RESUMEN
G-quadruplexes (G4s), which are known to have important roles in regulation of key biological processes in both normal and pathological cells, are the most actively studied non-canonical structures of nucleic acids. In this review, we summarize the results of studies published in recent years that change significantly scientific views on various aspects of our understanding of quadruplexes. Modern notions on the polymorphism of DNA quadruplexes, on factors affecting thermodynamics and kinetics of G4 folding-unfolding, on structural organization of multiquadruplex systems, and on conformational features of RNA G4s and hybrid DNA-RNA G4s are discussed. Here we report the data on location of G4 sequence motifs in the genomes of eukaryotes, bacteria, and viruses, characterize G4-specific small-molecule ligands and proteins, as well as the mechanisms of their interactions with quadruplexes. New information on the structure and stability of G4s in telomeric DNA and oncogene promoters is discussed as well as proof being provided on the occurrence of G-quadruplexes in cells. Prominence is given to novel experimental techniques (single molecule manipulations, optical and magnetic tweezers, original chemical approaches, G4 detection in situ, in-cell NMR spectroscopy) that facilitate breakthroughs in the investigation of the structure and functions of G-quadruplexes.
Asunto(s)
ADN/química , G-Cuádruplex , ARN/química , Animales , Bacterias/genética , Secuencia de Bases , Conformación de Ácido Nucleico , Telómero/químicaRESUMEN
Using UV and CD spectroscopy, we studied the thermodynamic stability and folding topology of G-quadruplexes (G4), formed by G-rich fragments in human microsatellites that differ in the number of guanosines within the repeating unit. The oligonucleotides d(GGGT)4 and d(GGT)4 were shown to form propeller-type parallel-stranded intramolecular G-quadruplexes. The G4 melting temperature is dramatically decreased (by more than 45°C) in the transition from the tri-G-tetrad to the bi-G-tetrad structure. d(GT)n-repeats do not form perfect G-quadruplexes (one-G-tetrad); folded G4-like conformation is not stable at room temperature and is not stabilized by monovalent metal ions. The minimum concentration of K+ that promotes quadruplex folding of d(GGT)4 was found to depend on the supporting Na+ concentration. It was demonstrated for the first time that the complementary regions flanking G4-motifs (as in d(CACTGG-CC-(GGGT)4-TA-CCAGTG)) cannot form a double helix in the case of a parallel G4 due to the steric remoteness, but instead destabilize the structure. Additionally, we investigated the effect of the described oligonucleotides on the activity of topoisomerase I, one of the key cell enzymes, with a focus on the relationship between the stability of the formed quadruplexes and the inhibition degree of the enzyme. The most active inhibitor with IC50 = 0.08 µM was the oligonucleotide d(CACTGG-CC-(GGGT)4-TA-CCAGTG), whose flanking G4-motif sequences reduced the extreme stability of G-quadruplex formed by d(GGGT)4.
Asunto(s)
ADN-Topoisomerasas de Tipo I/metabolismo , ADN/química , G-Cuádruplex , Repeticiones de Microsatélite , Dicroismo Circular , ADN/metabolismo , ADN-Topoisomerasas de Tipo I/genética , Guanina/química , Guanina/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Conformación de Ácido Nucleico , Espectrofotometría Ultravioleta , Temperatura , TermodinámicaRESUMEN
A number of thrombin-binding DNA aptamers have been developed during recent years. So far the structure of just a single one, 15-mer thrombin-binding aptamer (15TBA), has been solved as G-quadruplex. Structures of others, showing variable anticoagulation activities, are still not known yet. In this paper, we applied the circular dichroism and UV spectroscopy to characterize the temperature unfolding and conformational features of 31-mer thrombin-binding aptamer (31TBA), whose sequence has a potential to form G-quadruplex and duplex domains. Both structural domains were monitored independently in 31TBA and in several control oligonucleotides unable to form either the duplex region or the G-quadruplex region. The major findings are as follows: (1) both duplex and G-quadruplex domains coexist in intramolecular structure of 31TBA, (2) the formation of duplex domain does not change the fold of G-quadruplex, which is very similar to that of 15TBA, and (3) the whole 31TBA structure disrupts if either of two domains is not formed: the absence of duplex structure in 31TBA abolishes G-quadruplex, and vice versa, the lack of G-quadruplex folding results in disallowing the duplex domain.
