Probing the Structure of DNA Aptamers with a Classic Heterocycle. 英文参考文献.docVIP

Molecules 2004, 9, 67–85 molecules ISSN 1420-3049 Probing the Structure of DNA Aptamers with a Classic Heterocycle. Arthur E. Wood IV and G. Reid Bishop* Department of Chemistry and Biochemistry, Mississippi College, 200 South Capitol St., Clinton, MS 39058. USA. Tel. +1 (601)-925-7665, Fax +1 (601)-925-3993., * Author to whom correspondence should be addressed; email rbishop@ Received: 5 February 2004; in revised form: 9 February 2004 / Accepted: 15 February 2004 / Published: 28 February 2004 Abstract: DNA aptamers are synthetic, single-stranded DNA oligonucleotides selected by SELEX methods for their binding with specific ligands. Here we present ethidium binding results for three related DNA aptamers (PDB code: 1OLD, 1DB6, and 2ARG) that bind L-argininamide (L-Arm). The ligand bound form of each aptamers structure has been reported and each are found to be composed primarily of two domains consisting of a stem helical region and a loop domain that forms a binding pocket for the cognate ligand. Previous thermodynamic experiments demonstrated that the DNA aptamer 1OLD undergoes a large conformational ordering upon binding to L-Arm. Here we extend those linkage binding studies by examining the binding of the heterocyclic intercalator ethidium to each of the three aptamers by fluorescence and absorption spectrophotometric titrations. Our results reveal that ethidium binds to each aptamer with ?Gos in the range of -8.7 to -9.4 kcal/mol. The stoichiometry of binding is 2:1 for each aptamer and is quantitatively diminished in the presence of L-Arm as is the overall fluorescence intensity of ethidium. Together, these results demonstrate that a portion of the bound ethidium is excluded from the aptamer in the presence of a saturating amount of L-Arm. These results demonstrate the utility of ethidium and related compounds for the probing of non-conventional DNA st