Orderly microaggregates of G-/C-rich oligonucleotides associated with spermine

Spermine-induced orderly assembling properties of G-/C-rich oligonucleotides are investigated in dilute and crowding conditions. The first time we report that the parallel G-quadruplexes is preferential to condense into anisotropic microaggregates in the presence of spermine, whereas the hybrid-type and the antiparallel G-quadruplexes have no significant interactions with spermine; and spermine can induce the condensation of i-motif C-rich oligonucleotides other than the random coiled C-rich strands. Moreover, the condensation of C-rich oligonucleotides can be reversibly regulated by pH and temperature. G-/C-rich oligonucleotides exhibit the cholesteric liquid crystalline phase at low strand concentration in the presence of spermine under crowding conditions. The results illuminate that the parallel G-quadruplex and i-motifs are probably necessity conformations for G-/C-rich oligonucleotides that involved in the regulation of chromosome organization in living cells.     

Human telomeric DNA: G-quadruplex,i-motif and Watson-Crick double helix

Human telomeric DNA composed of (TTAGGG/CCCTAA)n repeats may form a classical Watson-Crick double helix. Each individual strand is also prone to quadruplex formation: the G-rich strand may adopt a G-quadruplex conformation involving G-quartets whereas the C-rich strand may fold into an i-motif based on intercalated C*C+ base pairs. Using an equimolar mixture of the telomeric oligonucleotides d[AGGG(TTAGGG)3] and d[(CCCTAA)3CCCT], we defined which structures existed and which would be the predominant species under a variety of experimental conditions. Under near-physiological conditions of pH, temperature and salt concentration, telomeric DNA was predominantly in a double-helix form. However, at lower pH values or higher temperatures, the G-quadruplex and/or the i-motif efficiently competed with the duplex. We also present kinetic and thermodynamic data for duplex association and for G-quadruplex/i-motif unfolding.     

The RNA i-motif

Oligodeoxynucleotides with stretches of cytidine residues associate into a four-stranded structure, the i-motif, in which two head-to-tail, intercalated, parallel-stranded duplexes are held together by hemiprotonated C.C+ pairs. We have investigated the possibility of forming an i-motif structure with C-rich ribonucleic acids. The four C-rich RNAs studied, r(UC5), r(C5), r(C5U) and r(UC3), associate into multiple intercalated structures at acidic pH. r(UC5) forms two i-motif structures that differ by their intercalation topologies. We report on a structural study of the main form and we analyze the small conformational differences found by comparison with the DNA i-motif. The stacking topology of the main structure avoids one of the six 2'-OH/2'-OH repulsive contacts expected in a fully intercalated structure. The C3'-endo pucker of the RNA sugars and the orientation of the intercalated C.C+ pairs result in a modest widening of the narrow grooves at the steps where the hydroxyl groups are in close contact. The free energy of the RNA i-motif, on average -4 kJ mol(-1) per C.C+ pair, is half of the value found in DNA i-motif structures.     

Cationic porphyrins promote the formation of i-motif DNA and bind peripherally by a nonintercalative mechanism

Telomeric C-rich strands can form a noncanonical intercalated DNA structure known as an i-motif. We have studied the interactions of the cationic porphyrin 5,10,15,20-tetra-(N-methyl-4-pyridyl)porphine (TMPyP4) with the i-motif forms of several oligonucleotides containing telomeric sequences. TMPyP4 was found to promote the formation of the i-motif DNA structure. On the basis of (1)H NMR studies, we have created a model of the i-motif-TMPyP4 complex that is consistent with all the available experimental data. Two-dimensional NOESY data prompted us to conclude that TMPyP4 binds specifically to the edge of the intercalated DNA core by a nonintercalative mechanism. Since we have shown that TMPyP4 binds to and stabilizes the G-quadruplex form of the complementary G-rich telomeric strand, this study raises the intriguing possibility that TMPyP4 can trigger the formation of unusual DNA structures in both strands of the telomeres, which may in turn explain the recently documented biological effects of TMPyP4 in cancer cells.     

