RESUMEN
Yersinia phage YerA41 is morphologically similar to jumbo bacteriophages. The isolated genomic material of YerA41 could not be digested by restriction enzymes, and used as a template by conventional DNA polymerases. Nucleoside analysis of the YerA41 genomic material, carried out to find out whether this was due to modified nucleotides, revealed the presence of a ca 1 kDa substitution of thymidine with apparent oligosaccharide character. We identified and purified the phage DNA polymerase (DNAP) that could replicate the YerA41 genomic DNA even without added primers. Cryo-electron microscopy (EM) was used to characterize structural details of the phage particle. The storage capacity of the 131 nm diameter head was calculated to accommodate a significantly longer genome than that of the 145 577 bp genomic DNA of YerA41 determined here. Indeed, cryo-EM revealed, in contrast to the 25 Å in other phages, spacings of 33-36 Å between shells of the genomic material inside YerA41 heads suggesting that the heavily substituted thymidine increases significantly the spacing of the DNA packaged inside the capsid. In conclusion, YerA41 appears to be an unconventional phage that packages thymidine-modified genomic DNA into its capsids along with its own DNAP that has the ability to replicate the genome.
Asunto(s)
Bacteriófagos , Bacteriófagos/química , Bacteriófagos/genética , Cápside , Microscopía por Crioelectrón , ADN Viral/genética , ADN Polimerasa Dirigida por ADN/genética , Genoma Viral/genética , TimidinaRESUMEN
18F-Labeled [60]fullerene-based molecular spherical nucleic acids (MSNAs), consisting of a human epidermal growth factor receptor 2 (HER2) mRNA antisense oligonucleotide sequence with a native phosphodiester and phosphorothioate backbone, were synthesized, site-specifically labeled with a positron emitting fluorine-18 and intravenously administrated via tail vein to HER2 expressing HCC1954 tumor-bearing mice. The biodistribution of the MSNAs was monitored in vivo by positron emission tomography/computed tomography (PET/CT) imaging. MSNA with a native phosphodiester backbone (MSNA-PO) was prone to rapid nuclease-mediated degradation, whereas the corresponding phosphorothioate analogue (MSNA-PS) with improved enzymatic stability showed an interesting biodistribution profile in vivo. One hour after the injection, majority of the radioactivity was observed in spleen and liver but also in blood with an average tumor-to-muscle ratio of 2. The prolonged radioactivity in blood circulation may open possibilities to the targeted delivery of the MSNAs.
Asunto(s)
Fulerenos , Neoplasias , Ácidos Nucleicos , Ratones , Humanos , Animales , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Distribución Tisular , Tomografía de Emisión de Positrones/métodos , Neoplasias/diagnóstico por imagen , Radioisótopos de Flúor , Línea Celular TumoralRESUMEN
5'-O-(2-Methoxyisopropyl) (MIP)-protected 2'-deoxynucleosides as chiral P(V)-building blocks, based on the limonene-derived oxathiaphospholane sulfide, were synthesized and used for the assembly of di-, tri-, and tetranucleotide phosphorothioates on a tetrapodal pentaerythritol-derived soluble support. The synthesis cycle consisted of two reactions and two precipitations: (1) the coupling under basic conditions, followed by neutralization and precipitation and (2) an acid catalyzed 5'-O-deacetalization, followed by neutralization and precipitation. The simple P(V) chemistry together with the facile 5'-O-MIP deprotection proved efficient in the liquid phase oligonucleotide synthesis (LPOS). Ammonolysis released nearly homogeneous Rp or Sp phosphorothioate diastereomers in ca. 80% yield/synthesis cycle.
