Biomedical subjects

Carmelo Di Primo

Publications and source records attributed to Carmelo Di Primo.

8 recordsLinked to original sources

PubMed2006

Aptamers targeted to an RNA hairpin show improved specificity compared to that of complementary oligonucleotides.

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.

Aptamers, Nucleotide↗

PubMed2006

Bimodal loop-loop interactions increase the affinity of RNA aptamers for HIV-1 RNA structures.

Multiple loop-loop interactions between adjacent RNA hairpins regulate gene expression in different organisms. To demonstrate that such natural interactions could be mimicked for generating RNA ligands that are able to recognize simultaneously at least two structured RNA targets, a double kissing complex model was designed. The target consisted of two HIV-1 transactivating responsive (TAR) RNA variants, BRU and MAL, connected by a non-nucleotidic linker. The double ligand was generated by combining the corresponding hairpin aptamers, R06BRU and R06MAL, identified previously by in vitro selection [Ducongé, F., and Toulmé, J. J (1999) RNA 5, 1605-1614]. The resulting interaction was analyzed by thermal denaturation monitored by UV spectroscopy, electrophoretic mobility shift assays (EMSAs), and surface plasmon resonance (SPR) experiments. The bimodal complex was characterized by a binding equilibrium constant increased by at least 1 order of magnitude compared to that of the complexes between the individual parent hairpins. This resulted from a slower dissociation rate. We then made use of such a strategy for targeting two structured functional motifs of the folded 5' untranslated region (5'UTR) of HIV-1. Two bivalent RNA ligands were designed that targeted simultaneously the TAR and dimerization initiation site (DIS) hairpins or the TAR and poly(A) ones. The results show that these ligands also displayed enhanced affinity for their target compared to the individual molecules. The work reported here suggests that bimodal structured RNA ligands might provide a way of increasing the affinity of aptamers for folded RNA targets.

Base Pairing↗

PubMed2004

LNA/DNA chimeric oligomers mimic RNA aptamers targeted to the TAR RNA element of HIV-1.

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.

Base Sequence↗

PubMed2004

Regulating eukaryotic gene expression with aptamers.

Aptamers are RNA or DNA oligonucleotides identified within a randomly synthesized library, through an in vitro selection procedure. The selected candidates display a pre-determined property of interest with respect to a given target. Successful selection has been carried out against targets ranging from small (amino acids, antibiotics) to macro-molecules (proteins, nucleic acids). They generally show an affinity in the nanomolar range and a high specificity of target recognition. Interestingly, aptamers selected against purified targets in the test tube retain their properties within cells. RNA aptamers can be generated in situ from an appropriate DNA construct or delivered as nuclease-resistant oligonucleotide analogues. For example, aptamers recognizing RNA structure through loop-loop interactions modulate the trans-activation of in vitro transcription mediated by the TAR RNA element of human immunodeficiency virus type 1. Consequently, they constitute both exquisite tools for functional genomics analysis and promising prototypes of therapeutic agents. Natural aptameric motifs have been identified within mRNA sequences, which upon binding to a metabolite control the expression of the encoded gene, which is generally involved in the biosynthesis of this particular metabolite.

Amino Acid Motifs↗

PubMed2003

Molecular dynamics reveals the stabilizing role of loop closing residues in kissing interactions: comparison between TAR-TAR* and TAR-aptamer.

A RNA aptamer (R06) raised against the trans- activation responsive (TAR) element of HIV-1 was previously shown to generate a loop-loop complex whose stability is strongly dependent on the selected G and A residues closing the aptamer loop. The rationally designed TAR* RNA hairpin with a loop sequence fully complementary to the TAR element, closed by U,A residues, also engages in a loop-loop association with TAR, but with a lower stability compared with the TAR-R06 complex. UV absorption monitored thermal denaturation showed that TAR-TAR*(GA), in which the U,A kissing residues were exchanged for G,A, is as stable as the selected TAR-R06 complex. Consequently, we used the TAR-TAR* structure deduced from NMR studies to model the TAR-R06 complex with either GA, CA or UA loop closing residues. The results of the molecular dynamics trajectories correlate well with the thermal denaturation experiments and show that the increased stability of the GA variant results from an optimized stacking of the bases at the stem-loop junction and from stable interbackbone hydrogen bonds.

Base Pairing↗

PubMed2002

2'-O-methyl-RNA hairpins generate loop-loop complexes and selectively inhibit HIV-1 Tat-mediated transcription.

The interaction of the TAR RNA element of human immunodeficiency virus type 1 (HIV-1) with a 2'-O-methyl analogue of an RNA hairpin aptamer previously identified by in vitro selection [Ducongé, F., and Toulmé, J. J. (1999) RNA 5, 1605-1614] was characterized by UV-monitored thermal denaturation and surface plasmon resonance experiments. The complex between TAR and this aptamer derivative displays stability (K(d) = 9.9 +/- 1.0 nM) and kinetic properties [k(on) = 9.0 +/- 0.3 M(-1) s(-1), k(off) = (8.9 +/- 0.6) x 10(-4) s(-1)] close to those of the parent RNA aptamer. The modified aptamer forms a "kissing" complex with TAR driven by the same key elements as the TAR-RNA aptamer one. In particular, the G and A residues closing the aptamer loop remain crucial for TAR-2'-O-methyl aptamer complexes. Moreover, the 2'-O-methyl aptamer analogue specifically inhibits Tat-mediated transcription in an in vitro assay more efficiently than the RNA aptamer. This is likely due to the increased lifetime of the former oligonucleotide in the cell-free extract. The 2'-O-methyl modification extends the range of molecules that can be used to target viral hairpin RNA through loop-loop interactions. More generally, this demonstrates the interest of SELEX for targeting RNA hairpins and understanding nucleic acid interactions.

Gene Expression Regulation, Viral↗

PubMed2002

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.

Amides↗

PubMed2002

In vitro selection of DNA aptamers against the HIV-1 TAR RNA hairpin.

In vitro selection was performed to identify DNA aptamers against the TAR RNA stem-loop structure of HIV-1. A counterselection step allowed the elimination of kissing complex-forming aptamers previously selected (Boiziau et al. J. Biol. Chem. 1999; 274:12730). This led to the emergence of oligonucleotides, most of which contained two consensus sequences, one targeted to the stem 3'-strand (5'-CCCTAGTTA) and the other complementary to the TAR apical loop (5'-CTCCC). The best aptamer could be shortened to a 19-mer oligonucleotide, characterized by a dissociation constant of 50 nM. A 16-mer oligonucleotide complementary to the TAR stem 3'-strand could also be derived from the identified aptamers, with an equal affinity (Kd = 50 nM). Experiments performed to elucidate the interaction between TAR and the aptamers (UV melting measures, enzymatic and chemical footprints) demonstrated that the TAR stem 5'-strand was not simply displaced as a result of the complex formation but unexpectedly remained associated on contact with the antisense oligonucleotide. We suggest that a multistranded structure could be formed.

DNA, Antisense↗