A colorimetric nanosensor based on a selective target-responsive aptamer kissing complex.

Herein, we report a novel approach for the design of a colorimetric aptasensor based on functionalized gold nanoparticle probes. This approach relies on the conjugation of nanoparticles by two functional DNA and RNA hairpins that engage specific kissing (loop-loop) interactions in response to the addition of a small analyte ligand, leading to particle aggregation and then red-to-purple colour change of the colloidal solution.

Design of a dual aptamer-based recognition strategy for human matrix metalloproteinase 9 protein by piezoelectric biosensors.

MMP-9, human matrix metalloproteinase 9, belongs to the family of zinc-dependent peptide-bond hydrolases and is involved in the degradation of the extracellular matrix (ECM). In clinics, it is well known that elevated MMP-9 serum levels are associated with cardiovascular dysfunctions, several aspects of the physiology and pathology of the central nervous system, neuropsychiatric disorders and degenerative diseases related to brain tumors, and excitotoxic/neuroinflammatory processes. Due to the large interest of diagnostics in this protein, efforts to set up sensitive methods to detect MMP-9 for early diagnosis of a number of metabolic alterations are rapidly increasing. In this panorama, biosensors could play a key role; therefore we explored for the first time the development of an aptamer-based piezoelectric biosensor for a sensitive, label free, and real time detection of MMP-9. The detecting strategy involved two different aptamers in a sandwich-like approach able to detect down to 100 pg mL(-1) (1.2 pM) of MMP-9 as detection limit in standard solution. As proof of principle, commercial serum was investigated in terms of possible interferents, their identification and role in MMP-9 detection. The estimated detection limit for MMP-9 is about 560 pg mL(-1) (6.8 pM) in untreated serum.

Driving in vitro selection of anti-HIV-1 TAR aptamers by magnesium concentration and temperature.

In vitro selection with either DNA or RNA libraries was performed against the TAR RNA element of HIV-1. The role of the selection conditions on the outcome of the selection was evaluated by varying the magnesium concentration and the temperature. The selection stringency was demonstrated to determine i) the affinity of the best identified aptamers for the TAR target, and ii) the type of interaction between the two partners. Selections performed with a DNA library under low (4 degrees C, 10 mM magnesium) and high stringency (23 degrees C, 3 mM magnesium) led to the emergence of "kissing aptamers"; but even if the motif interacting directly with the TAR loop were identical in the two kinds of aptamers, the consensus was extended from eight to thirteen nucleotides when the Mg(2+) concentration was decreased from 10 to 3 mM. Similar kissing aptamers were selected at 23 degrees C and 37 degrees C starting with two different RNA libraries under identical ionic conditions. In addition, selection performed at 37 degrees C yielded a significant proportion of antisense sequences. Only antisense RNAs complementary to the TAR loop competitively inhibited the association of a Tat peptide with TAR.

Modulation of RNA function by oligonucleotides recognizing RNA structure.

Numerous RNA structures are responsible for regulatory processes either because they constitute a signal, like the hairpins or pseudoknots involved in ribosomal frameshifting, or because they are binding sites for proteins such as the trans-activating responsive RNA element of the human immunodeficiency virus whose binding to the viral protein Tat and cellular proteins allows full-length transcription of the retroviral genome. Selective ligands able to bind with high affinity to such RNA motifs may serve as tools for dissecting the molecular mechanisms in which they are involved. Such ligands might also constitute prototypes of therapeutic agents when RNA structures play a role in the expression of dysfunctional genes or in the multiplication of pathogens. Different classes of ligands (aminoglycosides, interacalating agents, peptides) are of interest to this aim. However, oligonucleotides deserve particular consideration. They have been extensively used in the frame of the antisense strategy. The apparent simplicity of this rational approach is, at first sight, very attractive. Indeed, numerous successful studies have been published describing the efficient inhibition of translation, splicing, or reverse transcription in cell-free systems, in cultured cells, or in vivo by oligomers complementary to an RNA region. However, RNA structures restrict the access of the target site to the antisense sequence: The competition between the intramolecular association of RNA regions weakens or even abolishes the antisense effect. Various possibilities have been developed to circumvent this limitation. This includes both rational and combinatorial strategies. High-affinity oligomers were designed to invade the RNA structure. Alternatively, triplex-forming oligonucleotides (TFO) and aptamers may recognize the folded RNA motif. Whereas the use of TFOs is rather limited owing to the strong sequence constraints for triple-helix formation, in vitro selection offers a way to explore vast oligoribo or oligodeoxyribo libraries to identify strong, selective oligonucleotide binders. The candidates (aptamers) selected against the TAR RNA element of HIV-1, which form stable loop-loop (kissing) complexes with the target, provide interesting examples of oligonucleotides recognizing a functional RNA structure through an important contribution of tertiary interactions.

