MicroRNAs (miRNAs) hold potential as biomarkers for numerous cancer types, including myelodysplastic neoplasms (MDS). Here, we present a highly sensitive assay based on the oligonucleotide-triggered release of gold nanoparticles (AuNPs) for the detection of hsa-miR-451a with a surface plasmon resonance biosensor. The performance of the assay is in large part determined by the size and functional coating of AuNPs. Therefore, we investigate AuNPs in a size range from 43 to 170 nm, functionalized with thiol- or biotin-terminated oligonucleotides (AuNPsSdT or AuNPsBdT). Our study reveals that 103 nm AuNPsSdT are the best option to improve the assay performance due to their high colloidal stability, a release efficiency exceeding 90%, and a sensor response enhancement factor exceeding 105. We demonstrate that in conjunction with 103 nm AuNPsSdT, the AuNP release assay can detect hsa-miR-451a at levels down to 40 aM and quantify hsa-miR-451a physiological levels in human blood plasma. Moreover, we use the assay to demonstrate a significant down-regulation of hsa-miR-451a in blood plasma of MDS patients compared to healthy individuals, suggesting the potential relevance of hsa-miR-451a as a prospective MDS biomarker.
- Klíčová slova
- Large gold nanoparticles, Myelodysplastic neoplasms, Nanoparticle release assay, Surface plasmon resonance, miRNA detection,
- MeSH
- biosenzitivní techniky metody MeSH
- kovové nanočástice * chemie MeSH
- lidé MeSH
- limita detekce MeSH
- mikro RNA * krev genetika MeSH
- myelodysplastické syndromy * krev genetika diagnóza MeSH
- nádorové biomarkery krev MeSH
- oligonukleotidy chemie MeSH
- povrchová plasmonová rezonance MeSH
- zlato * chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- mikro RNA * MeSH
- MIRN451 microRNA, human MeSH Prohlížeč
- nádorové biomarkery MeSH
- oligonukleotidy MeSH
- zlato * MeSH
The KRAS mutation is a crucial biomarker for determining targeted cancer therapies, making its accurate and cost-effective detection vital for precision oncology. However, current methodologies, such as next-generation sequencing (NGS) or PCR-based methods, are often expensive and technically complex, limiting their accessibility. Here, we present a novel bioassay for KRAS G12V mutation analysis that combines rolling circle amplification (RCA) with locked nucleic acid (LNA)-modified magnetic beads, electrochemical detection using carbon electrode chips, and AI-assisted analysis via a logistic regression classifier. Our platform demonstrated exceptional selectivity in distinguishing the KRAS G12V mutation from wild-type (wt) sequences, enabling analysis <1 % of mutated DNA in a wt sample. We validated the bioassay on 7 cancer cell lines and 11 patient-derived samples, achieving results that perfectly correlated with NGS data. This innovative approach simplifies the workflow, reduces costs, and offers high sensitivity and specificity, making it a promising tool for clinical diagnostics and personalized cancer treatment strategies.
- Klíčová slova
- DNA point mutation, Electrochemistry, KRAS gene, Locked nucleic acid, Rolling circle amplification,
- MeSH
- biotest * metody MeSH
- bodová mutace * MeSH
- elektrochemické techniky * metody MeSH
- lidé MeSH
- mutační analýza DNA metody MeSH
- nádorové buněčné linie MeSH
- oligonukleotidy * chemie genetika MeSH
- protoonkogenní proteiny p21(ras) * genetika MeSH
- techniky amplifikace nukleových kyselin * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- KRAS protein, human MeSH Prohlížeč
- locked nucleic acid MeSH Prohlížeč
- oligonukleotidy * MeSH
- protoonkogenní proteiny p21(ras) * MeSH
PURPOSE: There are limited treatment options for advanced melanoma that have progressed during or after immune checkpoint inhibitor therapy. Intratumoral (IT) immunotherapy may improve tumor-specific immune activation by promoting local tumor antigen presentation while avoiding systemic toxicities. The phase 3 ILLUMINATE-301 study (ClinicalTrials.gov identifier: NCT03445533) evaluated tilsotolimod, a Toll-like receptor-9 agonist, with or without ipilimumab in patients with anti-PD-1 advanced refractory melanoma. METHODS: Patients with unresectable stage III-IV melanoma that progressed during or after anti-PD-1 therapy were randomly assigned 1:1 to receive 24 weeks of tilsotolimod plus ipilimumab or 10 weeks of ipilimumab alone. Nine IT injections of tilsotolimod were administered to a single designated lesion over 24 weeks. Intravenous ipilimumab 3 mg/kg was administered once every 3 weeks from week 2 in the tilsotolimod arm and week 1 in the ipilimumab arm. The primary end point was efficacy measured using objective response rate (ORR; independent review) and overall survival (OS). RESULTS: A total of 481 patients received tilsotolimod plus ipilimumab (n = 238) or ipilimumab alone (n = 243). ORRs were 8.8% in the tilsotolimod arm and 8.6% in the ipilimumab arm, with disease control rates of 34.5% and 27.2%, respectively. Median OS was 11.6 months in the tilsotolimod arm and 10 months in the ipilimumab arm (hazard ratio, 0.96 [95% CI, 0.77 to 1.19]; P = .7). Grade ≥3 treatment-emergent adverse events occurred in 61.1% and 55.5% of patients in the tilsotolimod and ipilimumab arms, respectively. CONCLUSION: Combining IT tilsotolimod with ipilimumab did not significantly improve the ORR or OS compared with ipilimumab alone in patients with anti-PD-1 advanced refractory melanoma.
