Oligonucleotide Concentration Research Articles

Bismuth-gold nanohybrid based nano photosensitizer to combat antimicrobial resistance

Antimicrobial resistance (AMR) leads to a decrease in the adequacy of antimicrobial agents and an increase in the rate of adverse effects and mortality. The main objective of this project is to investigate the synergistic effect of BiAu@NCLin-T1 and its substructures as an antimicrobial photodynamic therapy (aPDT) agent to combat microbial resistance. In addition, the effect of photothermal therapy (PTT) on some of the designed nanostructures at a temperature of 40 °C was also tested. The antimicrobial test was carried out using the growth curve method against Escherichia coli and Staphylococcus aureus. Computational methods were used to investigate the stability and entropy of oligonucleotide sequence structures. Various analyses were performed to identify the nanostructures, including Ultraviolet–visible (UV–vis) spectroscopy, Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), transmission electron microscopy (TEM), X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray analysis (EDS) and fluorescence analysis. The BiAu@NCLin-T1 appeared the significant aPDT impact against the gram-negative E.coli strain at two distinctive oligonucleotide concentrations (1, and 1.5 micromolar (µM)). Based on the results, the outlined nanostructures can act as a photosensitizer (PS), a photothermal treatment (PTT) agent, and an antimicrobial agent to combat resistant bacteria.

The Usefulness of Determining Plasma and Tissue Concentrations of Phosphorodiamidate Morpholino Oligonucleotides to Estimate Their Efficacy in Duchenne Muscular Dystrophy Patients.

Currently, four kinds of phosphorodiamidate morpholino oligomers (PMOs), such as viltolarsen, have been approved for the treatment of Duchenne muscular dystrophy (DMD); however, it is unclear whether human efficacy can be estimated using plasma concentrations. This study summarizes the tissue distribution of viltolarsen in mice and cynomolgus monkeys and evaluates the relationship between exposure and efficacy based on exon skipping. In the tissue distribution studies, all muscles in DMD-model mice showed higher concentrations of viltolarsen than those in wild-type mice and cynomolgus monkeys, and the concentrations in skeletal muscle were correlated with the exon-skipping efficiency in mice and cynomolgus monkeys. In addition, a highly sensitive bioanalytical method using liquid chromatography with tandem mass spectrometry shows promise for determining plasma concentrations up to a later time point, and the tissue (muscle)/plasma concentration ratio (Kp) in DMD-model mice was shown to be useful for predicting changes in pharmacodynamic (PD) markers in humans. Our results suggest that pharmacokinetic (PK)/PD analysis can be conducted by using the human PK profile or Kp values and skipping efficiency in DMD-model mice. This information will be useful for the efficient and effective development of PMOs as therapeutic agents. SIGNIFICANCE STATEMENT: We evaluated the relationship between the plasma or tissue concentrations and the efficiency of exon skipping for viltolarsen as an example phosphorodiamidate morpholino oligomers in the skeletal and cardiac muscle of mice and cynomolgus monkeys for pharmacokinetic/pharmacodynamic (PK/PD) analysis. The results suggest that PK/PD analysis can be conducted by using the human PK profile or tissue (muscle)/plasma concentration ratios and skipping efficiency in DMD-model mice.

Development and Validation of PCR Diagnostic Assays for Detection of Avibacterium paragallinarum and Ornithobacterium rhinotracheale.

PCR is the most effective method for detecting difficult-to-cultivate pathogens and pathogens that are part of mixed infections in animals, such as Ornithobacterium rhinotracheale, which causes bird ornithobacteriosis, or Avibacterium paragallinarum, which causes infectious coryza. In this work, we developed and validated two efficient and sensitive diagnostic assays for the rapid and accurate detection of A. paragallinarum and O. rhinotracheale DNA in bacterial isolates and clinical samples using real-time PCR with TaqMan-like probes. When designing the PCR assays, we performed in silico analysis, optimized DNA isolation methods and PCR conditions, and assessed the analytical and diagnostic performance of PCR. We designed primers and probes that have no mismatches with published whole-genome sequences of bacteria. The optimization of conditions showed that the PCR assays are sufficiently robust to changes in temperature and oligonucleotide concentration. The validation showed that the developed assays have high analytical and diagnostic sensitivity and specificity. These assays are expected to improve the differential diagnosis of respiratory diseases in chickens and turkeys.

