RESUMEN
The combination of fluorine labeling and pulsed electron-nuclear double resonance (ENDOR) is emerging as a powerful technique for obtaining structural information about proteins and nucleic acids. In this work, we explored the capability of Mims 19F ENDOR experiments on reporting intermolecular distances in trityl- and 19F-labeled DNA duplexes at three electron paramagnetic resonance (EPR) frequencies (34, 94, and 263 GHz). For spin labeling, we used the hydrophobic Finland trityl radical and hydrophilic OX063 trityl radical. Fluorine labels were introduced into two positions of a DNA oligonucleotide. The results indicated that hyperfine splittings visible in the ENDOR spectra are consistent with the most populated interspin distances between 19F and the trityl radical predicted from molecular dynamic (MD) simulations. Moreover, for some cases, ENDOR spectral simulations based on MD results were able to reproduce the conformational distribution reflected in the experimental ENDOR line broadening. Additionally, MD simulations provided more detailed information about the melting of terminal base pairs of the oligonucleotides and about the configuration of the trityls relative to a DNA end.
Asunto(s)
Flúor , Ácidos Nucleicos , Espectroscopía de Resonancia por Spin del Electrón , Marcadores de Spin , ADN , OligonucleótidosRESUMEN
Human apurinic/apyrimidinic endonuclease 1 (APE1) participates in the DNA repair system. It is believed that the main biological function of APE1 is Mg2+-dependent hydrolysis of AP-sites in DNA. On the base of structural data, kinetic studies, and mutation analysis, the key stages of APE1 interaction with damaged DNA were established. It has been shown recently that APE1 can act as an endoribonuclease that catalyzes mRNA hydrolysis at certain pyrimidine-purine sites and thus controls the level of certain transcripts. In addition, the presence of Mg2+ ions was shown to be not required for the endoribonuclease activity of APE1, in contrast to the AP-endonuclease activity. This indicates differences in mechanisms of APE1 catalysis on RNA and DNA substrates, but the reasons for these differences remain unclear. Here, the analysis of endoribonuclease hydrolysis of model RNA substrates with wild type APE1 enzyme and its mutant forms Y171F, R177F, R181A, D210N, N212A, T268D, M270A, and D308A, was performed. It was shown that mutation of Asn212, Asp210, and Tyr171 residues leads to the decrease of AP-endonuclease activity while endoribonuclease activity is retained. Also, T268D and M270A APE1 mutants lose specificity to pyrimidine-purine sequences. R177F and R181A did not show a significant decrease in enzyme activity, whereas D308A demonstrated a decrease of endoribonuclease activity.
RESUMEN
Human apurinic/apyrimidinic endonuclease 1 (APE1) participates in the DNA repair system. It is believed that the main biological function of APE1 is Mg^(2+)-dependent hydrolysis of AP-sites in DNA. On the base of structural data, kinetic studies, and mutation analysis, the key stages of APE1 interaction with damaged DNA were established. It has been shown recently that APE1 can act as an endoribonuclease that catalyzes mRNA hydrolysis at certain pyrimidine-purine sites and thus controls the level of certain transcripts. In addition, the presence of Mg^(2+) ions was shown to be not required for the endoribonuclease activity of APE1, in contrast to the AP-endonuclease activity. This indicates differences in mechanisms of APE1 catalysis on RNA and DNA substrates, but the reasons for these differences remain unclear. Here, the analysis of endoribonuclease hydrolysis of model RNA substrates with wild type APE1 enzyme and its mutant forms Y171F, R177F, R181A, D210N, N212A, T268D, M270A, and D308A, was performed. It was shown that mutation of Asn212, Asp210, and Tyr171 residues leads to the decrease of AP-endonuclease activity while endoribonuclease activity is retained. Also, T268D and M270A APE1 mutants lose specificity to pyrimidine-purine sequences. R177F and R181A did not show a significant decrease in enzyme activity, whereas D308A demonstrated a decrease of endoribonuclease activity.
Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa , Endonucleasas , Reparación del ADN/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Endorribonucleasas/genética , Humanos , Cinética , MutaciónRESUMEN
Human apurinic/apyrimidinic (AP) endonuclease APE1 is one of the participants in the DNA base excision repair. The main biological function of APE1 is to hydrolyze the phosphodiester bond on the 5'-side of the AP sites. It has been shown recently that APE1 acts as an endoribonuclease and can cleave mRNA, thereby controlling the level of some transcripts. The sequences of CA, UA, and UG dinucleotides are the cleavage sites in RNA. In the present work, we performed a comparative analysis of the cleavage efficiency of model RNA substrates with short hairpin structures in which the loop size and the location of the pyrimidine-purine dinucleotide sequence were varied. The effect of various divalent metal ions and pH on the efficiency of the endoribonuclease reaction was analyzed. It was shown that site-specific hydrolysis of model RNA substrates depends on the spatial structure of the substrate. In addition, RNA cleavage occured in the absence of divalent metal ions, which proves that hydrolysis of DNA- and RNA substrates occurs via different catalytic mechanisms.
RESUMEN
Progress in synthesis of novel fluorescent oligonucleotides has provided effective instruments for nucleic acid detection. Pyrene conjugated oligonucleotides have demonstrated their effectiveness as fluorescent hybridization probes. Here we describe the synthesis, isolation, and analysis of 5'-monopyrene and 5'-bispyrene conjugates of oligo(2'-O-methylribonucleotides) and their application as probes for fluorescent detection of mismatches in RNA targets.
Asunto(s)
Disparidad de Par Base/genética , Hibridación de Ácido Nucleico/métodos , Sondas de Oligonucleótidos/química , Sondas ARN/química , ARN/análisis , Colorantes Fluorescentes , Sondas de Oligonucleótidos/genética , Oligonucleótidos/genética , Pirenos/química , Piridinas/química , Sondas ARN/genética , Ribosa/análogos & derivados , Ribosa/química , Espectrometría de Fluorescencia/métodosRESUMEN
The ability of a series of novel modified external guide sequences (EGS oligonucleotides) to induce the hydrolysis of target RNA with bacterial ribonuclease P has been studied; the most efficient modification variants have been selected. We have found patterns of the oligonucleotide sugar-phosphate backbone modi-fications that enhance oligonucleotide stability in the biological environment and do not violate the ability to interact with the enzyme and induce the RNA hydrolysis. It has been shown that analogues of EGS oligonucleotides selectively modified at 2'-position (2'-O-methyl and 2'-fluoro) or at internucleotide phosphates (phosphoryl guanidines) can be used for the addressed cleavage of a model RNA target by bacterial RNase P. The ability of new phosphoryl guanidine analogues of oligodeoxyribonucleotides that are stable in biological media to induce the hydrolysis of target RNA with bacterial ribonuclease P has been shown for the first time. The modified EGS oligonucleotides with an optimal balance between functional activity and stability in biological media can be considered as potential antibacterial agents.
Asunto(s)
Oligonucleótidos/química , División del ARN , ARN/química , Ribonucleasa P/química , BacteriasRESUMEN
In this paper, we report structure-based rational design and physico-chemical and biological studies of novel pyrene excimer forming dual probes for visualization of intracellular RNAs. Herein, the probes based on 2'-O-methyl RNA with linkers of different structure and length between pyrene moiety and ribose are studied with respect to their hybridization and spectral properties. We found optimal linkers that provide more intense excimer emission (at â¼480nm) of RNA-bound probes; particularly, the length of the linker arm of the 3'-component of dual probes plays a key role in formation of pyrene excimer. Calculated molecular dynamics trajectories and probability distributions of pyrene-pyrene dimer formation upon hybridization of the dual probes with RNA target are in agreement with the obtained fluorescence spectroscopy data for the corresponding duplexes. Our study demonstrates the excellent binding properties of new dual probes to structured RNA and their feasibility for the visualization of intracellular RNA targets.
