In Vitro and in Vivo Activities of Oligodeoxynucleotide-Based Thrombin Inhibitors Containing Neutral Formacetal Linkages

Abstract

A series of 15-mer oligodeoxynucleotide analogues were synthesized, and their thrombin inhibitory activities in vitro and in vivo were evaluated. These oligodeoxynucleotide analogues share the same sequence (GGTTGGTGTGGTTGG) but have one or more phosphodiester linkages replaced by a neutral formacetal group. The results obtained from monosubstitutions show that no single phosphodiester group is critical for the thrombin inhibitory activity, suggesting that the interaction between the oligodeoxynucleotide and thrombin is based on a multiple-site charge-charge interaction. Analysis of the effects of different phosphodiester replacements indicates that the backside and left side of the chairlike structure formed by the molecule may be involved in binding with thrombin, presumably by having direct contacts with the anion-binding exosite of the enzyme. For the oligodeoxynucleotides containing two noncontiguous formacetal groups, the effect of the disubstitution is the sum of the effects obtained from the corresponding two monosubstitutions. Infusion of an oligodeoxynucleotide containing four formacetal groups into monkeys showed an increased in vivo anticoagulant effect and an extended in vivo half-life compared to the unmodified oligodeoxynucleotide.

A Fiber-Optic Microarray Biosensor Using Aptamers as Receptors

“…In addition, the aptamer extends farther away from the surface as it has a sixatom spacer at the 5Ј end. The 15-mer antithrombin aptamer folds into a chair-like structure with two Gtetrad stacks connected by two TT loops and a single TGT loop in solution (21)(22)(23). This tertiary structure has been reported to play an important role in binding to the anion-binding exosite on the serine protease thrombin, a key blood-coagulation enzyme.…”

The Role of Cell Surface Receptors in the Activation of Human B Cells by Phosphorothioate Oligonucleotides

“…In addition, it has been shown that interaction between ODNs and thrombin is based on multiple site charge-charge interactions. Neutralizing the negative charge of ODNs by replacing the negatively charged phosphodiester groups with neutral charged groups decreased their thrombin-inhibitory activities (33). Therefore, charge-charge interactions may play a role in binding of sODNs and ODNs to their receptors.…”

Polyvalent nucleic acid aptamers and modulation of their activity: a focus on the thrombin binding aptamer

“…Some researchers have exploited non-natural modifications of the nucleobases (Krawczyk et al, 1995;He et al, 1998a;Marathias et al, 1999;López de la Osa et al, 2006;Mendelboum Raviv et al, 2008;Nallagatla et al, 2009;Goji & Matsui, 2011), which included: 1) guanines modified with hydrophobic substituents in the N 2 and C 8 positions; 2) 6-thio-, 3) 8-amino-, 4) iso-, and 5) 8-bromo-guanine modifications; and 6) thymine with 4-thio-uracil substitutions. Other research groups modified the nucleotide backbone of TBA 15 by introducing valuable surrogates replacing the natural phosphodiester linkages, such as: 1) neutral formacetal groups (He et al, 1998b), 2) phosphorothioate linkages (Saccà et al, 2005;Pozmogova et al, 2010;Zaitseva et al, 2010), 3) 3′-3′ or 5′-5′ phosphodiester bonds (Martino et al, 2006;Esposito et al, 2007;Pagano et al, 2008;Russo Krauss et al, 2011), and 4) methylphosphonate bonds (Saccà et al, 2005), or the nucleoside moieties, with insertion within the backbone of: 5) 2′-deoxy-2′-fluoro-D-arabinonucleotides (2′F-araN) (Peng & Damha, 2007), 6) locked-nucleic acids Bonifacio et al, 2008), 7) unlocked nucleic acids (UNA) (Agarwal et al, 2011;Jensen et al, 2011;Pasternak et al, 2011), and 8) acyclic thymine nucleoside (Coppola et al, 2008) residues. Most of these chemical modifications do not result into relevant improvements in the anticoagulant properties of TBA, even if in some reports an increase of the overall stability of the G-quadruplex structure and/or of the affinity for thrombin is registered.…”