RESUMEN
Chemical modification of DNA is a common strategy to improve the properties of oligonucleotides, particularly for therapeutics and nanotechnology. Existing synthetic methods essentially rely on phosphoramidite chemistry or the polymerization of nucleoside triphosphates but are limited in terms of size, scalability, and sustainability. Herein, we report a robust alternative method for the de novo synthesis of modified oligonucleotides using template-dependent DNA ligation of shortmer fragments. Our approach is based on the fast and scaled accessibility of chemically modified shortmer monophosphates as substrates for the T3 DNA ligase. This method has shown high tolerance to chemical modifications, flexibility, and overall efficiency, thereby granting access to a broad range of modified oligonucleotides of different lengths (20 â 120 nucleotides). We have applied this method to the synthesis of clinically relevant antisense drugs and ultramers containing diverse modifications. Furthermore, the designed chemoenzymatic approach has great potential for diverse applications in therapeutics and biotechnology.
Asunto(s)
ADN Ligasas , ADN , Oligonucleótidos , Oligonucleótidos/química , Oligonucleótidos/síntesis química , ADN Ligasas/metabolismo , ADN/química , Moldes Genéticos , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/síntesis químicaRESUMEN
AIM: The availability of disease-modifying therapies and newborn screening programs for spinal muscular atrophy (SMA) has generated an urgent need for reliable prognostic biomarkers to classify patients according to disease severity. We aim to identify cerebrospinal fluid (CSF) prognostic protein biomarkers in CSF samples of SMA patients collected at baseline (T0), and to describe proteomic profile changes and biological pathways influenced by nusinersen before the sixth nusinersen infusion (T302). METHODS: In this multicenter retrospective longitudinal study, we employed an untargeted liquid chromatography mass spectrometry (LC-MS)-based proteomic approach on CSF samples collected from 61 SMA patients treated with nusinersen (SMA1 n=19, SMA2 n=19, SMA3 n=23) at T0 at T302. The Random Forest (RF) machine learning algorithm and pathway enrichment analysis were applied for analysis. RESULTS: The RF algorithm, applied to the protein expression profile of naïve patients, revealed several proteins that could classify the different types of SMA according to their differential abundance at T0. Analysis of changes in proteomic profiles identified a total of 147 differentially expressed proteins after nusinersen treatment in SMA1, 135 in SMA2, and 289 in SMA3. Overall, nusinersen-induced changes on proteomic profile were consistent with i) common effects observed in allSMA types (i.e. regulation of axonogenesis), and ii) disease severity-specific changes, namely regulation of glucose metabolism in SMA1, of coagulation processes in SMA2, and of complement cascade in SMA3. CONCLUSIONS: This untargeted LC-MS proteomic profiling in the CSF of SMA patients revealed differences in protein expression in naïve patients and showed nusinersen-related modulation in several biological processes after 10 months of treatment. Further confirmatory studies are needed to validate these results in larger number of patients and over abroader timeframe.
Asunto(s)
Aprendizaje Automático , Atrofia Muscular Espinal , Oligonucleótidos , Proteómica , Humanos , Proteómica/métodos , Atrofia Muscular Espinal/tratamiento farmacológico , Atrofia Muscular Espinal/líquido cefalorraquídeo , Atrofia Muscular Espinal/metabolismo , Oligonucleótidos/uso terapéutico , Masculino , Femenino , Biomarcadores/líquido cefalorraquídeo , Biomarcadores/metabolismo , Estudios Retrospectivos , Lactante , Estudios Longitudinales , Preescolar , Cromatografía Liquida/métodos , NiñoRESUMEN
Large synthetic oligonucleotides such as guide ribonucleic acid (gRNA), a critical reagent in clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing, have complex higher order structures (HOS) inherent in their design. In this study, we first developed a generic anion exchange chromatography (AEX) method for the comprehensive analysis of a 100mer single guide ribonucleic acid (sgRNA) impurity profiling. AEX demonstrated superior resolution compared to other common chromatographic methods employed for sgRNA analysis, such as Ion-Pairing Reversed Phase Liquid Chromatography (IP-RPLC) and Hydrophilic Interaction Chromatography (HILIC). Moreover, we discovered AEX's potential in probing the HOS of RNAs by adjusting the temperature and using organic additives. Our study also highlighted that sgRNA possesses a unique HOS distinctly different from other therapeutic nucleic acids, such as antisense oligonucleotides and messenger RNAs.