Asunto(s)
Aptámeros de Nucleótidos/química , ADN/química , G-Cuádruplex , Oligonucleótidos/química , Dicroismo Circular , ADN/metabolismo , Proteínas de Unión al ADN/química , Conformación de Ácido Nucleico , Espectroscopía de Fotoelectrones , Conformación Proteica , Estructura Secundaria de Proteína , Trombina/antagonistas & inhibidores , Trombina/química , Trombina/metabolismoRESUMEN
A spectroscopic assay for detection of extrahelical thymine residues in DNA heteroduplexes under their modification by potassium permanganate has been developed. The assay is based on increase in absorbance at 420 nm due to accumulation of thymidine oxidation intermediates and soluble manganese dioxide. The analysis was carried out using a set of 19-bp DNA duplexes containing unpaired thymidines opposite tetrahydrofuranyl derivatives mimicking a widespread DNA damage (apurinic (AP) sites) and a library of 50-bp DNA duplexes containing all types of base mismatches in different surroundings. The relation between the selectivity of unpaired T oxidation and the thermal stability of DNA double helix was investigated. The method described here was shown to discriminate between DNA duplexes with one or two AP sites and to reveal thymine-containing mismatches and all noncanonical base pairs in AT-surroundings. Comparative results of CCM analysis and the rapid photometric assay for mismatch detection are demonstrated for the first time in the same model system. The chemical reactivity of target thymines was shown to correlate with local disturbance of double helix at the mismatch site. As the spectroscopic assay does not require the DNA cleavage reaction and gel electrophoresis, it can be easily automated and used for primary screening of somatic mutations.
Asunto(s)
Análisis Mutacional de ADN/métodos , Ácidos Nucleicos Heterodúplex/química , Disparidad de Par Base , Ácidos Nucleicos Heterodúplex/genética , Oligodesoxirribonucleótidos/química , Oxidación-Reducción , Fotometría , Mutación Puntual , Permanganato de Potasio , Timidina/químicaRESUMEN
Novel modified DNA duplexes with single bridging 5'-SS-monophosphoryldithio links [-OP(=O)-O(-)-SS-CH(2)-] were synthesized by autoligation of an oligonucleotide 3'-phosphorothioate and a 5'-mercapto-oligonucleotide previously converted to a 2-pyridyldisulfide adduct. Monophosphoryldisulfide link formation is not a stringent template-dependent process under the conditions used and does not require strong binding of the reactive oligomers to the complementary strand. The modified internucleotide linkage, resembling the natural phosphodiester bond in size and charge density, is stable in water, easily undergoes thiol-disulfide exchange and can be specifically cleaved by the action of reducing reagents. DNA molecules containing an internal -OP(=O)-O(-)-SS-CH(2)- bridge are stable to spontaneous exchange of disulfide-linked fragments (recombination) even in the single-stranded state and are promising reagents for autocrosslinking with cysteine-containing proteins. The chemical and supramolecular properties of oligonucleotides with 5'-sulfhydryl groups were further characterized. We have shown that under the conditions of chemical ligation the 5'-SH group of the oligonucleotide has a higher reactivity towards N-hydroxybenzotriazole-activated phosphate in an adjacent oligonucleotide than does the OH group. This autoligation, unlike disulfide bond formation, proceeds only in the presence of template oligonucleotide, necessary to provide the activated phosphate in close proximity to the SH-, OH- or phosphate function.
