Abstract
In the search for optimized thrombin binding aptamers (TBAs), we herein describe the synthesis of a library of TBA analogues obtained by end-functionalization with the electron-rich 1,5-dialkoxy naphthalene (DAN) and the electron-deficient 1,8,4,5-naphthalenetetra-carboxylic diimide (NDI) moieties. Indeed, when these G-rich oligonucleotides were folded into the peculiar TBA G-quadruplex (G4) structure, effective donor–acceptor charge transfer interactions between the DAN and NDI residues attached to the extremities of the sequence were induced, providing pseudo-cyclic structures. Alternatively, insertion of NDI groups at both extremities produced TBA analogues stabilized by π–π stacking interactions. All the doubly-modified TBAs were characterized by different biophysical techniques and compared with the analogues carrying only the DAN or NDI residue and unmodified TBA. These modified TBAs exhibited higher nuclease resistance, and their G4 structures were markedly stabilized, as evidenced by increased Tm values compared to TBA. These favorable properties were also associated with improved anticoagulant activity for one DAN/NDI-modified TBA, and for one NDI/NDI-modified TBA. Our results indicated that TBA pseudo-cyclic structuring by ad hoc designed end-functionalization represents an efficient approach to improve the aptamer features, while pre-organizing and stabilizing the G4 structure but allowing sufficient flexibility to the aptamer folding, which is necessary for optimal thrombin recognition.
Highlights
Nucleic acid-based aptamers represent a promising class of short DNA or RNA molecules which, upon folding into peculiar three-dimensional arrangements, act as specific and high-affinity ligands for a wide range of biologically relevant targets [1,2,3]
To expand the structural diversity of thrombin binding aptamers (TBAs) derivatives, we took inspiration from a recent work by some of us [51], and in the current study, we developed novel modified TBA analogues end-functionalized with phosphodiester-linked donor–acceptor pairs based on the electron-rich 1,5-dialkoxy naphthalene (DAN) and electron-deficient
Scheme 1 reports the synthesis of the TBA-Np/Dp analogue
Summary
Nucleic acid-based aptamers represent a promising class of short DNA or RNA molecules which, upon folding into peculiar three-dimensional arrangements, act as specific and high-affinity ligands for a wide range of biologically relevant targets [1,2,3]. The practical applications of aptamers are primarily limited by their susceptibility to nuclease degradation, rapid blood/renal elimination or suboptimal thermal stability. In this context, the post-SELEX modification of aptamers is a valuable strategy to improve their features and to obtain more effective therapeutic agents [18,21,22,23,24,25,26,27]. Chemical modifications of nucleic acids considerably increase the functional and structural diversity of aptamer libraries and substantially improve their target affinities and/or biological properties
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