The C:C⁺ base pair

The hemi-protonated C:C⁺ base pairs are the key interactions for i-motif stability. The three hydrogen bonds of the C:C⁺ base pair confer a high stability. Computer calculations indicate that the base-pairing energy (BPE) for the C:C⁺ base pair is 169.7 kJ/mol, higher than BPEs of canonical Watson-Crick G·C (96.6 kJ/mol) and neutral C·C (68.0 kJ/mol) (26). The central hydrogen bonding in a hemiprotonated C:C⁺ (N3···H···N3) base pair has been described as a double-well potential where the proton delocalizes/oscillates between the two wells (27). Leroy et al. estimated the proton-transfer rate to be 8 × 10⁴ s⁻¹ (28). The NMR structural study of an intramolecular telomeric i-motif (PDB code: 1ELN) showed that the C:C⁺ base pairs are planar and the N3–N3 distance is around 2.6–2.8 Å (29). Most importantly, protonation at the N3 position produces a positively charged base pair. NMR spectroscopy and computational analyses performed by Lieblein et al. suggested that the N3···H⁺···N3 bonds possess an asymmetric double-well potential and that the proton in one C:C⁺ base pair tends to adopt a position that leads to the largest distance with respect to the proton of neighboring C:C⁺ base pair (27).

The effect of chemical modifications in the C:C⁺ base pairs has been investigated in different contexts. Wadkins et al. indicated that a cytosine modification might have different effects on i-motif stability depending on the environmental conditions (30). For instance, substitution of cytosine by its halogenated analogues (Figure ​(Figure2A),2A), such as 5-fluoro, 5-bromo and 5-iodo derivatives, increases stability of i-motifs at acidic conditions (31). Furthermore, cytosine methylation at position 5 leads to an increase in the pH of mid transition (i.e. pH_T) and T_m of i-motif structures (Δ pH_T = +0.11 for two sequences containing two 5-methylcytosine substitutions), while hydroxymethylation leads to a decrease in pH_T (ΔpH_T = –0.2) and T_m (Figure ​(Figure2A)2A) (32).

 

Figure 2.

Nucleoside modifications introduced in i-motif structures. (A) Nucleobase modifications. (B) Sugar modifications and (C) A nucleoside analogue possessing both sugar and nucleobase modifications. d: deoxyribose, r: ribose and ara: arabinose sugar.

Recently, Waller's group investigated the effect of 2′-deoxyriboguanylurea (GuaUre-dR) (Figure ​(Figure2A)2A) on human telomeric i-motif formation (33). GuaUre-dR is a breakdown product of decitabine, a cytidine analogue that acts as an epimutagen and a chemotherapeutic agent. Despite the fact that this analogue can form a base pair with cytosine without protonation, the modified telomeric sequences exhibited a decrease in the pH_T (5.8) compared to the unmodified i-motifs (pH_T = 6.1) (33). Mir et al. studied the effect of pseudoiso-deoxycytidine (psC) (Figure ​(Figure2A)2A) on the stability of head-to-head and head-to-tail dimeric i-motif structures (34). An increase of the stability of the studied i-motif structures was observed when the neutral psC:C base pair was located at the end of a C:C⁺ stack. However, protonated base pairs are required (psC:C⁺ or psC:psC⁺) when the psC modifications are located in central positions of the i-motif structures. The results from these two studies underline the importance of the electrostatic interactions in the i-motif stability, where the presence of positive charges in the core of the structures is a key factor for its stability.

Significant i-motif stabilizations have been reported recently by introducing phenoxazine 2′-deoxynucleotides (i-clamps) containing a C8-aminopropynol tether (Figure 2A). These nucleotides are able to form base pairs with protonated cytosines and, simultaneously, interact favorably with the phosphate backbone of the opposite strand. As in the case of psC substitutions, the effect depends on the position of the substitution, where more stabilization is observed when they are located at the 5′-end of the C-stacks (35).

Very recently, natural base lesions were introduced in the TAA loops and in the core cytosines of the human telomeric C-rich strand d(CCCTAA)₃CCCT (36). This study reveals that i-motifs containing apurinic sites (apA) and 8-oxoadenine (8oxoA) substituting adenine, and 5-hydroxymethyluracil (5hmU) (Figure ​(Figure2A)2A) substituting thymine exhibit thermal stabilities that depend on the position of these substitutions in the sequence. Thus, in comparison to the unmodified structure, ΔT_m values for i-motifs having apA substitutions ranges between −1.7 and +5.0°C, those having 8oxoA vary from −1.4 to +3.5°C, and ΔT_m for the ones containing 5hmU is +0.4°C on average. On the contrary, the presence of uracil instead of cytosine (due to the enzymatic or spontaneous deamination of cytosine) substantially reduces i-motif thermal stability. The extent of this destabilization depends on the position of the deaminated cytosines. Whereas the loss of outer cytosines produces a decrease of 11.6–17.0°C in melting temperatures, the destabilization produced by the substitution of inner cytosines is even more pronounced due to the loss of a C:C⁺ pair intercalated between two inversely oriented pairs (36).