Asunto(s)
Oligonucleótidos Fosforotioatos , Oligonucleótidos Fosforotioatos/química , EstereoisomerismoRESUMEN
Glyco-decorated spherical nucleic acids (SNAs) may be attractive delivery vehicles, emphasizing the sugar-specific effect on the outer sphere of the construct and at the same time hiding unfavorable distribution properties of the loaded oligonucleotides. As examples of such nanoparticles, tripodal sugar constituents of bleomycin were synthesized and conjugated with a fluorescence-labeled antisense oligonucleotide (AONARV7). Successive copper(I)-catalyzed azide-alkyne and strain-promoted alkyne-nitrone cycloadditions (SPANC) were utilized for the synthesis. Then, the glyco-AONARV7 conjugates were hybridized with complementary strands of a C60-based molecular spherical nucleic acid (i.e., a hybridization-mediated carrier). The formation and stability of these assembled glyco-decorated SNAs were evaluated by polyacrylamide gel electrophoresis (PAGE), UV melting profile analysis, and time-resolved fluorescence spectroscopy. Association constants were extracted from time-resolved fluorescence data. Preliminary cellular uptake experiments of the glyco-AONARV7 conjugates (120 nM solutions) and of the corresponding glyco-decorated SNAs (10 nM solutions) with human prostate cancer cells (PC3) showed an efficient uptake in each case. A marked variation in intracellular distribution was observed.
Asunto(s)
OroRESUMEN
A 2-trifluoromethylaniline C-nucleoside was synthesized, incorporated in the middle of an oligonucleotide, and mercurated. The affinity of the mercurated oligonucleotide toward complementary strands placing each of the canonical nucleobases opposite to the organomercury nucleobase analogue was examined by ultraviolet (UV), circular dichroism (CD), and 19F NMR spectroscopy analyses. According to the UV melting profile analysis, the organomercury nucleobase analogue showed increased affinities in the order T > G > C > A. The CD profiles indicated the typical B-type helix in each case. The 19F resonance signal proved sensitive for the local environmental changes, showing clearly distinct signals for the duplexes with different opposing nucleobases. Furthermore, valuable information on the mercurated oligonucleotide and its binding to complementary strands at varying temperature could be obtained by 19F NMR spectroscopy.
Asunto(s)
Mercurio , Nucleótidos , Emparejamiento Base , Dicroismo Circular , Espectroscopía de Resonancia Magnética , Conformación de Ácido Nucleico , OligonucleótidosRESUMEN
(2R,3S)-4-(Methoxyamino)butane-1,2,3-triol was converted into a protected phosphoramidite building block and incorporated into the middle of a short DNA oligonucleotide. O1 and O3 of the (2R,3S)-4-(methoxyamino)butane-1,2,3-triol were engaged in phosphodiester linkages, leaving O2 and the methoxyamino function available to form an N-methoxy-1,3-oxazinane ring through reaction with an aldehyde. In modified oligonucleotides thus obtained, the oxazinane ring formally replaces the furanose ring and the aldehyde, the base moiety of natural nucleosides. The feasibility of synthesizing base-modified oligonucleotides by this approach was demonstrated with several aromatic and aliphatic aldehydes featuring various functional groups.
Asunto(s)
Ácidos Nucleicos , Aldehídos , Butanos , ADN , Oligonucleótidos/metabolismoRESUMEN
The therapeutic efficacy and pharmacokinetics of antibody-drug conjugates (ADCs) in general, and antibody-oligonucleotide conjugates (AOCs) in particular, depend on the drug-to-antibody ratio (DAR) distribution and average value. The DAR is considered a critical quality attribute, and information pertaining to it needs to be gathered during ADC/AOC development, production, and storage. However, because of the high structural complexity of ADC/AOC samples, particularly in the initial drug-development stages, the application of the current state-of-the-art mass spectrometric approaches can be limited for DAR analysis. Here, we demonstrate a novel approach for the analysis of complex ADC/AOC samples, following native size-exclusion chromatography Orbitrap Fourier transform mass spectrometry (FTMS). The approach is based on the integration of the proteoform-level mass spectral peaks in order to provide an estimate of the DAR distribution and its average value with less than 10% error. The peak integration is performed via a truncation of the Orbitrap's unreduced time-domain ion signals (transients) before mass spectra generation via FT processing. Transient recording and processing are undertaken using an external data acquisition system, FTMS Booster X2, coupled to a Q Exactive HF Orbitrap FTMS instrument. This approach has been applied to the analysis of whole and subunit-level trastuzumab conjugates with oligonucleotides. The obtained results indicate that ADC/AOC sample purification or simplification procedures, for example, deglycosylation, could be omitted or minimized prior to the DAR analysis, streamlining the drug-development process.
