I-motifs are four-stranded non-canonical DNA structures formed by cytosine-rich
sequences and stabilised by hemiprotonated cytosine-cytosine+ base pairs. They have
biological functions relating to the transcription of genes, including many oncogenes,
meaning the reliable detection of i-motifs is essential, yet it is currently largely based on
the iMab antibody and its newer alternative, the iMbody nanobody. Recent studies,
however, have raised doubts about their structural specificity. In this thesis we
investigated the interaction between the iMbody nanobody and the model oligonucleotide
containing TCGCTCCC repeats, named VK2c, under conditions in which the
oligonucleotide is folded into an i-motif and conditions under which it is unfolded. The
structure of the DNA was monitored by circular dichroism (CD) spectroscopy, and the
binding was characterised thermodynamically by isothermal titration calorimetry (ITC),
both at pH 5 in acetate buffer and at pH 7 in phosphate buffer. A pH titration carried out
in both directions confirmed that the oligonucleotide adopts the characteristic i-motif
structure at pH 5 and is predominantly unfolded at pH 7, so that the two selected pH
values represent the two states well. ITC revealed exothermic, enthalpy driven binding at
both pH values, with a dissociation constant of 7.7 nM at pH 5 and 471 nM at pH 7, with
an apparent stoichiometry about three times higher at pH 7. The CD spectra of the
nanobody and oligonucleotide complexes were not additive, with a considerably larger
deviation at pH 5 than at pH 7; in the presence of excess nanobody the spectrum did not
shift towards the unfolded form, but in the opposite direction, towards longer
wavelengths. The iMbody nanobody therefore recognizes the i-motif preferentially but
not exclusively, which makes iMbody alone an unreliable tool for the structural detection
of i-motifs.
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