Sugar and phosphate backbone modifications

C-tract length

In general, under the same experimental conditions, the i-motif structure possessing a higher number of C:C⁺ base pairs would be more stable (52). Very recently, Waller's group and Burrows’ group investigated the effect of C-tract length on the folding of intramolecular i-motif structures under physiological conditions (16,17).

Waller's group studied several sequences containing at least four C-tracts with different lengths. Their results reveal that, in general, the change in pH_T increases as the number of cytosines per tract increases. For instance, pH_T increases from 6.1 for C₂(T₃C₂)₃ to 6.7 for C₃(T₃C₃)₃, to 7.1 for C₄(T₃C₄)₃ and 7.2 for C₅(T₃C₇)₅. A similar trend was observed for the thermal stability (T_m) where C₃(T₃C₃)₃ exhibited a T_m = 7°C while the T_ms of C₄(T₃C₄)₃ and C₅(T₃C₇)₅ were 15.8°C and 26.2°C, respectively. Increasing the C-tract length beyond five enhances the stability; however, it also leads to two melting transitions and to increased hysteresis between the folding and unfolding transitions suggesting the formation of two distinct species at pH 7.4. The sequence with the longest C-tract length tested was C₁₀(T₃C₁₀)₃ which exhibits a pH_T of 7.3. Despite that the tested sequences have tracts of four or more cytosines that fold into i-motif at neutral pH values, more recent studies show that sequences containing shorter C tracts also have the tendency to form stable i-motifs at neutral pH (53,54).

On the other hand, the Burrows group was interested in investigating the formation and stability of i-motif structures from several dC_n homo-oligonucleotides (n = 10–30) (17). Utilizing different pH-dependent methods an interesting trend was observed between chain length and stability. The highest thermal stabilities and pH_T (T_m > 37°C and pH_T > 7.2) were observed for dC_n strands of length 15, 19, 23 and 27 cytosines (i.e. 4n – 1). This led to identifying 4n – 1 as the ‘sweet spot’ for i-motif folding in deoxycytidine homopolymers. When T nucleotides were introduced to control the length of the i-motif core and to form loops of varying lengths, the most stable structures consisted of an even number of C:C⁺ base pairs in the core and three loops of only one nucleotide in length, further confirming the results obtained for dC_n homo-oligonucleotides. However, it is important to note that these results are not directly applicable to other heteronucleotidic sequences since the presence of loop-to-loop interactions play important roles on the stability of the structures as reviewed in the next section.

Connecting loops and capping interactions

Several systematic studies investigating the number and nature of different nucleobases in the i-motif loops have been undertaken recently in an attempt to comprehend the effect of the interactions between loop nucleobases on i-motif stability (55–57). Capping residues at the end of the i-motif core in addition to the length and nature of their connecting loops are very important factors for i-motif stability (58). Based on the length of the connecting loops, Brooks et al. divided intramolecular i-motif structures into two different classes. i-Motif structures possessing short loops (loop 1 (2-nt): loop 2 (3 to 4-nt): loop 3 (2-nt), i.e. 2:3–4:2) were classified as ‘class I’, while ‘class II’ i-motifs possess longer loops (6–8:2–5:6–7) (59). In general, very short loops, such as one nucleotide, favor the formation of mono and bimolecular i-motifs, whereas longer loops lead preferentially to the formation of intramolecular i-motif structures (60). Since longer loops might allow for extra stabilizing interactions, it has been proposed that class II i-motifs are more stable. However, there is a number of recent studies showing that class I i-motifs are stable at neutral or nearly neutral pH (53,61).

Thymidines are common capping residues in i-motifs since they can form T:T base pairs (Figure ​(Figure3A)3A) that are isomorphous to C:C⁺ base pairs and extend the i-motif core (61–63). In fact, T:T base pairs are not only good capping base pairs, but they even are tolerated in the middle of the C:C⁺ base pair stack (64). Hoogsteen and reverse Watson-Crick A:T base pairs (Figure ​(Figure3A)3A) in the loops of i-motifs have been found in the crystallographic structure of an oligonucleotide containing a single repeat of the human telomeric [d(TAACCC)] (65) and centromeric sequences (63) (Figure ​(Figure3A).3A). A:A (Figure ​(Figure3A)3A) and G:G base pairs have also been observed in several i-motif structures (66–68). This suggests it is not loop size per se but the precise sequence and the resulting interactions of the bases in the loop that are important for stabilization.

