4‐Thio‐deoxyuridylate‐modified thrombin aptamer and its inhibitory effect on fibrin clot formation, platelet aggregation and thrombus growth on subendothelial matrix

Abstract

Summary. Background: The consensus thrombin aptamer C15-mer is a single-stranded DNA of 15 nucleotides [d(GGTTGGTGTGGTTGG)] that was identified by the selection of thrombin-binding molecules from a large combinatorial library of oligonucleotides. It is capable of inhibiting thrombin at nanomolar concentrations through binding to a specific region within thrombin exosite 1. As has been shown in our earlier studies, the 4-thio-deoxyuridylate (s4dU)-containing oligonucleotides have high affinity for a number of proteins, due to the reduced hydrophilic character of the modified oligonucleotide. Methods: Three different analogs of the original thrombin-inhibiting sequence, in which some of the thymidylate residues were replaced by 4-thio-deoxyuridylates, were synthesized. The inhibitory effect of modified aptamers was tested on thrombin-catalyzed fibrin clot formation and fibrinopeptide A release from fibrinogen, thrombin-induced platelet aggregation/ secretion, and the formation of thrombus on coverslips coated with human collagen type III, thrombin-treated fibrinogen or subendothelial matrix of human microvascular endothelial cells. Results: As compared with the C15-mer, the analog with the sequence GG(s4dU)TGG(s4dU)G(s4dU)GGT(s4dU)GG (UC15-mer) showed a 2-fold increased inhibition of thrombincatalyzed fibrin clot formation, fibrinopeptide A release, platelet aggregation and secretion in human plasma and thrombus formation on thrombin-treated fibrinogen surfaces under flow conditions. Concerning the inhibition of thrombininduced fibrin formation from purified fibrinogen and activation of washed platelets, UC15-mer was 3-fold and twelve-fold more effective than C15-mer, respectively. Conclusion: The replacement of four thymidylate residues in C15-mer by 4-thio-deoxyuridylate resulted in a new thrombin aptamer with increased anticoagulant and antithrombotic properties.

Improving the Stability of Aptamers by Chemical Modification

“…An RNA aptamer targeting the Most of these are aided by the medicinal chemistry approach, with the fast development of organic synthesis serving as a toolbox [10]. As a result of chemical modifications, many exceptional characteristics besides stability such as improved specificity [11], increased affinity [12][13][14][15], enhanced delivery and prolonged plasma residence [7,[16][17][18][19][20][21][22][23] have been reported and summarized [1,[3][4][5]10,24,25], among which aptamers conjugated by polyethylene glycol significantly promote aptamer's bio-distribution to varied tissues and organs in vivo and increase their plasma retention time, with prolonged half-lives for blood clearance [16,22]. Aptamers can be also encapsulated in or attached to biodegradable polymers/proteins for sustained drug delivery and prolonged release in vivo [18][19][20][21].…”

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

“…In various strategies TBA 15 maintained its natural backbone and was only modified in the sequence: 1) by substituting several bases in the loops and/or in the G-quartets (Smirnov & Shafer, 2000; Saccà et (Uehara et al, 2008;Buff et al, 2010). 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.…”

Anomeric DNA quadruplexes

“…Typically, the modifications affected either central loop only or in combination with selected thymines in TT loops as described for isodeoxyguanosine, 2′-fluoroarabinose, 4-thio, and unlocked TBA analogs. 13,14,17,36 Because the central loop is the most exposed region in the TBA structure 8,35 its modification may considerably improve nuclease resistance of chimeric aptamers. Anomeric modification in the TGT loop was shown to dramatically increase nuclease resistance.…”

“…studies on TBA analogs containing 3′-3′ and 5′-5′ junctions, LNA residues, internucleotide phosphorothioates, 2′-fluoroarabinose, isodeoxyguanosine and several other modified nucleotides have been reported. [11][12][13][14][15][16][17][18] Enhanced binding affinity to thrombin and improved anticoagulant and antithrombotic properties have been observed for certain TBA modifications. [11][12][13] In this study, we report the modification of TBA with anomeric α-nucleotides (Fig.…”