Asunto(s)
Reactivos de Enlaces Cruzados/química , ADN/química , Oligodesoxirribonucleótidos/química , Tionucleótidos/química , Cromatografía Líquida de Alta Presión , Reactivos de Enlaces Cruzados/síntesis química , Reactivos de Enlaces Cruzados/metabolismo , ADN/síntesis química , Disulfuros/química , Ditiotreitol/química , Electroforesis en Gel de Poliacrilamida , Etildimetilaminopropil Carbodiimida/química , Modelos Químicos , Desnaturalización de Ácido Nucleico , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/metabolismo , Moldes GenéticosRESUMEN
A DNA third strand with a 3'-psoralen substituent was designed to form a triplex with the sequence downstream of the T.A mutant base pair of the human sickle cell beta-globin gene. Triplex-mediated psoralen modification of the mutant T residue was sought as an approach to gene repair. The 24-nucleotide purine-rich target sequence switches from one strand to the other and has four pyrimidine interruptions. Therefore, a third strand sequence favorable to two triplex motifs was used, one parallel and the other antiparallel to it. To cope with the pyrimidine interruptions, which weaken third strand binding, 5-methylcytosine and 5-propynyluracil were used in the third strand. Further, a six residue "hook" complementary to an overhang of a linear duplex target was added to the 5'-end of the third strand via a T(4) linker. In binding to the overhang by Watson-Crick pairing, the hook facilitates triplex formation. This third strand also binds specifically to the target within a supercoiled plasmid. The psoralen moiety at the 3'-end of the third strand forms photoadducts to the targeted T with high efficiency. Such monoadducts are known to preferentially trigger reversion of the mutation by DNA repair enzymes.
Asunto(s)
Furocumarinas , Globinas/genética , Hemoglobina Falciforme/genética , Mutación Puntual , Adenina , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Huella de ADN , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/química , Moldes Genéticos , TiminaRESUMEN
In contrast to shorter homologs which only form a single-stranded nucleic acid alpha-helix in acid solution at [Na+]/=0.2 M) the protonated base-backbone interactions are so weakened that duplex stability becomes increasingly dependent upon H-bonded base pairing and stacking and almost independent of pH. Between pH 6 and 8 this duplex structure is devoid of protonated dA residues and shows positive dependence of T m on ionic strength similar to that of DNA.
Asunto(s)
Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Composición de Base , Secuencia de Bases , Dicroismo Circular , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Desnaturalización de Ácido Nucleico , SodioRESUMEN
Cyanogen bromide has been found to induce the template-guided condensation of oligonucleotides only in the presence of N-substituted morpholines. Based on 31P, 1H and 13C NMR spectroscopy data, the mechanism of the phosphomonoester group activation by cyanogen bromide in N-substituted morpholine buffers is suggested. It has also been shown that BrCN can be used for the synthesis of oligonucleotide derivatives in aqueous solution.
Asunto(s)
Bromuro de Cianógeno/química , Evolución Molecular , Morfolinas/química , Oligodesoxirribonucleótidos/química , Oligonucleótidos/síntesis química , Tampones (Química) , ADN/química , Moldes GenéticosRESUMEN
An efficient method for producing the covalent closure of oligonucleotides on complementary templates by the action of BrCN was developed. A rational design of linear precursor oligonucleotides was studied, and the effect of factors such as oligonucleotide concentration and oligomer-template length ratio was evaluated. The efficiency of circularization was shown to correlate well with the secondary structure of the precursor oligomer (as predicted by a simple computer analysis), hairpin-like structures bearing free termini clearly favouring the circularization reaction. A novel idea, consisting of the incorporation of non-nucleotide insertions in the precursor oligomer (namely, 1,2-dideoxy-D-ribofuranose residues), may render this method universal and highly effective. An original set of assays was developed to confirm the circular structure of the covalently closed oligonucleotides.