 

Figure 3.

(A) Several base pairs other than C:C⁺ that have been observed in i-motif structures (from top to bottom; A:T Hoogsteen, T:T, A:T reverse Watson-Crick and A:A) (Left). Schematic view of the dimeric i-motif structures formed by the two variants of centromeric A-box sequence (63) showing the presence of T:T and A:T reverse Watson–Crick and Hoogsteen base pairs flanking the set of intercalated C:C⁺ base pairs (Right). The residue in blue denotes the only difference in the primary structure of the two sequences. (B) G:C:G:T minor groove tetrad (Top). Schematic representation of the minimal i-motif structure stabilized by two C:C⁺ base pairs and two flanking G:C:G:T minor groove tetrads (Bottom) and representation of these structures in tandem (Right) (53).

One of the capping interactions that provokes more significant effects in the i-motif stability is the formation of minor groove tetrads on the ends of the C:C⁺ tracts. The first example of this kind of interaction was found by Gallego et al. in the structure of the human B-box centromeric sequence, d(TCCCGTTTCCA) (66). This sequence forms a stable head-to-head dimeric i-motif that features a G:T:G:T tetrad between two lateral loops. The two G:T pairs are found in the minor groove side as previously found in other structures (69). Tetrads of the same family have been observed with Watson-Crick G:C base pairs (70) and a combination of G:C and G:T base pairs (69,71). Stabilization of i-motif structures through minor groove tetrads has been observed in other dimeric i-motif structures (72), and more recently in monomeric i-motifs able to form in tandem repeats (53) (Figure ​(Figure3B).3B). In the latter case, the stabilization provoked by two minor groove tetrads is dramatic, with pH_T values close to 8.0.

In cases where a loop connecting two C-tracts is long enough, the formation of other secondary structures, like stem-loop hairpins, is possible. This situation has been observed in a C-rich sequence located near the promoter region of the n-MYC gene (73), although its three-dimensional structure remains to be elucidated.

Existence of i-motif structures in vivo

The biological relevance of i-motif structures had been largely questioned due to the lack of experimental evidence of their existence in vivo and to the fact that the formation of these structures is favored at pH values more acidic than the intracellular pH. However, several recent studies have changed this paradigm.

A very interesting study on the stability of i-motifs inside cells was performed by Dzatko et al. who applied NMR spectroscopy in living mammalian cells in order to investigate the stability of i-motif structures in the cellular environment (77). Several i-motif forming sequences from different human promoter regions (DAP, HIF-1α, PDGF-A and JAZF1) conveniently labeled with a fluorophore were pre-folded as i-motifs and transfected into HeLa cells. Flow cytometry and confocal microscopy images indicated that the transfected oligonucleotides entered the cells and localized in the nucleus without compromising the cellular viability and the homeostasis of the intracellular pH (pH_i). The in-cell NMR spectra for DAP, PDGF-A and JAZF1 exhibited i-motif imino signals up to 35°C, suggesting the presence of folded i-motif structures at physiological conditions. In contrast, no i-motif-specific imino signals were detected for the HIF-1α sequence. Overall, these in-cell NMR spectra indicate that pre-formed i-motif structures introduced into the cells remain stable and persist in the complex intracellular environment of living HeLa cells.

Very recently, Christ's and Dinger's groups developed an antibody, named iMab, able to bind with high affinity and specificity to C-rich DNA sequences forming i-motif structures (6). These groups describe that this antibody is probably the definitive probe for i-motif formation in living human cells. First, the authors demonstrate that iMab antibody binds different well-defined i-motif structures while showing absence of binding to any other DNA structure such as a duplex, hairpin or G-quadruplex. The use of iMab for immunofluorescent staining in three different cell lines (MCF7, U2OS and HeLa) revealed punctuate foci that were attributed to the recognition of i-motifs structures in the nuclei of cells. Very interestingly, the study showed that the number of foci varied along the cell cycle. The authors evaluated the number of foci in cells arrested in three different stages of the cell cycle (early S phase and G0/G1 and G1/S boundaries) showing that the highest number of foci appeared at the G1/S boundary (Figure ​(Figure4)4) (6). This observation suggests that i-motif formation can be associated with transcription as late G1 phase is characterized by high transcriptional activity. In contrast, the significant decrease of iMab foci during S phase (Figure ​(Figure4)4) suggests that i-motif structures are resolved during DNA replication. Likewise, the number of G4 foci detected in cells by immunofluorescence assays with BG4 antibody also varies along the cell cycle (7,78). In contrast to i-motif, G4 detection was higher during S-phase whose major event is DNA replication. Altogether, these results indicate that i-motif and G4 structures are differently populated in diverse stages of the cell cycle, and suggests that they might play opposing roles in regulating gene expression. This study provides the strongest evidence, so far, for the existence of i-motif structures in vivo and their relevance in key biological processes.

