Different duplex/quadruplex junctions determine the properties of anti-thrombin aptamers with mixed folding

Abstract

Mixed duplex/quadruplex oligonucleotides have attracted great interest as therapeutic targets as well as effective biomedical aptamers. In the case of thrombin-binding aptamer (TBA), the addition of a duplex motif to the G-quadruplex module improves the aptamer resistance to biodegradation and the affinity for thrombin. In particular, the mixed oligonucleotide RE31 is significantly more effective than TBA in anticoagulation experiments and shows a slower disappearance rate in human plasma and blood. In the crystal structure of the complex with thrombin, RE31 adopts an elongated structure in which the duplex and quadruplex regions are perfectly stacked on top of each other, firmly connected by a well-structured junction. The lock-and-key shape complementarity between the TT loops of the G-quadruplex and the protein exosite I gives rise to the basic interaction that stabilizes the complex. However, our data suggest that the duplex motif may have an active role in determining the greater anti-thrombin activity in biological fluids with respect to TBA. This work gives new information on mixed oligonucleotides and highlights the importance of structural data on duplex/quadruplex junctions, which appear to be varied, unpredictable, and fundamental in determining the aptamer functional properties.

Improved RE31 Analogues Containing Modified Nucleic Acid Monomers: Thermodynamic, Structural, and Biological Effects

“…What is more, the lack of involvement in the intramolecular contacts of positions T 11 , T 15 , and T 20 makes them tolerant to chemical modification. 16 The characteristic structure of RE31 gives additional opportunities to increase its thermal stability via introduction of modified residues into the duplex fragment. It was previously proven that introduction of LNA residues into DNA or RNA duplexes increases their thermal stability and improves selectivity in comparison to the corresponding unmodified reference.…”

“…The above effect can be explained by involvement of these two residues in formation of hydrogen bonds between RE31 molecule and thrombin. 16 Therefore, the increased flexibility of the RE31 loops via insertion of UNA residues perturbs favorable intermolecular interactions. The simultaneous introduction of UNA-U at positions T 11 , T 15 and T 20 provoked the most significant reduction of RE31 anticoagulant properties (the AE for O7 was 0.65 s).…”

“…15 According to crystallographic data, it was shown that the duplex part of RE31 gradually transforms into the G-quadruplex structure with similarly oriented helical axes of the two fragments. 16 Therefore, the aptamer is believed to adopt a rod-like shape, which prevents simultaneous binding of both RE31 regions to a thrombin molecule. The binding to target protein involves only the G-quadruplex region and is assigned to take place in exosite I.…”

Through-bond effects in the ternary complexes of thrombin sandwiched by two DNA aptamers

“…However, the elucidation at the atomic level of the way in which both aptamers bind to the protein in a ternary complex may suggest new approaches for the design of thrombin inhibitors with enhanced specificity. It should be noted that while the positioning of HD1-like aptamers at exosite I is well established (24–26,39), HD22 was found to adhere to exosite II in an unexpected bent conformation (27), whose details could be finely regulated by the presence of the aptamer bound at exosite I.…”

Crystal structure of a DNA aptamer bound to PvLDH elucidates novel single-stranded DNA structural elements for folding and recognition

“…To further understand the various interactions involved between ssNAs and proteins, it is critical to determine the structures of protein:ssNA complexes. However, besides modified DNA such as SOMAmers 28 29 30 , only five non-redundant structures of protein:DNA aptamer complexes 31 32 33 34 35 36 have been identified.…”