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 derivatives are a potent approach to the intracellular delivery of nucleic acids. The binding of these derivatives to serum albumin is a determinant of their fate in the body, as its structure contains several sites of high affinity for hydrophobic compounds. This study focuses on the features of self-association and non-covalent interactions with human serum albumin of novel self-penetrating oligonucleotide derivatives. The study revealed that the introduction of a triazinyl phosphoramidate modification bearing two dodecyl groups at the 3' end region of the oligonucleotide sequence has a negligible effect on its affinity for the complementary sequence. Dynamic light scattering verified that the amphiphilic oligonucleotides under study can self-assemble into micelle-like particles ranging from 8 to 15 nm in size. The oligonucleotides with dodecyl groups form stable complexes with human serum albumin with a dissociation constant of approximately 10-6 M. The oligonucleotide micelles are simultaneously destroyed upon binding to albumin. Using an electrophoretic mobility shift assay and affinity modification, we examined the ability of DNA duplexes containing triazinyl phosphoramidate oligonucleotides to interact with Ku antigen and PARP1, as well as the mutual influence of PARP1 and albumin or Ku antigen and albumin upon interaction with DNA duplexes. These findings, together with the capability of dodecyl-containing derivatives to effectively penetrate different cells, such as HEK293 and T98G, indicate that the oligonucleotides under study can be considered as a platform for the development of therapeutic preparations with a target effect.
RESUMEN
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
This study describes the effective attack of oligonucleotides on the viral genome of highly pathogenic H5N1 influenza A virus (IAV) in vivo using for the first time the new delivery system consisting of biocompatible low-toxic titanium dioxide nanoparticles and immobilized polylysine-containing oligonucleotides with the native (ODN) and partially modified (ODNm) internucleotide bonds. Intraperitoneal injection of the TiO2â¢PL-ODN nanocomposite provided 65-70% survival of mice, while intraperitoneal or oral administration of TiO2â¢PL-ODNm was somewhat more efficient (~80% survival). The virus titer in the lung was reduced by two-three orders of magnitude. The nanocomposites are nontoxic to mice under the used conditions. TiO2 nanoparticles, unbound ODN, and the nanocomposite bearing the random oligonucleotide showed an insignificant protective effect, which indicates the ability of targeted oligonucleotides delivered in mice in the nanocomposites to site-specifically interact with complementary RNAs. The protection of oligonucleotides in nanocomposites by TiO2 nanoparticles and partial modification of the internucleotide bonds provides a continued presence of oligonucleotides in the body for the effective and specific action on the viral RNA. The proposed oligonucleotide delivery system can claim not only to effectively inhibit IAV genes but also to turn off other genes responsible for diseases caused by nucleic acids.
Asunto(s)
Antivirales/administración & dosificación , Portadores de Fármacos/química , Subtipo H5N1 del Virus de la Influenza A/efectos de los fármacos , Gripe Humana/tratamiento farmacológico , Oligodesoxirribonucleótidos Antisentido/administración & dosificación , Administración Oral , Animales , Modelos Animales de Enfermedad , Perros , Femenino , Genoma Viral/efectos de los fármacos , Humanos , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/virología , Inyecciones Intraperitoneales , Células de Riñón Canino Madin Darby , Masculino , Ratones , Nanocompuestos/química , ARN Viral/antagonistas & inhibidores , Titanio/química , Carga Viral/efectos de los fármacosRESUMEN
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
Novel alternatives to traditional antibiotics are now of great demand for the successful treatment of microbial infections. Here, we present the engineering and properties of new oligonucleotide inhibitors of RNase P, an essential bacterial enzyme. The series of 2'-O-methyl RNA (2'-OMe-RNA) and phosphoryl guanidine oligonucleotides were targeted to the substrate-binding region of M1 RNA subunit of the RNase P. Uniformly modified 2'-OMe RNA and selectively modified phosphoryl guanidine oligonucleotides possessed good stability in biological media and effectively inhibited RNase P. Their conjugates with transporting peptides were shown to penetrate bacterial cells (Escherichia coli and Acinetobacter baumannii) and inhibit bacterial growth.
RESUMEN
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
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
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.