 

Figure 4.

(A) Confocal microscopy images showing fluorescent foci of i-motif recognized by iMab antibody in the nuclei of HeLa cells arrested at different stages of cell cycle. Flow cytometry diagrams show the population of synchronized and propidium iodide stained cells in each stage. (B) Box plot graph showing the quantification of iMab foci in each phase. Boxes represent 25th to 75th percentile. Horizontal line and ‘+’ symbol inside the box represent medians and means, respectively. Whiskers indicate lower and highest values registered in each case. ***P < 0.001, ****P < 0.0001. Reprinted with permission from Zeraati, M., Langley, D.B., Schofield, P., Moye, A.L., Rouet, R., Hughes, W.E., Bryan, T.M., Dinger, M.E. and Christ, D. (2018) I-motif DNA structures are formed in the nuclei of human cells. Nature chemistry, 10, 631–637. Copyright (2018) Springer Nature.

Interaction of DNA i-motifs with ligands and proteins

Compared to the well-documented examples of G4 ligands, the discovery of specific i-motif binding ligands lags far behind. Several compounds such as TMPyP4 (79), bis-acridine (BisA) (80) and phenanthroline derivatives (74) have been evaluated as i-motif ligands. Some of them have a stabilizing effect, but they are not selective since they also bind other DNA structures. Likewise, some complexes with terbium and ruthenium metals have been studied as potential i-motif binders (81,82); however, they lack specificity and lead to a slight destabilization of i-motif structures.

Carboxyl-modified single-walled carbon nanotubes (C-SWNTs) are considered the first selective i-motif ligands. Addition of C-SWNTs leads to a remarkable increase of thermal stability of the intramolecular i-motif formed by the C-rich human telomeric sequence at acidic pH (83). Additionally, it was found that the presence of C-SWNTs induces the formation of this i-motif structure at pH 8.0 and inhibits duplex formation between complementary human telomeric C- and G-rich sequences. Through S1 nuclease assays and fluorescence changes of 2-aminopurine labeled loops, it was proposed that the nanotubes bind to the 5′-end of the major groove of the telomeric i-motif structure. The stabilization effect of the C-SWNTs was explained by the favorable electrostatic interactions between the C:C⁺ base pairs and the negatively charged C-SWNTs which substantially decrease the pK_a of the C:C⁺ base pairs (83).

Qu et al. investigated the biomedical effect of C-SWNTs on telomerase activity and telomere function (discussed later in this review). This study determined that i-motif structures formed in the presence of C-SWNTs could inhibit telomerase activity, interfere with telomere functions, and lead to senescence and apoptosis in cancer cells in vitro and in vivo (84).

Following the carbon nanotubes, another study has succeeded in identifying a selective i-motif ligand. After screening a library of 1990 compounds, Hurley et al. identified a compound (IMC-48) that binds and stabilizes the i-motif structure formed by the C-rich sequence of BCL2 gene promoter. In parallel, a similar compound (IMC-76) was found to favor an alternative hairpin conformation formed by the same sequence (Figure ​(Figure5).5). These molecules can be used to shift the dynamic equilibrium between the two structures formed by this BCL2 promoter sequence, a hairpin and an i-motif structure containing large loops (8:5:7) (85). IMC-48 binds within the central loop of the i-motif (Figure ​(Figure5A)5A) and is likely stabilized by stacking interactions with thymines. On the other hand, the binding site of IMC-76 was found between the WC hydrogen-bonded tracts in the hairpin structure (Figure ​(Figure5B).5B). Interestingly, the molecules have opposite effects, while IMC-48 leads to the activation of gene expression, IMC-76 markedly suppresses the levels of BCL2 mRNA.

 

Figure 5.

(A) Structures of IMC-48 and IMC-76 ligands, specific for BCL2 i-motif and BCL2 hairpin, respectively. (B) BCL2 promoter sequence can adopt two different major conformations which are in a pH-dependent equilibrium. Acidic conditions lead to formation of an i-motif whereas at pH 6.6 there is a mixture of hairpin and i-motif conformations. IMC-76 binds to the hairpin structure shifting the equilibrium towards the hairpin structure which leads to transcriptional repression (A to B) whereas IMC-48 binds to the central loop of the BCL2 i-motif stabilizing the structure that upon binding of hnRNP LL via RRMs 1 and 2 of the protein (A to C) promotes the unfolding of the i-motif and the activation of BCL2 transcription (C to D). Adapted with permission from Kang, H.J., Kendrick, S., Hecht, S.M. and Hurley, L.H. (2014) The Transcriptional Complex Between the BCL2 i-Motif and hnRNP LL Is a Molecular Switch for Control of Gene Expression That Can Be Modulated by Small Molecules. J Am Chem Soc, 136, 4172–4185. Copyright (2014) American Chemical Society.