Assessment of enzymatically synthesized DNA for gene assembly.

Phosphoramidite chemical DNA synthesis technology is utilized for creating de novo ssDNA building blocks and is widely used by commercial vendors. Recent advances in enzymatic DNA synthesis (EDS), including engineered enzymes and reversibly terminated nucleotides, bring EDS technology into competition with traditional chemical methods. In this short study, we evaluate oligos produced using a benchtop EDS instrument alongside chemically produced commercial oligonucleotides to assemble a synthetic gene encoding green fluorescent protein (GFP). While enzymatic synthesis produced lower concentrations of individual oligonucleotides, these were available in half the time of commercially produced oligonucleotides and were sufficient to assemble functional GFP sequences without producing hazardous organic chemical waste.

Experimental Tests of the Virtual Circular Genome Model for Nonenzymatic RNA Replication.

The virtual circular genome (VCG) model was proposed as a means of going beyond template copying to indefinite cycles of nonenzymatic RNA replication during the origin of life. In the VCG model, the protocellular genome is a collection of short oligonucleotides that map to both strands of a virtual circular sequence. Replication is driven by templated nonenzymatic primer extensions on a subset of kinetically trapped partially base-paired configurations, followed by the shuffling of these configurations to enable continued oligonucleotide elongation. Here, we describe initial experimental studies of the feasibility of the VCG model for replication. We designed a small 12-nucleotide model VCG and synthesized all 247 oligonucleotides of lengths 2 to 12 corresponding to this genome. We experimentally monitored the fate of individual labeled primers in the pool of VCG oligonucleotides following the addition of activated nucleotides and investigated the effect of factors such as oligonucleotide length, concentration, composition, and temperature on the extent of primer extension. We observe a surprisingly prolonged equilibration process in the VCG system that enables a considerable extent of reaction. We find that environmental fluctuations would be essential for continuous templated extension of the entire VCG system since the shortest oligonucleotides can only bind to templates at low temperatures, while the longest oligonucleotides require high-temperature spikes to escape from inactive configurations. Finally, we demonstrate that primer extension is significantly enhanced when the mix of VCG oligonucleotides is preactivated. We discuss the necessity of ongoing in situ activation chemistry for continuous and accurate VCG replication.

Flexible Cyclic Immunofluorescence (cyCIF) Using Oligonucleotide Barcoded Antibodies.

Advances in our understanding of the complex, multifaceted interactions between tumor epithelia, immune infiltrate, and tumor microenvironmental cells have been driven by highly multiplexed imaging technologies. These techniques are capable of labeling many more biomarkers than conventional immunostaining methods. However, multiplexed imaging techniques suffer from low detection sensitivity, cell loss-particularly in fragile samples-, and challenges with antibody labeling. Herein, we developed and optimized an oligonucleotide antibody barcoding strategy for cyclic immunofluorescence (cyCIF) that can be amplified to increase the detection efficiency of low-abundance antigens. Stained fluorescence signals can be readily removed using ultraviolet light treatment, preserving tissue and fragile cell sample integrity. We also extended the oligonucleotide barcoding strategy to secondary antibodies to enable the inclusion of difficult-to-label primary antibodies in a cyCIF panel. Using both the amplification oligonucleotides to label DNA barcoded antibodies and in situ hybridization of multiple fluorescently labeled oligonucleotides resulted in signal amplification and increased signal-to-background ratios. This procedure was optimized through the examination of staining parameters including staining oligonucleotide concentration, staining temperature, and oligonucleotide sequence design, resulting in a robust amplification technique. As a proof-of-concept, we demonstrate the flexibility of our cyCIF strategy by simultaneously imaging with the original oligonucleotide conjugated antibody (Ab-oligo) cyCIF strategy, the novel Ab-oligo cyCIF amplification strategy, as well as direct and indirect immunofluorescence to generate highly multiplexed images.