RNA and N3'-->P5' kissing aptamers targeted to the trans-activation responsive (TAR) RNA of the human immunodeficiency virus-1.

We used in vitro selection to identify RNA aptamers able to selectively bind to the TAR RNA motif of HIV-1, an unperfect RNA hairpin involved in the transcription of the retroviral genome. We selected aptameric RNA hairpins giving rise to kissing complexes with TAR. The N3'-->P5' phosphoramidate variant of the aptamer bind to TAR with a Kd in the low nanomolar range. However, only the RNA-RNA loop-loop complex is recognized by the Rop protein of E. coli which is specific for kissing complexes.

DNA aptamers selected against the HIV-1 RNase H display in vitro antiviral activity.

The DNA polymerase of the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is a target widely used to inhibit HIV-1 replication. In contrast, very few inhibitors of the RNase H activity associated with RT have been described, despite the crucial role played by this activity in viral proliferation. DNA ligands with a high affinity for the RNase H domain of HIV-1 RT were isolated by systematic evolution of ligands by an exponential enrichment strategy (SELEX), using recombinant RTs with or without the RNase H domain. The selected oligonucleotides (ODNs) were able to inhibit in vitro the HIV-1 RNase H activity, while no effect was observed on cellular RNase H. We focused our interest on two G-rich inhibitory oligonucleotides. Model studies of the secondary structure of these ODNs strongly suggested that they were able to form G-quartets. In addition to the inhibition of HIV-1 RNase H observed in a cell free system, these ODNs were able to strongly diminish the infectivity of HIV-1 in human infected cells. Oligonucleotides described here may serve as leading compounds for the development of specific inhibitors of this key retroviral enzyme activity.

A method to select chemically modified aptamers directly.

In vitro selection is a strategy to identify high-affinity ligands of a predetermined target among a large pool of randomized oligonucleotides. Most in vitro selections are performed with unmodified RNA or DNA sequences, leading to ligands of high affinity and specificity (aptamers) but of very short lifetime in the ex vivo and in vivo context. Only a very limited number of modified triphosphate nucleotides conferring nuclease resistance to the oligomer can be incorporated by polymerases. This encourages the development of alternative methods for the identification of nuclease-resistant aptamers. In this paper, we describe such a method. After selection of 2'O-methyl oligonucleotides against the TAR RNA structure of HIV-1, the complementary DNA sequences are fished out of a pool of randomized oligodeoxynucleotides by Watson-Crick hybridization. The DNA-fished sequences are amplified by PCR as double and single strands, the latter being used to fish back the chemically modified candidates from the initial library. This procedure allows an indirect amplification of the selected candidates. This enriched pool of modified sequences is then used for the next selection round against the target.

Comparative analysis of translation efficiencies of hepatitis C virus 5' untranslated regions among intraindividual quasispecies present in chronic infection: opposite behaviors depending on cell type.

Hepatitis C virus (HCV) RNA translation initiation is dependent on the presence of an internal ribosome entry site (IRES) that is found mostly in its 5' untranslated region (5' UTR). While exhibiting the most highly conserved sequence within the genome, the 5' UTR accumulates small differences, which may be of biological and clinical importance. In this study, using a bicistronic dual luciferase expression system, we have examined the sequence of 5' UTRs from quasispecies characterized in the serum of a patient chronically infected with HCV genotype 1a and its corresponding translational activity. Sequence heterogeneity between IRES elements led to important changes in their translation efficiency both in vitro and in different cell cultures lines, implying that interactions of RNA with related transacting factors may vary according to cell type. These data suggest that variants occasionally carried by the serum prior to reinfection could be selected toward different compartments of the same infected organism, thus favoring the hypothesis of HCV multiple tropism.

Eukaryotic ribonucleases HI and HII generate characteristic hydrolytic patterns on DNA-RNA hybrids: further evidence that mitochondrial RNase H is an RNase HII.

RNase H activities from HeLa cells (either of cytoplasmic or mitochondrial origin), and from mitochondria of beef heart and Xenopus ovaries, have been tested with RNA-DNA substrates of defined length (20 bp) and sequence. Substrates were either blunt-ended, or presented DNA or RNA overhangs. The hydrolysis profiles obtained at early times of the digestion showed a good correlation between the class of RNase H, either type I or II assigned according to biochemical parameters, whatever the organism. Consequently, the pattern of primary cuts can be considered as a signature of the predominant RNase H activity. For a given sequence, hydrolysis profiles obtained are similar, if not identical, for either blunt-ended substrates or those presenting overhangs. However, profiles showed variations depending on the sequence used. Of the three sequences tested, one appears very discriminatory, class I RNases H generating a unique primary cut 3 nt from the 3' end of the RNA strand, whereas class II RNases H generated two simultaneous primary cuts at 6 and at 8 nt from the 5' end of the RNA strand. Hydrolysis profiles further confirm the assignation of the mitochondrial RNase H activity from HeLa cells, beef heart and Xenopus oocytes to the class II.