Asunto(s)
ADN Circular/química , Oligonucleótidos/química , Secuencia de Bases , Bromuro de Cianógeno/química , ADN de Cadena Simple/química , Enlace de Hidrógeno , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Moldes GenéticosRESUMEN
Previously (Dolinnaya & Fresco, 1992), on the basis of an analysis of UV absorption and CD properties as a function of temperature and pH, the secondary structure of the deoxyoligonucleotide d(A(+)-G)10 was hypothesized to be helical and intramolecular in origin, being stabilized not by stacked bases or hydrogen-bonded base pairs but instead by ionic bonds between positively charged adenine residues and distal negatively charged phosphates. Several other properties are now shown to be consistent with this unusual type of structure. The molecular weight determined for d(A(+)-G)10 by sedimentation equilibrium is that of the single strand, and consistent with this, there is no molecular weight change on helix disruption. Formation of the d(A(+)-G)10 helix is accompanied by cooperative uptake of nine protons, corresponding to nine adenine residues that can form ionic bonds with all the available distal phosphates, i.e., the n+1 or the n+2 phosphates. The thermodynamic parameters of this helical structure obtained from both van't Hoff analysis of the melting of the structure and calorimetric measurements are in keeping with the ionic properties of the proposed structure. So are the dependence of its stability on pH and ionic strength, and also on oligomer length when compared with the behavior of d(A(+)-G)6. The possible role of this type of secondary structure in protein recognition of the single-stranded homopurine element of H-DNA is evaluated.
Asunto(s)
Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Secuencia de Bases , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Concentración de Iones de Hidrógeno , Cinética , Matemática , Datos de Secuencia Molecular , TermodinámicaRESUMEN
We have identified a type of secondary structure for the homopurine oligomer d(A-G)10 below pH 6 in 0.01 M Na+ that is characterized by intense CD but only minor hypochromicity. The stability of this helix, designated d(A(+)-G)10, does not depend on oligomer concentration and increases sharply as ionic strength or pH drops, reaching a maximum at 4.0 (melting temperature, 37 degrees C). The pKa for the transition, 5.3 at 25 degrees C and even higher with decreasing temperature and [Na+], is much higher than the intrinsic pKa values for dA or dG residues. While the dA residues are protonated in the helix, further protonation of the dG residues disrupts it. When observed at 280 nm, melting of the helix first results in hypochromicity due to stacking of extrahelical dG residues with neighboring dA residues. The character and temperature dependence of the CD spectra of the constituent dinucleoside monophosphates indicate minimal chirality and base overlap for the A+pG sequences in d(A(+)-G)10 but left-handed twist with some base overlap for the GpA+ sequences. The observed properties are best satisfied by a model for an intramolecular helix with limited base overlap, stabilized by ionic bonds between dA residues protonated at N-1 and downstream negatively charged phosphates brought close due to the backbone helical twist, while Gsyn residues lie external to the helix. This structure could provide additional stabilizing energy for biologically relevant protonated non-B-DNA structures adopted by homopurine.homopyrimidine sequences due to topological stress or specific protein binding.
Asunto(s)
Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Dicroismo Circular , Nucleótidos de Desoxiadenina/química , Nucleótidos de Desoxiguanina/química , Concentración de Iones de Hidrógeno , Desnaturalización de Ácido Nucleico , Espectrofotometría Ultravioleta , TermodinámicaRESUMEN
A synthetic ribooligonucleotide, r(CCAGACUGm-AAGAUCUGG), corresponding to the unmodified yeast tRNA(Phe) anticodon arm is shown to bind to poly(U) programmed small ribosomal subunits of both E. coli and rabbit liver with affinity two order less than that of a natural anticodon arm. Its deoxyriboanalogs d(CCAGACTGAAGATCTGG) and d(CCAGA)r(CUGm-AAGA)d(TCTGG), are used to study the influence of sugar-phosphate modification on the interaction of tRNA with programmed small ribosomal subunits. The deoxyribooligonucleotide is shown to adopt a hairpin structure. Nevertheless, as well as oligonucleotide with deoxyriboses in stem region, it is not able to bind to 30S or 40S ribosomal subunits in the presence of ribo-(poly(U] or deoxyribo-(poly (dT) template. The deoxyribooligonucleotide also has no inhibitory effect on tRNA(Phe) binding to 30S ribosomes at 10-fold excess over tRNA. Neomycin does not influence binding of tRNA anticodon arm analogs used. Complete tRNA molecule and natural modifications of anticodon arm are considered to stabilize the arm structure needed for its interaction with a programmed ribosome.
