RESUMEN
The i-motif is an intriguing non-canonical DNA structure, whose role in the cell is still controversial. Development of methods to study i-motif formation under physiological conditions in living cells is necessary to study its potential biological functions. The cytosine analog 1,3-diaza-2-oxophenoxazine (tCO) is a fluorescent nucleobase able to form either hemiprotonated base pairs with cytosine residues, or neutral base pairs with guanines. We show here that when tCO is incorporated in the proximity of a G:C:G:C minor groove tetrad, it induces a strong thermal and pH stabilization, resulting in i-motifs with Tm of 39ºC at neutral pH. The structural determination by NMR methods reveals that the enhanced stability is due to a large stacking interaction between the guanines of the tetrad with the tCO nucleobase, which forms a tCO:C+ in the folded structure at unusually-high pHs, leading to an increased quenching in its fluorescence at neutral conditions. This quenching is much lower when tCO is base-paired to guanines and totally disappears when the oligonucleotide is unfolded. By taking profit of this property, we have been able to monitor i-motif folding in cells.
Asunto(s)
Citosina , ADN , Emparejamiento Base , Citosina/análogos & derivados , ADN/química , Conformación de Ácido Nucleico , Oxazinas/química , Oxazinas/metabolismo , Células HeLa , Humanos , FluorescenciaRESUMEN
We study here a DNA oligonucleotide having the ability to form two different i-motif structures whose relative stability depends on pH and temperature. The major species at neutral pH is stabilized by two C:C+ base pairs capped by two minor groove G:C:G:C tetrads. The high pH and thermal stability of this structure are mainly due to the favorable effect of the minor groove tetrads on their adjacent positively charged C:C+ base pairs. At pH 5, we observe a more elongated i-motif structure consisting of four C:C+ base pairs capped by two G:T:G:T tetrads. Molecular dynamics calculations show that the conformational transition between the two structures is driven by the protonation state of key cytosines. In spite of large conformational differences, the transition between the acidic and neutral structures can occur without unfolding of the i-motif. These results represent the first case of a conformational switch between two different i-motif structures and illustrate the dramatic pH-dependent plasticity of this fascinating DNA motif.
Asunto(s)
ADN , G-Cuádruplex , Humanos , Conformación de Ácido Nucleico , ADN/química , Emparejamiento Base , Concentración de Iones de HidrógenoRESUMEN
Tetrads (or quartets) are arrangements of four nucleobases commonly involved in the stability of four-stranded nucleic acids structures. Four-stranded or quadruplex structures have attracted enormous attention in the last few years, being the most extensively studied guanine quadruplex (G-quadruplex). Consequently, the G-tetrad is the most common and well-known tetrad. However, this is not the only possible arrangement of four nucleobases. A number of tetrads formed by the different nucleobases have been observed in experimental structures. In most cases, these tetrads occur in the context of G-quadruplex structures, either inserted between G-quartets, or as capping elements at the sides of the G-quadruplex core. In other cases, however, non-G tetrads are found in more unusual four stranded structures, such as i-motifs, or different types of peculiar fold-back structures. In this report, we review the diversity of these non-canonical tetrads, and the structural context in which they have been found.
Asunto(s)
G-Cuádruplex , Guanina/química , Modelos Moleculares , Conformación de Ácido NucleicoRESUMEN
We report here the three-dimensional structure of an i-motif/duplex junction, determined by NMR methods at neutral pH. By including a minor groove tetrad at one side of the C:C+ stack of a monomeric i-motif, and a stem/loop hairpin at the other side, we have designed stable DNA constructs in which i-DNA and B-DNA regions coexist in a wide range of experimental conditions. This study demonstrates that i- and B-DNA are structurally compatible, giving rise to a distinctive fold with peculiar groove shapes. The effect of different residues at the i-motif/duplex interface has been explored. We also show that these constructs can be adapted to sequences of biological relevance, like that found in the promoter region of the KRAS oncogene.
RESUMEN
We report here the solution structure of several repetitive DNA sequences containing d(TCGTTCCGT) and related repeats. At physiological pH, these sequences fold into i-motif like quadruplexes in which every two repeats a globular structure is stabilized by two hemiprotonated C:C+ base pairs, flanked by two minor groove tetrads resulting from the association of G:C or G:T base pairs. The interaction between the minor groove tetrads and the nearby C:C+ base pairs affords a strong stabilization, which results in effective pHT values above 7.5. Longer sequences with more than two repeats are able to fold in tandem, forming a rosary bead-like structure. Bioinformatics analysis shows that these sequences are prevalent in the human genome, and are present in development-related genes.