Asunto(s)
Inmunoconjugados , Preparaciones Farmacéuticas , Análisis de Fourier , Inmunoconjugados/análisis , Espectrometría de Masas , OligonucleótidosRESUMEN
A highly nucleobase-discriminating metalated nucleoside analogue, 3-fluoro-2-mercuri-6-methylaniline, was incorporated into an oligonucleotide molecular beacon. Fluorescence emission spectra were measured after the addition of four different complementary strands, in which the nucleobase opposite the metalated analogue varies. The fluorescence results showed a clear binding selectivity at room temperature, in the order G>T>C>A. The selectivity is based on the different affinities between the metalated nucleoside analogue and the canonical nucleobases. The synthesized probe is capable of robust discrimination between the two purine as well as the two pyrimidine bases by fluorescence at room temperature, and more sophisticated temperature analysis allows clear separation of every canonical nucleobase. The probe would, hence, be a suitable method for the detection of single nucleotide polymorphisms.
Asunto(s)
Compuestos de Anilina/química , Colorantes Fluorescentes/química , Nucleótidos/química , Compuestos de Anilina/síntesis química , Conformación de Carbohidratos , Colorantes Fluorescentes/síntesis química , Espectrometría de FluorescenciaRESUMEN
An azide-functionalized 12-armed Buckminster fullerene has been monosubstituted in organic media with a substoichiometric amount of cyclooctyne-modified oligonucleotides. Exposing the intermediate products then to the same reaction (i.e., strain-promoted alkyne-azide cycloaddition, SPAAC) with an excess of slightly different oligonucleotide constituents in an aqueous medium yields molecularly defined monofunctionalized spherical nucleic acids (SNAs). This procedure offers a controlled synthesis scheme in which one oligonucleotide arm can be functionalized with labels or other conjugate groups (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTA, and Alexa-488 demonstrated), whereas the rest of the 11 arms can be left unmodified or modified by other conjugate groups in order to decorate the SNAs' outer sphere. Extra attention has been paid to the homogeneity and authenticity of the C60-azide scaffold used for the assembly of full-armed SNAs.
Asunto(s)
Fulerenos/química , Ácidos Nucleicos/química , Alquinos/química , Azidas/química , Catálisis , Química Clic , Cobre/química , Reacción de CicloadiciónRESUMEN
Oxazinomycin is a C-nucleoside antibiotic that is produced by Streptomyces hygroscopicus and closely resembles uridine. Here, we show that the oxazinomycin triphosphate is a good substrate for bacterial and eukaryotic RNA polymerases (RNAPs) and that a single incorporated oxazinomycin is rapidly extended by the next nucleotide. However, the incorporation of several successive oxazinomycins or a single oxazinomycin in a certain sequence context arrested a fraction of the transcribing RNAP. The addition of Gre RNA cleavage factors eliminated the transcriptional arrest at a single oxazinomycin and shortened the nascent RNAs arrested at the polythymidine sequences suggesting that the transcriptional arrest was caused by backtracking of RNAP along the DNA template. We further demonstrate that the ubiquitous C-nucleoside pseudouridine is also a good substrate for RNA polymerases in a triphosphorylated form but does not inhibit transcription of the polythymidine sequences. Our results collectively suggest that oxazinomycin functions as a Trojan horse substrate and its inhibitory effect is attributable to the oxygen atom in the position corresponding to carbon five of the uracil ring.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/química , ARN/química , Transcripción Genética/efectos de los fármacos , Uridina/análogos & derivados , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/genética , Oxígeno/química , Pseudomonas/química , ARN/genética , División del ARN/efectos de los fármacos , Streptomyces/química , Especificidad por Sustrato , Timidina/química , Timidina/genética , Transcripción Genética/genética , Factores de Elongación Transcripcional/genética , Uracilo/química , Uridina/síntesis química , Uridina/química , Uridina/farmacologíaRESUMEN
