The Importance of Phosphates for DNA G‐Quadruplex Formation: Evaluation of Zwitterionic G‐Rich Oligodeoxynucleotides

A quaternary ammonium butylsulfonyl phosphoramidate group (N+) was designed to replace all the phosphates in a G‐rich oligodeoxynucleotide d(TG4T), resulting in a formally charge‐neutral zwitterionic N+TG4T sequence. We evaluated the effects of N+phosphate modifications on the structural, thermodynamic and kinetic properties of the parallel G‐quadruplexes (G4) formed by TG4T and compared them to the properties of the recently published phosphoryl guanidine d(TG4T) (PG‐TG4T). Using size‐exclusion chromatography, we established that, unlike PG‐TG4T, which exists as a mixture of complexes of different molecularity in solution, N+TG4T forms an individual tetramolecular complex. In contrast to PG modifications that destabilized G4s, the presence of N+ modifications increased thermal stability relative to unmodified [d(TG4T)]4. The initial stage of assembly of N+TG4T proceeded faster in the presence of Na+ than K+ions and, similarly to PG‐TG4T, was independent of the salt concentration. However, after complex formation exceeded 75 %, N+TG4T in solution with Na+showed slower association than with K+. N+TG4T could also form G4s in solution with Li+ions at a very low strand concentration (10 μM); something that has never been reported for the native d(TG4T). Charge‐neutral PG‐G4s can invade preformed native G4s, whereas no invasion was observed between N+and native G4s, possibly due to the increased thermal stability of [N+TG4T]4. The N+ modification makes d(TG4T) fully resistant to enzymatic digestion, which could be useful for intracellular application of N+‐modified DNA or RNA.