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.

• 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

Base excision repair is one of the important DNA repair mechanisms in cells. The fundamental role in this complex process is played by DNA glycosylases. Here, we present a novel approach for the real-time measurement of uracil DNA glycosylase activity, which employs selected oligonucleotides immobilized on the surface of magnetic nanoparticles and Förster resonance energy transfer. We also show that the approach can be performed by surface plasmon resonance sensor technology. We demonstrate that the immobilization of oligonucleotides provides much more reliable data than the free oligonucleotides including molecular beacons. Moreover, our results show that the method provides the possibility to address the relationship between the efficiency of uracil DNA glycosylase activity and the arrangement of the used oligonucleotide probes. For instance, the introduction of the nick into oligonucleotide containing the target base (uracil) resulted in the substantial decrease of uracil DNA glycosylase activity of both the bacterial glycosylase and glycosylases naturally present in nuclear lysates.

• MeSH
• buněčné jádro metabolismus   MeSH
• lidé MeSH
• magnetické nanočástice oxidů železa MeSH
• oligonukleotidové sondy chemie   metabolismus   MeSH
• oprava DNA MeSH
• rezonanční přenos fluorescenční energie MeSH
• uracil-DNA-glykosidasa metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Oligonucleotides (OND) represent a promising therapeutic approach. However, their instability and low intestinal permeability hamper oral bioavailability. Well-established for oral delivery, self-emulsifying drug delivery systems (SEDDS) can overcome the weakness of other delivery systems such as long-term instability of nanoparticles or complicated formulation processes. Therefore, the present study aims to prepare SEDDS for delivery of a nonspecific fluorescently labeled OND across the intestinal Caco-2 monolayer. The hydrophobic ion pairing of an OND and a cationic lipid served as an effective hydrophobization method using either dimethyldioctadecylammonium bromide (DDAB) or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). This strategy allowed a successful loading of OND-cationic lipid complexes into both negatively charged and neutral SEDDS. Subjecting both complex-loaded SEDDS to a nuclease, the negatively charged SEDDS protected about 16% of the complexed OND in contrast to 58% protected by its neutral counterpart. Furthermore, both SEDDS containing permeation-enhancing excipients facilitated delivery of OND across the intestinal Caco-2 cell monolayer. The negatively charged SEDDS showed a more stable permeability profile over 120 min, with a permeability of about 2 × 10-7 cm/s, unlike neutral SEDDS, which displayed an increasing permeability reaching up to 7 × 10-7 cm/s. In conclusion, these novel SEDDS-based formulations provide a promising tool for OND protection and delivery across the Caco-2 cell monolayer.

• Publikační typ
• časopisecké články MeSH

Methods of artificial evolution such as SELEX and in vitro selection have made it possible to isolate RNA and DNA motifs with a wide range of functions from large random sequence libraries. Once the primary sequence of a functional motif is known, the sequence space around it can be comprehensively explored using a combination of random mutagenesis and selection. However, methods to explore the sequence space of a secondary structure are not as well characterized. Here we address this question by describing a method to construct libraries in a single synthesis which are enriched for sequences with the potential to form a specific secondary structure, such as that of an aptamer, ribozyme, or deoxyribozyme. Although interactions such as base pairs cannot be encoded in a library using conventional DNA synthesizers, it is possible to modulate the probability that two positions will have the potential to pair by biasing the nucleotide composition at these positions. Here we show how to maximize this probability for each of the possible ways to encode a pair (in this study defined as A-U or U-A or C-G or G-C or G.U or U.G). We then use these optimized coding schemes to calculate the number of different variants of model stems and secondary structures expected to occur in a library for a series of structures in which the number of pairs and the extent of conservation of unpaired positions is systematically varied. Our calculations reveal a tradeoff between maximizing the probability of forming a pair and maximizing the number of possible variants of a desired secondary structure that can occur in the library. They also indicate that the optimal coding strategy for a library depends on the complexity of the motif being characterized. Because this approach provides a simple way to generate libraries enriched for sequences with the potential to form a specific secondary structure, we anticipate that it should be useful for the optimization and structural characterization of functional nucleic acid motifs.

