RESUMEN
Currently, the CRISPR-Cas9 system serves as a prevalent tool for genome editing and gene expression regulation. Its therapeutic application is limited by off-target effects that can affect genomic integrity through nonspecific, undesirable changes in the genome. Various strategies have been explored to mitigate the off-target effects. Many approaches focus on modifying components of the system, namely, Cas9 and guide RNAs, to enhance specificity. However, a common challenge is that methods aiming to increase specificity often result in a significant reduction in the editing efficiency. Here, we introduce a novel approach to modifying crRNA to balance CRISPR-Cas9 specificity and efficiency. Our approach involves incorporating nucleoside modifications, such as replacing ribo- to deoxyribonucleosides and backbone modifications, using phosphoryl guanidine groups, specifically 1,3-dimethylimidazolidin-2-ylidene phosphoramidate. In this case, within the first 10 nucleotides from the 5' crRNA end, phosphodiester bonds are substituted with phosphoryl guanidine groups. We demonstrate that crRNAs containing a combination of deoxyribonucleosides and single or multiple phosphoryl guanidine groups facilitate the modulation of CRISPR-Cas9 system activity while improving its specificity in vitro.
Asunto(s)
Sistemas CRISPR-Cas , ADN , Edición Génica , Guanidina , ARN Guía de Sistemas CRISPR-Cas , ADN/química , ADN/metabolismo , Edición Génica/métodos , Guanidina/química , ARN Guía de Sistemas CRISPR-Cas/química , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , ARN/química , ARN/metabolismo , Humanos , Guanidinas/químicaRESUMEN
Numerous types of oligonucleotide modifications have been developed since automated synthesis of DNA/RNA became a common instrument in the creation of synthetic oligonucleotides. Despite the growing number of types of oligonucleotide modifications under development, only a few of them and, moreover, their combinations have been studied widely enough in terms of their influence on the properties of corresponding NA constructions. In the present study, a number of oligonucleotides with combinations of 3'-end lipophilic (a single cholesteryl or a pair of dodecyl residues) and phosphate backbone modifications were synthesized. The influence of the combination of used lipophilic groups with phosphate modifications of various natures and different positions on the efficiency of cell penetration was evaluated. The obtained results indicate that even a couple of phosphate modifications are able to affect a set of oligonucleotide properties in a complex manner and can remarkably change cellular uptake. These data clearly show that the strategy of using different patterns of modification combinations has great potential for the rational design of oligonucleotide structures with desired predefined properties.
Asunto(s)
Oligonucleótidos , Fosfatos , Transporte Biológico , ARNRESUMEN
Lipophilic oligonucleotide conjugates represent a powerful tool for nucleic acid cellular delivery, and many methods for their synthesis have been developed over the past few decades. In the present study, a number of chemical approaches for the synthesis of different fork- and comb-like dodecyl-containing oligonucleotide structures were performed, including use of non-nucleotide units and different types of phosphate modifications such as alkyl phosphoramidate, phosphoryl guanidine, and triazinyl phosphoramidate. The influence of the number of introduced lipophilic residues, their mutual arrangement, and the type of formed modification backbone on cell penetration was evaluated. The results obtained indicate great potential in the developed chemical approaches, not only for the synthesis of complex oligonucleotide structures but also for the fine-tuning of their properties.
Asunto(s)
Guanidinas , Oligonucleótidos , Oligonucleótidos/química , Guanidina/química , FosfatosRESUMEN
Serum albumin is currently in the focus of biomedical research as a promising platform for the creation of multicomponent self-assembling systems due to the presence of several sites with high binding affinity of various compounds in its molecule, including lipophilic oligonucleotide conjugates. In this work, we investigated the stoichiometry of the dodecyl-containing oligonucleotides binding to bovine and human serum albumins using an electrophoretic mobility shift assay. The results indicate the formation of the albumin-oligonucleotide complexes with a stoichiometry of about 1 : (1.25 ± 0.25) under physiological-like conditions. Using atomic force microscopy, it was found that the interaction of human serum albumin with the duplex of complementary dodecyl-containing oligonucleotides resulted in the formation of circular associates with a diameter of 165.5 ± 94.3 nm and 28.9 ± 16.9 nm in height, and interaction with polydeoxyadenylic acid and dodecyl-containing oligothymidylate resulted in formation of supramolecular associates with the size of about 315.4 ± 70.9 and 188.3 ± 43.7 nm, respectively. The obtained data allow considering the dodecyl-containing oligonucleotides and albumin as potential components of the designed self-assembling systems for solving problems of molecular biology, biomedicine, and development of unique theranostics with targeted action.
