Thrombin Binding Aptamer Analogues Research Articles

Improving the Biological Properties of Thrombin-Binding Aptamer by Incorporation of 8-Bromo-2'-Deoxyguanosine and 2'-Substituted RNA Analogues.

Thrombin-binding aptamer (TBA) is one of the best-known G-quadruplex (G4)-forming aptamers. By adopting its peculiar chair-like G4 structure, TBA can efficiently bind to thrombin, thus producing an anticoagulant effect. The major limit to its therapeutic application is represented by its poor thermal and biological resistance. Therefore, numerous research studies have been focused on the design of TBA analogues with chemical modifications to improve its pharmacokinetic and pharmacodynamic properties. To maintain the functional recognition to protein surface on which TBA anticoagulant activity depends, it is essential to preserve the canonical antiparallel topology of the TBA quadruplex core. In this paper, we have designed three TBA variants with modified G-tetrads to evaluate the effects of nucleobase and sugar moiety chemical modifications on biological properties of TBA, preserving its chair-like G-quadruplex structure. All derivatives contain 8-bromo-2'-deoxyguanosine (GBr) in syn positions, while in the anti-positions, locked nucleic acid guanosine (GLNA) in the analogue TBABL, 2'-O-methylguanosine (GOMe) in TBABM, and 2'-F-riboguanosine (GF) in TBABF is present. CD (Circular Dichroism), CD melting, 1H-NMR (Nuclear Magnetic Resonance), and non-denaturing PAGE (Polyacrylamide Gel Electrophoresis), nuclease stability, prothrombin time (PT) and fibrinogen-clotting assays have been performed to investigate the structural and biological properties of these TBA analogues. The most interesting results have been obtained with TBABF, which revealed extraordinary thermal stability (Tm approximately 40 °C higher than that of TBA), anticoagulant activity almost doubled compared to the original aptamer, and, above all, a never-observed resistance to nucleases, as 50% of its G4 species was still present in 50% FBS at 24 h. These data indicate TBABF as one of the best TBA analogue ever designed and investigated, to the best of our knowledge, overcoming the main limitations to therapeutic applications of this aptamer.

Structural and Binding Effects of Chemical Modifications on Thrombin Binding Aptamer (TBA)

The thrombin binding aptamer (TBA) is a promising nucleic acid-based anticoagulant. We studied the effects of chemical modifications, such as dendrimer Trebler and NHS carboxy group, on TBA with respect to its structures and thrombin binding affinity. The two dendrimer modifications were incorporated into the TBA at the 5′ end and the NHS carboxy group was added into the thymine residues in the thrombin binding site of the TBA G-quadruplex (at T4, T13 and both T4/T13) using solid phase oligonucleotide synthesis. Circular dichroism (CD) spectroscopy confirmed that all of these modified TBA variants fold into a stable G-quadruplex. The binding affinity of TBA variants with thrombin was measured by surface plasmon resonance (SPR). The binding patterns and equilibrium dissociation constants (KD) of the modified TBAs are very similar to that of the native TBA. Molecular dynamics simulations studies indicate that the additional interactions or stability enhancement introduced by the modifications are minimized either by the disruption of TBA–thrombin interactions or destabilization elsewhere in the aptamer, providing a rational explanation for our experimental data. Overall, this study identifies potential positions on the TBA that can be modified without adversely affecting its structure and thrombin binding preference, which could be useful in the design and development of more functional TBA analogues.

Structural properties and anticoagulant/cytotoxic activities of heterochiral enantiomeric thrombin binding aptamer (TBA) derivatives.

The thrombin binding aptamer (TBA) possesses promising antiproliferative properties. However, its development as an anticancer agent is drastically impaired by its concomitant anticoagulant activity. Therefore, suitable chemical modifications in the TBA sequence would be required in order to preserve its antiproliferative over anticoagulant activity. In this paper, we report structural investigations, based on circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR), and biological evaluation of four pairs of enantiomeric heterochiral TBA analogues. The four TBA derivatives of the d-series are composed by d-residues except for one l-thymidine in the small TT loops, while their four enantiomers are composed by l-residues except for one d-thymidine in the same TT loop region. Apart from the left-handedness for the l-series TBA derivatives, CD and NMR measurements have shown that all TBA analogues are able to adopt the antiparallel, monomolecular, ‘chair-like’ G-quadruplex structure characteristic of the natural D-TBA. However, although all eight TBA derivatives are endowed with remarkable cytotoxic activities against colon and lung cancer cell lines, only TBA derivatives of the l-series show no anticoagulant activity and are considerably resistant in biological environments.