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Oligonucleótidos , Cromatografía por Intercambio Iónico/métodos , Oligonucleótidos/química , Oligonucleótidos/análisis , ARN Guía de Sistemas CRISPR-Cas/química , Cromatografía de Fase Inversa/métodos , Interacciones Hidrofóbicas e Hidrofílicas , Aniones/químicaRESUMEN
Oligonucleotide drugs are shining in clinical therapeutics, but efficient and safe delivery systems severely limit their widespread use. A disulfide unit technology platform based on dynamic thiol exchange chemistry at the cell membrane has the potential for drug delivery. However, the alteration of the disulfide unit CSSC dihedral angle induced by different substituents directly affects the effectiveness of this technology and its stability. Previously, we constructed a trivalent low dihedral angle disulfide unit that can effectively promote the cellular uptake of small molecules. Here, we constructed a novel disulfide unit-masked oligonucleotide hybrid based on a low dihedral angle disulfide unit, motivated by prodrug design. Cellular imaging results showed that such a system exhibited superior cellular delivery efficiency than the commercial Lipo2000 without cytotoxicity. The thiol reagents significantly reduced its cellular uptake (57-74%), which proved to be endocytosis-independent. In addition, in vivo distribution experiments in mice showed that such systems can be rapidly distributed in liver tissues with a duration of action of more than 24 h, representing a potential means of silencing genes involved in the pathogenesis of liver-like diseases. In conclusion, this trivalent disulfide unit-masked system we constructed can effectively deliver large oligonucleotide drugs.
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Disulfuros , Oligonucleótidos , Disulfuros/química , Animales , Oligonucleótidos/química , Ratones , Humanos , Sistemas de Liberación de Medicamentos , Hígado/metabolismo , Hígado/efectos de los fármacosRESUMEN
Oligonucleotide therapeutics (OT) have emerged as promising drug modality for various intractable diseases. Recently, liquid chromatography-mass spectrometry (LC-MS) has been commonly employed for characterizing and quantifying OT in biological samples. Traditionally, the ion pairing-reverse phase (IP-RP) LC-MS method has been utilized in OT bioanalyses; however, this approach is associated with several limitations, including the memory effect and ion suppression effect of IP reagents. Therefore, this study aimed to develop a new RP-LC-MS method that eliminates the need for IP reagents. Our investigation revealed that ammonium bicarbonate was essential for the successful implementation of this nonIP-RP-LC-MS-based bioanalysis of OT. Moreover, the developed method demonstrated high versatility, accommodating the analysis of various natural or chemically modified oligonucleotides. The sensitivity of the method was further assessed using reconstituted plasma samples (the lower limit of quantification in this experiment was 0.5-1 ng/mL). In summary, the developed nonIP-RP-LC-MS method offers an easy, reliable, and cost-effective approach to the bioanalysis of OT.