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

This work presents the first structural analysis of an RNA-DNA complex consisting of an 18 nt RNA hairpin and a 20 nt DNA aptamer. The DNA molecule was previously selected, from a randomly synthesized library, against the transactivation response element (TAR) involved in transcriptional regulation of the HIV genome. The DNA aptamer used in the present study is an imperfect stem-loop with the sequence 5'-ACTCCCAT-3', characteristic of the selected candidates, in the apical loop. This octameric motif contains five bases complementary to the TAR loop sequence 5'-CUGGGA-3'. The use of homo- and heteronuclear NMR spectroscopy allowed assignment of the complex resonances and resolution of its secondary structure. Evidence is given for a kissing complex fold, which consists of a quasi-continuous helix formed by one stem of DNA, one stem of RNA and a central hybrid helix comprising 5 bp. Two out of helices residues of DNA and one of RNA connect the DNA-RNA loop-loop helix to the stem of either partner in the complex. In addition, two thymines of the DNA stem are engaged in a non-canonical T.T base pair.

Towards the selection of phosphorothioate aptamers optimizing in vitro selection steps with phosphorothioate nucleotides.

The high affinity of a given nucleic acid for a protein ligand can be used to isolate specific inhibitors of enzymes involved in pathological situations. The latter property is the basis of the SELEX (systematic evolution of ligands by exponential enrichment) technique. Recently, several potent nucleic acids inhibitors of HIV-1 replication have been isolated using the SELEX approach. However, phosphodiester oligodeoxynucleotides (PO-ODNs) were not used as antiviral agents because of their sensitivity to nucleases. Our goal in this work was to explore the possibility of selecting, from a fully substituted phosphorothioate library, oligonucleotides having both a strong affinity for HIV-1 reverse transcriptase (RT) and nuclease resistance. HIV-1 RT initiates in vivo reverse transcription from the 3' end of a host tRNALys. Although phosphorothioate ODNs (PS-ODNs) have been claimed to bind unspecifically to proteins, we have shown previously that an ODN corresponding to the acceptor stem of tRNALys was able to inhibit specifically HIV-1 replication in HIV-1 infected cells, without showing cytotoxicity up to 10 microM. As the SELEX strategy requires 'in vitro' transcription and reverse transcription of the selected DNA, we have assayed the available PS precursors as a model system by using PS-dNTPs and rNTPs. We have also developed an experimental procedure to optimize the incorporation of four PS-dNTPs during the PCR step of the SELEX approach. In the course of this work, we have showed that the PS-dGTP is a strong inhibitor of thermostable DNA polymerases as well as of HIV-1 RT.

4-amino-1H-benzo[g]quinazoline-2-one: a fluorescent analog of cytosine to probe protonation sites in triplex forming oligonucleotides.

We developed a new fluorescent analog of cytosine, the 4-amino-1H-benzo[g]quinazoline-2-one, which constitute a probe sensitive to pH. The 2'-O-Me ribonucleoside derivative of this heterocycle was synthesized and exhibited a fluorescence emission centered at 456 nm, characterized by four major excitation maxima (250, 300, 320 and 370 nm) and a fluorescence quantum yield of Phi = 0.62 at pH 7.1. The fluorescence emission maximum shifted from 456 to 492 nm when pH was decreased from 7.1 to 2.1. The pK(a) (4) was close to that of cytosine (4.17). When introduced in triplex forming oligonucleotides this new nucleoside can be used to reveal the protonation state of triplets in triple-stranded structures. Complex formation was detected by a significant quenching of fluorescence emission (approximately 88%) and the N-3 protonation of the quinazoline ring by a shift of the emission maximum from 485 to 465 nm. Using this probe we unambiguously showed that triplex formation of the pyrimidine motif does not require the protonation of all 4-amino-2-one pyrimidine rings.

Is a closing "GA pair" a rule for stable loop-loop RNA complexes?