Asunto(s)
Anticodón/metabolismo , Oligonucleótidos/metabolismo , ARN de Transferencia de Fenilalanina/metabolismo , Ribosomas/metabolismo , Animales , Anticodón/efectos de los fármacos , Anticodón/genética , Secuencia de Bases , Escherichia coli/metabolismo , Cinética , Hígado/metabolismo , Datos de Secuencia Molecular , Neomicina/farmacología , Conformación de Ácido Nucleico , Poli T/metabolismo , Poli U/metabolismo , ARN de Transferencia de Fenilalanina/efectos de los fármacos , ARN de Transferencia de Fenilalanina/genética , Conejos , Ribosomas/efectos de los fármacos , LevadurasRESUMEN
Both cyanogen bromide (BrCN) and 1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide may be used as coupling reagents for the template-directed assembly of DNA duplexes containing the sugar-phosphate backbone modification. Both reagents show similar ligation site structure-specific trend. Practical recommendations are given for selection of the condensing reagent depending on the properties of the duplex. Based on 31P NMR spectroscopy data, a scheme is suggested for BrCN activation of the nucleotide phosphomonoester group. Using both condensing reagents, we studied the condensation of oligonucleotides containing ribo-segments (from mononucleotide residue to full sequence) on the DNA template. Efficiency of the chemical ligation of RNA oligomers was shown to be much lower than that of DNA analogues. The coupling yield depends on the position of the RNA segment in the hybrid duplexes and on the position of the phosphate group in the nick.
Asunto(s)
Carbodiimidas/química , Bromuro de Cianógeno/química , ADN/química , Ácidos Nucleicos Heterodúplex , ARN/química , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Moldes GenéticosRESUMEN
A series of oligomeric double and triple helical DNAs with irregular sequences of homopurine and homopyrimidine strands were prepared. DNA triplexes were identified by CD spectroscopy and thermal denaturation profiles (biphasic helix-coil transition). Condensation of oligonucleotides on single and double-stranded DNA templates was performed using water-soluble carbodiimide, phosphodiester and pyrophosphate internucleotide bonds being newly formed. Such chemical ligation proved to be a sensitive monitor of changes in the sugar-phosphate backbone resulting from conversion of double to triple helix and of third-strand binding.
Asunto(s)
ADN/química , Conformación de Ácido Nucleico , Secuencia de Bases , Dicroismo Circular , Calor , Datos de Secuencia Molecular , Desnaturalización de Ácido Nucleico , Moldes GenéticosRESUMEN
Chemical ligation of oligonucleotides in double-stranded helices has been considered in its structural-kinetic aspect. A study was made of (i) two series of DNA duplexes with various arrangements of reacting groups in the ligation junction induced by mispairing or by alteration of furanose structure (the replacement of dT unit with rU, aU, IU, xU, dxT ones) and of (ii) eight synthetic water-soluble carbodiimides with different substituents at N1 and N3 atoms. We assumed that some information on the local structure of modified sites in the duplex can be obtained from kinetic parameters of oligonucleotide coupling reaction. The ratio of kinetic constants k3/(k2 + k3) for productive and nonproductive decomposition of the activated phosphomonoester derivative apparently reflects the reaction site structure: for a given duplex this parameter is virtually independent of the condensing agent composition. Based on the analysis of the chemical ligation kinetics a suggestion has been made about the conformation of some modified units in the double helix.
