Further evidence for the presence of pre-existing structure in the absence of ions comes from the absolute frequencies of the guanine carbonyl stretches. The frequency of nitrogenous base carbonyl stretches is sensitive to DNA conformation and is often used to determine DNA secondary structure. The guanine carbonyl stretching frequency is observed at 𝜔 𝛼 = 1671 -1688 cm -1 (GCO II), which is higher than a single strand guanine typically observed between 1660-1673 cm -1 , 27 which could suggest the presence of a pre-existing DNA Structure. In fact, circular dichroism measurements of MycL1, a similar G4-forming sequence to Myc2345 (see Figure in the Supplementary Information for sequence comparison), also suggest that some structure can be present in a G4 DNA sequence, even in the absence of potassium ions. Given the above indications that some structure resembling a G-quartet is present, even in the absence of potassium ions, the obvious question is whether the entire G4 structure may be pre-formed. Evidence for the absence of a fully stacked G4 complex in the absence of potassium ions comes from four observations. Firstly, in the geometry optimization that we did, a stacked quartet without potassium ions was not found to be energetically stable. This structure is unfavourable due to the repulsive forces between the 12 carbonyl oxygens. As this is a purely enthalpic effect and as entropic forces will only drive the structure apart in a real solvated system, we, therefore, consider the presence of a G4-like structure in the absence of potassium ions to be unlikely. The three other pieces of evidence for the absence of stacked G-quartets (the entire G4 structure) in the absence of potassium ions are the redshifts of particular frequencies observed when the potassium ions are added, the intensity changes for multiple peaks upon the addition of potassium and the formation of new spectral features already known to be associated only with fully stacked G4. We proceed to discuss these in more detail below.