• MeSH
• aptamery nukleotidové genetika   MeSH
• DNA katalytická genetika   MeSH
• genová knihovna * MeSH
• konformace nukleové kyseliny MeSH
• mutageneze MeSH
• nukleotidové motivy genetika   MeSH
• obrácené repetice genetika   MeSH
• párování bází MeSH
• pravděpodobnost MeSH
• řízená evoluce molekul metody   MeSH
• RNA katalytická genetika   MeSH
• syntetická biologie metody   MeSH
• techniky in vitro MeSH
• Publikační typ
• časopisecké články MeSH

A set of modified 2'-deoxyribonucleoside triphosphates (dNTPs) bearing a linear or branched alkane, indole or phenyl group linked through ethynyl or alkyl spacer were synthesized and used as substrates for polymerase synthesis of hypermodified DNA by primer extension (PEX). Using the alkyl-linked dNTPs, the polymerase synthesized up to 22-mer fully modified oligonucleotide (ON), whereas using the ethynyl-linked dNTPs, the enzyme was able to synthesize even long sequences of >100 modified nucleotides in a row. In PCR, the combinations of all four modified dNTPs showed only linear amplification. Asymmetric PCR or PEX with separation or digestion of the template strand can be used for synthesis of hypermodified single-stranded ONs, which are monodispersed polymers displaying four different substituents on DNA backbone in sequence-specific manner. The fully modified ONs hybridized with complementary strands and modified DNA duplexes were found to exist in B-type conformation (B- or C-DNA) according to CD spectral analysis. The modified DNA can be replicated with high fidelity to natural DNA through PCR and sequenced. Therefore, this approach has a promising potential in generation and selection of hypermodified aptamers and other functional polymers.

• MeSH
• adenin chemie   metabolismus   MeSH
• aptamery nukleotidové chemická syntéza   genetika   MeSH
• cytosin chemie   metabolismus   MeSH
• deoxyribonukleosidy chemie   genetika   metabolismus   MeSH
• dinukleosidfosfáty chemie   genetika   metabolismus   MeSH
• DNA-dependentní DNA-polymerasy genetika   metabolismus   MeSH
• DNA chemie   genetika   metabolismus   MeSH
• guanin chemie   metabolismus   MeSH
• hydrofobní a hydrofilní interakce MeSH
• párování bází MeSH
• polymerázová řetězová reakce MeSH
• polymery chemická syntéza   metabolismus   MeSH
• replikace DNA * MeSH
• sekvence nukleotidů MeSH
• uracil chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

V profylaxi aterotrombotických příhod má léčba dyslipidemie, zejména vysoké koncentrace aterogenního lipoproteinu o nízké hustotě (low density lipoprotein, LDL), klíčové postavení. Se stále se snižujícími cílovými hodnotami LDL cholesterolu je potřeba zavádět nové inovativní postupy. Ne vždy vystačíme se statiny a ezetimibem. Inhibitory proproteinové konvertázy subtilisin-kexinového typu 9 (PCSK9) na bázi monoklonálních protilátek jsou nákladné a rezervujeme je pro zvláště rizikové nemocné. Vývoj spěje k perorálně účinným inhibitorům konvertázy PCSK9 či k mikro-řetězcům kyseliny ribonukleové (ribonucleic acid, RNA) - inclisiranu, které brzdí transkripci genu a snižují nabídku vlastního izoenzymu PCSK9. Podobně se prověřuje efekt účinnější a bezpečnější inhibice syntézy cholesterolu. K užití, zpravidla v kombinaci se statiny, je schválena kyselina bempedová. Vedle vlastního lipoproteinu LDL je významný lipoprotein Lp(a). Ten akceleruje jak aterogenezi, tak inhibicí fibrinolýzy zvyšuje riziko trombotické okluze. V intervenci vysokých koncentrací Lp(a) se prověřuje účinnost oligonukleotidu inhibujícího syntézu apolipoproteinu (a) - pelacarsenu. Nálezy z prvých fází klinického hodnocení jsou slibné. Poslední strategií, kde je vidět výrazný pokrok, je léčba zaměřená na snížení hypertriglyceridemie. Dosavadní léčba fibráty sice snížila koncentraci triglyceridů, vliv na pokles vaskulárních příhod však nebyl přesvědčivě doložen. Prověřuje se účinnost a bezpečnost nových postupů. Ty jsou zaměřeny zejména na zvýšení aktivity lipoproteinové lipázy. Je však otázkou, zda snížení hypertriglyceridemie povede též ke zlepšení prognózy, přesvědčivý důkaz zatím podán nebyl.

