Bioactive and nuclease-resistant l -DNA ligand of vasopressin

Abstract

In vitro selection experiments have produced nucleic acid ligands (aptamers) that bind tightly and specifically to a great variety of target biomolecules. The utility of aptamers is often limited by their vulnerability to nucleases present in biological materials. One way to circumvent this problem is to select an aptamer that binds the enantiomer of the target, then synthesize the enantiomer of the aptamer as a nuclease-insensitive ligand of the normal target. We have so identified a mirror-image single-stranded DNA that binds the peptide hormone vasopressin and have demonstrated its stability to nucleases and its bioactivity as a vasopressin antagonist in cell culture.The enantiomer of an autogenously folding macromolecule will assume the mirror-image structure of the parent molecule, as has been documented for proteins and nucleic acids (1, 2). The corollary that the enantiomers of two complex-forming molecules will interact identically also has been demonstrated, for example by inverting all chiral centers in both an enzyme and its substrate (3). The principle has application to a problem in biotechnology, namely, that the peptide or nucleic acid ligands generated by phage display or in vitro selection are susceptible to enzymatic degradation; for example, a DNA oligonucleotide injected i.v. is degraded with a half-life of Ϸ5 min (4-7). The principle of chiral inversion can be coupled to the powerful reiterable selection methods to generate ligands that are insusceptible to degradative enzymes. First, a peptide or nucleic acid ligand is generated that binds the synthetic enantiomer of the target molecule. Then, the enantiomer of the selected ligand is synthesized from D amino acids or L nucleotides. The resulting reflected ligand will then bind and possibly inhibit the target molecule ( Fig. 1). This ''selectionreflection'' strategy has been used recently in the identification of a D peptide ligand of a Src homology 3 domain (8) and L-RNA ligands of arginine and adenosine (9, 10).In our study, the vertebrate hormone arginine vasopressin (VP), a 9-residue cyclic L peptide, was chosen as a target for its ease of bioassay, its ease of synthesis in all-D form, and its cyclic structure that restricts conformational degrees of freedom relative to a linear peptide and may thus allow selection of a tighter binding ligand. A stable ligand of VP could be useful as a diagnostic reagent or as a therapeutic antagonist in diseases associated with excessive levels of VP in blood or cerebrospinal fluid. DNA was chosen as ligand material because much higher pool diversity can be achieved than for peptide selection, and the chemical stability of DNA is superior to that of RNA.We have identified a mirror image DNA that binds VP and have shown that this ligand is bioactive, specifically antagonizing the VP response of cultured kidney cells. Selectionreflection thus joins the approach of using modified RNA polymerase substrates (11-14) as a viable means of coupling the power of in vitro selection to the production of sta...

DNA ligands that bind tightly and selectively to cellobiose

“…7) involves the iterative isolation of ligands out of the random sequence pool with affinity for a defined target molecule and PCR-based amplification of the selected RNA or DNA oligonucleotides after each round of isolation. This previously has been suggested as a route to drug discovery (7), with the possible use of nucleotide analogs to synthesize aptamers that are resistant to nucleolytic degradation (16). Targets for aptamer selection typically have been macromolecules with complex surfaces, but several cases of small molecule targets have been reported (17).…”

An l -RNA-based aquaretic agent that inhibits vasopressin in vivo

“…Truncation and site-directed modifications, including the introduction of an internal (18 atoms) PEG linker spanning the former 5Ј and 3Ј end yielded the final candidate sequence Spiegelmer NOX-F37. NOX-F37 and its terminally PEGylated derivative (40 kDa) display dissociation constants of 1.7 nM and 1.3 nM (measured at 37°C), respectively, which is three orders of magnitude better than the dissociation constant of a previously published 55-mer mirror-image DNA molecule that binds to AVP with a K d of 1.2 M (measured at room temperature) (29). In both cases, the binding to nonmodified AVP was determined in solution but with different assay formats (ITC vs. equilibrium dialysis).…”

Refilling drug delivery depots through the blood

“…The precise nature of the reduced effect is currently under investigation. Possible degradation could be overcome through the choice of ODNs with improved serum stability such as peptide nucleic acids (52) or mirror-image ODNs (53)(54)(55). Saturation of ODNs on the surface of the gels can potentially be overcome through the use of toe-hold exchange technology to remove bound ODNs from the gel to recycle the gel surface for reuse (56).…”