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

Abstract

A quaternary ammonium butylsulfonyl phosphoramidate group (N+) was designed to replace all the phosphates in a G-rich oligodeoxynucleotide d(TG₄ T), resulting in a formally charge-neutral zwitterionic N+TG₄ T sequence. We evaluated the effects of N+phosphate modifications on the structural, thermodynamic and kinetic properties of the parallel G-quadruplexes (G4) formed by TG₄ T and compared them to the properties of the recently published phosphoryl guanidine d(TG₄ T) (PG-TG₄ T). Using size-exclusion chromatography, we established that, unlike PG-TG₄ T, which exists as a mixture of complexes of different molecularity in solution, N+TG₄ T 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(TG₄ T)]₄ . The initial stage of assembly of N+TG₄ T proceeded faster in the presence of Na⁺ than K⁺ ions and, similarly to PG-TG₄ T, was independent of the salt concentration. However, after complex formation exceeded 75 %, N+TG₄ T in solution with Na⁺ showed slower association than with K⁺ . N+TG₄ T 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(TG₄ T). 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+TG₄ T]₄ . The N+ modification makes d(TG₄ T) fully resistant to enzymatic digestion, which could be useful for intracellular application of N+-modified DNA or RNA.

Keywords: DNA; G-quadruplexes; enzymatic stability; kinetics; modified phosphate; thermal stability.