Isolation of a fluorophore-specific DNA aptamer with weak redox activity

摘要

Background: In vitro selection experiments with pools of random-sequence nucleic acids have been used extensively to isolate molecules capable of binding specific ligands and catalyzing self-modification reactions. Results: In vitro selection from a random pool of single-stranded DNAs has been used to isolate molecules capable of recognizing the fluorophore sulforhodamine B with high affinity. When assayed for the ability to promote an oxidation reaction using the reduced form of a related fluorophore, dihydrotetramethylrosamine, a number of selected clones show low levels of catalytic activity. Chemical modification and site-directed mutagenesis experiments have been used to probe the structural requirements for fluorophore binding. The aptamer recognizes its ligand with relatively high affinity and is also capable of binding related molecules that share extended aromatic rings and negatively charged functional groups. Conclusions: A guanosine-rich single-stranded DNA is capable of binding fluorophores with relatively high affinity and of weakly promoting a multiple-turnover reaction. A simple motif consisting of a three-tiered G-quartet stacked upon a standard Watson-Crick duplex appears to be responsible for this activity. The corresponding sequence might provide a useful starting paint for the evolution of novel, improved deoxyribozymes that generate fluorescent signals by promoting multiple-turnover reactions. [References: 30]