I-motif Core Research Articles

Crystal Structure of an i-Motif from the HRAS Oncogene Promoter.

An i-motif is a non-canonical DNA structure implicated in gene regulation and linked to cancers. The C-rich strand of the HRAS oncogene, 5'-CGCCCGTGCCCTGCGCCCGCAACCCGA-3' (herein referred to as iHRAS), forms an i-motif in vitro but its exact structure was unknown. HRAS is a member of the RAS proto-oncogene family. About 19 % of US cancer patients carry mutations in RAS genes. We solved the structure of iHRAS at 1.77 Å resolution. The structure reveals that iHRAS folds into a double hairpin. The two double hairpins associate in an antiparallel fashion, forming an i-motif dimer capped by two loops on each end and linked by a connecting region. Six C-C+ base pairs form each i-motif core, and the core regions are extended by a G-G base pair and a cytosine stacking. Extensive canonical and non-canonical base pairing and stacking stabilizes the connecting region and loops. The iHRAS structure is the first atomic resolution structure of an i-motif from a human oncogene. This structure sheds light on i-motifs folding and function in the cell.

Microscopic Insight into pH-Dependent Conformational Dynamics and Noncanonical Base Pairing in Telomeric i-Motif DNA.

Gene regulatory functions of noncanonical i-motif DNA are associated with dynamic i-motif formation in the cellular environment and pH variation. With atomistic simulations, we show the dramatic influence of solvent pH on the conformational dynamics of biologically relevant telomeric i-motif DNA coupled with protonation of cytosine bases in different conformations. We rationalized the pH-dependent dynamics and conformational variability of the i-motif in terms of base pairing and specific loop motions. The human telomeric i-motif is found to acquire various metastable folded conformations at pH values near the pKa of cytosine with the formation of a noncanonical C:C W:W trans base pair along with the hemiprotonated C:C+ pairs in the i-motif core. pH-dependent dynamics and the local solvent structure of i-motif DNA imply that the presence of a cosolvent or molecular crowding can promote i-motif formation in vivo by changing the conformational fluctuations and hydration state of the structure.

DNA i-Motifs With Guanidino-i-Clamp Residues: The Counterplay Between Kinetics and Thermodynamics and Implications for the Design of pH Sensors

I-motif structures, adopted by cytosine-rich DNA strands, have attracted considerable interest as possible regulatory elements in genomes. Applied science exploits the advantages of i-motif stabilization under acidic conditions: i-motif-based pH sensors and other biocompatible nanodevices are being developed. Two key characteristics of i-motifs as core elements of nanodevices, i.e., their stability under physiological conditions and folding/unfolding rates, still need to be improved. We have previously reported a phenoxazine derivative (i-clamp) that enhances the thermal stability of the i-motif and shifts the pH transition point closer to physiological values. Here, we performed i-clamp guanidinylation to further explore the prospects of clamp-like modifications in i-motif fine-tuning. Based on molecular modeling data, we concluded that clamp guanidinylation facilitated interstrand interactions in an i-motif core and ultimately stabilized the i-motif structure. We tested the effects of guanidino-i-clamp insertions on the thermal stabilities of genomic and model i-motifs. We also investigated the folding/unfolding kinetics of native and modified i-motifs under moderate, physiologically relevant pH alterations. We demonstrated fast folding/unfolding of native genomic and model i-motifs in response to pH stimuli. This finding supports the concept of i-motifs as possible genomic regulatory elements and encourages the future design of rapid-response pH probes based on such structures. Incorporation of guanidino-i-clamp residues at/near the 5′-terminus of i-motifs dramatically decreased the apparent unfolding rates and increased the thermal stabilities of the structures. This counterplay between the effects of modifications on i-motif stability and their effects on kinetics should be taken into account in the design of pH sensors.

The structural diversity of C-rich DNA aggregates: unusual self-assembly of beetle-like nanostructures.

We studied the ability of oligonucleotides CnT25 (n = 2, 5, 7, 9, 12, 25) to form an intermolecular i-motif using circular dichroism, ultra-violet spectroscopy, nuclear magnetic resonance, high-resolution atomic force microscopy, high-performance liquid chromatography, and molecular dynamics simulations. The arrangement of single-stranded oligonucleotides in multimer i-motifs was very unusual: C-tracts of different oligonucleotides followed each other consecutively in order to fold into a closed intermolecular i-motif core with minimal loops (one cytidine in a loop spanning over a minor groove, three cytidines in a loop over a major groove); intact T-tracts protruded from predefined loci allowing visualization of beetle-like nanostructures by atomic force microscopy. The same structures were formed from analogous biotinylated oligonucleotides demonstrating one of the potential applications of such structures as carriers of multiple functional groups. Our findings open up possibilities for the rational design of pH-sensitive DNA aggregates and evaluation of the efficiency of their assembly.

