Single-stranded DNA structural diversity: TAGGGT from monomers to dimers to tetramer formation

Abstract

Rationale: DNA quadruplex structures have emerged as novel drug targets due to their role in preventing abnormal gene transcription and maintaining telomere stability. Trapped Ion Mobility Spectrometry–Mass Spectrometry (TIMS–MS), combined with theoretical modeling, is a powerful tool for studying the kinetic intermediates of DNA complexes formed in solution and interrogated in the gas phase after desolvation. Methods: A TAGGGT ssDNA sequence was purchased and studied in 10 mM ammonium acetate using nanospray electrospray ionization (nESI)-TIMS-MS in positive and negative ion mode. Collisional cross section (CCS) profiles were measured using internal calibration (Tune Mix). Theoretical structures were proposed based on molecular dynamics, charge location and geometry optimization for the most intense IMS bands based on the number of TAGGGT units, adduct form and charge states. Results: A distribution of monomeric, dimeric and tetrameric TAGGGT structures were formed in solution and separated in the gas phase based on their mobility and m/z value (e.g., [M + 2H]+2, [2M + 3H]+3, [M – 2H]−2, [2M – 3H]−3, [4M + 4H]+4, [4M + 3H + NH4]+4, [4M + 2H + 2NH4]+4 and [4M + H + 3NH4]+4). The high mobility resolution of the TIMS-MS analyzer permitted the observation of multiple CCS bands per molecular ion form. Comparison with theoretical candidate structures suggests that monomeric TAGGGT species are stabilized by A-T and G+-G interactions, with the size of the conformer influenced by the proton location. In the case of the TAGGGT quadruplex, the protonated species displayed a broad CCS distribution, while six discrete conformers were stabilized by the presence of ammonium ions (n = 1–3). Conclusions: This is the first observation of multiple conformations of TAGGGT complexes (n = 1, 2 and 4) in 10 mM ammonium acetate. Candidate structures with intramolecular interactions of the form of G+-G and traditional A-T base pairing agreed with the experimental trends. Our results demonstrate the structural diversity of TAGGGT monomers, dimers and tetramers in the gas phase beyond the previously reported solution structure, using 10 mM ammonium acetate to replicate biological conditions.