Asunto(s)
ADN/química , Genoma Humano , Secuencias Repetitivas de Ácidos Nucleicos , Emparejamiento Base , Secuencia de Bases , G-Cuádruplex , Humanos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Conformación de Ácido NucleicoRESUMEN
Human centromeric alpha-satellite DNA is composed of tandem arrays of two types of 171â bp monomers; type A and type B. The differences between these types are concentrated in a 17â 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.
Asunto(s)
Centrómero/química , Sustancias Intercalantes/química , Emparejamiento Base , Secuencia de Bases , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Moleculares , Conformación de Ácido NucleicoRESUMEN
Tau pre-mRNA contains a stem-loop structure involved in the regulation of the alternative splicing of tau protein. We describe here a new family of Tau RNA ligands selected by dynamic combinatorial chemistry based on the combination of ametantrone with small RNA-binding molecules. The most promising compound results from derivatization of one of the side chains of the anthraquinone ring with the small aminoglycoside neamine through a short spacer. This compound binds the RNA target with a high affinity in a preferred binding site, in which the heteroaromatic moiety intercalates in the bulged region of the stem-loop and its side chains and neamine interact with the major groove of the RNA. Importantly, binding of this compound to mutated RNA sequences involved in the onset of some tauopathies such as FTDP-17 restores their thermodynamic stability to a similar or even higher levels than that of the wild-type sequence, thereby revealing its potential as a modulator of Tau pre-mRNA splicing.
Asunto(s)
Empalme Alternativo , Mitoxantrona/análogos & derivados , Precursores del ARN/genética , ARN Mensajero/genética , Proteínas tau/genética , Espectroscopía de Resonancia Magnética , Mitoxantrona/químicaRESUMEN
Some DNA oligonucleotides can fold back and self-associate forming dimeric structures stabilized by intermolecular base pairs. The resulting antiparallel dimer is a tightly packed four-stranded structure formed by a core of minor groove tetrads connected by short loops of unpaired nucleotides. We have explored the sequential requirements for the loop residues and have found that this family of structures is only stable with one- and two-residue loops, with the stability of the former ones being only marginal. Two-residue loops with purines in the first position give rise to the most stable structures due to their enhanced stacking interaction with the adjacent minor groove tetrad. On the other hand, pyrimidines confer more stability than purines in the second position of the loop.
Asunto(s)
Oligonucleótidos/química , Dimerización , Modelos Moleculares , Conformación de Ácido Nucleico , TemperaturaRESUMEN
The repetitive DNA sequences found at telomeres and centromeres play a crucial role in the structure and function of eukaryotic chromosomes. This role may be related to the tendency observed in many repetitive DNAs to adopt non-canonical structures. Although there is an increasing recognition of the importance of DNA quadruplexes in chromosome biology, the co-existence of different quadruplex-forming elements in the same DNA structure is still a matter of debate. Here we report the structural study of the oligonucleotide d(TCGTTTCGT) and its cyclic analog d
Asunto(s)
G-Cuádruplex , Emparejamiento Base , Dimerización , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Desnaturalización de Ácido Nucleico , Motivos de Nucleótidos , Oligonucleótidos/química , ProtonesRESUMEN
Minor groove aligned tetrads resulting from the association of Watson-Crick base pairs stabilize a distinct class of four-stranded DNA structures, different from G-quadruplexes or i-motifs. These tetrads can be formed by several arrangements of G-C or A-T base pairs. Here we prove that minor groove tetrads can be also formed by G-T mismatches. In this manuscript we describe the dimeric solution structures of two cyclic oligonucleotides stabilized by intermolecular G-T non-canonical base pairs. In the dimeric structure of d
Asunto(s)
Emparejamiento Base , G-Cuádruplex , Oligonucleótidos/química , Dimerización , Conformación de Ácido NucleicoRESUMEN
In addition to the better known guanine-quadruplex, four-stranded nucleic acid structures can be formed by tetrads resulting from the association of Watson-Crick base pairs. When such association occurs through the minor groove side of the base pairs, the resulting structure presents distinctive features, clearly different from quadruplex structures containing planar G-tetrads. Although we have found this unusual DNA motif in a number of cyclic oligonucleotides, this is the first time that this DNA motif is found in linear oligonucleotides in solution, demonstrating that cyclization is not required to stabilize minor groove tetrads in solution. In this article, we have determined the solution structure of two linear octamers of sequence d(TGCTTCGT) and d(TCGTTGCT), and their cyclic analogue d
Asunto(s)
Citosina/química , ADN/química , G-Cuádruplex , Guanina/química , Emparejamiento Base , Dimerización , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Desnaturalización de Ácido Nucleico , Oligodesoxirribonucleótidos/químicaRESUMEN