- MeSH
- dospělí MeSH
- ipilimumab * aplikace a dávkování škodlivé účinky MeSH
- lidé středního věku MeSH
- lidé MeSH
- melanom * farmakoterapie patologie imunologie mortalita MeSH
- nádory kůže * farmakoterapie patologie imunologie MeSH
- oligonukleotidy MeSH
- protokoly protinádorové kombinované chemoterapie * terapeutické užití škodlivé účinky MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- klinické zkoušky, fáze III MeSH
- multicentrická studie MeSH
- randomizované kontrolované studie MeSH
- Názvy látek
- ipilimumab * MeSH
- oligonukleotidy MeSH
- tilsotolimod MeSH Prohlížeč
Aptamers are short DNA or RNA sequences that can fold into unique three-dimensional structures, enabling them to bind specifically to target molecules with high affinity, similar to antibodies. A distinctive feature of many aptamers is their ability to adopt a G-quadruplex (G4) fold, a four-stranded structure formed by guanine-rich sequences. While G4 formation has been proposed or demonstrated for some aptamers, we aimed to investigate how frequently quadruplex-prone motifs emerge from the SELEX process. To achieve this, we examined quadruplex candidate sequences from the UTexas Aptamer Database, which contains over 1400 aptamer sequences extracted from 400 publications spanning several decades. We analyzed the G4 and i-motif propensity of these sequences. While no likely i-motif forming candidates were found, nearly 1/4 of DNA aptamers and 1/6 of RNA aptamers were predicted to form G4 structures. Interestingly, many motifs capable of forming G4 structures were not previously reported or suspected. Out of 311 sequences containing a potential stable G4 motif, only 53 of them (17%) reported the word "quadruplex" in the corresponding article. We experimentally tested G4 formation for 30 aptamer sequences and were able to confirm G4 formation for all the sequences with a G4Hunter score of 1.31 or more. These observations suggest the need to reevaluate G4 propensity among aptamer sequences.
- MeSH
- aptamerová technika SELEX MeSH
- aptamery nukleotidové * chemie MeSH
- G-kvadruplexy * MeSH
- guanin chemie MeSH
- nukleotidové motivy MeSH
- sekvence nukleotidů MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- aptamery nukleotidové * MeSH
- guanin MeSH
Continuous in vivo monitoring of small molecule biomarkers requires biosensors with reversibility, sensitivity in physiologically relevant ranges, and biological stability. Leveraging the real-time, label-free detection capability of surface plasmon resonance (SPR) technology, a molecularly responsive hydrogel film is introduced to enhance small molecule sensitivity. This advanced biosensing platform utilizes split-aptamer-cross-linked hydrogels (aptagels) engineered using 8-arm poly(ethylene glycol) macromers, capable of directly and reversibly detecting vancomycin. Investigation through SPR and optical waveguide mode, along with quartz crystal microbalance with dissipation (QCM-D) monitoring, reveals that the reversible formation of analyte-induced ternary molecular complexes leads to aptagel contraction and significant refractive index changes. Optimization of aptamer cross-link distribution and complementarity of split-aptamer pairs maximizes conformational changes of the aptagel, demonstrating a detection limit of 160-250 nM for vancomycin (6-9 fold improvement over monolayer counterpart) with a broad linear sensing range up to 1 mM. The aptagel maintains stability over 24 h in blood serum and 5 weeks in diluted blood plasma (mimicking interstitial fluid). This structurally responsive aptagel platform with superior stability and sensitivity offers promising avenues for continuous in vivo monitoring of small molecules.