Centromeric Alpha-Satellite DNA Adopts Dimeric i-Motif Structures Capped by AT Hoogsteen Base Pairs.

Human centromeric alpha-satellite DNA is composed of tandem arrays of two types of 171 bp monomers; type A and type B. The differences between these types are concentrated in a 17 bp region of the monomer called the A/B box. Here, we have determined the solution structure of the C-rich strand of the two main variants of the human alpha-satellite A box. We show that, under acidic conditions, the C-rich strands of two A boxes self-recognize and form a head-to-tail dimeric i-motif stabilized by four intercalated hemi-protonated C:C(+) base pairs. Interestingly, the stack of C:C(+) base pairs is capped by T:T and Hoogsteen A:T base pairs. The two main variants of the A box adopt a similar three-dimensional structure, although the residues involved in the formation of the i-motif core are different in each case. Together with previous studies showing that the B box (known as the CENP-B box) also forms dimeric i-motif structures, our finding of this non-canonical structure in the A box shows that centromeric alpha satellites in all human chromosomes are able to form i-motifs, which consequently raises the possibility that these structures may play a role in the structural organization of the centromere.

Structural basis for the identification of an i-motif tetraplex core with a parallel-duplex junction as a structural motif in CCG triplet repeats.

CCG triplet repeats can fold into tetraplex structures, which are associated with the expansion of (CCG)n trinucleotide sequences in certain neurological diseases. These structures are stabilized by intertwining i-motifs. However, the structural basis for tetraplex i-motif formation in CCG triplet repeats remains largely unknown. We report the first crystal structure of a CCG-repeat sequence, which shows that two dT(CCG)3 A strands can associate to form a tetraplex structure with an i-motif core containing four C:C(+) pairs flanked by two G:G homopurine base pairs as a structural motif. The tetraplex core is attached to a short parallel-stranded duplex. Each hairpin itself contains a central CCG loop in which the nucleotides are flipped out and stabilized by stacking interactions. The helical twists between adjacent cytosine residues of this structure in the i-motif core have an average value of 30°, which is greater than those previously reported for i-motif structures.

C7GC4]4 Association into supra molecular i-motif structures

The self-associative properties of cytidine-rich oligonucleotides into symmetrical i-motif tetramers give to these oligonucleotides the capacity of forming supramolecular structures (sms) that have potential applications in the nanotechnology domain. In order to facilitate sms formation, oligonucleotides containing two cytidine stretches of unequal length (CnXCm) separated by a non-cytidine spacer were synthesized. They were designed to associate into a tetramer including an i-motif core built by intercalation of the C·C+ pairs of the longer C stretch with the two dangling non-intercalated strands of the shorter C stretch at each end. Gel filtration chromatography shows that the non-intercalated C-rich ends give to this structure the capacity of forming extremely stable sms. Using C7GC4 as a model, we find that the sms formation rate varies as the oligonucleotide concentration and increases at high temperature. Competitively with the tetramer involved in sms elongation, CnXCm oligonucleotides form i-motif dimers that compete with sms elongation. The dimer stability is strongly reduced when the pH is moved away from the cytidine pK. This results in an equilibrium shift towards the tetramer and in the acceleration of the sms formation rate. The chromatograms of the sms formed by C7GC4 indicate a broad distribution. In a 1.5 mM solution incubated at 37°C, the equilibrium distribution is centered on a molecular weight corresponding to the assembly of nine tetramers and the upper limit corresponds to 80 tetramers. The lifetime of this structure is about 4 days at 40°C, pH 4.6.

Structure, internal motions and association-dissociation kinetics of the i-motif dimer of d(5mCCTCACTCC).

