Locking up the AS1411 Aptamer with a Flanking Duplex: Towards an Improved Nucleolin-Targeting

We have designed AS1411-N6, a derivative of the nucleolin (NCL)-binding aptamer AS1411, by adding six nucleotides to the 50'-end that are complementary to nucleotides at the 3'-end forcing it into a stem-loop structure. We evaluated by several biophysical techniques if AS1411-N6 can adopt one or more conformations, one of which allows NCL binding. We found a decrease of polymorphism of G-quadruplex (G4)-forming sequences comparing to AS1411 and the G4 formation in presence of K+ promotes the duplex folding. We also studied the binding properties of ligands TMPyP4, PhenDC3, PDS, 360A, and BRACO-19 in terms of stability, binding, topology maintenance of AS1411N6, and NCL recognition. The melting experiments revealed promising stabilizer effects of PhenDC3, 360A, and TMPyP4, and the affinity calculations showed that 360A is the most prominent affinity ligand for AS1411-N6 and AS1411. The affinity determined between AS1411-N6 and NCL denoting a strong interaction and complex formation was assessed by PAGE in which the electrophoretic profile of AS1411-N6 showed bands of the dimeric form in the presence of the ligands and NCL.

Automated summary

AS1411-N6, a derivative of the nucleolin-binding aptamer AS1411, adopts a stem-loop structure by adding complementary nucleotides. The formation of G4 sequences decreases compared to AS1411, and G4 formation is promoted by K+. Ligands such as PhenDC3, 360A, and TMPyP4 show promising stabilizer effects on AS1411-N6.