Formation and Dissociation of the Interstrand i-Motif by the Sequences d(X_nC₄Y_m) Monitored with Electrospray Ionization Mass Spectrometry

Abstract

Abstract. Formation and dissociation of the interstrand i-motifs by DNA with the sequence d(X n C 4 Y m ) (X and Y represent thymine, adenine, or guanine, and n, m range from 0 to 2) are studied with electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), and UV spectrophotometry. The ion complexes detected in the gas phase and the melting temperatures (T m ) obtained in solution show that a non-C base residue located at 5′ end favors formation of the four-stranded structures, with T > A > G for imparting stability. Comparatively, no rule is found when a non-C base is located at the 3′ end. Detection of penta-and hexa-stranded ions indicates the formation of i-motifs with more than four strands. In addition, the i-motifs seen in our mass spectra are accompanied by single-, double-, and triple-stranded ions, and the trimeric ions were always less abundant during annealing and heat-induced dissociation process of the DNA strands in solution (pH=4.5). This provides a direct evidence of a strand-by-strand formation and dissociation pathway of the interstrand i-motif and formation of the triple strands is the rate-limiting step. In contrast, the trimeric ions are abundant when the tetramolecular ions are subjected to collision-induced dissociation (CID) in the gas phase, suggesting different dissociation behaviors of the interstrand i-motif in the gas phase and in solution. Furthermore, hysteretic UV absorption melting and cooling curves reveal an irreversible dissociation and association kinetic process of the interstrand i-motif in solution.

Assembly of supramolecular DNA complexes containing both G-quadruplexes and i-motifs by enhancing the G-repeat-bearing capacity of i-motifs

“…MS spectra showed abundant tetramolecular ions of d(TC 5 T) (including [4M] 5− at m/z 1592.69 and [4M] 4− at m/z 1991.12) in pH 4.5 NH 4 OAc buffer, containing 50 mM NH 4 + . These tetrameric species disappeared when the solution pH was adjusted to 9.0, indicating the formation and dissociation of tetrameric i-motifs (Supplementary Figure S1a and b) in accordance with our previous results (37). …”

“…Specifically, the tetrameric ions ([4M] 7− at m/z 1499.36, [4M] 6− at m/z 1749.36 and [4M] 5− at m/z 2099.42) in the MS spectra (Supplementary Figure S2a and b) and the CD maximum at 288 nm and minimum at 265 nm (Supplementary Figure S2c) all indicate the presence of i-motif tetramers (47) and that they are more prominent in d(TGTC 5 T). We have previously demonstrated that short C-rich strands facilely form duplexes with C•CH + pairs under acidic condition and can be observed along with tetrameric i-motifs (37). Consequently, the low-abundance duplex ions observed here are mainly C•CH + -containing duplexes (C-duplex) since there are insufficient complementary G and C bases between two oligonucleotides to create WC duplexes (Supplementary Figure S2a and b).…”

“…The samples were infused directly into ion source at a flow rate of 3 μl min −1 . Optimal soft ionization conditions for quadruplex structures were found using the oligomer d(G 4 T 4 G 4 ) to tune the instrument to produce abundant [(dG 4 T 4 G 4 ) 2 + 3NH 4 ] 5− ions as described previously (37,40). These conditions also produce good spectra for i-motifs (37).…”

“…Optimal soft ionization conditions for quadruplex structures were found using the oligomer d(G 4 T 4 G 4 ) to tune the instrument to produce abundant [(dG 4 T 4 G 4 ) 2 + 3NH 4 ] 5− ions as described previously (37,40). These conditions also produce good spectra for i-motifs (37). Before the MS analysis samples were diluted with an equal volume of methanol solution 60% in water in order to obtain a good spray (41).…”

“…Thymines are used as the ‘spacers’ because they stabilize i-motif structures either in the middle or at the end of C-rich strands (37,38), and they are less prone to stacking or pairing with guanines (39). The rational investigation of C-runs with spacer and flanking residues has directed us to model i-motifs that can bear longer G-runs for the formation of G4s.…”