Kinetic hybrid i-motifs: intercepting DNA with RNA to form a DNA(2)-RNA(2) i-motif

We have constructed and characterized a long-lived hybrid DNA(2)-RNA(2) i-motif that is kinetically formed by mixing equivalent amount of C-rich RNA (R) and C-rich DNA (D). Circular dichroism shows that these hybrids are distinct from their parent DNA(4) or RNA(4) i-motif. pH dependent CD and UV thermal melting experiments showed that the complexes were maximally stable at pH 4.5, the pK(a) of cytosine, consistent with the complex being held by CH(+)-C base pairs. Fluorescence studies confirmed their tetrameric nature and established the relative strand polarities of the RNA and DNA strands in the complex. These showed that in a hybrid D(2)R(2) i-motif two DNA strands occupy one narrow groove and the two RNA strands occupy the other. This suggests that even the sugar-sugar interactions are highly specific. Interestingly, this hybrid slowly disproportionates into DNA(4) i-motifs and ssRNA which would be valuable to study intermediates in DNA(4) i-motif formation.     

[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 (C_nXC_m) 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, C_nXC_m 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.     

Intramolecular i-motif structure at acidic pH for progressive myoclonus epilepsy (EPM1) repeat d(CCCCGCCCCGCG)n.

The most common mutation associated with Progressive Myoclonus Epilepsy (EPM1) of Unverricht-Lundberg type is the expansion of a dodecamer repeat, d(CCCCGCCCCGCG)n. We show that the C-rich strand of this repeat (2-3 copies) forms intercalated i-motif structure at acidic pH as judged by CD spectroscopy and anomalous gel electrophoretic mobility. The stability of the structure increases with the increase in the length of the repeat. Transient formation of stable, folded back structure like i-motif could play an important role in the mechanism of expansion of this repeat.     

Intramolecular folding of a fragment of the cytosine-rich strand of telomeric DNA into an i-motif.

In the recently discovered i-motif, four stretches of cytosine form two parallel-stranded duplexes whose C.C+ base pairs are fully intercalated. The i-motif may be recognized by characteristic Overhauser cross-peaks of the proton NMR spectrum, reflecting short H1'-H1' distances across the minor groove, and short internucleotide amino-proton-H2'/H2" across the major groove. We report the observation of such cross-peaks in the spectra of a fragment of the C-rich telomeric strand of vertebrates, d[CCCTAA]3CCC. The spectra also demonstrate that the cytosines are base-paired and that proton exchange is very slow, as reported previously for the i-motif. From UV absorbance and gel chromatography measurements, we assign these properties to an i-motif which includes all or nearly all the cytosines, and which is formed by intramolecular folding at slightly acid or neutral pH. A fragment of telomeric DNA of Tetrahymena, d[CCCCAA]3CCCC, has the same properties. Hence four consecutive C stretches of a C-rich telomeric strand can fold into an i-motif. Hypothetically, this could occur in vivo.     

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 eta-form) and lower (the lambda-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 lambda and (5mC1.C11)/(C7.C17)/(C2.C12)/(C6.C16) for form eta. We have solved the structure of the eta-form, and we present a model for the lambda-form. The switch from eta to lambda 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 eta-form by the inter-loop reverse Watson-Crick A3.U13 pair, whose dissociation constant is around 10(-2) at 0 degrees C, and in the lambda-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 lambda-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.     

Small circular DNAs for synthesis of the human telomere repeat: varied sizes, structures and telomere-encoding activities

We describe the construction, structural properties and enzymatic substrate abilities of a series of circular DNA oligonucleotides that are entirely composed of the C-rich human telomere repeat, (CCCTAA)_n. The nanometer-sized circles range in length from 36 to 60 nt, and act as templates for synthesis of human telomere repeats in vitro. The circles were constructed successfully by the application of a recently developed adenine-protection strategy, which allows for cyclization/ligation with T4 DNA ligase. Thermal denaturation studies showed that at pH 5.0, all five circles form folded structures with similar stability, while at pH 7.0 no melting transitions were seen. Circular dichroism spectra at the two pH conditions showed evidence for i-motif structures at the lower pH value. The series was tested as rolling circle templates for a number of DNA polymerases at pH = 7.3–8.5, using 18mer telomeric primers. Results showed that surprisingly small circles were active, although the optimum size varied from enzyme to enzyme. Telomeric repeats 1000 nt in length could be synthesized in 1 h by the Klenow (exo-) DNA polymerase. The results establish a convenient way to make long human telomeric repeats for in vitro study of their folding and interactions, and establish optimum molecules for carrying this out.     