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Oligonucleótidos , Oligonucleótidos/análisis , Oligonucleótidos/química , Humanos , Límite de Detección , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Espectrometría de Masas/métodos , Cromatografía de Fase Inversa/métodosRESUMEN
Objective: To summarize the effects of disease-modifying drugs for spinal muscular atrophy (SMA) on the ventilation support of type 1 children after acute respiratory failure. Methods: A case-control study was conducted, including the data of clinical characteristics, medication and ventilation supports of 38 SMA patients of type 1 with pneumonia and acute respiratory failure hospitalized in Children's Hospital Affiliated to Capital Institute of Pediatrics from January 2020 to July 2023. Children were divided into the treatment group and the untreated group based on whether they started and persisted in using Nusinersen or Risdiplam or not before hospitalization. The differences of ventilation support between the 2 groups were analyzed. The children of the treatment group were divided into the improved group and the unimproved group based on whether they could be avoid of prolonged dependence on continuous mechanical ventilation in the next six months after discharge. The differences in clinical characteristics between the two groups were analyzed. T-test and χ2 test were used for comparison. Results: Among the enrolled children, 19 were male and 19 were female. The age was 1.3 (0.6, 2.0) years at the time of hospitalization due to pneumonia. There were 26 cases in the treatment group and 12 cases in the untreated group. The treatment group had a higher proportion of patients without prolonged dependence on continuous mechanical ventilation in the next six months after discharge (69% (18/26) vs. 2/12, χ2=9.10, P<0.05). Eighteen children were improved among the treated group, while 8 children were not. The improved group had a larger age of first onset of acute respiratory failure (1.6 (0.4, 3.4) vs. 0.5 (0.3, 0.7) years, Z=2.07, P<0.05), a longer duration of medication taken before hospitalization (3.6 (2.4, 8.7) vs. 1.2 (1.2, 2.4) months, t=2.74, P<0.05), and a smaller proportion with underlying diseases (1/18 vs. 6/8, χ2=13.58, P<0.05). Conclusions: SMA disease-modifying drugs are useful for type 1 children to avoid of prolonged dependence on continuous mechanical ventilation after acute respiratory failure. The patients who take medication longer, or have acute respiratory failure for the first-time at an older age, or without underlying diseases are more likely to avoid of.
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Respiración Artificial , Insuficiencia Respiratoria , Atrofias Musculares Espinales de la Infancia , Humanos , Femenino , Masculino , Insuficiencia Respiratoria/terapia , Insuficiencia Respiratoria/etiología , Lactante , Estudios de Casos y Controles , Atrofias Musculares Espinales de la Infancia/terapia , Atrofias Musculares Espinales de la Infancia/tratamiento farmacológico , Preescolar , Oligonucleótidos/uso terapéutico , Neumonía , HospitalizaciónRESUMEN
Pulsed electron-electron double resonance (PELDOR) spectroscopy is a powerful method for determining nucleic acid (NA) structure and conformational dynamics. PELDOR with molecular dynamics (MD) simulations opens up unique possibilities for defining the conformational ensembles of flexible, three-dimensional, self-assembled complexes of NA. Understanding the diversity and structure of these complexes is vital for uncovering matrix and regulative biological processes in the human body and artificially influencing them for therapeutic purposes. To explore the reliability of PELDOR and MD simulations, we site-specifically attached nitroxide spin labels to oligonucleotides, which form self-assembled complexes between NA chains and exhibit significant conformational flexibility. The DNA complexes assembled from a pair of oligonucleotides with different linker sizes showed excellent agreement between the distance distributions obtained from PELDOR and calculated from MD simulations, both for the mean inter-spin distance and the distance distribution width. These results prove that PELDOR with MD simulations has significant potential for studying the structure and dynamics of conformational flexible complexes of NA.
Asunto(s)
Simulación de Dinámica Molecular , Oligonucleótidos , Marcadores de Spin , Espectroscopía de Resonancia por Spin del Electrón/métodos , Oligonucleótidos/química , Conformación de Ácido Nucleico , ADN/químicaRESUMEN
The increasing need for mass spectrometric analysis of RNA molecules calls for a better understanding of their gas-phase fragmentation behaviors. In this study, we investigate the effect of terminal phosphate groups on the fragmentation spectra of RNA oligonucleotides (oligos) using high-resolution mass spectrometry (MS). Negative-ion mode collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) were carried out on RNA oligos containing a terminal phosphate group on either end, both ends, or neither end. We find that terminal phosphate groups affect the fragmentation behavior of RNA oligos in a way that is dependent on the precursor charge state and the oligo length. Specifically, for precursor ions of RNA oligos of the same sequence, those with 5'- or 3'-phosphate, or both, have a higher charge state distribution and lose the phosphate group(s) in the form of a neutral (H3PO4 or HPO3) or an anion ([H2PO4]- or [PO3]-) upon CID or HCD. Such a neutral or charged loss is most conspicuous for precursor ions of an intermediate charge state, e.g., 3- for 4-nt oligos or 4- and 5- for 8-nt oligos. This decreases the intensity of sequencing ions (a-, a-B, b-, c-, d-, w-, x-, y-, z-ions) and hence is unfavorable for sequencing by CID or HCD. Removal of terminal phosphate groups by calf intestinal alkaline phosphatase improved MS analysis of RNA oligos. Additionally, the intensity of a fragment ion at m/z 158.925, which we identified as a dehydrated pyrophosphate anion ([HP2O6]-), is markedly increased by the presence of a terminal phosphate group. These findings expand the knowledge base necessary for software development for MS analysis of RNA.