Reversible Photocontrol of Thrombin Activity by Replacing Loops of Thrombin Binding Aptamer using Azobenzene Derivatives.

The photoisomerization of azobenzenes provides a general means for the photocontrol of many important biomolecular structures and organismal functions. For temporal and spatial control activity of thrombin binding aptamer (TBA) by light, azobenzene derivatives were carefully selected as light-triggered molecular switches to replace TT loops and the TGT loop of TBA to reversibly control enzyme activity. These molecules interconverted between the trans and cis states under alternate UV and visible light irradiation, which consequently triggered reversible formation of G-quadruplex morphology. In addition, we investigated the impact of three azobenzene derivatives on stability, thrombin binding ability, and anticoagulant properties. The result showed that 4,4'-bis(hydroxymethyl)azobenzene at the TGT loop position significantly photoregulated affinity to thrombin and blood clotting in human plasma, which provided a successful strategy to control blood clotting in human plasma and a further evidence for design of TBA analogues with pivotal positions of modifications.

Thrombin binding aptamer analogues containing inversion of polarity sites endowed with antiproliferative and anti-motility properties against Calu-6 cells

BackgroundAlthough the thrombin binding aptamer (TBA) is endowed with both anticoagulant and antiproliferative properties, it is possible to reduce the first and enhance the second one by suitable chemical modifications. MethodsTwo oligonucleotides (TBA353 and TBA535) based on the TBA sequence (GGTTGGTGTGGTTGG) and containing inversion of polarity sites have been investigated by CD, UV and electrophoretic techniques for their ability to form G-quadruplex structures. Furthermore, their anticoagulant (PT assay), antiproliferative (MTT assay) and anti-motility (wound healing assay) properties against Calu-6 cells have been tested and compared with TBA. ResultsCD, UV and electrophoresis data indicate that both ODNs are able to form G-quadruplex structures. Particularly, results suggest that TBA535 adopts a G-quadruplex structure characterized by a loop arrangement different from that of TBA. Both TBA analogues drop the anticoagulant activity. However, TBA535 is endowed with a significant antiproliferative activity against lung cancer Calu-6 cells. Importantly, both TBA and TBA535 possess a remarkable anti-motility property against the same cell line. ConclusionsBoth TBA analogues TBA353 and TBA535 are able to form G-quadruplex structures with no anticoagulant activity. However only TBA535 is endowed with noteworthy antiproliferative and anti-motility properties against lung cancer Calu-6 cells. General significanceThe switching from the anticoagulant to antiproliferative property can be obtained also in TBA derivatives not adopting the “chair-like” G-quadruplex structure typical of TBA. Furthermore, results have highlighted an unprecedented anti-cell-motility property of TBA and TBA535 reinforcing the potential of these ODNs as anticancer drugs.

Development of an Efficient G-Quadruplex-Stabilised Thrombin-Binding Aptamer Containing a Three-Carbon Spacer Molecule.

The thrombin‐binding aptamer (TBA), which shows anticoagulant properties, is one of the most studied G‐quadruplex‐forming aptamers. In this study, we investigated the impact of different chemical modifications such as a three‐carbon spacer (spacer‐C3), unlocked nucleic acid (UNA) and 3′‐amino‐modified UNA (amino‐UNA) on the structural dynamics and stability of TBA. All three modifications were incorporated at three different loop positions (T3, T7, T12) of the TBA G‐quadruplex structure to result in a series of TBA variants and their stability was studied by thermal denaturation; folding was studied by circular dichroism spectroscopy and thrombin clotting time. The results showed that spacer‐C3 introduction at the T7 loop position (TBA‐SP7) significantly improved stability and thrombin clotting time while maintaining a similar binding affinity as TBA to thrombin. Detailed molecular modelling experiments provided novel insights into the experimental observations, further supporting the efficacy of TBA‐SP7. The results of this study could provide valuable information for future designs of TBA analogues with superior thrombin inhibition properties.

