Abstract
In this paper, we report our investigations on five T30175 analogues, prepared by replacing sequence thymidines with abasic sites (S) one at a time, in comparison to their natural counterpart in order to evaluate their antiproliferative potential and the involvement of the residues not belonging to the central core of stacked guanosines in biological activity. The collected NMR (Nuclear Magnetic Resonance), CD (Circular Dichroism), and PAGE (Polyacrylamide Gel Electrophoresis) data strongly suggest that all of them adopt G-quadruplex (G4) structures strictly similar to that of the parent aptamer with the ability to fold into a dimeric structure composed of two identical G-quadruplexes, each characterized by parallel strands, three all-anti-G-tetrads and four one-thymidine loops (one bulge and three propeller loops). Furthermore, their antiproliferative (MTT assay) and anti-motility (wound healing assay) properties against lung and colorectal cancer cells were tested. Although all of the oligodeoxynucleotides (ODNs) investigated here exhibited anti-proliferative activity, the unmodified T30175 aptamer showed the greatest effect on cell growth, suggesting that both its characteristic folding in dimeric form and its presence in the sequence of all thymidines are crucial elements for antiproliferative activity. This straightforward approach is suitable for understanding the critical requirements of the G-quadruplex structures that affect antiproliferative potential and suggests its application as a starting point to facilitate the reasonable development of G-quadruplexes with improved anticancer properties.
Highlights
DNA and RNA aptamers are small oligonucleotide ligands that bind their target, such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms, with remarkable affinity and specificity
The design of these analogues was founded on the plainness that G-quadruplex aptamers could be especially appropriate to be analyzed by this strategy, because it is rightful to think that most of the structural stability is based on a scaffold of stacked G-tetrads, while the loop residues, being projected externally, are more likely involved in the interaction with the target and, prone to be substituted by an abasic site in order to study in depth their role and function in the structure/activity relationship
The synthesis of the derivatives containing S is easy and founded on standard procedures, and the collected NMR, CD, and Polyacrylamide Gel Electrophoresis (PAGE) data strongly suggest that all of them adopt G4 structures strictly similar to those of the parent aptamer, with the ability to fold into a dimeric structure formed by the same two G-quadruplexes, each characterized by parallel strands, three all-anti G-tetrads and four one-thymidine loops
Summary
DNA and RNA aptamers are small oligonucleotide ligands that bind their target, such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms, with remarkable affinity and specificity. It is noteworthy that the presence of one- or two-residue loops linking the G-runs and protruding outwardly is characteristic of most of G4 aptamers whose structure has been ascertained or hypothesized, such as the thrombin-targeting anticoagulant aptamers TBA (Thrombin Binding Aptamer) and NU172 [7,8], the anti-HIV integrase aptamers T30923 [G3(TG3)3T] [9] and T30175 [GTG2(TG3)3T] [10], and the nucleolin-targeting antiproliferative aptamer AS1411 [11] Keeping in mind their topologies [12], it is very plausible that most of the structural stability of these G4 aptamers is due to the compact core formed by the stacked G-tetrads, while the external more accessible loop residues are mostly involved in the interaction with the target protein. In order to evaluate the T30175 antiproliferative potential and the involvement of the residues not belonging to the central core of stacked guanosines in the biological activity, we investigated the structural and biological properties of five T30175 analogues prepared using a single residue replacement approach of sequence thymidines with abasic sites (S) in comparison to the unmodified original aptamer (Table 1)
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