Parallel self-associated structures formed by T,C-rich sequences at acidic pH

Oligonucleotides of nonregular heteropyrimidine sequences incorporating or not incorporating purine residues 5'-d(ACTCCCTTCTCCTCTCTA), 5'-d(ACTCCCTGGTCCTCTCTA), 5'-d(TCTCTCCTGGTCCCTCC), and 5'-d(TCTCTCCTCTTCCCTCC) can form self-associated parallel-stranded (ps) structures at pH 4-5.5. The ps structures were identified by studying at neutral and acidic pH UV melting transitions, FTIR spectra, and fluorescence of pyrene-labeled oligonucleotides as well as by chemical joining of 5'-phosphorylated oligonucleotides. A gel electrophoresis run for oligonucleotides 5'-d(TCTCTCCTCTTCCCTCC) and 5'-d(ACTCCCTTCTCCTCTCTA) has shown the formation of homoduplexes at low DNA strand concentrations. Ps structures are held by C-C(+) base pairs and have N- and S-types of sugar puckering as detected by FTIR spectroscopy in the millimolar concentration range. Guanine inserts as well as thymine and purine inserts into an oligomeric cytosine sequence make the formation of the tetraplex i-motif unfavorable. MvaI restriction endonuclease, which recognizes the CCT/AGG sequence in DNA, does not cleave parallel pseudosubstrates.     

Formation of the G-quadruplex and i-motif structures in retinoblastoma susceptibility genes (Rb)

The formation of G-quadruplex and i-motif structures in the 5′ end of the retinoblastoma (Rb) gene was examined using chemical modifications, circular dichroism (CD) and fluorescence spectroscopy. It was found that substitutions of 8-methylguanine at positions that show syn conformations in antiparallel G-quadruplexes stabilize the structure in the G-rich strand. The complementary C-rich 18mer forms an i-motif structure, as suggested by CD spectroscopy. Based on the C to T mutation experiments, C bases participated in the C–C⁺ base pair of the i-motif structure were determined. Experiments of 2-aminopurine (2-AP) substitution reveal that an increase of fluorescence in the G-quadruplex relative to duplex is attributed to unstacked 2-AP within the loop of G-quadruplex. The fluorescence experiments suggest that formation of the G-quadruplex and i-motif can compete with duplex formation. Furthermore, a polymerase arrest assay indicated that formation the G-quadruplex structure in the Rb gene acts as a barrier in DNA synthesis.     

Participation of yeast inosine 5'-monophosphate dehydrogenase in an in vitro complex with a fragment of the C-rich telomeric strand

As part of our investigation of the i-motif, an intercalated structure formed by C-rich nucleic acid sequences, we searched for proteins of Saccharomyces cerevisiae which could associate with a sequence of the C-rich telomeric strand, d((CCCACA)(3)CCC). A gel retardation assay of yeast protein extract, in conditions where the DNA fragment folds into an intramolecular i-motif, shows formation of one major retarded band. The retarding factor was further characterized by a differential affinity procedure using streptavidin beads coated (or not coated) with the biotin-labeled DNA fragment. Differentially bound proteins were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and identified by mass spectroscopy and Edman degradation as Imd2p, Imd3p and Imd4p. These highly similar (>95%) proteins are analogs of the two human NAD-dependent inosine 5'-monophosphate dehydrogenases (IMPDH) which occur as tetramers. The mass of the protein, as determined by gel exclusion chromatography, is about 250 kDa and is compatible with an IMPDH tetramer, but other compositions, involving non-IMPDH components, are not excluded. We note that the genes coding for Imd2p and Imd3p are located close to the telomere, and could therefore be subject to silencing by the telomere position effect.     