Backbone modified TBA analogues endowed with antiproliferative activity

BackgroundThe thrombin binding aptamer (TBA) is endowed with antiproliferative properties but its potential development is counteracted by the concomitant anticoagulant activity. MethodsFive oligonucleotides (ODNs) based on TBA sequence (GGTTGGTGTGGTTGG) and containing l-residues or both l-residues and inversion of polarity sites have been investigated by NMR and CD techniques for their ability to form G-quadruplex structures. Furthermore, their anticoagulant (PT assay) and antiproliferative properties (MTT assay), and their resistance in fetal bovine serum have been tested. ResultsCD and NMR data suggest that the investigated ODNs are able to form right- and left-handed G-quadruplex structures. All ODNs do not retain the anticoagulant activity characteristic of TBA but are endowed with a significant antiproliferative activity against two cancerous cell lines. Their resistance in biological environment after six days is variable, depending on the ODN. ConclusionsA comparison between results and literature data suggests that the antiproliferative activity of the TBA analogues investigated could depends on two factors: a) biological pathways and targets different from those already identified or proposed for other antiproliferative G-quadruplex aptamers, and b) the contribution of the guanine-based degradation products. General significanceModified TBA analogues containing l-residues and inversion of polarity sites lose the anticoagulant activity but gain antiproliferative properties against two cancer cell lines. This article is part of a Special Issue entitled “G-quadruplex” Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Synthesis of New DNA G‐Quadruplex Constructs with Anthraquinone Insertions and Their Anticoagulant Activity

Abstract1,4‐Dihydroxyanthraquinone and 1,8‐dihydroxyanthraquinone were alkylated with 3‐bromopropan‐1‐ol and subsequently transformed into the corresponding DMT protected phosphoramidite building blocks for insertion into loops of the G‐quadruplex of the thrombin binding aptamer (TBA). The 1,4‐disubstituted anthraquinone linker led to a significant stabilization of the G‐quadruplex structure upon replacing a T in each of two neighboring lateral TT loops and a 26.2° increase in thermal melting temperature was found. CD Spectra of the modified quadruplexes confirmed anti‐parallel conformations in all cases under potassium buffer conditions as previously observed for TBA. Although the majority of the anthraquinone modified TBA analogues showed a decrease in clotting times in a fibrinogen clotting assay when compared to TBA, modified aptamers containing a 1,8‐disubstituted anthraquinone linker replacing G8 or T9 in the TGT loop showed improved anticoagulant activities. Molecular modeling studies explained the increased thermal melting temperatures of anthraquinone‐modified G‐quadruplexes.

5-Hydroxymethyl-2'-deoxyuridine residues in the thrombin binding aptamer: investigating anticoagulant activity by making a tiny chemical modification.

We report an investigation into analogues of the thrombin binding aptamer (TBA). Individual thymidines were replaced by the unusual residue 5-hydroxymethyl-2'-deoxyuridine (hmU). This differs from the canonical thymidine by a hydroxyl group on the 5-methyl group. NMR and CD data clearly indicate that all TBA derivatives retain the ability to fold into the "chair-like" quadruplex structure. The presence of the hmU residue does not significantly affect the thermal stability of the modified aptamers compared to the parent, except for analogue H9, which showed a marked increase in melting temperature. Although all TBA analogues showed decreased affinities to thrombin, H3, H7, and H9 proved to have improved anticoagulant activities. Our data open up the possibility to enhance TBA biological properties, simply by introducing small chemical modifications.

A Mini-Library of TBA Analogues Containing 3′-3′ and 5′-5′ Inversion of Polarity Sites

Several researches have been devoted to structure-activity relationship and to post-SELEX modifications of the thrombin binding aptamer (TBA), one of the first aptamers discovered by the SELEX methodology. However, no studies on TBA dealing with the effects of introduction of inversion of polarity sites have been reported yet. In this frame, we have undertaken the synthesis and the study of a mini-library composed of several TBA analogues containing a 3′-3′ or a 5′-5′ inversion of polarity site at different positions into the sequence. Particularly, in this article, we present preliminary results about their structural and biological properties.