Modified RNA sequence pools for<i>in vitro</i>selection

Lin, Yun; Qiu, Qiu; Gill, Stanley C.; Jayasena, Sumedha D.

doi:10.1093/nar/22.24.5229

Cited by 180 publications

(129 citation statements)

References 38 publications

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“…In vitro selection is a combinatorial technique that generates receptors, ligands, and catalysts from nucleic acid libraries containing as many as 10 14 -10 16 different molecules and is a powerful method to explore the sequence space of biopolymers in search of functional domains. Some effort has been devoted to developing in vitro selection processes that use modified monomer triphosphates as substrates for polymerases (27)(28)(29)(30)(31)(32)(33)(34)(35)(36), and several in vitro selection experiments have been undertaken to select for catalysts or aptamers whose functions depend on the presence of modified nucleotides (37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48), with notable success (39,40,(43)(44)(45)(46)(47). On the other hand, modified building blocks have not proven to always be critical in obtaining superior receptors or catalysts (40,42,48,49).…”

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confidence: 99%

A Novel, Modification-Dependent ATP-Binding Aptamer Selected from an RNA Library Incorporating a Cationic Functionality

et al. 2003

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An analogue of uridine triphosphate containing a cationic functional group was incorporated into a degenerate RNA library by enzymatic polymerization. In vitro selection experiments using this library yielded a novel receptor that binds ATP under physiological pH and salt conditions in a manner completely dependent on the presence of the cationic functionality. The consensus sequence and a secondary structure model for the ATP binding site were obtained by the analysis of functional sequences selected from a partially randomized pool based on the minimal parental sequence. Mutational studies of this receptor indicated that several of the modified uridines are critical for ATP binding. Analysis of the binding of ATP analogues revealed that the modified RNA receptor makes numerous contacts with ATP, including interactions with the triphosphate group. In contrast, the aptamer repeatedly isolated from natural RNA libraries does not interact with the triphosphate group of ATP. The incorporation of a cationic amine into nucleic acids clearly allows novel interactions to occur during the molecular recognition of ligands, which carries interesting implications for the RNA world hypothesis. In addition, new materials generated from such functionalized nucleic acids could be useful tools in research and diagnostics.

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A Novel, Modification-Dependent ATP-Binding Aptamer Selected from an RNA Library Incorporating a Cationic Functionality

et al. 2003

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“…Selectionreflection thus joins the approach of using modified RNA polymerase substrates (11)(12)(13)(14) as a viable means of coupling the power of in vitro selection to the production of stable bioactive ligands.…”

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Bioactive and nuclease-resistant l -DNA ligand of vasopressin

Williams

Liu

Schumacher

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

199

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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...

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“…SELEX allows for easy identification of aptamer sequences that can target a broad range of proteins and molecules under a variety of conditions, allowing for selection of binding that is responsive to factors such as pH, temperature, salt, etc. 44--46 As research on aptamers expands and modification of aptamers with appropriate ionizable groups becomes increasingly viable 47,48 for pH--dependent behavior, our platform can be used for such targets, as well. In addition, G--quartet binding, which is involved in the aptamer--thrombin binding in this work, is evident in many other sequence--target configurations, making our proof--of--concept likely applicable to these other biomolecules as well.…”

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confidence: 99%