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Aniones , Fosfatos , ARN , Aniones/química , Fosfatos/química , ARN/química , ARN/análisis , Oligonucleótidos/química , Oligonucleótidos/análisis , Espectrometría de Masas/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
Clinical trials with treatments inhibiting myostatin pathways to increase muscle mass are currently ongoing in spinal muscular atrophy. Given evidence of potential myostatin pathway downregulation in Spinal Muscular Atrophy (SMA), restoring sufficient myostatin levels using disease-modifying treatments (DMTs) might arguably be necessary prior to considering myostatin inhibitors as an add-on treatment. This retrospective study assessed pre-treatment myostatin and follistatin levels' correlation with disease severity and explored their alteration by disease-modifying treatment in SMA. We retrospectively collected clinical characteristics, motor scores, and mysotatin and follistatin levels between 2018 and 2020 in 25 Belgian patients with SMA (SMA1 (n = 13), SMA2 (n = 6), SMA 3 (n = 6)) and treated by nusinersen. Data were collected prior to treatment and after 2, 6, 10, 18, and 30 months of treatment. Myostatin levels correlated with patients' age, weight, SMA type, and motor function before treatment initiation. After treatment, we observed correlations between myostatin levels and some motor function scores (i.e., MFM32, HFMSE, 6MWT), but no major effect of nusinersen on myostatin or follistatin levels over time. In conclusion, further research is needed to determine if DMTs can impact myostatin and follistatin levels in SMA, and how this could potentially influence patient selection for ongoing myostatin inhibitor trials.
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Folistatina , Atrofia Muscular Espinal , Miostatina , Índice de Severidad de la Enfermedad , Humanos , Miostatina/metabolismo , Miostatina/antagonistas & inhibidores , Masculino , Femenino , Atrofia Muscular Espinal/tratamiento farmacológico , Atrofia Muscular Espinal/metabolismo , Folistatina/metabolismo , Oligonucleótidos/uso terapéutico , Estudios Retrospectivos , Preescolar , Niño , Lactante , AdolescenteRESUMEN
Facioscapulohumeral muscular dystrophy (FSHD) is an inherited myopathy, characterized by progressive and asymmetric muscle atrophy, primarily affecting muscles of the face, shoulder girdle, and upper arms before affecting muscles of the lower extremities with age and greater disease severity. FSHD is a disabling condition, and patients may also present with various extramuscular symptoms. FSHD is caused by the aberrant expression of double homeobox 4 (DUX4) in skeletal muscle, arising from compromised epigenetic repression of the D4Z4 array. DUX4 encodes the DUX4 protein, a transcription factor that activates myotoxic gene programs to produce the FSHD pathology. Therefore, sequence-specific oligonucleotides aimed at reducing DUX4 levels in patients is a compelling therapeutic approach, and one that has received considerable research interest over the last decade. This review aims to describe the current preclinical landscape of oligonucleotide therapies for FSHD. This includes outlining the mechanism of action of each therapy and summarizing the preclinical results obtained regarding their efficacy in cellular and/or murine disease models. The scope of this review is limited to oligonucleotide-based therapies that inhibit the DUX4 gene, mRNA, or protein in a way that does not involve gene editing.
