Oligonucleotides In Solution Research Articles

Control of cell penetration enhancer shielding and endosomal escape-kinetics crucial for efficient and biocompatible siRNA delivery

Although cationic liposomes are efficient carriers for nucleic acid delivery, their toxicity often hampers the clinical translation. Polyethylene glycol (PEG) coating has been largely used to improve their stability and reduce toxicity. Nevertheless, it has been found to decrease the transfection process. In order to exploit the advantages of cationic liposomes and PEG decoration for nucleic acid delivery, liposomes decorated with tetraArg-[G-1]-distearoyl glycerol (Arg4-DAG) dendronic oligo-cationic lipid enhancer (OCE) and PEG-lipid have been investigated. Non decorated or OCE-decorated lipoplexes (OCEfree-LPX and OCE-LPX, respectively) were obtained by lipid film hydration using oligonucleotide (ON) solutions. PEG and OCE/PEG decorated lipoplexes (PEG-OCEfree-LPX and PEG-OCE-LPX, respectively) were obtained by post-insertion of 2 or 5 kDa PEG-DSPE on preformed lipoplexes. The OCE decoration yielded lipoplexes with size of about 240 nm, 84% loading efficiency at 10 N/P ratio, ten times higher than OCEfree-LPX, and prevented the ON release when incubated with physiological heparin concentration or with plasma. The PEG decoration reduced the zeta potential, enhanced the lipoplex stability in serum and decreased both hemolysis and cytotoxicity, while it did not affect the lipoplex size and ON loading. With respect to OCEfree-LPX, the OCE-LPX remarkably associated with cells and were taken up by different cancer cell lines (HeLa and MDA-MB-231). Interestingly, 2 or 5 kDa PEG decoration did not reduce either the cell interaction or the cell up-take of the cationic lipoplexes. With siRNA as a payload, OCE enabled efficient internalization, but endosomal release was hampered. Post-transfection treatment with the lysosomotropic drug chloroquine allowed to identify the optimal time point for endosomal escape. Chloroquine treatment after 12 to 20 h of LPX pre-incubation enabled siRNA mediated target knockdown indicating that this is the time window of endo-lysosomal processing. This indicates that OCE can protect siRNA from lysosomal degradation for up to 20 h, as shown by these rescue experiments.

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.

The interaction of dengue virus capsid protein with negatively charged interfaces drives the in vitro assembly of nucleocapsid-like particles.

Dengue virus (DENV) causes a major arthropod-borne viral disease, with 2.5 billion people living in risk areas. DENV consists in a 50 nm-diameter enveloped particle in which the surface proteins are arranged with icosahedral symmetry, while information about nucleocapsid (NC) structural organization is lacking. DENV NC is composed of the viral genome, a positive-sense single-stranded RNA, packaged by the capsid (C) protein. Here, we established the conditions for a reproducible in vitro assembly of DENV nucleocapsid-like particles (NCLPs) using recombinant DENVC. We analyzed NCLP formation in the absence or presence of oligonucleotides in solution using small angle X-ray scattering, Rayleigh light scattering as well as fluorescence anisotropy, and characterized particle structural properties using atomic force and transmission electron microscopy imaging. The experiments in solution comparing 2-, 5- and 25-mer oligonucleotides established that 2-mer is too small and 5-mer is sufficient for the formation of NCLPs. The assembly process was concentration-dependent and showed a saturation profile, with a stoichiometry of 1:1 (DENVC:oligonucleotide) molar ratio, suggesting an equilibrium involving DENVC dimer and an organized structure compatible with NCLPs. Imaging methods proved that the decrease in concentration to sub-nanomolar concentrations of DENVC allows the formation of regular spherical NCLPs after protein deposition on mica or carbon surfaces, in the presence as well as in the absence of oligonucleotides, in this latter case being surface driven. Altogether, the results suggest that in vitro assembly of DENV NCLPs depends on DENVC charge neutralization, which must be a very coordinated process to avoid unspecific aggregation. Our hypothesis is that a specific highly positive spot in DENVC α4-α4’ is the main DENVC-RNA binding site, which is required to be firstly neutralized to allow NC formation.

Scanning single-molecule counting system for Eprobe with highly simple and effective approach.