Asunto(s)
ADN/química , Ácidos Nucleicos Heterodúplex/química , Autorradiografía , Secuencia de Bases , Carbodiimidas/química , Electroforesis en Gel de Poliacrilamida , Cinética , Datos de Secuencia MolecularRESUMEN
Cyanogen bromide was found to condense oligodeoxyribonucleotides on a complementary template in aqueous solution. Optimum conditions for this vigorous and effective reaction were developed. CNBr proved to be useful for incorporation of phosphoramidate or pyrophosphate internucleotide bonds in DNA duplexes.
Asunto(s)
Bromuro de Cianógeno/farmacología , ADN/efectos de los fármacos , Desoxirribonucleasas de Localización Especificada Tipo II , Oligodesoxirribonucleótidos , Secuencia de Bases , Fenómenos Químicos , Química , ADN Ligasas/metabolismo , Enzimas de Restricción del ADN/metabolismo , Oligodesoxirribonucleótidos/metabolismoRESUMEN
The efficiency of chemical ligation method have been demonstrated by assembling a number of DNA duplexes with modified sugar phosphate backbone. Condensation on a tetradecanucleotide template of hexa(penta)- and undecanucleotides differing only in the terminal nucleoside residue have been performed using water-soluble carbodiimide as a condensing agent. As was shown by comparing the efficiency of chemical ligation of single-strand breaks in those duplexes, the reaction rate rises 70 or 45 times if the 3'-OH group is substituted with an amino or phosphate group (the yield of products with a phosphoramidate or pyrophosphate bond is 96-100% in 6 d). Changes in the conformation of reacting groups caused by mismatched base pairs (A.A, A.C) as well as the hybrid rU.dA pair or an unpaired base make the template-directed condensation less effective. The thermal stability of DNA duplexes was assayed before and after the chemical ligation. Among all of the modified duplexes, only the duplex containing 3'-rU in the nick was found to be a substrate of T4 DNA ligase.
Asunto(s)
ADN , Oligodesoxirribonucleótidos , Composición de Base , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/síntesis química , Radioisótopos de Fósforo , FosforilaciónRESUMEN
Self-association of a decanucleotide d(TGGCCAAGCTp) in an aqueous solution is shown by UV spectroscopy, CD and sedimentation analysis to yield a pseudopolymeric (concatemeric) duplex having a geometry similar to that of DNA B-type. It is demonstrated that in conditions when the concatemeric duplex is stable a water-soluble carbodiimide induces efficient polymerization of the 3'- or 5'-phosphorylated decanucleotide, and the resulting polymers d(TGGCCAAGCTp)2-10 contain only natural phosphodiester bonds. In conditions optimal for template-guided polymerization of d(TGGCCAAGCTp) the overall yield of 20-100-member polynucleotides exceeds 90%. The obtained polymeric duplexes are cleaved by restriction endonuclease Alu II, Bsu RI, and Hind III to corresponding decamers which were isolated and sequenced.
Asunto(s)
Oligodesoxirribonucleótidos/síntesis química , Oligonucleótidos/síntesis química , Secuencia de Bases , Dicroismo Circular , Enzimas de Restricción del ADN , Cinética , Conformación de Ácido Nucleico , Espectrofotometría Ultravioleta , Moldes GenéticosRESUMEN
A new class of synthetic DNA duplexes containing repeating oligonucleotide sequences, double-helical concatemers, is characterized. The UV-absorption and circular dichroism of a concatemer formed in self-association of d(T-G-C-A-C-A-T-G) have been studied. The thermodynamical parameters of complex formation are the following: delta Ho1 = -9.2 +/- 0.3 kcal/mol, delta So1 = -27 +/- 1 e.u. The data obtained show that pseudopolymeric duplexes having structures that are similar to DNA-B-type helices are formed in solutions of d(T-G-C-A-C-A-T-G). Polymerization of 32P-d(pT-G-C-A-C-A-T-G) induced by water-soluble carbodiimide has been carried out under the conditions of concatemer stability. The yield of the dimer, a 16-member oligonucleotide, was 13%.