Asunto(s)
Proteínas de Homeodominio , Distrofia Muscular Facioescapulohumeral , Distrofia Muscular Facioescapulohumeral/genética , Distrofia Muscular Facioescapulohumeral/terapia , Distrofia Muscular Facioescapulohumeral/metabolismo , Humanos , Animales , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Oligonucleótidos/uso terapéutico , Modelos Animales de Enfermedad , Músculo Esquelético/metabolismo , Músculo Esquelético/patologíaRESUMEN
The utilization of TiO2 nanolayers that possess excellent biocompatibility and physical properties in DNA sensing and sequencing remains largely to be explored. To examine their applicability in gene sequencing, a comprehensive study on the interaction of DNA oligonucleotides with TiO2 nanolayers was performed through adsorption and desorption experiments. TiO2 nanolayers with 10 nm thickness were fabricated via magnetron sputtering onto a 6-inch silicon wafer. A simple chip block method, validated via quartz crystal microbalance experiments with dissipation monitoring (QCM-D), was proposed to study the adsorption behaviors and interaction mechanisms under a variety of critical influencing factors, including DNA concentration, length, and type, adsorption time, pH, and metal ions. It is determined that the adsorption takes 2 h to reach saturation in the MES solution and the adsorption capacity is significantly enhanced by lowering the pH due to the isoelectric point being pH = 6 for TiO2. The adsorption percentages of nucleobases are largely similar in the MES solution while following 5T = 5G > 5C > 5A in HEPES buffer for an adsorption duration of 2.5 h. Through pre-adsorption experiments, it is deduced that DNA oligonucleotides are horizontally adsorbed on the nanolayer. This further demonstrates that mono-, di-, and tri-valent metal ions promote the adsorption, whereas Zn2+ has strong adsorption by inducing DNA condensation. Based on the desorption experiments, it is revealed that electrostatic force dominates the adsorption over van der Waals force and hydrogen bonds. The phosphate group is the main functional group for adsorption, and the adsorption strength increases with the length of the oligonucleotide. This study provides comprehensive data on the adsorption of DNA oligonucleotides onto TiO2 nanolayers and clarifies the interaction mechanisms therein, which will be valuable for applications of TiO2 in DNA-related applications.
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ADN , Oligonucleótidos , Titanio , Titanio/química , Adsorción , Oligonucleótidos/química , ADN/química , Concentración de Iones de Hidrógeno , Nanoestructuras/química , Tecnicas de Microbalanza del Cristal de CuarzoRESUMEN
Spinal muscular atrophy (SMA) is a prevalent paediatric neuromuscular disorder characterised by muscle weakness and atrophy resulting from degeneration of spinal cord anterior horn α motor neurons. Gene therapy formulations exhibit varying benefits and limitations, driving the need for patient-friendly treatment options tailored to specific populations. The objective of this meta-analysis was to assess the effectiveness of gene therapy for motor function in children with SMA. The analysis encompassed a total of 719 participants from six randomised controlled trials (RCTs) conducted between 2017 and 2023. Among the studies, one demonstrated a significant and large standardised effect size (Cohen's d) favouring nusinersen in terms of Hammersmith Functional Motor Scale - Expanded (HFMSE) (d = 0.97) and revised upper limb module (RULM) (d = 0.96). Additionally, another study showed a moderate standardised effect size (Cohen's d) in favour of nusinersen concerning Hammersmith Infant Neurological Examination-Section 2 (HINE-2) (d = 0.48). However, it is important to note that further research with a longer duration of observation is required to strengthen the evidence. Key Words: Spinal muscular atrophy, Nusinersen, Risdiplam, Motor function, Cohen's d.