Here, we report a rapid and ultra-sensitive detection technique for fluorescent molecules called scanning single molecular counting (SSMC). The method uses a fluorescence-based digital measurement system to count single molecules in a solution. In this technique, noise is reduced by conforming the signal shape to the intensity distribution of the excitation light via a circular scan of the confocal region. This simple technique allows the fluorescent molecules to freely diffuse into the solution through the confocal region and be counted one by one and does not require statistical analysis. Using this technique, 28 to 62 aM fluorescent dye was detected through measurement for 600 s. Furthermore, we achieved a good signal-to-noise ratio (S/N = 2326) under the condition of 100 pM target nucleic acid by only mixing a hybridization-sensitive fluorescent probe, called Eprobe, into the target oligonucleotide solution. Combination of SSMC and Eprobe provides a simple, rapid, amplification-free, and high-sensitive target nucleic acid detection system. This method is promising for future applications to detect particularly difficult to design primers for amplification as miRNAs and other short oligo nucleotide biomarkers by only hybridization with high sensitivity.

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.

Pillar[5]arenes as potential personage for DNA compactization and gene therapy

Here we demonstrated that pillar[5]arenes with counterions I− and Cl− show the ability to plasmid compactization and increasing bacterial transformation efficiently. Pillar[5]arenes have been tested for binding with palindromic decamer oligonucleotide and interacting with plasmid DNA. The complexation of pillar[5]arene with oligonucleotide has been shown by NMR- and CD-spectroscopy. Pillar[5]arenes form complexes with oligonucleotide in solution in the 1:1 or 1:2 stoichiometry. Molecular modeling allowed to constructs the models of these complexes. Pillar[5]arene interaction with the plasmid DNA have been studied using atomic force microscopy. According to AFM images pillar[5]arene-I− and pillar[5]arene-Cl− packed up the plasmid DNA to aggregates with diameter about 100 nm with different morphology. An increase in DNA transformation efficiency into bacterial cells has been shown for pillar[5]arenes with counterions I− and Cl−.

Technical Considerations for Use of Oligonucleotide Solution API.

The most common approach for the manufacture of oligonucleotides includes isolation of the active pharmaceutical ingredient (API) via lyophilization to provide a solid product, which is then dissolved to provide an aqueous formulation. It is well known from the development and manufacture of large molecules (“biologics”) that API production does not always require isolation of solid API before drug product formulation, and this article provides technical considerations for the analogous use of oligonucleotide API in solution. The primary factor considered is solution stability, and additional factors such as viscosity, concentration, end-to-end manufacturing, microbiological control, packaging, and storage are also discussed. The technical considerations discussed in this article will aid the careful evaluation of the relative advantages and disadvantages of solution versus powder API for a given oligonucleotide drug substance.

(Invited) Understanding the Role of Protein Corona on Oligonucleotide Recognition Efficiency in Fluorescent Flares

Nanoparticle-based biosensors are developed and implemented to address a wide range of biomedical questions in bioimaging, molecular diagnostics, targeted drug delivery and controlled release, or photothermal cancer therapy. In this respect, gold nanoparticles provide physicochemical and optical properties that make them particularly interesting. Their relevant, and often unique, plasmonic response has enabled their integration with various transduction modalities (e.g. colorimetric, fluorescence, Raman) and led to the development of extremely sensitive and selective detection platforms. Added advantages associated with gold nanoparticles are their excellent biocompatibility and low toxicity compared to some other nanomaterials (notably quantum dots and silver nanoparticles) when used for sensing and imaging purposes in vivo.In recent years, the performance of gold nanoparticles in the biological milieu has attracted substantial interest. It is well known that, when employing nanoparticles in in vivo studies, proteins present in the biological environment contribute to opsonization, a process that leads to the recognition and attack by macrophages and systematic clearance. For in vitro applications, this process can affect instead the performance of the nanoparticle-based sensors, by reducing in particular sensitivity and selectivity. In both instances, the culprit is the rapid formation of a protein corona around the nanoparticle, which hinders the interaction of any responsive moiety conjugated to its surface with the target molecules present in the surrounding environment. In this talk, I will discuss how the formation of protein corona affects the response of fluorescent flares as they are employed to identify target oligonucleotides in solution. By monitoring the fluorescent response that originates upon hybridization of the nanoflares with their targets, we determined that protein corona, regardless of the identity of the proteins employed to mimic the biological environment, negatively impacts the hybridization dynamics, leading to a reduction in sensitivity by almost 20%, compared to the 2 nM LODs in the absence of corona. We also observed that the presence of protein corona leads to reduced resistance to nucleases, with different effects that depend on the size of the specific proteins, leading us to hypothesize the importance of steric hindrance. Finally, and surprisingly, we observed that polyethylene glycol (PEG) functionalization does not substantially improve the performance of the flares, with further reduced sensitivity due to the lower number of recognition moieties that can be accommodated on the nanoparticle’s surface when PEG is also introduced. We believe that these results will offer further quantitative insight useful for the development of in vivo and in vitro probes based on gold nanoparticles.