α-1,6-Fucosyltransferase Is Essential for Myogenesis in Zebrafish

Glycosylation is an important mechanism regulating various biological processes, including intercellular signaling and adhesion. α-1,6-fucosyltransferase (Fut8) belongs to a family of enzymes that determine the terminal structure of glycans. Fut8 is widely conserved from Caenorhabditis elegans to humans, and its mutants have been reported in humans, mice, and zebrafish. Although mutants show various symptoms, such as spinal deformity and growth retardation, its effects on skeletal muscles are unknown. We aimed to elucidate the function of Fut8 in skeletal muscle using zebrafish and C2C12 cells for evaluation. We observed that most fut8a morphants died at 2 days post-fertilization (dpf) or in earlier developmental stages even at low concentrations of morpholino oligonucleotides (MOs). Mutant juveniles also had small body sizes, and abnormal myocepta and sarcomere structures, suggesting that Fut8a plays important roles in myogenesis. Moreover, treatment of C2C12 cells with 2-fluorofucose (2FF), a fucosylation inhibitor, during cell differentiation dramatically reduced the expression of myogenic genes, such as Myomaker and other myogenic fusion genes, and inhibited myotube formation. These results indicate that Fut8 is an important factor in myogenesis, and myofusion in particular.

Liquid crystal phase formation and non-Newtonian behavior of oligonucleotide formulations

Viscosity behavior of liquid oligonucleotide therapeutics and its dependence on formulation properties has been poorly studied to date. We observed a high increase in viscosity and solidification of therapeutic oligonucleotide formulations with increasing oligonucleotide concentration creating challenges during drug product manufacturing. In this study, we characterized the viscosity behavior of three different single strand DNA oligonucleotides based on oligonucleotide concentration and formulation composition. We subsequently studied the underlying mechanism for increased viscosity at higher oligonucleotide concentrations by dynamic light scattering (DLS), 1H nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), and polarized light microscopy. Viscosity was highly dependent on formulation composition, oligonucleotide sequence, and concentration, and especially dependent on the presence and combination of different individual ions, such as the presence of sodium chloride in the formulation. In samples with elevated viscosity, the viscosity behavior was characterized by non-Newtonian, shear-thinning flow behavior. We further studied these samples by DLS and 1H NMR, which revealed the presence of supra-molecular assemblies, and further characterization by polarized light and DSC characterized these assemblies as liquid crystals in the formulation. The present study links the macroscopic viscosity behavior of oligonucleotide formulations to the formation of supra-molecular assemblies and to the presence of liquid crystals, and highlights the importance of formulation composition selection for these therapeutics.

DNA Nanostructure-Guided Plasmon Coupling Architectures

DNA Nanostructure-Guided Plasmon Coupling Architectures

Intramolecular Folding of PolyT Oligonucleotides Induced by Cooperative Binding of Silver(I) Ions.

Ag+-bridged T-Ag+-T was recently discovered in a Ag+-DNA nanowire crystal, but it was reported that Ag+ had little to no affinity to T nucleobases and T-rich oligonucleotides in solution. Therefore, the binding mode for the formation of this type of novel metallo base pair in solution is elusive. Herein, we demonstrate that Ag+ can interact with polyT oligonucleotides once the concentration of Ag+ in solution exceeds a threshold value. The threshold value is independent of the concentration of the polyT oligonucleotide but is inversely proportional to the length of the polyT oligonucleotide. The polyT oligonucleotides are intramolecularly folded due to their positively cooperative formation and the stack of T-Ag+-T base pairs, resulting in the 5'- and 3'-ends being in close proximity to each other. The intramolecular Ag+-folded polyT oligonucleotide has a higher thermal stability than the duplex and can be reversibly modulated by cysteine.

Virtual Circular Genome Model for Prebiotically Plausible Nonenzymatic RNA Replication

Nonenzymatic RNA replication is thought to have been an essential step on the path to the emergence of the first ribozymes during the origin of life. However, current nonenzymatic RNA replication models are limited by the extent and fidelity of primer extension chemistry, and the difficulty of copying the strands of the product duplex. Here we discuss the virtual circular genome (VCG) model, which is based on a collection of short oligonucleotides that map onto both strands of one or more virtual circular sequences. Replication is driven by annealing of the oligonucleotides to allow for templated primer extension or ligation, followed by rearrangement to allow additional oligonucleotide elongation. Every oligonucleotide in the system can act as a primer, template, or downstream helper. Therefore, the genomic replication process is distributed across the population. Over multiple generations, this model can be viewed as a flow of material from shorter to longer oligonucleotides that cover the genome. We have monitored the fate of individual primers in the pool of oligonucleotides, and have investigated factors such as oligo length, concentration, temperature, and metal catalyst on the primer extension yield. We propose several strategies that may lead to fast and accurate primer extension in the VCG model, and which may eventually enable cycles of genome replication and the evolution of ribozymes within replicating protocells.