At slightly acidic pH, the association of two d(5mCCTCACTCC) strands results in the formation of an i-motif dimer. Using NMR methods, we investigated the structure of [d(5mCCTCACTCC)]2, the internal motion of the base pairs stacked in the i-motif core, the dimer formation and dissociation kinetics versus pH. The excellent resolution of the 1H and 31P spectra provided the determination of dihedral angles, which together with a large set of distance restraints, improve substantially the definition of the sugar-phosphate backbone by comparison with previous NMR studies of i-motif structures. [d(5mCCTCACTCC)]2 is built by intercalation of two symmetrical hairpins held together by six symmetrical C•C+ pairs and by pair T7•T7. The hairpin loops that are formed by a single residue, A5, cross the narrow grooves on the same side of the i-motif core. The base pair intercalation order is C9•C9+/5mC1•5mC1+/C8•C8+/C2•C2+/T7.T7/C6•C6+/C4•C4+. The T3 bases are flipped out in the wide grooves. The core of the structure includes four long-lived pairs whose lifetimes at 15°C range from 100 s (C8•C8+) to 0.18 s (T7•T7). The formation rate and the lifetime of [d(5mCCTCACTCC)]2 were measured between pH 6.8 and 4.8. The dimer formation rate is three to four magnitude orders slower than that of a B-DNA duplex. It depends on pH, as it must occur for a bimolecular process involving non cooperative association of neutral and protonated residues. In the range of pH investigated, the dimer lifetime, 500 s at 0°C, pH 6.8, varies approximately as 10−pH.

Structure of a C-rich strand fragment of the human centromeric satellite III: a pH-dependent intercalation topology

Repetitive DNA sequences may adopt unusual pairing arrangements. At acid to neutral pH, cytidine-rich DNA oligodeoxynucleotides can form the i-motif structure in which two parallel-stranded duplexes with C·C+ pairs are intercalated head-to-tail. The i-motif may be formed by multimeric associations or by intra-molecular folding, depending on the number of cytidine tracts, the nucleotide sequences between them, and the experimental conditions.We have found that a natural fragment of the human centromeric satellite III, d(CCATTCCATTCCTTTCC), can form two monomeric i-motif structures that differ in their intercalation topology and that are favored at pH values higher (the η-form) and lower (the λ-form) than 4.6. The change in intercalation may be related to adenine protonation in the loops.We studied the uridine derivative methylated on the first cytidine base, d(5mCCATTCCAUTCCUTTCC), whose proton spectrum is better resolved. The intercalation topologies are (C7·C17)/(5mC1·C11)/(C6·C16)/(C2·C12) for form λ and (5mC1·C11)/(C7·C17)/(C2·C12)/(C6·C16) for form η. We have solved the structure of the η-form, and we present a model for the λ-form. The switch from η to λ involves disruption of the i-motif. In both forms, the central AUT linker crosses the wide groove, and the first and the third linkers loop across the minor grooves. The i-motif core is extended in the η-form by the inter-loop reverse Watson-Crick A3·U13 pair, whose dissociation constant is around 10−2 at 0 °C, and in the λ-form by the interloop T5·T15 pair.In contrast, d(5mCCATTCCTTACCTTTCC) folds into a pH-independent structure that has the same intercalation topology as the λ-form. The i-motif core is extended below by the interloop T5·T15 pair and closed on top by the T8·A10 pair.Thus, the C-rich strand of the human satellite III tandem repeats, like the G-rich strand, can fold into various compact structures. The relevance of these features to centromeric function remains unknown.

Intramolecular i-Motif Structures of Telomeric DNA

The i-motif is an intercalated structure formed by association in a head to tail orientation of two parallel duplexes whose strands are held together by hemiprotonated C·C+ pairs. The i-motif may be formed by a single strand containing four cytidine repeats, by association of two strands containing two cytidine repeats or by four strands containing a single cytidine stretch. The repeated C-rich sequences of centromeric and telomeric regions can potentially fold into an intramolecular i-motif. We have investigated by NMR spectroscopy the structure of d(CCCTA2CCCTA2CCCTA2CCCT), a fragment of the vertebrate telomere. It includes an i-motif core of six intercalated C·C+ pairs. At one end (the “top”), the central TA2 linker loops across one of the narrow grooves, and the core is extended by base stacking in the loop. At the bottom, where the two other TA2 linkers loop across the wide grooves, the NMR spectra reveal motions in the microsecond to millisecond scale. The pseudo-symmetry of the structure, which results in degenerate spectra and poor resolution, was broken by appropriate substitution of T by U and of C by 5-methylcytidine (5mC). This allowed us to solve the structure of d(CCCTA25mCCCTA2CCCUA2CCCT). The motion is restricted to a flip of A18 around the glycosidic bond. Returning to the pseudo-symmetrical sequence, we find that each of the bottom loops switches between the structures of the first and third loops of the non-symmetrical sequence. We also analyzed the effects of the loop sequence and of the length of the C-stretches on the topology and stability of the intramolecular i-motif structure.

