mediated transcription in an in vitro assay, which marks this nuclease-resistant aptamer as a relevant candidate for experiments in cells.

Targeting RNA structures by antisense oligonucleotides.

The presence of folded regions in RNA competes with the binding of a complementary oligonucleotide, resulting in a weak antisense effect. Due to the key role played by a number of RNA structures in the natural regulation of gene expression it might be of interest to design antisense sequences able to selectively interact with such motifs in order to interfere with the biological processes they mediate. Different possibilities have been explored. A high affinity oligomer will disrupt the structure; if the target structure is solved one can take advantage of unpaired bases (bulges, loops) to minimize the thermodynamic cost of the binding. Alternatively, the folded structure can be accommodated within the complex via the formation of a local triple helix. Oligomers able to adapt to the RNA structure (aptamers) can be extracted by in vitro selection from randomly synthesized libraries comprising several billions of sequences.

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

Transactivation-response element (TAR) is a stable stem–loop structure of HIV RNA, which plays a crucial role during the life cycle of the virus. The apical loop of TAR acts as a binding site for essential cellular cofactors required for the replication of HIV. High-affinity aptamers directed against the apical loop of TAR have been identified by the SELEX approach. The RNA aptamers with the highest affinity for TAR fold as hairpins and form kissing complexes with the targeted RNA through loop–loop interactions. The aptamers with the strongest binding properties all possess a GA base pair combination at the loop-closing position. Using liquid-crystal NMR methodology, we have obtained a structural model in solution of a TAR–aptamer kissing complex with an unprecedented accuracy. This high-resolution structure reveals that the GA base pair is unilaterally shifted toward the 5′ strand and is stabilized by a network of intersugar hydrogen bonds. This specific conformation of the GA base pair allows for the formation of two supplementary stable base-pair interactions. By systematic permutations of the loop-closing base pair, we establish that the identified atomic interactions, which form the basis for the high stability of the complex, are maintained in several other kissing complexes. This study rationalizes the stabilizing role of the loop-closing GA base pairs in kissing complexes and may help the development or improvement of drugs against RNA loops of viruses or pathogens as well as the conception of biochemical tools targeting RNA hairpins involved in important biological functions.

SELEX and dynamic combinatorial chemistry interplay for the selection of conjugated RNA aptamers

SELEX (for Systematic Evolution of Ligands by Exponential enrichment) has proven to be extraordinarily powerful for the isolation of DNA or RNA aptamers that bind with high affinity and specificity to a wide range of molecular targets. However, the modest chemical functionality of nucleic acids poses some limits on the versatility of aptamers as binders and catalysts. To further improve the properties of aptamers, additional chemical diversity must be introduced. The design of chemical modifications is not a trivial task. Recently, dynamic combinatorial chemistry (DCC) has been introduced as an alternative to traditional combinatorial chemistry. DCC employs equilibrium shifting to effect molecular evolution of a dynamic combinatorial library of molecules. Herein, we describe an original process that combines DCC and SELEX for the in vitro selection of modified aptamers which are conjugated to chemically diverse small-molecules. Its successful application for the selection of small-molecule conjugated RNA aptamers that bind tightly to the transactivation-response (TAR) element of HIV-1 is presented.

Systematic screening of LNA/2′-O-methyl chimeric derivatives of a TAR RNA aptamer

We synthesized and evaluated by surface plasmon resonance 64 LNA/2′‐O‐methyl sequences corresponding to all possible combinations of such residues in a kissing aptamer loop complementary to the 6‐nt loop of the TAR element of HIV‐1. Three combinations of LNA/2′‐O‐methyl nucleoside analogues where one or two LNA units are located on the 3′ side of the aptamer loop display an affinity for TAR below 1 nM, i.e. one order of magnitude higher than the parent RNA aptamer. One of these combinations inhibits the TAR‐dependent luciferase expression in a cell assay.