Expression of GM content in mass fraction from digital PCR data.

Nowadays the quantification of the content of genetically modified (GM) constituents in food or feed products is performed by using either quantitative real-time PCR (qPCR) or digital PCR (dPCR). The latter is increasingly used. Therefore, experimental protocols for the quantification of 52 GM events authorised in the EU have been converted into a digital format and minimum performance characteristics for dPCR methods are detailed. Because of the need to harmonise the transformation of PCR results between two different measurement scales, 50 conversion factors for Certified Reference Materials (CFCRM) have been experimentally determined by three and sometimes four independent expert laboratories. The uncertainty of each CFCRM has been estimated to express dPCR results in mass fraction with a consistent uncertainty contribution. In 38 out of 58 cases, the validated qPCR methods (for 52 event-specific and 6 taxon-specific measurements) could easily be transferred into dPCR methods by using the same oligo sequences, final oligo concentration or annealing temperatures for the dPCR procedure. Laboratories have nevertheless used different strategies to improve the resolution or to reduce the so-called rain in their dPCR outcome. Those modifications were needed for PCR procedures that could not be converted without changes into a digital format. Therefore, exclusion/quality criteria such as the maximum rate of partitions with intermediate fluorescence “rain”, the minimum resolution and repeatability are suggested for dPCR methods. The CFCRM determined in this study were generally in agreement with the declared zygosity of the GM parental donor for hemizygous maize events. In a limited number of GM events the CFCRM values were significantly different when measured with different maize-specific (ZmAdh1 or hmgA) genes.

Multiplex qPCR assay to determine Leishmania infantum load in Lutzomyia longipalpis sandfly samples.

The study aimed to develop a multiplex qPCR to detect Leishmania infantum load in different sandfly sample settings using Leishmania kDNA and sandfly vacuolar ATPase (VATP) subunit C as internal control gene. The amplification of Lutzomyia longipalpis VATP gene was evaluated together with Leishmania infantum kDNA in a multiplex reaction. The concentration of VATP gene oligonucleotides was adjusted until no statistically significant difference was observed between all multiplex standard curves and singleplex curves, that is, only kDNA amplification. Limit of detection (LoD) was measured using a probit model and a cut-off defined by receiver operating characteristic analysis. Limit of quantification (LoQ) was assessed by a linear model using the coefficient of variation threshold of 25%. After assuring VATP gene amplification, its primer-probe concentrations were best at 100 nM/10nM, respectively. The cut-off Cq value for L. infantum kDNA was defined as 35.46 with 100% of sensitivity and specificity. A total of 95% LoD was determined to be of 0.162 parasites while LoQ was 5.858. Our VATP/kDNA multiplex qPCR assay shows that it can be used to evaluate both DNA integrity and determine L. infantum load in L. longipalpis even for low yielded samples, that is, individual midguts.

Nanoparticle-Assisted Nuclear Relaxation-Based Detection of Oligonucleotides.

Nuclear relaxation-based techniques rely on the capability of paramagnetic center to affect the transverse relaxation time (T2) of the water protons. These changes are sensitive to the microenvironment of the paramagnetic center, which can be used to detect a variety of targets. In this work, we present an "on-off" oligonucleotide detection scheme in aqueous solutions, which uses gadolinium phthalocyanine (GdTcPc)-grafted silica nanoparticles as the paramagnetic centers. A probe oligonucleotide strand was conjugated to the GdTcPc to act as a recognition element. In the presence of the target oligonucleotide, which was complementary to the probe, an increase in the ΔT2 value, which was measured by subtracting the characteristic T2 value of the background solution from that of the sample using a benchtop relaxometer, was observed. The magnitude of this increase was proportional to the target oligonucleotide concentration. A linear range was obtained from 30 to 140nM, with a detection limit of 15nM. The developed nuclear relaxation-based detection scheme is shown to be a simple, fast, and selective method to detect oligonucleotide and could be useful in point-of-care diagnostic applications.