RNA hairpin aptamers specific for the trans-activation-responsive (TAR) RNA element of human immunodeficiency virus type 1 were identified by in vitro selection (Ducongé, F., and Toulmé, J. J. (1999) RNA 5, 1605-1614). The high affinity sequences selected at physiological magnesium concentration (3 mm) were shown to form a loop-loop complex with the targeted TAR RNA. The stability of this complex depends on the aptamer loop closing "GA pair" as characterized by preliminary electrophoretic mobility shift assays. Thermal denaturation monitored by UV-absorption spectroscopy and binding kinetics determined by surface plasmon resonance show that the GA pair is crucial for the formation of the TAR-RNA aptamer complex. Both thermal denaturation and surface plasmon resonance experiments show that any other "pairs" leads to complexes whose stability decreases in the order AG > GG > GU > AA > GC > UA >> CA, CU. The binding kinetics indicate that stability is controlled by the off-rate rather than by the on-rate. Comparison with the complex formed with the TAR* hairpin, a rationally designed TAR RNA ligand (Chang, K. Y., and Tinoco, I. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8705-8709), demonstrates that the GA pair is a key determinant which accounts for the 50-fold increased stability of the TAR-aptamer complex (K(d) = 2.0 nm) over the TAR-TAR* one (K(d) = 92. 5 nm) at physiological concentration of magnesium. Replacement of the wild-type GC pair next to the loop of RNA I' by a GA pair stabilizes the RNA I'-RNA II' loop-loop complex derived from the one involved in the control of the ColE1 plasmid replication. Thus, the GA pair might be the preferred one for stable loop-loop interactions.

Aptamers: selected oligonucleotides for therapy.

In vitro selection constitutes a unique way to explore vast libraries (up to 10(14) to 10(15) different molecules) of randomly synthesized nucleic acids. Selected RNA or DNA sequences (aptamers) may help to understand the molecular interactions and processes of interest for human diseases. Aptamers can be chemically-modified for improved efficiency and use in complex biological media. Moreover, aptamers selected in vitro may retain their activity in vivo and thus offer novel perspectives for gene therapy and the design of new drugs.

Inhibition of in vitro and ex vivo translation by a transplatin-modified oligo(2'-O-methylribonucleotide) directed against the HIV-1 gag-pol frameshift signal.

A 2'-O-methylribooligonucleotide containing a G1.U.G3 triad modified by trans-diamminedichloro-platinum(II) was targeted to the RNA region responsible for the gag-pol frameshifting during translation of the HIV-1 mRNA. The binding of the platinated oligonucleotide to its target RNA induced a rearrangement of the (G1, G3)-intrastrand crosslink, leading to the formation of an intermolecular oligonucleotide-RNA G-A crosslink. This resulted in the selective arrest of translation of a luciferase gene placed downstream of the HIV-1 frameshift signal both in a cell-free extract (rabbit reticulocyte lysate) and in RNA-transfected cells. A specific inhibition of luciferase activity was still observed when the oligonucleotide-RNA complex was not pre-formed prior to either translation or transfection. Moreover, a selective inhibition was also observed when the oligonucleotide and the plasmid DNA encoding the luciferase and bearing the RNA gag- pol frameshifting signal were co-transfected in NIH 3T3 cultured cells. Therefore the intra-strand-->interstrand conversion of the platinum crosslink kinetically competes with the translation machinery and blocks the polypeptide elongation. These transplatin-modified oligonucleotides which operate within a live cell on a 'real-time' basis and do not need an external triggering signal constitute a promising new class of selective reactive probes.

In vitro selection identifies key determinants for loop-loop interactions: RNA aptamers selective for the TAR RNA element of HIV-1.

We selected RNA aptamers specific for the trans-activation responsive (TAR) RNA, a stem-loop structure crucial for the transcription of the integrated genome of the human immunodeficiency virus. Most of the selected sequences could be folded as imperfect hairpins and displayed a 5'-GUCCCAGA-3' consensus motif constituting the apical loop. The six central bases of this consensus sequence are complementary to the entire TAR loop, leading to the formation of TAR RNA-aptamer "kissing" complexes. The consensus G and A residues closing the aptamer loop contributed to the high affinity (Kd = 30 nM at 23 degrees C) of the aptamers for the TAR RNA. This G A pair was shown to be crucial for binding to TAR at a low magnesium concentration. The selection also identified 5'-PuPy and 5'-PyPu base pairs at alpha and beta positions of the stem, next to the loop, respectively. This strategy offered a way to identify key determinants of loop-loop interactions and to generate high affinity ligands of TAR RNA structure.

Binding of chemically-modified oligonucleotides to the double-stranded stem of an RNA hairpin.

We monitored the binding of triplex-forming oligopyrimidines to the double-stranded stem of the RNA hairpin responsible for the gag-pol frameshift in HIV-1. Whereas the substitution of 5, propynyl-C for C had a limited effect, the use of a Peptide Nucleic Acid 12mer led to a drastic reduction in the stability of the oligomer/RNA complex.

Antisense effects of oligonucleotides complementary to the hairpin of the Leishmania mini-exon RNA.

We investigated the binding and the translation inhibitory properties of hexadecamers complementary to the mini-exon sequence of the protozoan parasite Leishmania amazonensis. This targeted RNA region folds into a hairpin. Large differences were observed in the antisense properties of the different oligomers although their binding to RNA always requires the disruption of the stem region.