Oligonucleotides modified by a 2'-deoxy-2'-(N-methoxyamino) ribonucleotide react readily with aldehydes in slightly acidic conditions to yield the corresponding N-(methoxy)oxazolidine-linked oligonucleotide-conjugates. The reaction is reversible and dynamic in slightly acidic conditions, while the products are virtually stable above pH 7, where the reaction is in a ''switched off-state''. Small molecular examinations have demonstrated that aldehyde constituents affect the cleavage rate of the N-(methoxy)oxazolidine-linkage. This can be utilized to adjust the stability of this pH-responsive cleavable linker for drug delivery applications. In the present study, Fmoc-ß-Ala-H was immobilized to a serine-modified ChemMatrix resin and used for the automated assembly of two peptidealdehydes and one aldehyde-modified peptide nucleic acid (PNA). In addition, a triantennary N-acetyl-d-galactosamine-cluster with a ß-Ala-H unit has been synthesized. These aldehydes were conjugated via N-(methoxy)oxazolidine-linkage to therapeutically relevant oligonucleotide phosphorothioates and one DNA-aptamer in 19-47% isolated yields. The cleavage rates of the conjugates were studied in slightly acidic conditions. In addition to the diverse set of conjugates synthesized, these experiments and a comparison to published data demonstrate that the simple conversion of Gly-H to ß-Ala-H residue resulted in a faster cleavage of the N-(methoxy)oxazolidine-linker at pH 5, being comparable (T0.5 ca 7 h) to hydrazone-based structures.
Asunto(s)
Aldehídos/química , Hidrazonas/química , Oligonucleótidos/química , Oxazoles/química , Ácidos Nucleicos de Péptidos/química , Estructura MolecularRESUMEN
γ-Modified (i.e., (S)-aminomethyl, (S)-acetamidomethyl, (R)-4-(hydroxymethyl)triazol-1-ylmethyl, and (S)-guanidinylmethyl) triplex-forming peptide nucleic acids (TFPNAs) were synthesized and the effect of the backbone modifications on the binding to a miR-215 model was studied. Among the modifications, an appropriate pattern of three γ-(S)-guanidinylmethyl modifications increased the affinity and Hoogsteen-face selectivity for the miR-215 model without ternary (PNA)2 /RNA complex formation. Moreover, the γ-(S)-guanidinylmethyl groups were observed to facilitate internalization of the TFPNAs into living PC-3 prostate cancer cells.
Asunto(s)
Guanidina/química , MicroARNs/metabolismo , Conformación de Ácido Nucleico , Ácidos Nucleicos de Péptidos/química , Ácidos Nucleicos de Péptidos/metabolismoRESUMEN
A 3-fluoro-6-methylaniline nucleoside was synthesized and incorporated into an oligonucleotide, and its ability to form mercury-mediated base pairs was studied. UV melting experiments revealed increased duplex stability with thymine, guanine, and cytosine opposite to the probe and a clear nucleobase-specific binding preference (T > G > C > A). Moreover, the 3-fluoro group was utilized as a spin label that showed distinct 19F NMR resonance shifts depending on the complementary nucleobase, providing more detailed information on Hg(II)-mediated base pairing.
Asunto(s)
Compuestos de Anilina/química , Emparejamiento Base/efectos de los fármacos , Sondas de ADN/química , Nucleótidos/química , Sitios de Unión , Imagen por Resonancia Magnética con Fluor-19 , Mercurio/farmacologíaRESUMEN
MicroRNAs (miRNAs) are endogenous, small, noncoding ribonucleic acids (RNAs) that bind to the 3' untranslated regions of messenger RNAs (mRNAs) and induce translational repression or mRNA degradation. Although numerous studies have reported that miRNAs are of potential use for disease diagnostics and gene therapy, little is known about their fates in vivo. This study elucidated the whole-body distributions and kinetics of intravenously administered miRNA-targeting molecules in vivo by positron emission tomography (PET) imaging. A 22-mer sequence targeting miR-15b was conjugated with three different chelators and labeled with gallium-68 (68Ga). These tracers were compared with a scrambled 22-mer sequence; 22-mer with two single base substitutions; anti-miR-34 22-mer; hexathymidylate (T6), a 6-mer sequence; and an unconjugated chelator. miR-15b was chosen as a target because it is important for bone remodeling. All three 68Ga-labeled anti-miR-15b molecules had similar biodistributions and kinetics, and they all accumulated in the bones, kidneys, and liver. The bone accumulation of these tracers was the highest in the epiphyses of long tubular bones, maxilla, and mandible. By contrast, the scrambled 22-mer sequence, the 6-mer, and the unconjugated chelator did not accumulate in bones. PET imaging successfully elucidated the distributions and kinetics of 68Ga-labeled chelated miRNA-targeting molecules in vivo. This approach is potentially useful to evaluate new miRNA-based drugs.