Hybrid DNA i-motif: Aminoethylprolyl-PNA (pC5) enhance the stability of DNA (dC5) i-motif structure

This report describes the synthesis of C-rich sequence, cytosine pentamer, of aep-PNA and its biophysical studies for the formation of hybrid DNA:aep-PNAi-motif structure with DNA cytosine pentamer (dC₅) under acidic pH conditions. Herein, the CD/UV/NMR/ESI-Mass studies strongly support the formation of stable hybrid DNA i-motif structure with aep-PNA even near acidic conditions. Hence aep-PNA C-rich sequence cytosine could be considered as potential DNA i-motif stabilizing agents in vivo conditions.     

Crystal violet as an i-motif structure probe for reversible and label-free pH-driven electrochemical switch

A simple pH-induced electrochemical switch based on an i-motif structure is developed by using crystal violet as a selective electrochemical probe for the i-motif structure. Thiol-modified cytosine-rich single-strand oligonucleotide (C-rich ssDNA) can be self-assembled on the gold electrode surface via gold–sulfur interaction. Crystal violet is employed as an electrochemical probe for the i-motif structure because of its capability of binding with the i-motif structure through an end-stacking mode. In acidic aqueous solution, crystal violet may approach the electrode surface owing to the formation of the i-motif structure, resulting in an obvious signal, so-called “ON” state. Whereas in neutral or basic aqueous solution, the i-motif structure unfolds to dissociative single strand, which causes crystal violet to leave from the electrode surface, and a weak signal is obtained, so-called “OFF” state. In addition, in the range of pH 4.6–7.3, the increase in current has a good linear relationship (R = 0.989) with pH value in the testing solutions. This pH-driven electrochemical switch has the advantages of simplicity, sensitivity, high selectivity, and good reversibility. Furthermore, it provides a possible platform for pH measurement.     

pH-dependent assembly of DNA-gold nanoparticles based on the i-motif

Oligonucleotides containing stretches of 2 '-deoxycytidine residues were immobilized on 15 nm gold nanoparticles. Under acidic pH conditions a reversible supramolecular assembly is formed, induced by the formation of the tetrameric i-motif structure. The replacement of 2 '-deoxycytidine by 5-propynyl-2 '-deoxycytidine (dC*) leads to novel i-motif structures. Oligonucleotides incorporating multiple residues of dC* were immobilized on 15 nm gold nanoparticles and are able to aggregate into i-motif structures even at non-optimal pH values.     

pH-Dependant fluorescence switching of an i-motif structure incorporating an isomeric azobenzene/pyrene fluorophore

In this study, we synthesized an Azo-py phosphoramidite, featuring azobenzene and pyrene units, as a novel fluorescent and isomeric (trans- and cis-azobenzene units) material, which we incorporated in an i-motif DNA sequence. We then monitored the structural dynamics and changes in fluorescence as the modified DNA sequences transformed from single strands at pH 7 to i-motif quadruplex structures at pH 3. After incorporating Azo-py into the 4A loop position of an i-motif sequence, dramatic changes in fluorescence occurred as the DNA structures changed from single-strands to i-motif quadruplex structures. Interestingly, the cis form of Azo-py induced a more stable i-motif structure than did the trans form, as confirmed from circular dichroism spectra and melting temperature data. The absorption and fluorescence signals of these Azo-py-incorporated i-motif systems exhibited switchable and highly correlated signaling patterns. Such isomeric structures based on Azo-py might find potential applications in biology, where control over stable i-motif quadruplex structures might be performed with switchable fluorescence signaling.     

Chemical modification of pyrimidine TFOs: effect on i-motif and triple helix formation

In order to form more stable triple helical structures or to prevent their degradation in cells, oligonucleotide analogs are routinely used, either in the backbone or among the bases. The target sequence chosen for this study is a 16-base-long oligopurine-oligopyrimidine region present in the human neurotrophin 4/5 gene. Seven different chemical modifications were tested for their effect on (i) triple helix formation and (ii) i-DNA stability. i-DNA is a tetrameric structure involving hemiprotonated C x C+ base pairs, which may act as a competing structure for triplex formation, especially in the case of a cytosine-rich third strand. At acid pH, oligophosphoramidates formed the most stable triple helix, whereas oligonucleotides including 5-propynyl-dU formed a stable i-motif which precluded triplex formation. Only two candidates stabilized triple helices at neutral pH: oligonucleotides with phosphoramidate linkage and phosphodiester oligonucleotides containing 5-methyl-dC and 5-propynyl-dU.