期刊
MOLECULES
卷 27, 期 20, 页码 -出版社
MDPI
DOI: 10.3390/molecules27206781
关键词
G-quadruplex aptamer; ligands; aptamer-ligand interactions; biophysical techniques
资金
- FCT/MCT [UIDB/00709/2020, POCI-01-0145-FEDER-022122, ROTEIRO/0031/2013-PINFRA/22161/2016]
- FEDER through COMPETE 2020
- POCI
- PORL
- PIDDAC
- Inserm
- INCa G4 Access
- ANR [ANR-20-CE12-0023]
- Symbit project from the ERDF [CZ.02.1.01/0.0/0.0/15_003/0000477]
- FCT [UIDP/00709/2020, CENTRO-01-0145-FEDER-181235]
In this study, the formation and stability of G-quadruplex structure of a G-rich DNA aptamer AT11-L2 were explored under different experimental conditions, along with its interactions with various G4 ligands and nucleolin (NCL). The results showed that AT11-L2 adopted different topologies depending on the KCl concentration, and the addition of ligands significantly stabilized the G-quadruplex structure. Furthermore, a specific interaction between AT11-L2 G-quadruplex and NCL was identified, and co-localization of these two molecules was observed in certain cell lines.
In this work we explore the structure of a G-rich DNA aptamer termed AT11-L2 (TGGTGGTGGTTGTTGTTGGTGGTGGTGGT; derivative of AT11) by evaluating the formation and stability of G-quadruplex (G4) conformation under different experimental conditions such as KCl concentration, temperature, and upon binding with a variety of G4 ligands (360A, BRACO-19, PDS, PhenDC3, TMPyP4). We also determined whether nucleolin (NCL) can be a target of AT11-L2 G4. Firstly, we assessed by circular dichroism, UV and NMR spectroscopies the formation of G4 by AT11-L2. We observed that, for KCl concentrations of 65 mM or less, AT11-L2 adopts hybrid or multiple topologies. In contrast, a parallel topology predominates for buffer containing 100 mM of KCl. The T-m of AT11-L2 in 100 mM of KCl is 38.9 degrees C, proving the weak stability of this sequence. We also found that upon titration with two molar equivalents of 360A, BRACO-19 and PhenDC3, the G4 is strongly stabilized and its topology is maintained, while the addition of 3.5 molar equivalents of TMPyP4 promotes the disruption of G4. The K-D values between AT11-L2 G4, ligands and NCL were obtained by fluorescence titrations and are in the range of mu M for ligand complexes and nM when adding NCL. In silico studies suggest that four ligands bind to the AT11-L2 G4 structure by stacking interactions, while the RBD1,2 domains of NCL interact preferentially with the thymines of AT11-L2 G4. Finally, AT11-L2 G4 co-localized with NCL in NCL-positive tongue squamous cell carcinoma cell line.
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