Asunto(s)
Diseño de Fármacos , Sondas de Oligonucleótidos/química , ARN/química , Espectrometría de Fluorescencia , Relación Estructura-ActividadRESUMEN
Conjugates of pyrimidine triplex forming 3'-protected oligo(2'-O-methylribonucleotides) with minor groove binders (MGB) and triplex specific intercalator benzoindoloquinoline (BIQ) at 5'-terminus were synthesized. The conjugates formed stable complexes with target dsDNA by simultaneous binding both in its minor and major grooves and BIQ intercalation. The dissociation constants and thermal stability of the conjugate complexes with model dsDNA corresponding to polypurine tract (PPT) of genes nef and pol from HIV proviral genome were determined. Conjugation of oligo(2'-O-methylribonucleotides) with MGB and intercalator increased the stability of the triple complexes with dsDNA at pH 7.2 and 37 degrees C. Intercalator introduction accelerates the process of complex formation. Dose-dependent arrest of the in vitro transcription was demonstrated when a 780 b.p. DNA fragment containing the polypurine tract was transcribed under the control of T7 promoter in the presence of different concentrations of conjugates of oligo(2'-O-methylribonucleotides) containing MGB and BIQ intercalator.
Asunto(s)
ADN/química , Pirimidinas/química , Ribonucleótidos/síntesis química , Transcripción Genética , Conformación de Ácido Nucleico , Ribonucleótidos/químicaRESUMEN
A series of novel fluorescent conjugates of oligo(2'-O-methylribonucleotides) containing the insertions of 2'bispyrenylmethylphosphorodiamidate derivatives of ribonucleosides (Urd, Cyd) were synthesized and their fluorescent properties were investigated. The possibility of detection of RNA transcript of 5'-terminal fragment of mRNA of multidrug resistance gene mdr1 (1-678) in solution using synthesized multipyrene probes was demonstrated.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Resistencia a Múltiples Medicamentos/genética , Colorantes Fluorescentes/síntesis química , Sondas de Oligonucleótidos/síntesis química , ARN Mensajero/análisis , Región de Flanqueo 5'/genética , Animales , Citidina/análogos & derivados , Citidina/síntesis química , Humanos , Técnicas In Vitro , Mutagénesis Insercional , Patología Molecular , Uridina/análogos & derivados , Uridina/síntesis químicaRESUMEN
5'-Pyrenylmethylphosphoramidite and 5'-bispyrenylmethylphosphordiamidite derivatives of oligo(2'-O-methylribonucleotides) and their analogues with thymidine attached at their 3'-termini by a 3'-3'-phosphodiester internucleotide bond (inverted thymidine) were synthesized. The effect of the pyrene residue(s) on the thermal stability of duplexes of the modified oligonucleotides with RNA and DNA was studied. A possibility of detection of hybridization of 5'-mono- and 5'-bispyrenyl derivatives with RNA and DNA targets in solution was demonstrated according to the changes in fluorescence. 5'-Pyrenylmethylphosphoramidite derivatives of oligo(2'-O-methylribonucleotides) and their inverted analogues were shown to serve as sensitive probes for the detection of oligonucleotide substitutions in RNA and DNA by the method of thermal denaturation of the formed duplexes detected according to changes in their fluorescence.
Asunto(s)
Colorantes Fluorescentes/síntesis química , Oligorribonucleótidos/síntesis química , Pirenos/síntesis química , Colorantes Fluorescentes/química , Fluorometría , Desnaturalización de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/química , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos/síntesis química , Sondas de Oligonucleótidos/química , Oligorribonucleótidos/química , Pirenos/químicaRESUMEN
A series of octa (2-O-methylribonucleotides) with an additional 3'-terminal deoxynucleoside (T, dC, dA or dG) linked by the 3'-3' (inverted) bond was synthesized. The exceptional stability of these oligomers to a 3'-exonuclease (SVP) and nucleases in culture medium containing 10% heat-inactivated fetal calf serum was demonstrated. It was shown that the addition of the 3'-dangling inverted deoxynucleoside increases substantially the thermal stability of the duplexes of oligo(2'-O-methylribonucleotides) with complementary RNA and DNA in the case of a relatively weak terminal AmU(T) pair and enhances the mismatch sensitivity.
