Functional isoDNA aptamers: modified thrombin binding aptamers with a 2′-5′-linked sugar-phosphate backbone (isoTBA)

Abstract

The regioisomeric 3'-deoxy-2'-5'-linked thrombin binding DNA aptamers (isoTBAs) were chemically synthesized and their ability to form unimolecular anti-parallel G-quadruplexes in the presence of K(+) ions was evaluated. These modified sequences retain the function of the native thrombin binding aptamer (TBA), exhibit better stability against exonuclease and are capable of slowing down the process of blood clotting.

2′‐5′‐Isomerically Linked Thrombin‐Binding Aptamer (isoTBA) Forms a Stable Unimolecular Parallel G‐Quadruplex in the Presence of Sr2+ Ions

“…In the presence of Na + and K + ions, the typical antiparallel topology was observed for both, TBA and isoTBA, as reported earlier . Both displayed maxima centered at 292 nm and minima near 260 nm.…”

“…Negligible hysteresis (Δ T m ≈ 0 °C, Table and Figure S5, SI) was observed, in agreement with a unimolecular complex with Sr 2+ for TBA and isoTBA. Further, when the UV‐ T m measurements were carried out at a higher strand concentration, including a ten‐fold excess (Figures S5, S6, SI), no appreciable differences were observed (Δ T m =+2 to −2 °C), and also as observed earlier for TBA and isoTBA with K + , again suggesting the unimolecularity of the quadruplex. The data are listed in Table .…”

“…A number of modifications in the sugar‐phosphate backbone of TBA have been reported with a view to enhancing its properties . We earlier reported an isoTBA oligomer wherein the natural 3′‐5′‐phosphodiester backbone linkages were replaced by the isomeric 2′‐5′‐phosphodiesters, and showed that it could fold into a unimolecular antiparallel G‐quadruplex structure, similar to that observed for TBA and also effect anti‐coagulation . The isomeric backbone in this oligomer leads to further enhancement in the nuclease resistance properties of the G‐quadruplex and is therefore, attractive in the context of application in biological systems.…”

In What Ways Do Synthetic Nucleotides and Natural Base Lesions Alter the Structural Stability of G-Quadruplex Nucleic Acids?

“…When the T7 and T9 were replaced by U nucleotides, the stability increased up to 45°C. On the other hand, the iso TBAs exhibited higher stability against exonucleases and were capable of retaining the biological function of the native TBA, that is, slowing down the process of blood clotting [ 240 ]. The antiparallel chair-type TBA GQ, whose nucleotides are connected via 3′-5′ phosphodiester linkages, folded into parallel GQ when the 2-3-2-nucleotide-long loops (TT…TGT…TT) were shortened.…”

Towards applications of synthetic genetic polymers in diagnosis and therapy

“…In addition to sugar chirality, Gunjal et al and Kolganova et al have shown that alternative regio-isomerisation of the RNA backbone linkages provides further routes to improved stability; the classic G-quadruplex thrombin binding DNA motif (TBA) could be partially converted to 2 0 -5 0 linked 'isoDNA' [13] or a-anomers [14], with subsequent improved exonuclease resistance, although once again complete conversion without loss of anticoagulant function was not possible. Such regio-isomerisation is likely to come at the price of duplex stability [15] and may thus be less suitable for nucleic acid folds that are less stable than G-quadruplexes.…”