Label-Free Homogeneous microRNA Detection in Cell Culture Medium Based on Graphene Oxide and Specific Fluorescence Quenching.

Label-free homogeneous optical detection of low concentration of oligonucleotides using graphene oxide in complex solutions containing proteins remains difficult. We used a colloidal graphene oxide (GO) as a fluorescent probe quencher to detect microRNA-21 spiked-in cell culture medium, overcoming previously reported problematic aspects of protein interference with graphene oxide. We used a “turn off” assay for specific quenching-based detection of oligo DNA-microRNA hybridization in solution. A fluorescein conjugated 30-mer single-stranded DNA (ssDNA) probe was combined with a complementary synthetic microRNA (18 nucleotides) target. The probe-target hybridization was detected by specific quenching due to photoinduced electron transfer (PET). On the next step, GO captures and quenches the unhybridized probe by fluorescence resonance energy transfer (FRET) in the presence of cell culture medium supplemented with platelet lysate, 0.1% sodium dodecyl sulfate (SDS), 0.1% Triton X-100 and 50% formamide. This resulted in sensitive measurement of the specific probe-target complexes remaining in solution. The detection is linear in the range of 1 nM and 8 nM in a single 100 μL total volume assay sample containing 25% cell culture medium supplemented with platelet lysate. We highlight a general approach that may be adopted for microRNA target detection within complex physiological media.

In-silico amplification of randomly selected male - specific region – Y (MSY) genes as candidate genes for the selection of fertile indigenous bulls for artificial insemination

Molecular markers-based selection is a novel tool for unbundling complexities associated with the selection of animals for breeding improvement programmes because these markers could contribute to full exploitation of animal potential prior to enrollment for improvement programmes. This study examined in-silico polymerase chain reaction amplification of some selected Male Specific region Y (MSY) genes in genome assemblies including Bos_taurus_UMD_3.1.1/bosTau8 and ARS-UCD1.2/bosTau9;BaylorBtau_4.6.1/bosTau7, Bos_taurus_UMD_3.1/bosTau6, and Baylor4.0/bosTau4. The results of the amplification showed that 12 out of the selected 30 genes were expressed at an average primer melting temperature of 61.78 oC; while salt and annealing oligo concentrations were 50 mM and 50 nM per reaction, respectively. Out of expressed genes; 6, 5 and 1 genes were expressed in genome assemblies Bos_taurus_UMD_3.1.1/bosTau8, ARS-UCD1.2/bosTau9, and BaylorBtau_4.6. 1/bosTau7, respectively. The study concluded that the expressed genes are potential markers for selection of fertile bulls and the primers used are suitable for in-vivo annotation of the genes in the prospective bull selection process for breeding programmes.

High-Performance Estimation of Lead Ion Concentration Using Smartphone-Based Colorimetric Analysis and a Machine Learning Approach.

Traditional methods for detection of lead ions in water samples are costly and time-consuming. In this work, an accurate smartphone-based colorimetric sensor was developed utilizing a novel machine learning algorithm. In the presence of Pb2+ ions in the solution of specifically functionalized gold nanoparticles, the color of solution turns from red to purple. Indeed, the color variation of the solution is proportional to Pb2+ concentration. The smartphone camera captures the corresponding color change, and the image is processed by an efficient artificial intelligence protocol. The nonlinear regression approach was used for concentration estimation, in which the parameters of the proposed model are obtained using a new feature extraction algorithm. In prediction of Pb2+ concentration, the average absolute error and root-mean-square error were 0.094 and 0.124, respectively. The influence of pH of the medium, temperature, oligonucleotide concentration, and reaction time on the performance of the proposed sensor was carefully investigated and understood to achieve the best sensor response. This novel sensor exhibited good linearity for the detection of Pb2+ in the concentration range of 0.5–2000 ppb with a detection limit of 0.5 ppb.

Detection of gene mutation responsible for Huntington's disease by terahertz attenuated total reflection microfluidic spectroscopy.