Asunto(s)
ARN/química , Ribonucleótidos/química , Disparidad de Par Base , Secuencia de Bases , ADN/química , ADN Complementario/metabolismo , Desoxirribonucleótidos/química , Cinética , Modelos Químicos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex , Hibridación de Ácido Nucleico , Oligonucleótidos/química , Oligorribonucleótidos/química , ARN Complementario/química , Temperatura , TermodinámicaRESUMEN
A new type of fluorescent nucleic acid probes, 2-bis-pyrene-modified oligonucleotides, is described. Preparation of these conjugates involves attachment of two pyrene moieties to the 2'-phosphate group introduced into any position within a sequence by solid-phase phosphoramidite synthesis. Good hybridization properties of the 2'-bis-pyrene probes, their nuclease resistance and sensitivity of fluorescence to the type of complementary nucleic acid have been demonstrated.
Asunto(s)
Colorantes Fluorescentes/farmacología , Ácidos Nucleicos/química , Sondas de Oligonucleótidos/química , Oligonucleótidos/química , ADN/química , Transferencia Resonante de Energía de Fluorescencia , Concentración de Iones de Hidrógeno , Modelos Químicos , Biología Molecular/métodos , Hibridación de Ácido Nucleico , Fosfatos/química , Pirenos/química , ARN/química , Espectrometría de Fluorescencia , TemperaturaRESUMEN
Site-specific photomodification of the 5'-terminal fragment of MDR1 mRNA by perfluoroarylazide derivatives of 2'-O-modified (2'-O-methyl or 2'-O-tetrahydropyranyl) oligoribo- and oligodeoxyribonucleotides was investigated. The conjugates built of 2'-O-modified oligoribonucleotides demonstrate beneficial features compared with their deoxyribo analogs: the extent of RNA modification by 2'-O-modified oligoribonucleotides and oligodeoxyribonucleotide conjugates was 40- 50% and 20%, respectively.
Asunto(s)
Azidas/química , Genes MDR/genética , Oligorribonucleótidos/química , ARN Mensajero/genética , Emparejamiento Base , Oligorribonucleótidos/genética , Fotoquímica , ARN Mensajero/metabolismo , Ribonucleasas/metabolismoRESUMEN
Design, synthesis, physico-chemical and in vitro biological studies of new pyrimidine oligo(2'-O-methylribonucleotide) conjugates with oligocarboxamide minor groove binders (MGB) and benzoindoloquinoline intercalator (BIQ) are described. These conjugates formed stable triple helices with the target double-stranded DNA and inhibited its in vitro transcription upon binding.
Asunto(s)
ADN/metabolismo , Ribonucleótidos/metabolismo , Secuencia de Bases , ADN/química , Espectrofotometría UltravioletaRESUMEN
New conjugates containing two parallel or antiparallel carboxamide minor groove binders (MGB) attached to the same terminal phosphate of one oligonucleotide strand were synthesized. The conjugates interact with their target DNA stronger than the individual components. Effect of conjugated MGB on DNA duplex and triplex stability and their sequence specificity was demonstrated on the short oligonucleotide duplexes and on the triplex formed by model 16-mer oligonucleotide with HIV polypurine tract.
Asunto(s)
ADN/química , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Emparejamiento Base , Sitios de Unión , Citosina , ADN Viral/química , Guanina , VIH/genética , Indicadores y ReactivosRESUMEN
A new method for functionalization of oligonucleotides by addition of aminoalkyl derivatives to the intermolecular phosphorus atom of the oligonucleotide N3'-P5' phosphoramidate bond in the presence of triphenylphosphine, 4-dimethylaminopyridine, and 2,2'-dipyridyl disulfide was suggested. The reaction proceeded with both low-molecular alkylamines (1,6-diaminohexane or N,N-dimethyl-1,3-diaminopropane) and a ligand in minor groove containing a aminoalkyl group.