Asunto(s)
Huesos/diagnóstico por imagen , Riñón/diagnóstico por imagen , Hígado/diagnóstico por imagen , MicroARNs/farmacocinética , Tomografía de Emisión de Positrones/métodos , ARN Mensajero/metabolismo , Animales , Quelantes/química , Femenino , Radioisótopos de Galio/química , Cinética , Masculino , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
Glycosidic (ß-1''â6, 3' and 4') site isomers of neomycin B (i.e., neobiosamine (ß-1''â6, 3' and 4') neamines) have been synthesized in a straightforward manner. Peracetylated neomycin azide was used as a common starting material to obtain neobiosamine glycosyl donor and 6, 3',4'-tri-O-acetyl neamine azide that after simple protecting group manipulation was converted to three different glycosyl acceptors (i.e., 5,6,4'-, 5,3',4'- and 5,6,3'-tri-O-acetyl neamine azide). Glycosylation between the neobiosamine glycosyl donor and the neamine-derived acceptors gave the protected pseudo-tetrasaccharides, which were converted, via global deprotection (deacetylation and reduction of the azide groups), to the desired site isomers of neomycin. The effect of these aminoglycosides on the RNA and DNA triplex stability was studied by UV-melting profile analysis.
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Antibacterianos/síntesis química , Antibacterianos/farmacología , ADN/química , Framicetina/síntesis química , Framicetina/farmacología , Conformación de Ácido Nucleico/efectos de los fármacos , ARN/química , Antibacterianos/química , Cromatografía Líquida de Alta Presión , Framicetina/química , Isomerismo , Temperatura de Transición/efectos de los fármacosRESUMEN
Azidopropyl-modified precursors of chondroitin sulfate (CS) tetrasaccharides have been synthesized, which, after facile conversion to final CS structures, may be conjugated with alkyne-modified target compounds by a one-pot "click"-ligation. RP HPLC was used for the monitoring of the key reaction steps (protecting group manipulation and sulfation) and purification of the CS precursors (as partially protected form, bearing the O-Lev, O-benzoyl, and N-trichloroacetyl groups and methyl esters). Subsequent treatments with aqueous NaOH, concentrated ammonia, and acetic anhydride (i.e., global deprotection and acetylation of the galactosamine units) converted the precursors to final CS structures. The azidopropyl group was exposed to a strain-promoted azide-alkyne cycloaddition (SPAAC) with a dibenzylcyclooctyne-modified carboxyrhodamine dye to give labeled CSs. Conjugation with a 5'-cyclooctyne-modified oligonucleotide was additionally carried out to show the applicability of the precursors for the synthesis of biomolecular hybrids.
Asunto(s)
Sulfatos de Condroitina/química , Química Clic/métodos , Colorantes Fluorescentes/química , Oligonucleótidos/química , Alquinos , Azidas/química , Sulfatos de Condroitina/síntesis química , Reacción de CicloadiciónRESUMEN
Triplex-forming peptide nucleic acids (TFPNAs) were targeted to double-helical regions of 19 F-labeled RNA hairpin models (a UA-rich duplex with a hexaethylene glycol (heg) loop and a microRNA model, miR-215). In addition to conventional UV- and circular dichroism (CD)-based detection, binding was monitored by 19 Fâ NMR spectroscopy. Detailed information on the stoichiometry and transition between the triple-helical peptide nucleic acid (PNA)/RNA and (PNA)2 /RNA binding modes could be obtained. γ-(R)-Hydroxymethyl-modified thymine-1-yl- and 2-aminopyridin-3-yl-acetyl derivatives of TFPNAs were additionally synthesized, which were targeted to the same RNA models, and the effect of the γ-(R)-hydroxymethyl group on binding was studied. An appropriate pattern of γ-(R)-hydroxymethyl modifications reduced the stability of the ternary complex and preferred stoichiometric binding to the miR-215 model.