- MeSH
- aptamery nukleotidové * chemie MeSH
- biosenzitivní techniky * metody MeSH
- hydrogely * chemie MeSH
- lidé MeSH
- mikrorovnovážné techniky křemenného krystalu MeSH
- polyethylenglykoly chemie MeSH
- povrchová plasmonová rezonance * metody MeSH
- vankomycin * analýza krev MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- aptamery nukleotidové * MeSH
- hydrogely * MeSH
- polyethylenglykoly MeSH
- vankomycin * MeSH
BACKGROUND: The genetic and epigenetic alterations observed in acute myeloid leukemia (AML) contribute to its heterogeneity, influencing disease progression response to therapy, and patient outcomes. The use of antisense oligonucleotides (ASOs) technology allows for the design of oligonucleotide inhibitors based on gene sequence information alone, enabling precise targeting of key molecular pathways or specific genes implicated in AML. METHODS AND RESULTS: Midostaurin, a FLT3 specific inhibitor and ASOs targeting particular genes, exons, or mutations was conducted using AML models. This ASOs treatment was designed to bind to exon 7 of the MBNL1 (muscleblind-like) gene. Another target was the FLT3 gene, focusing on two aspects: (a) FLT3-ITD (internal tandem duplication), to inhibit the expression of this aberrant gene form, and (b) the FLT3 in general. Treated and untreated cells were analyzed using quantitative PCR (qPCR), dot blot, and Raman spectroscopy. This study contrasts midostaurin with ASOs that inhibit FLT3 protein production or its isoforms via mRNA degradation. A trend of increased FLT3 expression was observed in midostaurin-treated cells, while ASO-treated cells showed decreased expression, though these changes were not statistically significant. CONCLUSIONS: In AML, exon 7 of MBNL1 is involved in several cellular processes and in this study, exon 7 of MBNL1 was targeted for method optimization, with the highest block of the exon 7 gene variant observed 48 h post-transfection. Midostaurin, a multitargeted kinase inhibitor, acts against the receptor tyrosine kinase FLT3, a critical molecule in AML pathogenesis. While midostaurin blocks FLT3 signaling pathways, it paradoxically increases FLT3 expression.
- Klíčová slova
- Acute myeloid leukemia, Antisense oligonucleotides, FLT3, MBNL1, Target specific therapy,
- MeSH
- akutní myeloidní leukemie * genetika farmakoterapie MeSH
- antisense oligonukleotidy * farmakologie genetika MeSH
- exony genetika MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- proteiny vázající RNA genetika metabolismus MeSH
- regulace genové exprese u leukemie účinky léků MeSH
- staurosporin * analogy a deriváty farmakologie MeSH
- tyrosinkinasa 3 podobná fms * genetika antagonisté a inhibitory metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antisense oligonukleotidy * MeSH
- FLT3 protein, human MeSH Prohlížeč
- MBNL1 protein, human MeSH Prohlížeč
- midostaurin MeSH Prohlížeč
- proteiny vázající RNA MeSH
- staurosporin * MeSH
- tyrosinkinasa 3 podobná fms * MeSH
A portfolio of six modified 2'-deoxyribonucleoside triphosphate (dNTP) derivatives derived from 5-substituted pyrimidine or 7-substituted 7-deazapurine bearing different carbohydrate units (d-glucose, d-galactose, d-mannose, l-fucose, sialic acid and N-Ac-d-galactosamine) tethered through propargyl-glycoside linker was designed and synthesized via the Sonogashira reactions of halogenated dNTPs with the corresponding propargyl-glycosides. The nucleotides were found to be good substrates for DNA polymerases in enzymatic primer extension and PCR synthesis of modified and hypermodified DNA displaying up to four different sugars. Proof of concept binding study of sugar-modified oligonucleotides with concanavalin A showed positive effect of avidity and sugar units count.
- Klíčová slova
- Carbohydrates, DNA synthesis, Glycopolymers, Lectins, Nucleotides,
- MeSH
- DNA-dependentní DNA-polymerasy metabolismus chemie MeSH
- DNA * chemie metabolismus MeSH
- glykosidy chemie chemická syntéza MeSH
- konformace nukleové kyseliny MeSH
- konkanavalin A chemie metabolismus MeSH
- monosacharidy * chemie chemická syntéza MeSH
- oligonukleotidy chemie chemická syntéza metabolismus MeSH
- pyrimidiny chemie chemická syntéza MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA-dependentní DNA-polymerasy MeSH
- DNA * MeSH
- glykosidy MeSH
- konkanavalin A MeSH
- monosacharidy * MeSH
- oligonukleotidy MeSH
- pyrimidiny MeSH
Colorimetric assays in which the color of a solution changes in the presence of an input provide a simple and inexpensive way to monitor experimental readouts. In this study we used in vitro selection to identify a self-phosphorylating kinase deoxyribozyme that produces a colorimetric signal by converting the colorless substrate pNPP into the yellow product pNP. The minimized catalytic core, sequence requirements, secondary structure, and buffer requirements of this deoxyribozyme, which we named Apollon, were characterized using a variety of techniques including reselection experiments, high-throughput sequencing, comparative analysis, biochemical activity assays, and NMR. A bimolecular version of Apollon catalyzed multiple turnover phosphorylation and amplified the colorimetric signal. Engineered versions of Apollon could detect oligonucleotides with specific sequences as well as several different types of nucleases in homogenous assays that can be performed in a single tube without the need for washes or purifications. We anticipate that Apollon will be particularly useful to reduce costs in high-throughput screens and for applications in which specialized equipment is not available.