Asunto(s)
Oligonucleótidos , Albúmina Sérica , Animales , Bovinos , Humanos , Oligonucleótidos/química , Albúmina Sérica/metabolismo , Microscopía de Fuerza Atómica , Ensayo de Cambio de Movilidad ElectroforéticaRESUMEN
Antisense gapmer oligonucleotides containing phosphoryl guanidine (PG) groups, e.g., 1,3-dimethylimidazolidin-2-imine, at three to five internucleotidic positions adjacent to the 3' and 5' ends were prepared via the Staudinger chemistry, which is compatible with conditions of standard automated solid-phase phosphoramidite synthesis for phosphodiester and, notably, phosphorothioate linkages, and allows one to design a variety of gapmeric structures with alternating linkages, and deoxyribose or 2'-O-methylribose backbone. PG modifications increased nuclease resistance in serum-containing medium for more than 21 days. Replacing two internucleotidic phosphates by PG groups in phosphorothioate-modified oligonucleotides did not decrease their cellular uptake in the absence of lipid carriers. Increasing the number of PG groups from two to seven per oligonucleotide reduced their ability to enter the cells in the carrier-free mode. Cationic liposomes provided similar delivery efficiency of both partially PG-modified and unmodified oligonucleotides. PG-gapmers were designed containing three to four PG groups at both wings and a central "window" of seven deoxynucleotides with either phosphodiester or phosphorothioate linkages targeted to MDR1 mRNA providing multiple drug resistance of tumor cells. Gapmers efficiently silenced MDR1 mRNA and restored the sensitivity of tumor cells to chemotherapeutics. Thus, PG-gapmers can be considered as novel, promising types of antisense oligonucleotides for targeting biologically relevant RNAs.
RESUMEN
The use of cancer chemotherapy sensitizers is a promising approach to induce the effect of clinically used anticancer treatments. One of the interesting targets is Tyrosyl-DNA Phosphodiesterase 1 (Tdp1), a DNA-repair enzyme, that may prevent the action of clinical Topoisomerase 1 (Top1) inhibitors, such as topotecan (Tpc). Tdp1 eliminates covalent Top1-DNA (Top1c) complexes that appear under the action of topotecan and determines the cytotoxic effect of this drug. We hypothesize that Tdp1 inhibition would sensitize cells towards the effect of Tpc. Herein, we report the synthesis and study of lipophilic derivatives of purine nucleosides that efficiently suppress Tdp1 activity, with IC50 values in the 0.3-22.0 µM range. We also showed that this compound class can enhance DNA damage induced by topotecan in vitro by Comet assay on human cell lines HeLa and potentiate the antitumor effect of topotecan in vivo on a mice ascitic Krebs-2 carcinoma model. Thereby, this type of compound may be useful to develop drugs, that sensitize the effect of topotecan and reduce the required dose and, as a result, side effects.
Asunto(s)
Hidrolasas Diéster Fosfóricas , Topotecan , Animales , Ratones , Humanos , Topotecan/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Nucleósidos de Purina , Relación Estructura-Actividad , Inhibidores de Topoisomerasa I/farmacología , Esterasas/metabolismo , Daño del ADN , ADN , ADN-Topoisomerasas de Tipo I/metabolismoRESUMEN
Small interfering RNA (siRNA) is the most important tool for the manipulation of mRNA expression and needs protection from intracellular nucleases when delivered into the cell. In this work, we examined the effects of siRNA modification with the phosphoryl guanidine (PG) group, which, as shown earlier, makes oligodeoxynucleotides resistant to snake venom phosphodiesterase. We obtained a set of siRNAs containing combined modifications PG/2'-O-methyl (2'-OMe) or PG/2'-fluoro (2'-F); biophysical and biochemical properties were characterized for each duplex. We used the UV-melting approach to estimate the thermostability of the duplexes and RNAse A degradation assays to determine their stability. The ability to induce silencing was tested in cultured cells stably expressing green fluorescent protein. The introduction of the PG group as a rule decreased the thermodynamic stability of siRNA. At the same time, the siRNAs carrying PG groups showed increased resistance to RNase A. A gene silencing experiment indicated that the PG-modified siRNA retained its activity if the modifications were introduced into the passenger strand.
