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 therma...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-10-01
|
| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/24/21/15529 |
| _version_ | 1797631861254520832 |
|---|---|
| author | Antonella Virgilio Daniela Benigno Carla Aliberti Valentina Vellecco Mariarosaria Bucci Veronica Esposito Aldo Galeone |
| author_facet | Antonella Virgilio Daniela Benigno Carla Aliberti Valentina Vellecco Mariarosaria Bucci Veronica Esposito Aldo Galeone |
| author_sort | Antonella Virgilio |
| collection | DOAJ |
| description | 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 (G<sup>Br</sup>) in syn positions, while in the anti-positions, locked nucleic acid guanosine (G<sup>LNA</sup>) in the analogue TBABL, 2’-O-methylguanosine (G<sup>OMe</sup>) in TBABM, and 2’-F-riboguanosine (G<sup>F</sup>) 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 (T<sub>m</sub> 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. |
| first_indexed | 2024-03-11T11:29:45Z |
| format | Article |
| id | doaj.art-6a75b8c3905a41229b7d777978f38eb0 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T11:29:45Z |
| publishDate | 2023-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-6a75b8c3905a41229b7d777978f38eb02023-11-10T15:04:17ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-10-0124211552910.3390/ijms242115529Improving the Biological Properties of Thrombin-Binding Aptamer by Incorporation of 8-Bromo-2′-Deoxyguanosine and 2′-Substituted RNA AnaloguesAntonella Virgilio0Daniela Benigno1Carla Aliberti2Valentina Vellecco3Mariarosaria Bucci4Veronica Esposito5Aldo Galeone6Department of Pharmacy, University of Naples Federico II, 80131 Napoli, ItalyDepartment of Pharmacy, University of Naples Federico II, 80131 Napoli, ItalyDepartment of Pharmacy, University of Naples Federico II, 80131 Napoli, ItalyDepartment of Pharmacy, University of Naples Federico II, 80131 Napoli, ItalyDepartment of Pharmacy, University of Naples Federico II, 80131 Napoli, ItalyDepartment of Pharmacy, University of Naples Federico II, 80131 Napoli, ItalyDepartment of Pharmacy, University of Naples Federico II, 80131 Napoli, ItalyThrombin-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 (G<sup>Br</sup>) in syn positions, while in the anti-positions, locked nucleic acid guanosine (G<sup>LNA</sup>) in the analogue TBABL, 2’-O-methylguanosine (G<sup>OMe</sup>) in TBABM, and 2’-F-riboguanosine (G<sup>F</sup>) 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 (T<sub>m</sub> 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.https://www.mdpi.com/1422-0067/24/21/15529G-quadruplexthrombin binding aptamer8-bromo-2′-deoxyguanosineRNA analoguesanticoagulant activity |
| spellingShingle | Antonella Virgilio Daniela Benigno Carla Aliberti Valentina Vellecco Mariarosaria Bucci Veronica Esposito Aldo Galeone Improving the Biological Properties of Thrombin-Binding Aptamer by Incorporation of 8-Bromo-2′-Deoxyguanosine and 2′-Substituted RNA Analogues International Journal of Molecular Sciences G-quadruplex thrombin binding aptamer 8-bromo-2′-deoxyguanosine RNA analogues anticoagulant activity |
| title | Improving the Biological Properties of Thrombin-Binding Aptamer by Incorporation of 8-Bromo-2′-Deoxyguanosine and 2′-Substituted RNA Analogues |
| title_full | Improving the Biological Properties of Thrombin-Binding Aptamer by Incorporation of 8-Bromo-2′-Deoxyguanosine and 2′-Substituted RNA Analogues |
| title_fullStr | Improving the Biological Properties of Thrombin-Binding Aptamer by Incorporation of 8-Bromo-2′-Deoxyguanosine and 2′-Substituted RNA Analogues |
| title_full_unstemmed | Improving the Biological Properties of Thrombin-Binding Aptamer by Incorporation of 8-Bromo-2′-Deoxyguanosine and 2′-Substituted RNA Analogues |
| title_short | Improving the Biological Properties of Thrombin-Binding Aptamer by Incorporation of 8-Bromo-2′-Deoxyguanosine and 2′-Substituted RNA Analogues |
| title_sort | improving the biological properties of thrombin binding aptamer by incorporation of 8 bromo 2 deoxyguanosine and 2 substituted rna analogues |
| topic | G-quadruplex thrombin binding aptamer 8-bromo-2′-deoxyguanosine RNA analogues anticoagulant activity |
| url | https://www.mdpi.com/1422-0067/24/21/15529 |
| work_keys_str_mv | AT antonellavirgilio improvingthebiologicalpropertiesofthrombinbindingaptamerbyincorporationof8bromo2deoxyguanosineand2substitutedrnaanalogues AT danielabenigno improvingthebiologicalpropertiesofthrombinbindingaptamerbyincorporationof8bromo2deoxyguanosineand2substitutedrnaanalogues AT carlaaliberti improvingthebiologicalpropertiesofthrombinbindingaptamerbyincorporationof8bromo2deoxyguanosineand2substitutedrnaanalogues AT valentinavellecco improvingthebiologicalpropertiesofthrombinbindingaptamerbyincorporationof8bromo2deoxyguanosineand2substitutedrnaanalogues AT mariarosariabucci improvingthebiologicalpropertiesofthrombinbindingaptamerbyincorporationof8bromo2deoxyguanosineand2substitutedrnaanalogues AT veronicaesposito improvingthebiologicalpropertiesofthrombinbindingaptamerbyincorporationof8bromo2deoxyguanosineand2substitutedrnaanalogues AT aldogaleone improvingthebiologicalpropertiesofthrombinbindingaptamerbyincorporationof8bromo2deoxyguanosineand2substitutedrnaanalogues |