Several small molecule ligands have been investigated in an attempt to expand the i-motif-specific ligand library such as the type 2 topoisomerase inhibitor mitoxantrone (86), the para-isomer of the peptidomimetic ligand PBP1 (87), which leads to the upregulation of BCL2 gene expression, and thiazole orange (88). Recently, Shu et al. developed a series of acridone derivatives and determined that one of these derivatives, B19, is capable of binding to and stabilizing the i-motif formed in the c-MYC promoter resulting in downregulation of gene expression and eventually to tumor cell death (89). The characterization of more i-motif structures at high resolution as well as its stabilization at neutral pH will contribute to increase the number of ligands that stabilize i-motifs.

Poly-C-binding proteins (PCBP) are proteins that interact with C-rich DNA sequences and play a fundamental role in regulating gene expression. The PCBP family consists of the hnRNP K (heterogeneous nuclear ribonucleoprotein K), αCP1-4, and αCP-KL proteins (90). However, in most cases, it is not clear whether these proteins bind to a given i-motif structure or to the C-rich strand resulting from i-motif unfolding. In an early study, Marsich et al. discovered a highly cytosine-specific protein in human HeLa cells (91). They did not determine the identity of the protein; however, they were able to show that the protein is specific for the human telomeric sequence, d(CCCTAA)_n, containing at least four cytosine tracts (92). Later, Lacroix et al. found two proteins that bind telomeric C-rich sequences, hnRNP K and ASF/SF2 (93).

In a recent report, Niu et al. identified BmILF protein of Bombyx mori insect as an i-motif binding protein (94). By pull-down and EMSA assays, the authors demonstrated that BmILF binds to an i-motif structure formed at acidic pH by a C-rich sequence present in the BmPOUM2 gene promoter.

The BCL2 activating transcription factor heterogeneous nuclear ribonucleoprotein LL (hnRNP LL) is one of the very few i-motif binding proteins that have been studied in depth. It was identified through a pull-down assay aimed to find proteins involved in transcription and showing affinity for the i-motif structure formed by the BCL2 promoter oncogene (95). The hnRNP LL protein is a paralog of hnRNP L, which is a pre-mRNA splicing factor capable of binding and stabilizing BCL2 mRNA (95). hnRNP LL was found to bind specifically to i-motif structures as no binding was observed to either BCL2 promoter forming a duplex and to mutated single strand DNA unable to fold into an i-motif. The protein has four RNA recognition motifs (RRMs) and the BCL2 promoter sequence presents two consensus sequences that can be recognized by hnRNP LL RRMs. These consensus sequences correspond to those in the two lateral loops of the i-motif folding. Through EMSA experiments and luciferase reporter assays it was determined that hnRNP LL binds to the two lateral loops of the BCL2 i-motif (Figure ​(Figure5C)5C) by means of two of its four RRMs. Interestingly, CD and bromine footprinting experiments show that the binding of the protein unfolds the i-motif structures leading to single- stranded sequences. Therefore, the i-motif structure is the most kinetically favorable conformation for protein binding. The unfolding of the i-motif structures upon protein binding provides the more thermodynamically favored single-stranded conformation (Figure ​(Figure5D).5D). The protein remains bound to the single strand DNA and leads to the activation of BCL2 gene transcription. The discovery of the hnRNP LL as an i-motif binding protein able to activate gene transcription brings i-motif structures into focus as protein recognition sites capable of participating in regulation of gene expression.

Inhibition of telomerase activity

Mammalian telomeric DNA is composed of tandem repeats of the unit 5′-ATTGGG-3′/3′-TAACCC-5′. Importantly, the G-rich strand is a few hundreds of nucleotides longer than its complementary resulting in a G-rich ssDNA 3′-overhang that may form G4 structures. Studies have shown that the stabilization of certain human telomeric G4 topologies with ligands might lead to the inhibition of telomerase activity (96–98); however, the effect of targeting the complementary C-rich strand has not been investigated in depth. Thus, the study conducted by Qu et al. in 2012 was the first to investigate telomerase activity on i-motifs formed by C-rich human telomeric sequences stabilized by C-SWNT (84). As mentioned earlier, C-SWNTs were found to induce duplex dissociation and to stabilize human telomeric i-motifs at physiological pH and temperature under molecular crowding conditions. Likewise, C-SWNTs induce the formation of G4 structures on the complementary G-rich strand (83). In the presence of the C-SWNTs, telomerase activity is inhibited; thereby suggesting that the G4 stabilized on the leading strand can no longer be elongated by telomerase. Further investigations into the effect of C-SWNTs on cell growth and telomere structure and function suggest that the inhibition of cellular growth produced by C-SWNTs was a consequence of an impact on telomere structure rather than on telomerase activity. The persistence of i-motif and G4 structures, leads to telomere uncapping and release of telomere-binding proteins, resulting in telomere dysfunction. Telomere dysfunction induces DNA damage response and activates DNA repair pathways, which in turn trigger cell cycle arrest, senescence, and apoptosis (83).