Asunto(s)
Oligodesoxirribonucleótidos/genética , ARN Bicatenario/antagonistas & inhibidores , ARN Interferente Pequeño/genética , Ribonucleasas/genética , Guanidina/química , Humanos , Oligodesoxirribonucleótidos/antagonistas & inhibidores , Oligodesoxirribonucleótidos/farmacología , Interferencia de ARN , ARN Bicatenario/química , ARN Interferente Pequeño/química , ARN Interferente Pequeño/farmacología , Ribonucleasa Pancreática/química , Ribonucleasa Pancreática/genética , Ribonucleasas/química , TermodinámicaRESUMEN
The conjugation of lipophilic groups to oligonucleotides is a promising approach for improving nucleic acid-based therapeutics' intracellular delivery. Lipid oligonucleotide conjugates can self-aggregate in aqueous solution, which gains much attention due to the formation of micellar particles suitable for cell endocytosis. Here, we describe self-association features of novel "like-a-brush" oligonucleotide conjugates bearing three dodecyl chains. The self-assembly of the conjugates into 30-170 nm micellar particles with a high tendency to aggregate was shown using dynamic light scattering (DLS), atomic force (AFM), and transmission electron (TEM) microscopies. Fluorescently labeled conjugates demonstrated significant quenching of fluorescence intensity (up to 90%) under micelle formation conditions. The conjugates possess increased binding affinity to serum albumin as compared with free oligonucleotides. The dodecyl oligonucleotide conjugate and its duplex efficiently internalized and accumulated into HepG2 cells' cytoplasm without any transfection agent. It was shown that the addition of serum albumin or fetal bovine serum to the medium decreased oligonucleotide uptake efficacy (by 22.5-36%) but did not completely inhibit cell penetration. The obtained results allow considering dodecyl-containing oligonucleotides as scaffold compounds for engineering nucleic acid delivery vehicles.
RESUMEN
Biological activity of antisense oligonucleotides (asON), especially those with a neutral backbone, is often attenuated by poor cellular accumulation. In the present proof-of-concept study, we propose a novel delivery system for asONs which implies the delivery of modified antisense oligonucleotides by so-called transport oligonucleotides (tON), which are oligodeoxyribonucleotides complementary to asON conjugated with hydrophobic dodecyl moieties. Two types of tONs, bearing at the 5'-end up to three dodecyl residues attached through non-nucleotide inserts (TD series) or anchored directly to internucleotidic phosphate (TP series), were synthesized. tONs with three dodecyl residues efficiently delivered asON to cells without any signs of cytotoxicity and provided a transfection efficacy comparable to that achieved using Lipofectamine 2000. We found that, in the case of tON with three dodecyl residues, some tON/asON duplexes were excreted from the cells within extracellular vesicles at late stages of transfection. We confirmed the high efficacy of the novel and demonstrated that MDR1 mRNA targeted asON delivered by tON with three dodecyl residues significantly reduced the level of P-glycoprotein and increased the sensitivity of KB-8-5 human carcinoma cells to vinblastine. The obtained results demonstrate the efficacy of lipophilic oligonucleotide carriers and shows they are potentially capable of intracellular delivery of any kind of antisense oligonucleotides.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Sistemas de Liberación de Medicamentos , Neoplasias/tratamiento farmacológico , Oligonucleótidos Antisentido/genética , ARN Mensajero/antagonistas & inhibidores , Vinblastina/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP/antagonistas & inhibidores , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/química , Humanos , Neoplasias/genética , Neoplasias/patología , ARN Mensajero/genética , Células Tumorales Cultivadas , Vinblastina/administración & dosificación , Vinblastina/químicaRESUMEN
This article reports experimental data related to the research article entitled "Prevention of DNA multimerization using phosphoryl guanidine primers during isothermal amplification with Bst exo- DNA polymerase" (R.R. Garafutdinov, A.R. Sakhabutdinova, M.S. Kupryushkin, D.V. Pyshnyi, 2020) [1]. Here, multimerization efficiency in terms of Tt (time-to-threshold) values obtained for artificial DNA templates with the different nucleotide sequences during isothermal amplification with Bst exo- DNA polymerase is given. Data on the influence of phosphoryl guanidine primers (PGO) on multimerization for the LTc template which has shown high efficiency of multimerization are presented as well.