An intramolecular i-motif: the solution structure and base-pair opening kinetics of d(5mCCT3CCT3ACCT3CC)

We present a high-definition structure of d(5mCCT3CCT3ACCT3CC), a DNA sequence which resembles a four-times repeat of the C-rich strand of telomeres and centromeres. The structure is monomeric. The CC stretches form four hemi-protonated C·C base-pairs, belonging to two parallel-stranded duplexes which intercalate head-to-tail into an i-motif core. The four grooves of the core are similar to those observed previously in i-motif tetramers, with P-P distances around 0.9 nm and 1.4 nm for the narrow and wide grooves, respectively. At 0°C, the structure is formed even at pH 7, despite the required protonation of cytidine pairs, suggesting that it may be biologically relevant.The intercalation topology of the i-motif core is read off the pattern of inter-residue cross-peaks along each groove: between H1′ protons across the narrow grooves, and between amino and H2′ protons across the wide grooves. In the hemi-protonated C·C pairs, the imino proton is shared equally between the two bases, as shown by the equal intensities of the NOESY cross-peaks between the imino proton and the two cis amino protons of the pair. Short inter-sugar distances and the direction of CH1′ bonds are consistent with CH1′⋯O4′ hydrogen bonds across the narrow grooves, as suggested by Berger et al. (1996). Proc. Natl. Acad. Sci. USA, 93, 12116–12121.At one extremity of the i-motif core, the T3A linker loops across one of the two wide grooves. It extends the core by stacking of A11, which also forms a strongly propeller-twisted reverse-Hoogsteen pair with T8. At the other extremity, the two T3linkers loop side by side across the two narrow grooves, extending the core by stacking of a T5·T16 pair which connects the two linkers. In this T·T pair between parallel strands, the hydrogen bonds are from imino proton to O4, and the base-pair lifetime is 6 ms at 0°C. The structures of segments 1 to 7 and 12 to 18, which form the i-motif core and the T3loops, are related by a 2-fold pseudo-symmetry: the geometries and environment are so similar that the NOESY spectra are barely resolved.These various interactions illustrate how linker sequences may affect the stability, intercalation topology and folding pattern of the intramolecular i-motif.

Solution structure and base pair opening kinetics of the i-motif dimer of d(5mCCTTTACC): a noncanonical structure with possible roles in chromosome stability

Repetitive cytosine-rich DNA sequences have been identified in telomeres and centromeres of eukaryotic chromosomes. These sequences play a role in maintaining chromosome stability during replication and may be involved in chromosome pairing during meiosis. The C-rich repeats can fold into an 'i-motif' structure, in which two parallel-stranded duplexes with hemiprotonated C.C+ pairs are intercalated. Previous NMR studies of naturally occurring repeats have produced poor NMR spectra. This led us to investigate oligonucleotides, based on natural sequences, to produce higher quality spectra and thus provide further information as to the structure and possible biological function of the i-motif. NMR spectroscopy has shown that d(5mCCTTTACC) forms an i-motif dimer of symmetry-related and intercalated folded strands. The high-definition structure is computed on the basis of the build-up rates of 29 intraresidue and 35 interresidue nuclear Overhauser effect (NOE) connectivities. The i-motif core includes intercalated interstrand C.C+ pairs stacked in the order 2*.8/1.7*/1*.7/2.8* (where one strand is distinguished by an asterisk and the numbers relate to the base positions within the repeat). The TTTA sequences form two loops which span the two wide grooves on opposite sides of the i-motif core; the i-motif core is extended at both ends by the stacking of A6 onto C2.C8+. The lifetimes of pairs C2.C8+ and 5mC1.C7+ are 1 ms and 1 s, respectively, at 15 degrees C. Anomalous exchange properties of the T3 imino proton indicate hydrogen bonding to A6 N7 via a water bridge. The d(5mCCTTTTCC) deoxyoligonucleotide, in which position 6 is occupied by a thymidine instead of an adenine, also forms a symmetric i-motif dimer. However, in this structure the two TTTT loops are located on the same side of the i-motif core and the C.C+ pairs are formed by equivalent cytidines stacked in the order 8*.8/1.1*/7*.7/2.2*. Oligodeoxynucleotides containing two C-rich repeats can fold and dimerize into an i-motif. The change of folding topology resulting from the substitution of a single nucleoside emphasizes the influence of the loop residues on the i-motif structure formed by two folded strands.