Intermolecular interactions between structured RNA play key roles in the regulation of gene expression. In Escherichia coli, the replication of the ColE1 plasmid is regulated by the interaction of two RNA transcripts, RNA I and RNA II, which fold as hairpins (1, 2). The interaction starts with base pairing between the complementary loops of these RNAs and leads to the formation of a double-stranded RNA along the entire length of RNA I, thus disrupting the RNA II hybridization with the template DNA required for replication (3,4). Even if the stability of these so-called "kissing" complexes is primarily based on loop complementarity (2), factors such as the orientation of the loops (5), the loop closing base pair, and the sequence of each stem next to the loop (6), are crucial for stability. For instance, the loop inversion 5Ј to 3Ј induces a 350-fold increased stability of the RNA I-RNA II complex.In HIV-1, 1 the dimerization of the genomic RNA involves the formation of a loop-loop complex between two structured regions (7). The dimerization initiation site of HIV-1 RNA folds as a hairpin, which is closed by a noncanonical AA pair. The 9-nt-long loop contains a 6-nt self-complementary sequence flanked by two 5Ј and one 3Ј purines, which, together with loop complementarity, are crucial for the dimerization process (8). Non-Watson-Crick interactions in RNA molecules have been also reported in the viral RNA element bound by the Rev protein of HIV-1 (9), in GRNA tetraloops (10, 11), tRNAs (12-14), and tandem mismatches within duplexes (15-17). All these results indicate that interactions other than canonical base pairs contribute to the structural diversity displayed by RNAs, which, as a matter of fact, is crucial for activity. The contribution of such noncanonical interactions can actually be conveniently explored by in vitro selection, since neither the structure of the target nor the interactions between the target and the interacting aptamer need to be known (18,19). This strategy was successfully used in our laboratory to identify DNA aptamers against DNA (20 -22) and RNA hairpin structures (23).Recently, RNA aptamers specific for the trans-activationresponsive (TAR) RNA (24) were selected by in vitro selection (25). The TAR RNA element is a 59-nt-long imperfect stem loop structure located at the 5Ј end of the retroviral RNA. A 3-nt bulge in the upper part of the hairpin constitutes part of the binding site of the viral protein Tat, which recruits cyclin T 1 . Together with additional TAR-bound cellular proteins, this complex prevents abortion of the transcription of the retroviral genome. Therefore, TAR plays a key role in the life cycle of HIV-1 and constitutes a valid target for the development of ligands, which could inhibit its interaction with viral and cellular proteins, thus ultimately preventing the development of the virus. The isolated high affinity anti-TAR aptamers were shown to fold as imperfect hairpins and form kissing complexes with the targeted RNA at physiological magnesium concentration. The ...

Absorbance and fluorescence spectra of bacterial cytochrome P-450cam and cytochrome P-450lin have been studied as a function of pressure. These pressure-induced spectral perturbations fall into two categories, which are interpreted as resulting from denaturation domains and are discussed in terms of protein structural dynamics. The results presented herein support a view that these two bacterial cytochromes have large structural differences and suggest a picture in which the gellike cortex of each protein may play an essential role in stability and function.

One of the major limitations of the use of phosphodiester oligonucleotides in cells is their rapid degradation by nucleases. To date, several chemical modifications have been employed to overcome this issue but insufficient efficacy and/or specificity have limited their in vivo usefulness. In this work conformationally restricted nucleotides, locked nucleic acid (LNA), were investigated to design nuclease resistant aptamers targeted against the HIV-1 TAR RNA. LNA/DNA chimeras were synthesized from a shortened version of the hairpin RNA aptamer identified by in vitro selection against TAR. The results indicate that these modifications confer good protection towards nuclease digestion. Electrophoretic mobility shift assays, thermal denaturation monitored by UV-spectroscopy and surface plasmon resonance experiments identified LNA/DNA TAR ligands that bind to TAR with a dissociation constant in the low nanomolar range as the parent RNA aptamer. The crucial G, A residues that close the aptamer loop remain a key structural determinant for stable LNA/DNA chimera-TAR complexes. This work provides evidence that LNA modifications alternated with DNA can generate stable structured RNA mimics for interacting with folded RNA targets.

Aptamers interacting with RNA hairpins through loop-loop (so-called kissing) interactions have been described as an alternative to antisense oligomers for the recognition of RNA hairpins. R06, an RNA aptamer, was previously shown to form a kissing complex with the TAR (trans-activating responsive) hairpin of HIV-1 RNA (Ducongé and Toulmé (1999) RNA 5, 1605). We derived a chimeric locked nucleic acid (LNA)/DNA aptamer from R06 that retains the binding properties of the originally selected R06 aptamer. We demonstrated that this LNA/DNA aptamer competes with a peptide of the retroviral protein Tat for binding to TAR, even though the binding sites of the two ligands do not overlap each other. This suggests that upon binding, the aptamer TAR adopts a conformation that is no longer appropriate for Tat association. In contrast, a LNA/DNA antisense oligomer, which exhibits the same binding constant and displays the same base-pairing potential as the chimeric aptamer, does not compete with Tat. Moreover, we showed that the LNA/DNA aptamer is a more specific TAR binder than the LNA/DNA antisense sequence. These results demonstrate the benefit of reading the three-dimensional shape of an RNA target rather than its primary sequence for the design of highly specific oligonucleotides.