RESUMEN
Over the last two decades, isothermal amplification of nucleic acids has gained more attention due to a number of advantages over the widely used polymerase chain reaction. For isothermal amplification, DNA polymerases with strand-displacement activity are needed, and Bst exo- polymerase is one of the most commonly used. Unfortunately, Bst exo- causes nonspecific DNA amplification (so-called multimerization) under isothermal conditions that results in undesirable products (multimers) consisting of tandem nucleotide repeats. Multimerization occurs only for short ssDNA or primer dimers, and the efficiency of multimerization depends significantly on the reaction conditions, but slightly depends on the sequence of DNA templates. In this study we report the prevention of DNA multimerization using a new type of modified oligonucleotide primers with internucleosidic phosphates containing 1,3-dimethyl-2-imino-imidazolidine moieties (phosphoryl guanidine (PG) groups). Primers with one, two or three PG groups located at the 3'- or 5'-ends or in the middle of the primers were designed. It turned out, such bulky groups interfere with the moving of Bst exo- polymerase along DNA chains. However, one modified phosphate does not notably affect the efficiency of polymerization, and the elongation is completely inhibited only when three contiguous modifications occur. Multimerization of the linear ssDNA templates is blocked by three modifications in the middle of both primers whereas specific amplification of the circular ssDNA by rolling circle amplification is not inhibited. Thus, incorporation of three PG groups is sufficient to prevent multimerization and allows to create improved primers for reliable isothermal amplification with Bst exo- DNA polymerase.
Asunto(s)
ADN Circular/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Técnicas de Amplificación de Ácido Nucleico/métodos , Oligonucleótidos/metabolismo , Replicación del ADN , PolimerizacionRESUMEN
This article presents new data on the properties of the diastereomers of a mono-substituted phosphoryl guanidine trideoxyribonucleotides d(TpCp*A) [1,2]. The data include information on isolation, identification, treatment with snake venom phosphodiesterase and structural analysis by 1D and 2D NMR spectroscopy and restrained molecular dynamics analysis. The data can be used for preparation, analysis, application of phosphoryl guanidine oligonucleotide and for development of new nucleic acids derivatives. This data article is associated with the manuscript titled "Diastereomers of a mono-substituted phosphoryl guanidine trideoxyribonucleotide: isolation and properties" [1].
RESUMEN
Recently, a new type of nucleic acid analogues with modified phosphate group, namely, phosphoryl guanidine oligonucleotides, has been described. In the present work, we assess the difference between diastereomers of a mono-substituted phosphoryl guanidine oligonucleotide and analyze their resistance to nuclease digestion. Individual diastereomers ('fast' and 'slow') of a trideoxynucleotide d (TpCp*A) were isolated by reverse-phase HPLC. Snake venom phosphodiesterase digestion showed that the native trideoxynucleotide was fully degraded after 30 min, whereas both 'fast' and 'slow' diastereomers of d (TpCp*A) were not completely digested even after 7 days. UV and CD spectra revealed similarities in the structure of the diastereomers. Structural analysis by 1D and 2D NMR spectroscopy also uncovered significant similarity in the properties of Rp and Sp diastereomers. Structural analysis of nuclear Overhauser effect spectroscopy (NOESY) data and restrained molecular dynamics methods showed very flexible single-stranded oligonucleotide structures. Detailed computational analysis of restraint penalty energies via restrained molecular dynamics simulations with the 2D NMR interproton distance data allowed us to conclude that most likely, the 'fast' isomer is the Sp diastereomer, and the 'slow' isomer is the Rp diastereomer.
Asunto(s)
Guanidina/química , Oligonucleótidos/química , Fosfatos/química , Dicroismo Circular , Guanidina/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Oligonucleótidos/aislamiento & purificación , Hidrolasas Diéster Fosfóricas/metabolismo , Espectrofotometría Ultravioleta , Estereoisomerismo , TermodinámicaRESUMEN
DNA damage can affect various regulatory elements of the genome, with the consequences for DNA structure, dynamics, and interaction with proteins remaining largely unexplored. We used solution NMR spectroscopy, restrained and free molecular dynamics to obtain the structures and investigate dominant motions for a set of DNA duplexes containing CpG sites permuted with combinations of 5-methylcytosine (mC), the primary epigenetic base, and 8-oxoguanine (oxoG), an abundant DNA lesion. Guanine oxidation significantly changed the motion in both hemimethylated and fully methylated DNA, increased base pair breathing, induced BIâBII transition in the backbone 3' to the oxoG and reduced the variability of shift and tilt helical parameters. UV melting experiments corroborated the NMR and molecular dynamics results, showing significant destabilization of all methylated contexts by oxoG. Notably, some dynamic and thermodynamic effects were not additive in the fully methylated oxidized CpG, indicating that the introduced modifications interact with each other. Finally, we show that the presence of oxoG biases the recognition of methylated CpG dinucleotides by ROS1, a plant enzyme involved in epigenetic DNA demethylation, in favor of the oxidized DNA strand. Thus, the conformational and dynamic effects of spurious DNA oxidation in the regulatory CpG dinucleotide can have far-reaching biological consequences.
