Biostable aptamers with antagonistic properties to the neuropeptide nociceptin/orphanin FQ.

The neuropeptide nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the opioid receptor-like 1 (ORL1) receptor, has been shown to play a prominent role in the regulation of several biological functions such as pain and stress. Here we describe the isolation and characterization of N/OFQ binding biostable RNA aptamers (Spiegelmers) using a mirror-image in vitro selection approach. Spiegelmers are L-enantiomeric oligonucleotide ligands that display high affinity and specificity to their targets and high resistance to enzymatic degradation compared to D-oligonucleotides. A representative Spiegelmer from the selections performed was size-minimized to two distinct sequences capable of high affinity binding to N/OFQ. The Spiegelmers were shown to antagonize binding of N/OFQ to the ORL1 receptor in a binding-competition assay. The calculated IC(50) values for the Spiegelmers NOX 2149 and NOX 2137a/b were 110 nM and 330 nM, respectively. The competitive antagonistic properties of these Spiegelmers were further demonstrated by their effective and specific inhibition of G-protein activation in two additional models. The Spiegelmers antagonized the N/OFQ-induced GTPgammaS incorporation into cell membranes of a CHO-K1 cell line expressing the human ORL1 receptor. In oocytes from Xenopus laevis, NOX 2149 showed an antagonistic effect to the N/OFQ-ORL 1 receptor system that was functionally coupled with G-protein-regulated inwardly rectifying K(+) channels.

Identification of epitope-like consensus motifs using mRNA display.

The mRNA display approach to in vitro protein selection is based upon the puromycin-mediated formation of a covalent bond between an mRNA and its gene product. This technique can be used to identify peptide sequences involved in macromolecular recognition, including those identical or homologous to natural ligand epitopes. To demonstrate this approach, we determined the peptide sequences recognized by the trypsin active site, and by the anti-c-Myc antibody, 9E10. Here we describe the use of two peptide libraries of different diversities, one a constrained library based on the trypsin inhibitor EETI-II, where only the six residues in the first loop were randomized (6.4 x 10(7) possible sequences, 6.0 x 10(11) sequences in the library), the other a linear-peptide library with 27 randomized amino acids (1.3 x 10(35) possible sequences, 2 x 10(13) sequences in the library). The constrained library was screened against the natural target of wild-type EETI, bovine trypsin, and the linear library was screened against the anti-c-myc antibody, 9E10. The analysis of selected sequences revealed minimal consensus sequences of PR(I,L,V)L for the first loop of EETI-II and LISE for the 9E10 epitope. The wild-type sequences, PRILMR for the first loop of EETI-II and QKLISE for the 9E10 epitope, were selected with the highest frequency, and in each case the complete wild-type epitope was selected from the library.

GnRH binding RNA and DNA Spiegelmers: a novel approach toward GnRH antagonism.

Mirror-image oligonucleotide ligands (Spiegelmers) that bind to the pharmacologically relevant target gonadotropin-releasing hormone I (GnRH) with high affinity and high specificity have been identified using the Spiegelmer technology. GnRH is a decapeptide that plays an important role in mammalian reproduction and sexual maturation and is associated with several benign and malignant diseases. First, aptamers that bind to D-GnRH with dissociation constants of 50-100 nM were isolated out of RNA and DNA libraries. The respective enantiomers of the DNA and RNA aptamers were synthesized, and their binding to L-GnRH was shown. These Spiegelmers bind to L-GnRH with similar affinity to that of the corresponding aptamers that bind to D-GnRH. We further demonstrated dose-dependent inhibition of GnRH-induced Ca(2+) release in Chinese hamster ovary cells that were stably transfected with the human GnRH receptor.

Aptamers and aptazymes: accelerating small molecule drug discovery.

Synthetic nucleic acid ligands, known as aptamers, are versatile tools that can greatly enhance the efficiency of modern drug development. Exhibiting binding characteristics comparable to or even better than monoclonal antibodies, these ligands can be used as detection probes, highly efficient inhibitors of protein function or specific competitors in high-throughput screening (HTS) assays. Thus, aptamer technology can be exploited to address the growing demand for multi-parallel analysis of proteomes, functional prioritization of potential drug targets and accelerated small molecule lead identification. The unique advantages of this technology are the rapid automated generation of sophisticated ligands against almost any target molecule and the convenient structural or chemical modification of the nucleic acid probes. Depending on the strategy, an RNA aptamer can be expressed transgenically to investigate and inactivate an endogenous protein in an animal model, or it can be designed to function as a highly sensitive nucleic acid biosensor. More recently, the technology has been extended to directly link functional target validation with HTS, accelerating the process of drug discovery.

Aptamers as tools for target prioritization and lead identification.

The increasing number of potential drug target candidates has driven the development of novel technologies designed to identify functionally important targets and enhance the subsequent lead discovery process. Highly specific synthetic nucleic acid ligands--also known as aptamers--offer a new exciting route in the drug discovery process by linking target validation directly with HTS. Recently, aptamers have proven to be valuable tools for modulating the function of endogenous cellular proteins in their natural environment. A set of technologies has been developed to use these sophisticated ligands for the validation of potential drug targets in disease models. Moreover, aptamers that are specific antagonists of protein function can act as substitute interaction partners in HTS assays to facilitate the identification of small-molecule lead compounds.