I-motif in centromeric sequences

The centromere is the chromosomal region on which the kinetochore, a key multiprotein complex for chromosome segregation, assembles during cell division. In most organisms, centromeres contain large arrays of tandemly repeated DNA sequences (DNA satellite). Centromeric DNA sequences substantially vary among species and can also be different in chromosomes of the same organism. In the absence of a shared genetic motif defining the centromere, the presence of nucleosomes containing the centromere-specific histone H3 variant (CENP-A) is recognized as the essential epigenetic feature of centromeric chromatin. However, the possible role of the centromeric DNA sequences in directing the formation of such specialized chromatin has been suggested and is an attractive matter of debate (99,100). Interestingly, Kasinathan and Henikoff proposed in a very recent report that the predominant formation of non-B DNA structures in centromeric DNA sequences might be the basis for the constitution of centromeric chromatin (101).

In humans, a 171 bp DNA called alphoid satellite is tandemly repeated along the centromeres (Figure ​(Figure6A).6A). The alphoid satellite is an AT rich sequence that frequently contains a 17-bp GC rich segment whose sequence has two variants known as CENP-B box and A box (Figure ​(Figure6A).6A). Interestingly, the CENP-B box, the sequence specifically bound by centromeric protein CENP-B, is absent in lower primates and in human Y chromosome. Gallego et al. found that whereas the G-rich sequence of CENP-B box folds into a structure stabilized by canonical base pairs, its complementary C-rich sequence forms a dimeric i-motif at acidic pH (Figure ​(Figure6B)6B) (66,102). Likewise, the C-rich sequences of the two variants of A box were reported to fold into dimeric i-motif structures (Figure ​(Figure3A)3A) (63). The dimeric i-motif structures of truncated versions of both CENP-B box and A box having 11 residues were determined at atomic resolution by NMR spectroscopy (63,66) (Figure ​(Figure6B).6B). The main difference between both structures is the relative disposition of the loops being at the same side of the structure (face-to-face topology) in CENP-B box i-motif and at opposite sides in A box (head-to-tail topology) (Figure ​(Figure6B6B).

 

Figure 6.

(A) Human chromosome showing the centromere in grey and the structure of the human centromeric alpha satellite DNA. The 171 bp monomers (green arrows) are AT-rich sequences that can contain either A box (red and black) or CENP-B box (orange and black). The monomers are tandemly repeated and form a higher order repeat, which in turn appears repeated along the centromere. (B) Schematic and cartoon structures of the dimeric i-motifs formed by truncated A box (PDB ID: 2MRZ) and CENP-B box (PDB ID: 1C11). Cytosines are represented in grey, thymines in green, adenines in magenta and guanines in blue. (C) Proposed model for nucleosome organization at the centromere. The i-motifs formed by C-rich sequences of A and CENP-B boxes (orange) would maintain the structural organization of the centromere.

The centromeric region of chromosome 3 of Droshophila melanogaster contains the dodeca satellite DNA, which is composed by tandem repeats of 11/12 bp (CCCGTACTGGT/CCCGTACTCGGT). The capability of these sequences to fold into i-motif structures has been also evaluated. Sequences derived from both 11 bp and 12 bp repeat units were able to fold into dimeric i-motif structures in vitro at acidic pH (103).

These observations lead to propose a possible role for i-motif structures as a structural element providing long-range interactions between laterally associated centromeric nucleosomes (63) (Figure ​(Figure6C).6C). The presence of the above mentioned i-motif forming sequences at the entrance and exit of the nucleosome would facilitate their participation in dimeric i-motifs formation (Figure ​(Figure6C).6C). Thus, not only non-B DNA structures may have a role in directing centromere location (101) but also in providing particular architectural features to the centromere.

Transcriptional regulation of gene expression

A substantial amount of data suggests that i-motif structures may be involved in regulation of transcription. A recent report showing i-motif foci in vivo determined that number of i-motif foci were higher during G1/S phase of the cell cycle (Figure ​(Figure4)4) in which the transcription activity is higher (6). This fact, together with the evidence described below suggest that these structures may have been selected as recognition motifs for proteins involved in the activation of the transcriptional machinery.