Asunto(s)
Metilación de ADN , ADN/genética , Epigénesis Genética , Estrés Oxidativo , Proteínas de Arabidopsis/metabolismo , Islas de CpG/genética , ADN/química , Enzimas/química , Genoma , Guanina/análogos & derivados , Guanina/química , Humanos , Espectroscopía de Resonancia Magnética , Metilación , Simulación de Dinámica Molecular , Proteínas Nucleares/metabolismo , Conformación Proteica , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Temperatura , TermodinámicaRESUMEN
This paper explores the potential of a modified phosphotriester approach to the synthesis of 5'-phosphoramidate derivatives of DNA and RNA oligonucleotides. The modification of 5'-deprotected support-bound oligonucleotides is done in two steps: i) conversion of the 5'-OH group of an oligonucleotide into an activated phosphodiester, and ii) treatment of the activated phosphodiester with an aminocompound. The approach is efficient and compatible with conventional solid phase oligonucleotide synthesis. It can be used for the conjugation of therapeutically relevant oligonucleotides with functional moieties or carrier constructions, which are to be removed after endocytosis.
Asunto(s)
ADN/química , Oligonucleótidos/síntesis química , Compuestos Organofosforados/síntesis química , ARN/química , Estructura Molecular , Técnicas de Síntesis en Fase SólidaRESUMEN
SDS-PAGE is considered to be a universal method for size-based separation and analysis of proteins. In this study, we applied the principle of SDS-PAGE to the analysis of new entirely uncharged nucleic acid (NA) analogues, - phosphoryl guanidine oligonucleotides (PGOs). The procedure was also shown to be suitable for morpholino oligonucleotides (PMOs) and peptide nucleic acids (PNAs). It was demonstrated that SDS can establish hydrophobic interactions with these types of synthetic NAs, giving them a net negative charge and thus making these molecules mobile in polyacrylamide slab gels under the influence of an electric field.
Asunto(s)
Electroforesis en Gel de Poliacrilamida/métodos , Oligonucleótidos/análisis , Oligonucleótidos/química , Conformación de Ácido NucleicoRESUMEN
Triarylmethyl (trityl, TAM) radicals are a promising class of spin labels for nanometer-scale distance measurements in biomolecules at physiological temperatures. However, to date, existing approaches to site-directed TAM labeling of DNA have been limited to label attachment at the termini of oligonucleotides, thus hindering a majority of demanded applications. Herein, we report a new versatile strategy for TAM attachment at arbitrary sites of nucleic acids. It utilizes an achiral non-nucleoside phosphoramidite monomer for automated solid-phase synthesis of oligonucleotides, which are then postsynthetically functionalized with TAM. We demonstrate a synthesis of a set of oligonucleotide complexes that are TAM-labeled at internal or terminal sites, as well as the possibility of measuring interspin distances up to â¼5-6 nm at 298 K using double quantum coherence electron paramagnetic resonance (EPR). Implementation of the developed approach strongly broadens the scope of nucleic acids and nucleoprotein complexes available for nanoscale structural EPR studies at room temperatures.