Terahertz absorption spectroscopy based on attenuated total reflection (ATR) from a microfluidic sample cell was designed and implemented to detect gene mutations leading to Huntington's disease (HD). The self-developed compact ATR microfluidic system was employed to detect two groups of base-repeated DNA molecules combined with a terahertz time-domain spectrometer in a marker-free manner. The first group featured different repetition patterns of oligonucleotide fragments, and the second group included the HD gene. For the oligonucleotides of different repetition patterns, there were significant differences among the three oligonucleotides with three repeats of the double bases, which could be unambiguously classified and identified; For the HD gene, it was found that the magnitude of the terahertz absorption coefficients of the four oligonucleotide solutions was, in ascending order, CAG-4 < CAG-16 < CAG-32 < CAG-40 (the numbers are the repeat times of the CAG base segment, with 40 repeats belonging to the HD gene), when the concentration of oligonucleotide was 1 mg/mL. Principal component analysis result indicated that the spectral differences of the four oligonucleotide solutions with different CAG repeat times were statistically significant and clearly distinguishable. These results demonstrate the potential of terahertz spectroscopy as a noninvasive, unmarked, fast and low-cost assay for gene diagnosis and clinical disease detection.

Development of orally administered insulin-loaded polymeric-oligonucleotide nanoparticles: statistical optimization and physicochemical characterization

Introduction Therapeutic peptides are administered via parenteral route due to poor absorption in the gastrointestinal (GI) tract, instability in gastric acid, and GI enzymes. Polymeric drug delivery systems have achieved significant interest in pharmaceutical research due to its feasibility in protecting proteins, tissue targeting, and controlled drug release pattern. Materials and methods In this study, the size, polydispersity index, and zeta potential of insulin-loaded nanoparticles were characterized by dynamic light scattering and laser Doppler micro-electrophoresis. The main and interaction effects of chitosan concentration and Dz13Scr concentration on the physicochemical properties of the prepared insulin-loaded nanoparticles (size, polydispersity index, and zeta potential) were evaluated statistically using analysis of variance. A robust procedure of reversed-phase high-performance liquid chromatography was developed to quantify insulin release in simulated GI buffer. Results and discussion: We reported on the effect of two independent parameters, including polymer concentration and oligonucleotide concentration, on the physical characteristics of particles. Chitosan concentration was significant in predicting the size of insulin-loaded CS-Dz13Scr particles. In terms of zeta potential, both chitosan concentration and squared term of chitosan were significant factors that affect the surface charge of particles, which was attributed to the availability of positively-charged amino groups during interaction with negatively-charged Dz13Scr. The excipients used in this study could fabricate nanoparticles with negligible toxicity in GI cells and skeletal muscle cells. The developed formulation could conserve the physicochemical properties after being stored for 1 month at 4 °C. Conclusion The obtained results revealed satisfactory results for insulin-loaded CS-Dz13Scr nanoparticles (159.3 nm, pdi 0.331, −1.08 mV). No such similar study has been reported to date to identify the main and interactive significance of the above parameters for the characterization of insulin-loaded polymeric-oligonucleotide nanoparticles. This research is of importance for the understanding and development of protein-loaded nanoparticles for oral delivery.

Imbibition of Femtoliter-Scale DNA-Rich Aqueous Droplets into Porous Nylon Substrates by Molecular Printing.

This work presents the first reported imbibition mechanism of femtoliter (fL)-scale droplets produced by microchannel cantilever spotting (μCS) of DNA molecular inks into porous substrates (hydrophilic nylon). Differently from macroscopic or picoliter droplets, the downscaling to the fL-size leads to an imbibition process controlled by the subtle interplay of evaporation, spreading, viscosity, and capillarity, with gravitational forces being quasi-negligible. In particular, the minimization of droplet evaporation, surface tension, and viscosity allows for a reproducible droplet imbibition process. The dwell time on the nylon surface permits further tuning of the droplet lateral size, in accord with liquid ink diffusion mechanisms. The functionality of the printed DNA molecules is demonstrated at different imbibed oligonucleotide concentrations by hybridization with a fluorolabeled complementary sequence, resulting in a homogeneous coverage of DNA within the imbibed droplet. This study represents a first step toward the μCS-enabled fabrication of DNA-based biosensors and microarrays into porous substrates.