The BCL2 oncogene promotes cell survival and proliferation through an anti-apoptotic mechanism. It is overexpressed in some cancer cells, while its under-expression leads to neurodegenerative diseases (104). As discussed earlier, the C-rich sequence of the BCL2 promoter folds into a hairpin and an i-motif structures that are in dynamic equilibrium (105). Two back-to-back studies by Hurley's group showcase two compounds (Figure ​(Figure5)5) and a protein capable of modulating BCL2 transcription in vitro and in vivo by specifically targeting either the i-motif or the hairpin form in dynamic equilibrium (85,95). Apparent stabilization of the i-motif structure via IMC-48 compound leads to significant upregulation of BCL2. On the contrary, apparent stabilization of the flexible hairpin species via IMC-76 compound leads to transcriptional repression in lymphoma cell lines (Figure ​(Figure5B)5B) (85).

In a consecutive study, the Hurley's group determined the effect of the hnRNP LL protein on BCL2 transcription. Two of the four RNA recognition motifs (RRMs) in hnRNP LL are required for stable binding to a single-stranded RNA or DNA. The lateral loops of BCL2 i-motif possess sequences capable of binding to the RRMs in hnRNP LL (Figure ​(Figure5C).5C). These studies demonstrate that hnRNP LL activates transcription by recognizing and unfolding the BCL2 i-motif (Figure ​(Figure5).5). The presence of IMC-48 shifts the equilibrium in favor of the i-motif and therefore increases the i-motif population available for binding to hnRNP LL. The research group demonstrated that IMC-48 binds to the central loop of the BCL2 i-motif, followed by recognition and binding of hnRNP LL to the two lateral loops. Consequently, hnRNP LL binding leads to i-motif unfolding which, in turn, leads to transcriptional activation. In conclusion, these studies demonstrate the effect of two small molecules (IMC-76 and IMC-48) and a transcriptional factor (hnRNP LL) on the relative population of i-motif and its impact on gene expression.

Recently, a study conducted by Muniyappa and co-authors demonstrated the potential of the C-rich sequences of the PI and PII promoters of human acetyl-CoA carboxylase 1 (ACC1) gene to fold into intramolecular i-motif structures at neutral pH under molecular crowding conditions (106). The authors state that, experiments in HeLa cells including i-motif-forming sequences in a promoter region regulating luciferase transcription lead to a decrease in protein expression and suggest a significant role of i-motif-forming sequences in the regulation of ACC1 gene expression. However, the authors also claim that G4 and i-motif structures in plasmids containing wild type sequences exhibit a cooperative effect leading to a decrease in luciferase expression. Therefore, it is unclear whether i-motif or G4 structures are responsible for transcriptional repression. This ambiguity is addressed in a recent study on the PDGFR-β promoter region (107) where point mutations of the G4 structures led to upregulation of gene expression. This upregulation is due to the effect introduced by the G4 mutations on the i-motif-forming strand.

In a similar study conducted by Niu et al., it was reported that the binding of BmILF protein to an i-motif activates the transcription of BmPOUM2, a Bombyx mori gene involved in developmental processes during metamorphosis. Luciferase expression assays demonstrated that vectors lacking the i-motif-forming sequence in the promoter of the gene resulted in significant decreasing of luciferase activity. Additionally, by using oligonucleotides complementary to the i-motif and G4 forming sequences present in the promoter, it was determined that only the hybridization of i-motif forming sequence with complementary oligonucleotides produced significant decrease in promoter activity. The authors propose that during transcription, the dsDNA at the promoter is melted and the i-motif is formed and bound by BmILF, which may recruit other factors to activate BmPOUM2 expression.

Burrows et al. investigated the effect that modified G4 and i-motif-forming sequences from VEGF gene promoter have on luciferase and renilla expression levels (108). In addition, this study reveals that sequences containing nucleobases produced during oxidative stress such as 8-oxo-7,8-dihydroguanine lead to the up- or down-regulation of transcription depending on whether the modified nucleobases are located on the coding or on the template strand of the promoter, respectively.

Kendrick et al. used IMC-76 in combination with ellipticine (GQC-05) in order to simultaneously target BCL2 and MYC oncogene promoters in diffuse large B-cell lymphoma (DLBCL) (109). GQC-05 stabilizes the G4 formed by G-rich MYC promoter sequence, supposedly acting as an ‘off-switch’ to impede gene expression (110). The simultaneous stabilization of the hairpin over the i-motif (via IMC-76) in the BCL2 promoter and the G4 (via GQC-05) in the MYC promoter decreased the mRNA levels of both genes and enhanced the sensitivity of DLBCL cells towards cyclophosphamide (a chemotherapeutic drug). This study was the first to simultaneously target two different DNA secondary structures, i.e. i-motifs and G4s, leading to dual transcriptional repression and providing an effective approach to treat aggressive malignancies (109).