Asunto(s)
ADN/química , Sondas Moleculares/química , Oligodesoxirribonucleótidos/química , Marcadores de Spin , Compuestos de Tritilo/química , Espectroscopía de Resonancia por Spin del Electrón , Simulación de Dinámica Molecular , Oligodesoxirribonucleótidos/síntesis química , TemperaturaRESUMEN
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) processes DNA 3'-end-blocking modifications, possesses DNA and RNA 3'-nucleosidase activity and is also able to hydrolyze an internal apurinic/apyrimidinic (AP) site and its synthetic analogs. The mechanism of Tdp1 interaction with DNA was analyzed using pre-steady state stopped-flow kinetics with tryptophan, 2-aminopurine and Förster resonance energy transfer fluorescence detection. Phosphorothioate or tetramethyl phosphoryl guanidine groups at the 3'-end of DNA have been used to prevent 3'-nucleosidase digestion by Tdp1. DNA binding and catalytic properties of Tdp1 and its mutants H493R (Tdp1 mutant SCAN1) and H263A have been compared. The data indicate that the initial step of Tdp1 interaction with DNA includes binding of Tdp1 to the DNA ends followed by the 3'-nucleosidase reaction. In the case of DNA containing AP site, three steps of fluorescence variation were detected that characterize (i) initial binding the enzyme to the termini of DNA, (ii) the conformational transitions of Tdp1 and (iii) search for and recognition of the AP-site in DNA, which leads to the formation of the catalytically active complex and to the AP-site cleavage reaction. Analysis of Tdp1 interaction with single- and double-stranded DNA substrates shows that the rates of the 3'-nucleosidase and AP-site cleavage reactions have similar values in the case of single-stranded DNA, whereas in double-stranded DNA, the cleavage of the AP-site proceeds two times faster than 3'-nucleosidase digestion. Therefore, the data show that the AP-site cleavage reaction is an essential function of Tdp1 which may comprise an independent of AP endonuclease 1 AP-site repair pathway.
Asunto(s)
Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Ácido Apurínico/química , Ácido Apurínico/metabolismo , Sitios de Unión/genética , ADN/química , ADN/genética , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Transferencia Resonante de Energía de Fluorescencia , Humanos , Hidrólisis , Cinética , Mutación , Conformación de Ácido Nucleico , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/genética , Polinucleótidos/química , Polinucleótidos/metabolismo , Unión Proteica , Especificidad por SustratoRESUMEN
Human apurinic/apyrimidinic endonuclease APE1 is one of the key enzymes of the base excision DNA repair system. The main biological function of APE1 is the hydrolysis of the phosphodiester bond on the 5'-side of an apurinic/apyrimidinic site (AP-site) to give the 5'-phosphate and 3'-hydroxyl group. It has long been known that AP-sites have mutagenic and cytotoxic effects and their accumulation in DNA is a potential hazard to the cell lifecycle. The structural and biochemical studies of APE1 are complicated by its high catalytic activity towards the AP-site and its cyclic or acyclic analogues. This work has focussed on the design, synthesis and analysis of oligonucleotide derivatives as potentially unreactive APE1 substrates. We have shown that the replacement of oxygen atoms in the phosphate group on the 5'-side from the AP-site analogue tetrahydrofuran (F) considerably decreases the rate of enzymatic hydrolysis of modified oligonucleotides. We have calculated that a N3'-P5' phosphoramidate linkage is hydrolysed about 30 times slower than the native phosphodiester bond while phosphorothioate or primary phosphoramidate linkages are cleaved more than three orders of magnitude slower. The value of IC50 of the oligonucleotide duplex containing a primary phosphoramidate linkage is 2.5 × 10(-7) M, which is in accordance with the APE1 association constant of DNA duplexes containing AP-sites. Thus, it is demonstrated that oligonucleotide duplexes with chemical modifications could be used as unreactive substrates and potential competitive inhibitors of APE1.
Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa/antagonistas & inhibidores , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Oligonucleótidos/química , Oligonucleótidos/farmacología , Catálisis , ADN/química , ADN/metabolismo , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Humanos , Hidrólisis , Estructura Molecular , Oligonucleótidos/síntesis química , Fosfatos/química , Unión ProteicaRESUMEN
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) promotes catalytic scission of a phosphodiester bond between the 3'-end of DNA and the hydroxyl group of a tyrosine residue, as well as cleaving off a variety of other 3'-terminal phosphate-linked DNA substituents. We have shown recently that Tdp1 can initiate an apurinic/apyrimidinic (AP) site repair pathway that is independent from the one mediated by AP endonuclease 1 (APE1). Until recently, there was no method available of tracking the AP-site cleaving activity of Tdp1 by real-time fluorescence assay. In the present study we demonstrate a highly specific real-time detection of the AP-site cleaving activity of Tdp1 which allows one to distinguish it from the activity of APE1 by using a short hairpin oligonucleotide with a 1,12-dodecanediol loop, a 5'-fluorophore, and a 3'-quencher. Specific phosphodiesterase activity of Tdp1, which is usually able to remove quencher from the 3'-end of DNA, was suppressed in our approach by introducing a noncleavable phosphate group mimic between the 3'-end and the quencher. As a nondigestible 3'-phosphate analogue, we have used a new uncharged tetramethyl phosphoryl guanidine (Tmg) group, which is resistant to 3'-phosphodiesterase cleavage.