Quantitative Analysis of Receptors for Adenosine Nucleotides Obtained via In Vitro Selection from a Library Incorporating a Cationic Nucleotide Analog

Abstract

5-(3"-Aminopropynyl)-2'-deoxyuridine (dJ), a modified nucleoside with a side chain carrying a cationic functional group, was incorporated into an oligonucleotide library, which was amplified using the Vent DNA polymerase in a polymerase chain reaction (PCR). When coupled to an in vitro selection procedure, PCR amplification generated receptors that bind ATP. This is the first example of an in vitro selection generating oligonucleotide receptors where the oligonucleotide library has incorporated a cationic nucleotide functionality. The selection yielded functionalized receptors having sequences differing from a motif known to arise in a standard selection experiment using only natural nucleotides. Surprisingly, both the natural and the functionalized motifs convergently evolved to bind not one, but two ATP molecules cooperatively. Likewise, the affinity of the receptors for ATP had converged; in both cases, the receptors are half saturated at the 3 mM concentrations of ATP presented during the selection. The convergence of phenotype suggests that the outcome of this selection experiment was determined by features of the environment during which selection occurs, in particular, a highly loaded affinity resin used in the selection step. Further, the convergence of phenotype suggests that the optimal molecular phenotype has been achieved by both selections for the selection conditions. This interplay between environmental conditions demanding a function of a biopolymer and the ability of the biopolymer to deliver that function is strictly analogous to that observed during natural selection, illustrating the nature of life as a self-sustaining chemical system capable of Darwinian evolution.

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

“…Overall, the introduction of an amino functionality appears to have made surprisingly little difference to the outcome of these selection experiments. Both the modified RNA and DNA pools still allowed the emergence of the same receptor structures that were seen from the unmodified RNA and DNA pools (40,(56)(57)(58)62). We suspect that the full potential of these modifications to influence the results of a selection might not be seen until selective pressures are applied that cannot be overcome by normal RNA and DNA.…”

“…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). For example, comparable or better Diels-Alderase ribozymes were obtained from pools assembled with natural nucleotides (49) vs corresponding modified pools (41).…”

Recent advances in the study of biomolecular interactions by capillary electrophoresis

“…ACE has been applied to study the binding of ATP to DNAs [112] having the sequences of a Lin-Patel-Huizenga-Szostak motif A and B. A sigmoidal binding curve, 15-Amino-acid peptides CZE [123] characteristic of a 1:2 complex, was found for both the A and B interaction with ATP.…”

Expanding the structural and functional diversity of RNA: analog uridine triphosphates as candidates for in vitro selection of nucleic acids

“…Additionally, Helene and co-workers have simultaneously incorporated two modified deoxynucleotides into a DNA transcript (12). Recently an amino-functionalized deoxyuridine was used to select for a DNA-based ATP aptamer (13). Similarly, a DNA aptamer has been selected in which 5-(1-pentynyl)-2′-deoxyuridine was essential for binding (14).…”