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. 2019 Jul 26;47(13):6590-6605.
doi: 10.1093/nar/gkz522.

Study of conformational transitions of i-motif DNA using time-resolved fluorescence and multivariate analysis methods

Sanae Benabou  1 , Cyril Ruckebusch  2 , Michel Sliwa  2 , Anna Aviñó  3 , Ramon Eritja  3 , Raimundo Gargallo  1 , Anna de Juan  1
Affiliations

Study of conformational transitions of i-motif DNA using time-resolved fluorescence and multivariate analysis methods

Sanae Benabou et al. Nucleic Acids Res. .

Abstract

Recently, the presence of i-motif structures at C-rich sequences in human cells and their regulatory functions have been demonstrated. Despite numerous steady-state studies on i-motif at neutral and slightly acidic pH, the number and nature of conformation of this biological structure are still controversial. In this work, the fluorescence lifetime of labelled molecular beacon i-motif-forming DNA sequences at different pH values is studied. The influence of the nature of bases at the lateral loops and the presence of a Watson-Crick-stabilized hairpin are studied by means of time-correlated single-photon counting technique. This allows characterizing the existence of several conformers for which the fluorophore has lifetimes ranging from picosecond to nanosecond. The information on the existence of different i-motif structures at different pH values has been obtained by the combination of classical global decay fitting of fluorescence traces, which provides lifetimes associated with the events defined by the decay of each sequence and multivariate analysis, such as principal component analysis or multivariate curve resolution based on alternating least squares. Multivariate analysis, which is seldom used for this kind of data, was crucial to explore similarities and differences of behaviour amongst the different DNA sequences and to model the presence and identity of the conformations involved in the pH range of interest. The results point that, for i-motif, the intrachain contact formation and its dissociation show lifetimes ten times faster than for the open form of DNA sequences. They also highlight that the presence of more than one i-motif species for certain DNA sequences according to the length of the sequence and the composition of the bases in the lateral loop.

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Figures

Figure 1.
Figure 1.
(A) Cytosine-protonated cytosine base pair; (B) Schematic process of the unfolding of the intramolecular i-motif structures; (C) Sequences studied in this work. Underlined bases are those that could be involved in the formation of C+ · C base pairs; (D) Hypothetical scheme of the intramolecular i-motif structures adopted by the sequences proposed to be studied.
Figure 2.
Figure 2.
Scheme of data structure for PCA analysis.
Figure 3.
Figure 3.
Fluorescence emission spectra (excitation 540 nm) of F-T10 (A) and F-TT-Q (B) sequences in solutions at different pH values. DNA concentration: 1 μM at 25°C.
Figure 4.
Figure 4.
Results of the mathematical fitting of the decay traces of F-T10 sequence. (A) Raw fluorescence decay, fitted decay and IRF decay at pH 5.0. (B) Residuals. (C) Autocorrelation of residuals. (D) Raw and fitted data of fluorescence decay profiles as a function of pH. (E) Distribution of the fractional amplitude of each lifetime contribution as a function of the pH of the control sequence F-T10.
Figure 5.
Figure 5.
Raw and fitted traces at different pH of F-TT-Q (A) and F-AA-Q (B) sequences. Distribution of the fractional amplitude of each lifetime with the pH of the F-TT-Q (C) and F-AA-Q (D) sequences.
Figure 6.
Figure 6.
Raw and fitted traces at different pH of the F-nmyc-Q (A) and F-nmycM-Q (B) sequences. Distribution of the fractional amplitude of each lifetime with the pH of the F-nmyc-Q (C) and F-nmycM-Q (D) sequences.
Figure 7.
Figure 7.
Fluorescence decay traces of DNA sequences and related PCA results. (A) Raw data of fluorescence decays of control (F-T10) and DNA sequences (F-T10, F-TT-Q, F-AA-Q, F-nmyc-Q and F-nmycM-Q) at different pH values. (B) Scores plot, (C) Loading of PC1 and (D) Loading of PC2.
Figure 8.
Figure 8.
Local PCA of F-TT-Q, F-AA-Q, F-nmyc-Q and F-nmycM-Q sequences at pH 6, 5 and 4 classifying by sequences. (A) Scores of PC1 versus PC2, (B) Scores of PC1 versus PC2 versus PC3, (C) Loading of PC1, (D) Loading of PC2 and (E) Loading of PC3.
Figure 9.
Figure 9.
MCR-ALS analysis of the decays of F-TT-Q and F-AA-Q sequences. (A) Pure spectra of F-TT-Q, (B) Concentration profiles of F-TT-Q, (C) Pure spectra of F-AA-Q and (D) Concentration profiles of F-AA-Q. Every point in concentration profiles represents the scatter in concentration at a certain pH and emission wavelength for the different decay traces acquired in the same experimental conditions.
Figure 10.
Figure 10.
MCR-ALS analysis of the decays of F-nmyc-Q and F-nmycM-Q sequences. (A) Pure spectra of F-TT-Q, (B) Concentration profiles of F-TT-Q, (C) Pure spectra of F-AA-Q and (D) Concentration profiles of F-AA-Q.
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