A DNA Aptamer That Binds Adenosine and ATP

Abstract

We have used in vitro selection to isolate adenosine/ATP-binding DNA sequences from a pool of approximately 2 x 10(14) different random-sequence single-stranded DNA molecules. One of these aptamers has been characterized and binds adenosine in solution with a dissociation constant of 6 +/- 3 microM. Experiments with ATP analogs indicate that functional groups on both the base and the sugar of ATP are involved in the ligand/aptamer interaction. The binding domain of this aptamer was localized to a 42 base sequence by deletion analysis. A pool of mutagenized versions of this sequence was then synthesized and screened for functional adenosine binding sequences; comparison of the selected variants revealed two highly conserved guanosine-rich regions, two invariant adenosine residues, and two regions of predominantly Watson--Crick covariation. This data led us to propose a model of the ATP-binding DNA structure which is based on a stable framework composed of two stacked G-quartets. The two highly conserved adenosine residues may stack between the top G-quartet and the two short stems, forming a pocket in which the adenosine or ATP ligand binds. Site-directed mutagenesis, base analog substitution studies, and the design of highly divergent but functional sequences provide support for this model.

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.…”

Advances in Aptamer-Based Biosensors for Food Safety

“…After several rounds, this pool is cloned, sequenced, and characterized to find aptamers with the desired properties. Targets for which aptamers can be developed are varied and range from small molecules (Huizenga & Szostak, 1995), to proteins and even whole cells. The in vitro nature of the selection process allows for the discovery of aptamers for even non-immunogenic or highly toxic substances.…”

Protein detection via direct enzymatic amplification of short DNA aptamers

“…(1,2) Targets to which aptamers have successfully been generated against include proteins, small molecules and RNA. (3)(4)(5)(6)(7)(8) Aptamers have been suggested as valuable alternatives to antibodies in biodetection and diagnostic applications due to their ease of discovery, their stability, and robust methods for their synthesis. (9)(10)(11)(12) A wealth of sensitive aptamer-based biodetection approaches have been reported in which aptamers were labeled with molecules such as redox probes, fluorescent dyes, or nanocrystals to be an integral part of signal transduction.…”