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Oligonucleótidos , Atrofias Musculares Espinales de la Infancia , Humanos , Oligonucleótidos/uso terapéutico , Atrofias Musculares Espinales de la Infancia/tratamiento farmacológico , Atrofias Musculares Espinales de la Infancia/fisiopatología , Terapia Genética , Resultado del Tratamiento , Ensayos Clínicos Controlados Aleatorios como Asunto , Niño , Compuestos Azo , PirimidinasRESUMEN
7-Deaza-2'-deoxyisoguanosine forms stable inverse Watson-Crick base pairs with 5-methyl-2'-deoxyisocytidine and purine-purine base pairs with 2'-deoxyguanosine or 5-aza-7-deaza-2'-deoxyguanosine. Both base pairs expand the genetic coding system. The manuscript reports on the functionalization of these base pairs with halogen atoms and clickable side chains introduced at 7-position of the 7-deazapurine base. Oligonucleotides containing the functionalized base pairs were prepared by solid-phase synthesis. To this end, a series of phosphoramidites were synthesized and clickable side chains with short and long linkers were incorporated in oligonucleotides. Fluorescent pyrene conjugates were obtained by postmodification. Functionalization of DNA with a single inverse Watson-Crick base pair by halogens or clickable residues has only a minor impact on duplex stability. Pyrene click adducts increase (long linker) or decrease (short linker) the double helix stability. Stable hybrid duplexes were constructed containing three consecutive purine-purine pairs of 7-functionalized 7-deaza-2'-deoxyisoguanine with guanine or 5-aza-7-deazaguanine in the center and Watson-Crick pairs at both ends. The incorporation of a hybrid base pair tract of 7-deaza-2'-deoxyisoguanosine/5-aza-7-deaza-2'-deoxyguanosine pairs stabilizes the double helix strongly. Fluorescence intensity of pyrene short linker adducts increased when the 7-deazapurine base was positioned opposite to 5-methylisocytosine (inverse base pair) compared to purine-purine base pairs with guanine or 5-aza-7-deazaguanine in opposite positions. For long liker adducts, the situation is more complex. Circular dichroism (CD) spectra of purine DNA differ to those of Watson-Crick double helices and are indicative for the new DNA constructs. The impact of 7-deaza-2'-deoxyisoguanine base pair functionalization is studied for the first time and all experimental details are reported to prepare DNA functionalized at the 7-deazaisoguanine site. The influence of single and multiple incorporations on DNA structure and stability is shown. Clickable residues introduced at the 7-position of the 7-deazaisoguanine base provide handles for Huisgen-Sharpless-Meldal click cycloadditions without harming the stability of purine-pyrimidine and purine-purine base pairs. Other chemistries might be used for bioconjugation. Our investigation paves the way for the functionalization of a new DNA related recognition system expanding the common Watson-Crick regime.
Asunto(s)
Emparejamiento Base , ADN , Purinas , Purinas/química , ADN/química , Guanosina/química , Guanosina/análogos & derivados , Pirenos/química , Oligonucleótidos/química , Desoxiguanosina/química , Desoxiguanosina/análogos & derivadosRESUMEN
Telomerase, crucial for maintaining telomere length, is an attractive target for cancer therapy due to its role in cellular immortality. Despite three decades of research efforts, no small-molecule telomerase inhibitors have been clinically approved, highlighting the extensive challenges in developing effective telomerase-based therapeutics. This review examines conventional and emerging methods to measure telomerase activity and discusses existing inhibitors, including oligonucleotides and small molecules. Furthermore, this review highlights recent breakthroughs in structural studies of telomerase using cryo-electron microscopy, which can facilitate improved structure-based drug design. Altogether, advancements in structural methodologies and high-throughput screening offer promising prospects for telomerase-based cancer therapeutic development.