In the prophylaxis of atherothrombotic events, the treatment of dyslipidemia, especially the effort to reduce the high concentration of atherogenic low-density lipoprotein (LDL), plays a key role. With ever-decreasing LDL-cholesterol targets, new innovative approaches need to be introduced. Statins and ezetimibe are not always sufficient. Monoclonal antibody-based subtilisin-kexin type 9 (PCSK9) proprotein convertase inhibitors are expensive and are reserved for particularly at-risk patients. Development leads to orally active inhibitors of PCSK9 convertase or to ribonucleic acid (RNA) - inclisirane micro-chains, which inhibit gene transcription and reduce the supply of the PCSK9 isoenzyme itself. Similarly, the effect of more effective and safer inhibition of cholesterol synthesis is being investigated. Bempedic acid is approved for use, usually in combination with statins. In addition to the LDL lipoprotein itself, the Lp (a) lipoprotein is important. It accelerates both atherogenesis and increases the risk of thrombotic occlusion by inhibiting fibrinolysis. In the intervention of high concentrations of Lp (a), the efficacy of an oligonucleotide inhibiting the synthesis of apolipoprotein (a) - pelacarsene is examined. The findings from the first phases of the clinical trial are promising. The last strategy, where significant progress is seen, is treatment to reduce hypertriglyceridemia. Although current fibrate treatment has reduced triglyceride levels, the effect on the decrease in vascular events has not been convincingly demonstrated. The effectiveness and safety of new procedures are being tested. These are mainly aimed at increasing lipoprotein lipase activity. However, the question is whether reducing pertriglyceridemia will also improve the prognosis, no convincing evidence has been provided yet.

• Klíčová slova
• bempedová kyselina, inclisiran, mipomersen, pelacarsen, evinacumab, volanesorsen,
• MeSH
• angiopoetinu podobné proteiny antagonisté a inhibitory   MeSH
• antisense oligonukleotidy terapeutické užití   MeSH
• apolipoprotein B-100 antagonisté a inhibitory   metabolismus   MeSH
• apolipoprotein C-III antagonisté a inhibitory   MeSH
• aterosklerotický plát patofyziologie   prevence a kontrola   MeSH
• ateroskleróza metabolismus   patofyziologie   prevence a kontrola   MeSH
• cholesterol metabolismus   škodlivé účinky   MeSH
• deriváty kyseliny fibrové farmakologie   terapeutické užití   MeSH
• dyslipidemie * farmakoterapie   patofyziologie   MeSH
• HDL-cholesterol metabolismus   účinky léků   MeSH
• hydroxymethylglutaryl-CoA-reduktasy metabolismus   účinky léků   MeSH
• hypertriglyceridemie farmakoterapie   MeSH
• kardiovaskulární nemoci prevence a kontrola   MeSH
• kyselina eikosapentaenová farmakologie   terapeutické užití   MeSH
• LDL-cholesterol metabolismus   škodlivé účinky   účinky léků   MeSH
• lidé MeSH
• lipoprotein (a) antagonisté a inhibitory   metabolismus   účinky léků   MeSH
• malá interferující RNA terapeutické užití   MeSH
• monoklonální protilátky terapeutické užití   MeSH
• niacin terapeutické užití   MeSH
• PCSK9 inhibitory MeSH
• proproteinkonvertasa subtilisin/kexin typu 9 účinky léků   MeSH
• rizikové faktory kardiovaskulárních chorob MeSH
• triglyceridy metabolismus   škodlivé účinky   MeSH
• Check Tag
• lidé MeSH