Asunto(s)
MicroARNs/química , Ácidos Nucleicos de Péptidos/química , Secuencia de Bases , Dicroismo Circular , Flúor/química , MicroARNs/metabolismo , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , Ácidos Nucleicos de Péptidos/síntesis química , Ácidos Nucleicos de Péptidos/metabolismo , Espectrofotometría UltravioletaRESUMEN
Phosphoramidite building blocks of ribostamycin (3 and 4), that may be incorporated at any position of the oligonucleotide sequence, were synthesized. The building blocks, together with a previously described neomycin-modified solid support, were applied for the preparation of aminoglycoside-2'-O-methyl oligoribonucleotide fusions. The fusions were used to clamp a single strand DNA sequence (a purine-rich strand of c-Myc promoter 1) to form triple helical 2'-O-methyl RNA/DNA-hybrid constructs. The potential of the aminoglycoside moieties to stabilize the triple helical constructs were studied by UV-melting profile analysis.
Asunto(s)
Aminoglicósidos/química , ADN de Cadena Simple/química , Oligorribonucleótidos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myc , Humanos , Oligorribonucleótidos/síntesis química , Oligorribonucleótidos/químicaRESUMEN
In this report, we investigate the efficiency and selectivity of a Zn2+-dependent peptide nucleic acid-based artificial ribonuclease (PNAzyme) that cleaves RNA target sequences. The target RNAs are varied to form different sizes (3 and 4 nucleotides, nt) and sequences in the bulge formed upon binding to the PNAzyme. PNAzyme-promoted cleavage of the target RNAs was observed and variation of the substrate showed a clear dependence on the sequence and size of the bulge. For targets that form 4-nt bulges, we identified systems with an improved efficacy (an estimated half-life of ca 7-8 h as compared to 11-12 h for sequences studied earlier) as well as systems with an improved site selectivity (up to over 70% cleavage at a single site as compared to 50-60% with previous targets sequences). For targets forming 3-nt bulges, the enhancement compared to previous systems was even more pronounced. Compared to a starting point of targets forming 3-nt AAA bulges (half-lives of ca 21-24 h), we could identify target sequences that were cleaved with half-lives three times lower (ca 7-8 h), i.e., at rates similar to those found for the fastest 4-nt bulge system. In addition, with the 3-nt bulge RNA target site selectivity was improved even further to reach well over 80% cleavage at a specific site.
Asunto(s)
Oligonucleótidos/química , Ácidos Nucleicos de Péptidos/química , ARN/química , Ribonucleasas/metabolismo , Conformación de Ácido NucleicoRESUMEN
meso-Tris(pyridin-4-yl)(4-carboxyphenyl)porphyrin and 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (Photochlor, HPPH) were amide-coupled to 1R,2S,3R,4R-2,3-dihydroxy-4-(hydromethyl)-1-aminocyclopentane and immobilized via an ester linkage to long chain alkyl amine-derivatized controlled pore glass (LCAA-CPG). The applicability of these supports (5 and 6) for the synthesis of porphyrin conjugates with oligomeric biomolecules was demonstrated using an automated phosphoramidite coupling chemistry. Cleavage from the support with concentrated ammonia gave the products, viz., porphyrin conjugates of oligonucleotides (7-9) and dendritic glycoclusters (10-13) and a cyclooctyne derivative (14) in 23-58% yield. In addition, the synthesized cyclooctyne derivative of meso-tris(pyridin-4-yl)(4-carboxyphenyl)porphyrin (14) was conjugated with an azidopropyl-modified hyaluronic acid (19). The hyaluronic acid-porphyrin conjugate (15) was radiolabeled with (64)Cu and its (15[(64)Cu]) receptor binding affinity to CD44-expressing tumor cells was evaluated.