- MeSH
- DNA katalytická * chemie metabolismus MeSH
- fosforylace MeSH
- kolorimetrie * metody MeSH
- konformace nukleové kyseliny MeSH
- oligonukleotidy chemie MeSH
- substrátová specifita MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA katalytická * MeSH
- oligonukleotidy MeSH
Ion-pairing reversed-phase liquid chromatography was utilized for the analysis of native and phosphorothioated oligonucleotides differing in the length (2-6mers and 21mer) and the number and position of phosphorothioate modifications. We investigated the influence of counterion (acetate vs. hexafluoroisopropanol) on the adsorption of eleven alkylamines on the stationary phases. A stronger adsorption of charged alkylamines on octadecyl- and phenyl-based stationary phases led to greater retention of oligonucleotides, and the adsorption of alkylamines was promoted with greater concentration of hexafluoroisopropanol in the mobile phase. Selected amines (triethylamine, dipropylamine, hexylamine) were used to study the resolution of n and n-x mers (main peak and its impurities shortened at 5´end), and diastereomeric separation of phosphorothioated oligonucleotides. The results confirmed a crucial role of alkylamine and counterion choice on the diastereomeric separation. The increasing hydrophobicity of alkylamine led to diminished diastereomeric selectivity which produced narrower phosphorothioated oligonucleotides peaks and led to improved n/n-x separation. Using hexafluoroisopropanol instead of acetate as counterion further enhances this effect (except for 100 mM concentration of hexafluoroisopropanol in combination with highly hydrophobic hexylamine). The elevated column temperature led to suppression of the diastereomeric resolution and improved resolution of n and n-x mers oligonucleotides. Baseline separation of oligonucleotides with different number of phosphorothioate linkages was achieved; this may be useful for therapeutic oligonucleotide analysis.
- Klíčová slova
- C18 column, Hexafluoroisopropanol, Ion-pairing reversed-phase chromatography, Phenyl column, Phosphorothioated oligonucleotides,
- MeSH
- adsorpce MeSH
- aminy chemie MeSH
- chromatografie s reverzní fází * metody MeSH
- fluorované uhlovodíky MeSH
- fosforothioátové oligonukleotidy * chemie izolace a purifikace MeSH
- hydrofobní a hydrofilní interakce MeSH
- propanoly chemie MeSH
- stereoizomerie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- aminy MeSH
- fluorované uhlovodíky MeSH
- fosforothioátové oligonukleotidy * MeSH
- hexafluoroisopropanol MeSH Prohlížeč
- propanoly MeSH
Effective translation of rare disease diagnosis knowledge into therapeutic applications is achievable within a reasonable timeframe; where mutations are amenable to current antisense oligonucleotide technology. In our study, we identified five distinct types of abnormal splice-causing mutations in patients with rare genetic disorders and developed a tailored antisense oligonucleotide for each mutation type using phosphorodiamidate morpholino oligomers with or without octa-guanidine dendrimers and 2'-O-methoxyethyl phosphorothioate. We observed variations in treatment effects and efficiencies, influenced by both the chosen chemistry and the specific nature of the aberrant splicing patterns targeted for correction. Our study demonstrated the successful correction of all five different types of aberrant splicing. Our findings reveal that effective correction of aberrant splicing can depend on altering the chemical composition of oligonucleotides and suggest a fast, efficient, and feasible approach for developing personalized therapeutic interventions for genetic disorders within short time frames.
- MeSH
- antisense oligonukleotidy * terapeutické užití genetika MeSH
- genetické nemoci vrozené genetika terapie MeSH
- lidé MeSH
- morfolino terapeutické užití genetika MeSH
- mutace * MeSH
- sestřih RNA * MeSH
- vzácné nemoci * genetika farmakoterapie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antisense oligonukleotidy * MeSH
- morfolino MeSH