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Descubrimiento de Drogas , Inhibidores Enzimáticos , Telomerasa , Telomerasa/antagonistas & inhibidores , Telomerasa/metabolismo , Humanos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Descubrimiento de Drogas/métodos , Microscopía por Crioelectrón , Neoplasias/tratamiento farmacológico , Diseño de Fármacos , Telómero/metabolismo , Oligonucleótidos/química , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéuticoRESUMEN
Antibody-oligonucleotide conjugates are a promising class of therapeutics for extrahepatic delivery of small interfering ribonucleic acids (siRNAs). These conjugates can be optimized for improved delivery and mRNA knockdown (KD) through understanding of structure-activity relationships. In this study, we systematically examined factors including antibody isotype, siRNA chemistry, linkers, conjugation chemistry, PEGylation, and drug-to-antibody ratios (DARs) for their impact on bioconjugation, pharmacokinetics (PK), siRNA delivery, and bioactivity. Conjugation site (cysteine, lysine, and Asn297 glycan) and DAR proved critical for optimal conjugate PK and siRNA delivery. SiRNA chemistry including 2' sugar modifications and positioning of phosphorothioates were found to be critical for delivery and duration of action. By utilizing cleavable and noncleavable linkers, we demonstrated the impact of linkers on PK and mRNA KD. To achieve optimal properties of antibody-siRNA conjugates, a careful selection of siRNA chemistry, DAR, conjugation sites, linkers, and antibody isotype is necessary.
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Inmunoconjugados , ARN Interferente Pequeño , ARN Interferente Pequeño/química , Relación Estructura-Actividad , Inmunoconjugados/química , Inmunoconjugados/farmacología , Humanos , Animales , Desarrollo de Medicamentos , Oligonucleótidos/química , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Antibody-oligonucleotide conjugates (AOCs) are promising treatments for Duchenne muscular dystrophy (DMD). They work via induction of exon skipping and restoration of dystrophin protein in skeletal and heart muscles. The structure-activity relationships (SARs) of AOCs comprising antibody-phosphorodiamidate morpholino oligomers (PMOs) depend on several aspects of their component parts. We evaluate the SAR of antimouse transferrin receptor 1 antibody (αmTfR1)-PMO conjugates: cleavable and noncleavable linkers, linker location on the PMO, and the impact of drug-to-antibody ratios (DARs) on plasma pharmacokinetics (PK), oligonucleotide delivery to tissues, and exon skipping. AOCs containing a stable linker with a DAR9.7 were the most effective PMO delivery vehicles in preclinical studies. We demonstrate that αmTfR1-PMO conjugates induce dystrophin protein restoration in the skeletal and heart muscles of mdx mice. Our results show that αmTfR1-PMO conjugates are a potentially effective approach for the treatment of DMD.
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Ratones Endogámicos mdx , Morfolinos , Distrofia Muscular de Duchenne , Animales , Morfolinos/química , Morfolinos/farmacología , Morfolinos/farmacocinética , Relación Estructura-Actividad , Ratones , Distrofia Muscular de Duchenne/tratamiento farmacológico , Desarrollo de Medicamentos , Distrofina/metabolismo , Distrofina/genética , Inmunoconjugados/química , Inmunoconjugados/farmacología , Inmunoconjugados/farmacocinética , Humanos , Masculino , Ratones Endogámicos C57BL , Oligonucleótidos/química , Oligonucleótidos/farmacocinética , Músculo Esquelético/metabolismo , Receptores de Transferrina/metabolismo , Receptores de Transferrina/inmunologíaRESUMEN
Adipose tissue, or fat tissue, can now be classified as an endocrine organ as it responds to stimuli by secreting a range of hormones, termed adipokines, which regulate the functions of various other tissues and organs. Because novel adipokines continue to be discovered and characterized by researchers, there is an enduring need for the development of new analytical assays that target these hormones. Discovered recently, asprosin is an adipokine hormone secreted by white adipose tissue (WAT) during fasting which has been implicated for its important effects on the liver, skeletal muscle, hypothalamus, pancreas, and possibly other tissues. While standard immunoassays have been developed, the continued surge in research on asprosin's function would greatly benefit from an assay with homogeneous, mix-and-read workflow, and the nanomolar clinical range makes this goal more feasible. In this work, we developed such an assay for asprosin using our thermofluorimetric analysis (TFA) methods with antibody-oligonucleotide conjugate probes. The assay, achievable in less than one hour, was successfully validated by quantifying native levels of asprosin in human serum collected from fasting, nonfasting, type II diabetic, and obese donors.