Four-stranded nucleic acid structures are central to many processes in biology and in supramolecular chemistry. It has been shown recently that four-stranded DNA structures are not only limited to the classical guanine quadruplex but also can be formed by tetrads resulting from the association of Watson-Crick base pairs. Such an association may occur through the minor or the major groove side of the base pairs. Structures stabilized by minor groove tetrads present distinctive features, clearly different from the canonical guanine quadruplex, making these quadruplexes a unique structural motif. Within our efforts to study the sequence requirements for the formation of this unusual DNA motif, we have determined the solution structure of the cyclic oligonucleotide dpCCGTCCGT by two-dimensional NMR spectroscopy and restrained molecular dynamics. This molecule self-associates, forming a symmetric dimer stabilized by two G:C:G:C tetrads with intermolecular G-C base pairs. Interestingly, although the overall three-dimensional structure is similar to that found in other cyclic and linear oligonucleotides of related sequences, the tetrads that stabilize the structure of dpCCGTCCGT are different to other minor groove G:C:G:C tetrads found earlier. Whereas in previous cases the G-C base pairs aligned directly, in this new tetrad the relative position of the two base pairs is slipped along the axis defined by the base pairs. This is the first time that a quadruplex structure entirely stabilized by slipped minor groove G:C:G:C tetrads is observed in solution or in the solid state. However, an analogous arrangement of G-C base pairs occurs between the terminal residues of contiguous duplexes in some DNA crystals. This structural polymorphism between minor groove GC tetrads may be important in stabilization of higher order DNA structures.
Asunto(s)
ADN/química , Fenómenos Químicos , Química Física , G-Cuádruplex , Cinética , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular/métodos , Conformación de Ácido NucleicoRESUMEN
We have investigated the molecular interaction between cyclic and linear oligonucleotides. We have found that short cyclic oligonucleotides can induce hairpinlike structures in linear DNA fragments. By using NMR and CD spectroscopy we have studied the interaction of the cyclic oligonucleotide d
Asunto(s)
ADN/metabolismo , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , Oligonucleótidos/metabolismo , Emparejamiento Base , ADN/química , Dimerización , Oligonucleótidos/químicaRESUMEN
A protocol for the straightforward preparation of small circular oligodeoxyribonucleotides (2-28 nt) is reported. The assembly of the oligonucleotide chain (standard phosphoramidite chemistry) and cyclization by the phosphotriester method take place on a tailor-made nucleotide-derivatized solid support. Although cyclization yields are moderate, the procedure exploits a synthesis design that allows selective cleavage of the circular oligonucleotide from the support, which facilitates isolation of the target molecule by simple filtration.
Asunto(s)
Oligonucleótidos/síntesis química , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de PoliacrilamidaRESUMEN
The structures and stabilities of cyclic DNA octamers of different sequences have been studied by NMR and CD spectroscopy and by restrained molecular dynamics. At low oligonucleotide concentrations, some of these molecules form stable monomeric structures consisting of a short stem of two base pairs connected by two mini-loops of two residues. To our knowledge, these dumbbell-like structures are the smallest observed to date. The relative stabilities of these cyclic dumbbells have been established by studying their melting transitions. Dumbbells made up purely of GC stems are more stable than those consisting purely of AT base pairs. The order of the base pairs closing the loops also has an important effect on the stabilities of these structures. The NMR data indicate that there are significant differences between the solution structures of dumbbells with G-C base pairs in the stem compared to those with A-T base pairs. In the case of dumbbells with G-C base pairs, the residues in the stem form a short segment of a BDNA helix stabilized by two Watson-Crick base pairs. In contrast, in the case of d
Asunto(s)
Emparejamiento Base , ADN/química , Conformación de Ácido Nucleico , Composición de Base , Secuencia de Bases , Dicroismo Circular , Ciclización , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Desnaturalización de Ácido Nucleico , Oligonucleótidos/química , Temperatura , TermodinámicaRESUMEN
The solution structure of a cyclic oligonucleotide d
Asunto(s)
ADN/química , Emparejamiento Base , G-Cuádruplex , Cinética , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , Oligonucleótidos/química , Soluciones , TermodinámicaRESUMEN
A successful application is reported of the multivariate curve resolution alternating least-squares method (MCR-ALS) for the analysis of nucleic acid melting and salt-induced transitions. Under conditions where several structures co-exist in a conformational equilibrium, MCR-ALS analysis of the UV and circular dichroism (CD) spectra at different temperatures, ionic strength and oligonucleotide concentration allows for the resolution of concentration profiles and pure spectra of the different species. The methodology is illustrated by the case of the cyclic oligonucleotide d