Secondary Structure Folding

The functionality of the GI-Nc depends on the abilities of the G-quadruplex and i-motif to maintain and change their conformations after endocytosis; it is crucial to confirm their folded structures under different environmental conditions. 

In this experiment, GI, GcI, and IcG strands are solubilized in buffers of varying pH and potassium ion concentration before circular dichroism is used to visualize their secondary structures.


G-quadruplexes are four-stranded helical structures formed from guanine-rich regions of DNA in the presence of monovalent cations such as sodium or potassium [1]. I-motifs are also four-stranded DNA structures formed from cytosine-rich regions in acidic conditions due to the hemi-protonation of cytosine [2]. These two structures, in the context of the GI-Nc, are responsible for the binding and release of doxorubicin, and the binding of zinc phthalocyanine.

In order to assess their formation in different conditions three different sequences were used: the GI, containing the G-quadruplex (G) and i-motif (I); the GcI, containing only the G-quadruplex; and the IcG, containing only the i-motif. We anticipate the GI would alter conformation with changes in both pH and cation concentration. The GcI was expected to fold differently only with changes in cation concentration while the IcG was expected to fold differently only with changes in pH.

Image 1: Folded i-motif (Day et. al.) (top) and G-quadruplex (Rhodes et. al.) (bottom) sequences


To evaluate the conformations of the G-quadruplex and i-motif under different pH and potassium conditions.

Techniques Used

Circular Dichroism

Circular dichroism (CD) is a spectroscopic method which utilizes the differential absorption of polarized light to study the structure of optically active chiral molecules, such as DNA. Different DNA molecules preferentially absorb either right or left circularly polarized light. This generates elliptically polarized light, which we can measure and use to characterize the structure of folded DNA [3].


Phosphate buffer base from “Duplex Formation and Verification” was adjusted to pHs of 7.2 and 5.5 using HCl. From the two buffers, an aliquot was taken and potassium chloride was added to a final concentration of 50mM. Hence, a set of four buffers was prepared:

  • pH 7.2, K+
  • pH 7.2, K-
  • pH 5.5, K+
  • pH 5.5, K-

GI, GcI, and IcG sequences were diluted to 100 µM accordingly:

pH 7.2 w/ K+

pH 7.2 w/o K+

pH 5.5 w/ K+

pH 5.5 w/o K+












These stock solutions were diluted to 20 µM before running in a Jasco CD J-815 spectrophotometer. The lower end of the spectra analyzed was set at 230nm and the higher end at 350nm with an interval of 1nm; the maximum y-value was 22.37 and the minimum was -10.98. More details can be found on our raw data spreadsheet below.


Image 2: Graphs generated from circular dichroism readings comparing (A) GI and GcI in the presence of potassium at different pHs, (B) GI at different concentrations of potassium and pHs, and (C) GI and IcG at pH 5.5 under different potassium concentrations

In Image 2, graph A, there is a shift in peak location for the GI sequence at two different pH’s, but no shift for the GcI sequence. The change in pH does not impact the secondary structure of GcI. 

Meanwhile, graph B shows that the GI is impacted by changes in both pH and potassium ion concentration. There is a peak shift between sequences at pH 7.2 (green and pink) and sequences at pH 5.5 (purple and red). The addition of potassium ions results in the formation of a smaller peak at approximately 260 nm.

In graph C, all four main peaks align, but the addition of potassium to the GI sequence results in the generation of a smaller peak at 260 nm. This ascertains that the IcG is unaffected by changes in potassium concentration, but the GI is affected.


It is apparent that the GI alters conformation with changes in either pH or potassium concentration, but the GcI and IcG fold differently only with differences in potassium concentration and pH respectively. This is demonstrated by the GI and GcI sequences peaking at 260 nm in the presence of potassium, denoting G-quadruplex formation, and the GI and IcG sequences peak shifts when the pH drops to 5.5 to showcase i-motif formation. However, the peaks generated by the GcI and IcG do not change when the pH or the potassium concentration is altered respectively.

  1. D. Rhodes, & Lipps, H. J. (2015). G-quadruplexes and their regulatory roles in biology, Nucleic Acids Research, 43(18), 8627–8637.
  2. Abou Assi, H., Garavís, M., González, C., & Damha, M. J. (2018). i-Motif DNA: structural features and significance to cell biology. Nucleic acids research, 46(16), 8038–8056. doi:10.1093/nar/gky735
  3. Libretexts. (2019, September 30). Circular Dichroism. Retrieved from,

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