Thermal and pH Stabilities of i‐DNA: Confronting in vitro Experiments with Models and In‐Cell NMR Data

Abstract

Recent studies indicate that i-DNA, afour-stranded cytosine-richD NA also knowna st he i-motif,i sa ctually formed in vivo;however,asystematic study on sequence effects on stability has been missing. Herein, an unprecedented number of different sequences ( 271) bearing four runs of 3-6 cytosines with different spacer lengths has been tested. While i-DNAstability is nearly independent on total spacer length, the central spacer plays as pecial role on stability.S tability also depends on the length of the C-tracts at both acidic and neutral pHs.T his study provides ag lobal picture on i-DNAs tability thanks to the large size of the introduced data set;i tr eveals unexpected features and allows to conclude that determinants of i-DNAstability do not mirror those of G-quadruplexes.Our results illustrate the structural roles of loops and C-tracts on i-DNAs tability,c onfirm its formation in cells,a nd allow establishing rules to predict its stability.

Effects of Sequence and Base Composition on the CD and TDS Profiles of i‐DNA

“…This is in agreement with the pH and thermal stabilities reported in the companion paper. [24] Then, the CD spectra of these 40 samples were normalized by the signal intensity at 264 nm (Figure S7) and the resulting spectra were submitted to PCA. As observed for the previously normalized 180 CD spectra, the samples having al onger central spacer are characterized by al ower ratio between the intensity of the bands at 288 and 264 nm.…”

“…Ther eason of this can be explained taking into account the structural requisites for the formation of an i-DNAstructure. In particular,t he central spacer is often responsible for the formation of aloop that spans the major groove of the i-DNA (see companion paper [24] )and that this loop requires at least 3 residues. [23] If the central spacer contains al ower number of residues,the i-DNAstructure can be formed in any case using some Cs of the adjacent C-tracts.Inthis case,alower number of C:C + pairs are formed and the structure will contain some unpaired Cs.O bviously,t he CD spectrum of the sample will proportionally contain information about both paired and unpaired Cs in the structure.Asalready mentioned, the C:C + base pairs in the stem provide aCDspectrum having anegative and apositive band at 264 and 288 nm, respectively,while cytosines not involved in the pairing are characterized by aC Ds pectrum having only ap ositive band centered around…”

“…[23] In the companion paper of this investigation, some of us have explored the simultaneous variation of both C-tract length and loop arrangements. [24] In particular,b ya nalyzing af irst set of 180 different DNAs equences (Table S1), the contribution of C-tracts and spacer length on i-DNAstability was explored. Thegeneral formula of the majority of i-DNAforming oligos employed in that study is C (3)(4)(5)(6) T (1)(2)(3)(4)(5)(6) C (3)(4)(5)(6) T (1)(2)(3)(4)(5)(6) C (3)(4)(5)(6) T (1)(2)(3)(4)(5)(6) C (3)(4)(5)(6) .W ei nvestigated sequences with cytosine tracts of equal length made of 3upto6Cs and three spacers made of 1u pt o6thymines,i ns uch ac ombination to have from 4t o1 2t otal thymines.T he samples were named according to the following rationale:a"T" was used as prefix because the three spacers were composed of thymines only; three consecutive numbers were used to describe the lengths of the three spacers in the 5' to 3' direction;w hile the suffix referred to the length of the C-tracts ("-3", "-4", "-5" or "-6"' for C 3 , C 4 , C 5 ,a nd C 6 ,r espectively).…”

“…In particular, ultraviolet (UV) and circular dichroism (CD) spectroscopies at different pH and temperature values were used to evaluate the thermal and pH stability of each i-DNA, as described in the companion paper. [24] In the present work, we analyze the CD spectral profiles and UV thermal difference spectra (TDS) of these 255 samples by means of multivariate data analysis to detect hidden but potentially informative bands in the spectra of the i-DNA-forming sequences.I ndeed, to our knowledge,o nly the well-known i-DNAc haracteristic bands of the CD (positive at 288 and negative at 264 nm) and TDS (positive at 240 and negative at 295 nm) spectra have been considered so far, limiting the informative power of the CD and UV-absorbance spectroscopies.…”

Insights into I-motif stabilization by high resolution primer extension assays: Its strengths and limitations

“…The most important factor is so far the pH of the environment, in which iM formation is heavily dependent on. Only slight pH changes affect iM folding and stability (Cheng et al, 2021a;Cheng et al, 2021b;Mergny et al, 1995). Other factors, such as temperature, ionic strength and crowding conditions have also positive/negative impact on the stability of the iM.…”