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Conformationally rigid nucleoside probes help understand the role of sugar pucker and nucleobase orientation in the thrombin-binding aptamer.

ABSTRACT: Modified thrombin-binding aptamers carrying 2'-deoxyguanine (dG) residues with locked North- or South-bicyclo[3.1.0]hexane pseudosugars were synthesized. Individual 2'-deoxyguanosines at positions dG5, dG10, dG14 and dG15 of the aptamer were replaced by these analogues where the North/anti and South/syn conformational states were confined. It was found that the global structure of the DNA aptamer was, for the most part, very accommodating. The substitution at positions 5, 10 and 14 with a locked South/syn-dG nucleoside produced aptamers with the same stability and global structure as the innate, unmodified one. Replacing position 15 with the same South/syn-dG nucleoside induced a strong destabilization of the aptamer, while the antipodal North/anti-dG nucleoside was less destabilizing. Remarkably, the insertion of a North/anti-dG nucleoside at position 14, where both pseudosugar conformation and glycosyl torsion angle are opposite with respect to the native structure, led to the complete disruption of the G-tetraplex structure as detected by NMR and confirmed by extensive molecular dynamics simulations. We conclude that conformationally locked bicyclo[3.1.0]hexane nucleosides appear to be excellent tools for studying the role of key conformational parameters that are critical for the formation of a stable, antiparallel G-tetrad DNA structures.

SUBMITTER: Saneyoshi H

PROVIDER: S-EPMC2761269 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Conformationally rigid nucleoside probes help understand the role of sugar pucker and nucleobase orientation in the thrombin-binding aptamer.

Saneyoshi Hisao H Mazzini Stefania S Aviñó Anna A Portella Guillem G González Carlos C Orozco Modesto M Marquez Víctor E VE Eritja Ramon R

Nucleic acids research 20090720 17

Modified thrombin-binding aptamers carrying 2'-deoxyguanine (dG) residues with locked North- or South-bicyclo[3.1.0]hexane pseudosugars were synthesized. Individual 2'-deoxyguanosines at positions dG5, dG10, dG14 and dG15 of the aptamer were replaced by these analogues where the North/anti and South/syn conformational states were confined. It was found that the global structure of the DNA aptamer was, for the most part, very accommodating. The substitution at positions 5, 10 and 14 with a locked ...[more]

PMID: 19620215

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Project description:Aptamers are functional nucleic acids that bind to a range of targets (small molecules, proteins or cells) with a high affinity and specificity. Chemically-modified aptamers are of interest because the incorporation of novel nucleobase components can enhance aptamer binding to target proteins, while fluorescent base analogues permit the design of functional aptasensors that signal target binding. However, since optimally modified nucleoside designs have yet to be identified, information about how to fine tune aptamer stability and target binding affinity is required. The present work uses molecular dynamics (MD) simulations to investigate modifications to the prototypical thrombin-binding aptamer (TBA), which is a 15-mer DNA sequence that folds into a G-quadruplex structure connected by two TT loops and one TGT loop. Specifically, we modeled a previously synthesized thymine (T) analog, namely 5-furyl-2'-deoxyuridine (5FurU), into each of the six aptamer locations occupied by a thymine base in the TT or TGT loops of unbound and thrombin bound TBA. This modification and aptamer combination were chosen as a proof-of-principle because previous experimental studies have shown that TBA displays emissive sensitivity to target binding based on the local environment polarity at different 5FurU modification sites. Our simulations reveal that the chemically-modified base imparts noticeable structural changes to the aptamer without affecting the global conformation. Depending on the modification site, 5FurU performance is altered due to changes in the local environment, including the modification site structural dynamics, degree of solvent exposure, stacking with neighboring bases, and interactions with thrombin. Most importantly, these changes directly correlate with the experimentally-observed differences in the stability, binding affinity and emissive response of the modified aptamers. Therefore, the computational protocols implemented in the present work can be used in subsequent studies in a predictive way to aid the fine tuning of aptamer target recognition for use as biosensors (aptasensors) and/or therapeutics.

Dataset Information

Conformationally rigid nucleoside probes help understand the role of sugar pucker and nucleobase orientation in the thrombin-binding aptamer.

Publications

Conformationally rigid nucleoside probes help understand the role of sugar pucker and nucleobase orientation in the thrombin-binding aptamer.

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