Following the above mentioned reports, several studies demonstrate how G4s and i-motifs act as on/off molecular switches for the regulation of tyrosine hydroxylase (Th) (111), MYC (112), platelet-derived growth factor receptor β (PDGFR-β) (107), and KRAS promoters (113). For instance, in the case of the MYC promoter it is the extent of transcriptionally induced negative superhelicity that determines whether MYC expression can be turned on. The authors propose that binding of SP1 to its promoter binding sites induces negative superhelicity, which in turn drives duplex melting at 4CT or 5CT GC-rich elements. The mechanism by which the protein contributes to increase negative superhelicity is not known. At low levels of SP1 there is only sufficient unwinding of the duplex to expose the 4CT element which contains six C-tracts that provides the two hnRNP K binding sites in the lateral loops of the i-motif (Figure ​(Figure7C7C to A). However, this provides insufficient binding strength for the hnRNP K–i-motif complex to compete with nucleolin, which binds to the G4 structure to repress gene transcription (i.e. acting as an OFF switch). However, with increased concentrations of SP1 available to bind to the duplex DNA, this results in increased transcriptionally induced negative superhelicity and the two additional C-tracts become available (Figure ​(Figure7C7C to B). This provides an additional CT binding site for hnRNP K uniquely found in the 5CT elements. Ultimately, two steps are needed to activate transcription, the first being recognition by hnRNP K of the two CT elements displayed in the lateral loops of the i-motif. In the second step, unfolding of the i-motif occurs, providing access to the third CT element, which is uniquely found in the 5CT elements. Now with three KH domains from the hnRNP K bound to the 3CT elements, this thermodynamically stable complex is able to compete with nucleolin–G4 complex and MYC transcription is turned on (112).

 

Figure 7.

Proposed scheme for the molecular mechanosensor mechanism for differential control of MYC expression through the NHE III₁. (A) Low SP1 occupancy of duplex promoter binding sites (as shown in (C), upper) results in low negative supercoiling and basal levels of transcription from the P1 and P2 promoters. In this scenario, hnRNP K can only access the central and lateral loops in the i-motif that bind to two of the KH domains; as such only the 4CT element is accessed, and nucleolin binding predominates over hnRNP K binding to the equilibrating G-quadruplex and i-motif, which are mutually exclusive. (B) At higher occupancy levels of SP1 to the duplex promoter binding sites (as shown in (C), lower), which results in enhanced negative supercoiling, the 5CT element is now fully melted, and hnRNP K forms a thermodynamically stable complex binding through the addition of the CT element. The binding affinity of hnRNP K to the unfolded i-motif and the additional CT element involving three KH domains now exceeds that of nucleolin to the G-quadruplex, and MYC expression from P1 and P2 is at peak levels. Reprinted with permission from Sutherland, C., Cui, Y.X., Mao, H.B. and Hurley, L.H. (2016) A Mechanosensor Mechanism Controls the G-Quadruplex/i-Motif Molecular Switch in the MYC Promoter NHE III1. J Am Chem Soc, 138, 14138–14151. Copyright (2016) American Chemical Society.

Regulation of DNA biosynthesis

Very recently, Sugimoto and co-authors investigated the effect of several non-canonical DNA structures on DNA replication by the Klenow fragment (KF) of DNA polymerase (114). Different i-motif-forming sequences inserted in the template strand of the replication reaction were found to stall DNA polymerase and thus impede DNA replication or repair. The stalling effect produced by i-motifs on replication was higher than that of other structures with similar thermodynamic stability such us hairpins or mixed-type G4s. This is justified by the unique intercalating topology of base pairs in i-motif structures, which complicate its unwinding and subsequent replication by DNA polymerase. As mentioned earlier, the base pairs in an i-motif structure are intercalated between two parallel duplexes and lack the base stacking interactions like in antiparallel duplexes (Figure ​(Figure1B).1B). Therefore, unzipping is particularly disfavored since consecutive base pairs belong to different duplexes. In addition, the arrangement of the loops in the i-motif structure might cause steric hindrance for polymerase binding. These two factors may contribute to the fact that i-motif unwinding by KF requires higher activation energy than that of other non-canonical structures. These data suggest that i-motifs could modulate DNA replication in vivo and pose a greater impact compared to other secondary structures.

The authors would like to acknowledge Professor Daniel Christ and Professor Laurence Hurley for providing the original copy of Figures ​Figures4,4, ​,55 and 7 to be used in this review.