Taking advantage of surface-enhanced Raman scattering (SERS) methodology with its unique ability to collect abundant intrinsic fingerprint information and noninvasive data acquisition we set up a SERS-based approach for recognition of physically induced DNA damage with further incorporation of artificial neural network (ANN). As a proof-of-concept application, we used the DNA molecules, where the one oligonucleotide (OND) was grafted to the plasmonic surface while complimentary OND was exposed to UV illumination with various exposure doses and further hybridized with the grafted counterpart. All SERS spectra of entrapped DNA were collected by several operators using the portable spectrometer, without any optimization of measurements procedure (e.g., optimization of acquisition time, laser intensity, finding of optimal place on substrate, manual baseline correction, etc.) which usually takes a significant amount of operator's time. The SERS spectra were employed as input data for ANN training, and the performance of the system was verified by predicting the class labels for SERS validation data, using a spectra dataset, which has not been involved in the training process. During that phase, accuracy higher than 98% was achieved with a level of confidence exceeding 95%. It should be noted that utilization of the proposed functional-SERS/ANN approach allows identifying even the minor DNA damage, almost invisible by control measurements, performed with common analytical procedures. Moreover, we introduce the advanced ANN design, which allows not only classifying the samples but also providing the ANN analysis feedback, which associates the spectral changes and chemical transformations of DNA structure.

• MeSH
• biosenzitivní techniky * MeSH
• DNA chemie   izolace a purifikace   MeSH
• kovové nanočástice chemie   MeSH
• neuronové sítě MeSH
• oligonukleotidy chemie   MeSH
• poškození DNA * MeSH
• Ramanova spektroskopie * MeSH
• zlato chemie   MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as "two-donor floxing" method). RESULTS: We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. CONCLUSION: We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.

• MeSH
• alely * MeSH
• blastocysta metabolismus   MeSH
• CRISPR-Cas systémy genetika   MeSH
• faktorová analýza statistická MeSH
• mikroinjekce MeSH
• myši knockoutované MeSH
• protein 2 vázající methyl-CpG genetika   metabolismus   MeSH
• protein Cas9 metabolismus   MeSH
• regresní analýza MeSH
• reprodukovatelnost výsledků MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

The unavailability of simple, quick, and sensitive genetic-based molecular diagnostic techniques has become the main driving force for inventing new approaches in the era of quantum dots (QDs): a new class of fluorescent probes with fascinating optical electronic properties. Using the unique size-dependent light-emitting properties of QDs, we have developed a QD-based ultrasensitive technique which removes the necessity for the genetic amplification step required in almost all types of molecular-based diagnostic techniques. The selectivity of the new approach is warranted by the careful design of a pair of specific oligonucleotide probes, chemically modified at their 5'-ends. Our results indicated the selective detection of Salmonella typhi in an assay time of 50 min with a limit of detection (LOD) of 2 CFU/mL. The rapidity, selectivity, and sensitivity and the low assay cost make the new diagnostic technique a promising new tool for laboratory and field-based approaches to molecular diagnosis of health-threatening pathogens. Graphical abstract.

• MeSH
• břišní tyfus diagnóza   mikrobiologie   MeSH
• diagnostické techniky molekulární přístrojové vybavení   metody   MeSH
• DNA bakterií chemie   genetika   MeSH
• fluorescenční barviva chemie   MeSH
• kvantové tečky chemie   MeSH
• lidé MeSH
• limita detekce MeSH
• oligonukleotidové sondy chemie   genetika   MeSH
• Salmonella typhi chemie   genetika   izolace a purifikace   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH

Cíl: Cílem této práce bylo vyvinout nanoléčivo založené na specifickém transportu pomocí oligonukleotidové próby (komplementární k BRCA1 genové mutaci) s předpokládaným účinkem cytostatika bez výrazných toxických efektů k nenádorovým buňkám. Použitým cytostatikem bylo antracyklinové antibiotikum doxorubicin. Přes svou terapeutickou účinnost však vykazuje vysokou kardiotoxicitu. Možnost, jak zvýšit terapeutické okno, představují nanotransportéry. Metodika: Doxorubicin a oligonukleotidová próba byly navázány k fullerenům. Doba modifikace nanotransportérů byla 24 h. Nanotransportér byl následně charakterizován pomocí biofyzikálních technik (SEM, dynamického rozptylu světla, spektrální a elektrochemické metody). Pomocí elektrochemické a fluorescenční analýzy byl vzniklý komplex nanotransportéru potvrzen. Výsledky: Doxorubicin se záporně nabitým povrchem interaguje s kladně nabitými fullereny elektrostatickou interakcí a tato interakce byla potvrzena na základě elektronové mikroskopie (SEM), elektrochemicky (změna potenciálu byla 35 mV) a měřením dynamického rozptylu světla (změna ζ-potenciálu byla 22 mV). Ke komplexu byla amidovou vazbou připojena oligonukleotidová proba, která je komplementární k mutované sekvenci BRCA genu. Pokles fluorescenčního signálu nanokonstruktu o 80 % indikoval navázání oligonukleotidu. Pro prokázání funkčnosti byla navržena magnetická zlatá nanočástice modifikovaná komplementární sekvencí k testovanému nanotransportéru. Závěr: Předpokládáme, že navržený nanokonstrukt bude možné využít pro DNA cílené směřování protinádorového léčiva k buňkám s mutací v genu BRCA. Nanotransportér má tyto základní vlastnosti: a) fulleren vykazuje vysokou afinitu k buňce a proniká cytoplazmatickou membránou; b) doxorubicin se uvolní do cytoplazmy v nádorových buňkách díky nízkému pH; c) celý konstrukt je směřován cíleně na mutovanou sekvenci genu BRCA; d) zlatá nanočástice zesiluje cytotoxický efekt

Objective: Main goal of this project was to develop a nanotransporter based on a targeted delivery using oligonucleotide probe, which is complementary to a mutated BRCA1 gene sequence with the expected effect of a cytostatic without significant toxic effects. The mentioned cytostatic is often used doxorubicin. Despite its therapeutic efficacy, however, it exhibits high cardiotoxicity. Nanotransporters represent the possibility of increasing the therapeutic window. Methods: Doxorubicin and oligonucleotide probe were bound to fullerenes. The modification time was 24 hours. The nanotransporter was subsequently characterized by biophysical techniques (SEM, dynamic light scattering, spectral and electrochemical methods.) Using the electrochemical and fluorescence analysis the formation of nanotransporter was confirmed. Results: Doxorubicin with a negatively charged surface interacts with positively charged fullerenes by electrostatic interaction and this interaction was confirmed by on the basis of electron microscopy (SEM), electrochemically (change of potential was 35 mV) and by measurement of dynamic light scattering (change of ζ-potential was 22 mV). Oligonucleotide probe, which is complementary to the BRCA mutated gene sequence, was bound to the complex by an amide bond. The drop of the fluorescence signal by 80% indicated binding of the oligonucleotide. To demonstrate the functionality, a magnetic gold nanoparticle, modified by the complementary sequence to the tested nanotransporter, was developed. Conclusion: We assume, that the proposed nanotransporter will be used for DNA targeted delivery of the antitumor drug to the cells with BRCA mutated genes. The nanotransporter has these basic characteristics: a) fullerene has a high affinity to the cell and penetrates the cell membrane; b) doxorubicin is released into cytoplasm in tumour cells due to low pH; c) the entire construct is targeted to the BRCA mutated gene sequence; d) the gold nanoparticle enhances the cytotoxic effect.

• MeSH
• doxorubicin aplikace a dávkování   terapeutické užití   toxicita   MeSH
• fullereny terapeutické užití   MeSH
• geny BRCA1 MeSH
• geny BRCA2 MeSH
• kovové nanočástice využití   MeSH
• lékové transportní systémy MeSH
• lidé MeSH
• mutace genetika   MeSH
• nádory prsu * terapie   MeSH
• nanomedicína * MeSH
• oligonukleotidy terapeutické užití   MeSH
• pilotní projekty MeSH
• protinádorová antibiotika MeSH
• zlato terapeutické užití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH