Abstract
In this work we examined the properties of thrombin-binding aptamer (TBA) modified by the introduction of inversion of polarity sites (IPS) in order to assess the effect of modification on the activation of TBA to serve as DNAzyme with peroxidase-like activity. Two oligonucleotides were designed to possess one (IPS1) or three (IPS2) inversion sites. TBA typically forms antiparallel G-quadruplexes with two G-tetrads, which exhibits very low DNAzyme peroxidise activity. DNAzyme activity is generally attributed to parallel G-quadruplexes. Hence, inversion of polarity was introduced in the TBA molecule to force the change of G-quadruplex topology. All oligonucleotides were characterized using circular dichroism and UV-Vis melting profiles. Next, the activity of the DNAzymes formed by studied oligonucleotides and hemin was investigated. The enhancement of peroxidase activity was observed when inversion of polarity was introduced. DNAzyme based on IPS2 showed the highest peroxidase activity in the presence of K+ or NH4+ ions. This proves that inversion of polarity can be used to convert a low-activity DNAzyme into a DNAzyme with high activity. Since TBA is known for its anticoagulant properties, the relevant experiments with IPS1 and IPS2 oligonucleotides were performed. Both IPS1 and IPS2 retain some anticoagulant activity in comparison to TBA in the reaction with fibrinogen. Additionally, the introduction of inversion of polarity makes these oligonucleotides more resistant to nucleases.
Highlights
We propose the formation of a 3 + 1 structure C (Scheme 1) for IPS1 in sodium and potassium ions and a similar 3 + 1 structure D for IPS2 at sodium ions
The IPS1 formed an antiparallel 3 + 1 structure with one propeller and two lateral loops [17], whereas the IPS2 adopted an antiparallel G4 structure but with all propeller loops. This topology facilitates the binding of hemin and explains the high activity of the IPS2
Our experiments proved that the introduction of inversion of polarity sites can drastically enhance the peroxidase activity of DNAzyme
Summary
Thrombin-binding aptamers are important in the medical and clinical fields. Nucleic acid equivalents of antibodies, are single stranded DNA or RNA molecules that can selectively bind with other molecules [2]. They are selected through the SELEX procedure (Systematic Evolution of Ligands by EXponential enrichment) and each year new aptamers for biologically significant molecules are developed e.g., adenosine, vascular endothelial growth factor or cancer cells [3,4,5]. The G-quadruplex (G4 DNA) is an advanced structure formed by single-stranded nucleic acid stabilized by Hoogsteen hydrogen bonds, electrostatic interactions, and hydrophobic stacking between guanosine residues [8]
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