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Fibrilina-1 , Humanos , Fibrilina-1/química , Inmunoensayo/métodos , Anticuerpos/inmunología , Anticuerpos/química , Oligonucleótidos/química , AdipoquinasRESUMEN
The genome contains numerous regulatory elements that may undergo complex interactions and contribute to the establishment, maintenance, and change of cellular identity. Three-dimensional genome organization can be explored with fluorescence in situ hybridization (FISH) at the single-cell level, but the detection of small genomic loci remains challenging. Here, we provide a rapid and simple protocol for the generation of bright FISH probes suited for the detection of small genomic elements. We systematically optimized probe design and synthesis, screened polymerases for their ability to incorporate dye-labeled nucleotides, and streamlined purification conditions to yield nanoscopy-compatible oligonucleotides with dyes in variable arrays (NOVA probes). With these probes, we detect genomic loci ranging from genome-wide repetitive regions down to non-repetitive loci below the kilobase scale. In conclusion, we introduce a simple workflow to generate densely labeled oligonucleotide pools that facilitate detection and nanoscopic measurements of small genomic elements in single cells.
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Hibridación Fluorescente in Situ , Oligonucleótidos , Hibridación Fluorescente in Situ/métodos , Humanos , Oligonucleótidos/genética , Genómica/métodos , Análisis de la Célula Individual/métodos , Colorantes Fluorescentes/químicaRESUMEN
Antisense oligonucleotides (ASOs) are crucial for biological applications as they bind to complementary RNA sequences, modulating protein expression. ASOs undergo synthetic modifications like phosphorothioate (PS) backbone and locked nucleic acid (LNA) to enhance stability and specificity. Tandem mass spectrometry (MS) techniques were employed to study gapmer ASOs, which feature a DNA chain within RNA segments at both termini, revealing enhanced cleavages with ultraviolet photodissociation (UVPD) and complementary fragment ions from collision-induced dissociation (CID) and electron detachment dissociation (EDD). 2DMS, a data-independent analysis technique, allowed for comprehensive coverage and identification of shared fragments across multiple precursor ions. EDD fragmentation efficiency correlated with precursor ion charge states, with higher charges facilitating dissociation due to intramolecular repulsions. An electron energy of 22.8 eV enabled electron capture and radical-based cleavage. Accumulating multiple scans and generating average spectra improved signal intensity, aided by denoising algorithms. Data analysis utilised a custom Python script capable of handling modifications and generating unique mass lists.
Asunto(s)
Oligonucleótidos , Espectrometría de Masas en Tándem , Espectrometría de Masas en Tándem/métodos , Oligonucleótidos/química , Oligonucleótidos Antisentido/químicaRESUMEN
Deoxyribonucleic acid triplexes have potential roles in a range of biological processes involving gene and transcriptional regulation. A major challenge in exploiting the formation of these higher-order structures to target genes in vivo is their low stability, which is dependent on many factors including the length and composition of bases in the sequence. Here, different DNA base modifications have been explored, primarily using native mass spectrometry, in efforts to enable stronger binding between the triplex forming oligonucleotide (TFO) and duplex target sites. These modifications can also be used to overcome pyrimidine interruptions in the duplex sequence in promoter regions of genomes, to expand triplex target sequences for antigene therapies. Using model sequences with a single pyrimidine interruption, triplex forming oligonucleotides containing locked nucleic acid base modifications were shown to have a higher triplex binding propensity than DNA-only and dSpacer-containing TFOs. However, the triplex forming ability of these systems was limited by the competitive formation of multiple higher order assemblies. Triplex forming sequences that correspond to specific gene targets from the Pseudomonas aeruginosa genome were also investigated, with LNA-containing TFOs the only variant able to form triplex using these sequences. This work indicates the advantages of utilizing synthetically modified TFOs to form triplex assemblies in vivo for potential therapeutic applications and highlights the advantages of native mass spectrometry for the study of their formation.