Light-Triggered Cellular Delivery of Oligonucleotides

The major challenge in the therapeutic applicability of oligonucleotide-based drugs is the development of efficient and safe delivery systems. The carriers should be non-toxic and stable in vivo, but interact with the target cells and release the loaded oligonucleotides intracellularly. We approached this challenge by developing a light-triggered liposomal delivery system for oligonucleotides based on a non-cationic and thermosensitive liposome with indocyanine green (ICG) as photosensitizer. The liposomes had efficient release properties, as 90% of the encapsulated oligonucleotides were released after 1-minute light exposure. Cell studies using an enhanced green fluorescent protein (EGFP)-based splicing assay with HeLa cells showed light-activated transfection with up to 70%–80% efficacy. Moreover, free ICG and oligonucleotides in solution transfected cells upon light induction with similar efficacy as the liposomal system. The light-triggered delivery induced moderate cytotoxicity (25%–35% reduction in cell viability) 1–2 days after transfection, but the cell growth returned to control levels in 4 days. In conclusion, the ICG-based light-triggered delivery is a promising method for oligonucleotides, and it can be used as a platform for further optimization and development.

Binding of CTAB to Single Stranded DNA

Oligonucleotides will aggregate in the presence of a cationic surfactant in a similar way to DNA condensation, which occurs during the transportation of nucleic acids through the cell membrane. Aggregates formed from oligonucleotides of 20 to 100 bases in length in the presence of the surfactant, CTAB (cetyl trimethylammonium bromide) have been studied using UV spectroscopy and fluorescence polarization by titrating oligonucleotide solutions with CTAB. Results indicate that the onset of a signal change (optical density or polarization) occurs when the ratio of CTAB to DNA nucleotides reaches approximately 0.7. This ratio is consistent with previous literature studies and has been attributed to the formation of aggregates triggered by the presence of a surfactant such as CTAB. Measurements from UV spectroscopy showed a second onset occurring at a ratio of about 2 that may be attributed to aggregate precipitation. The saturation binding curves obtained using both techniques were sigmoidal in shape implying that the process is cooperative. A variety of models were explored to glean additional information on the binding including the association constant between CTAB and a DNA nucleotide, the Hill coefficient, and the number of CTAB molecules bound per nucleotide.

Methods of Synthesis and Characterization of Conductive DNA Nanowires Based on Metal Ion-Mediated Base Pairing for Single-Molecule Electronics

Advances in the field of molecular electronics have made possible the directmeasurement of charge transport across single molecules. In particular, workon DNA oligomers has demonstrated that this weakly-conducting biomoleculecan be functionalized through metal-mediated nucleobase pairing in order tosignificantly increase electron mobility across the molecule. The introductionof interacting stacks of single metal ions inside the DNA helix is an attractiveplatform for assay and optimization; for this reason we present a protocolfor the production and processing of nanowires with a metal base pair forsingle-molecule applications. In particular, we describe the construction of DNA duplex wires with a cytosine-Ag+-cytosine base pair (dC:Ag+:dC).A thorough investigation of buffer components suggests the use of divalentmagnesium counterions to stabilize highly mismatched oligonucleotides insolution. We further analyse cleaning and processing of thin gold films forbatch-fabrication of conductive imaging substrates for use in conductivescanning probe assays of single-molecule conductivity. With a clear path toelectrical assays, we suggest that the C:Ag+:C orthogonal nucleotide pair andother similar chemistries may provide a foundation for molecular electroniccomponents in integrated devices

Scheme of the Complex Formation of DNA Telomeric Sequence with TMPyP4 Porphyrine

The model of the complex formation in a DNA oligonucleotide solution in the presence of TMPyP4 porphyrine has been recognized using a matrix method basing on the experimentally observed composition-property features. The experimental data on UV spectroscopic titration of G4 Tel22 oligonucleotide by TMPyP4 porphyrine have been interpreted in terms of equilibrium complex formation of the ligand with the macromolecule at four independent binding sites (coordination vacancies). The suggested matrix model has considered the mutual influence of the ligands bound at each of the Tel22 coordination vacancies. Sequential attachment of the TMPyP4 ligand to the Tel22(TMPyP4)n complex has affected the shape of the UV absorption spectrum of the earlier bound ligands. The cross-validation of the conventional stepwise complex formation model and the matrix binding model has confirmed the validity of the new interpretation.

Injectability as a function of viscosity and dosing materials for subcutaneous administration

Injectability is a term related to the ease of parenteral administration of a dosing solution, and includes dose preparation, dose administration, ergonomics related to these procedures, pain of injection, and other adverse events at the injection site. This article focuses on force measurements related to injectability, namely: force to expel syringe contents (expulsion force – a mimic for in vivo injection force), needle-penetration force, and needle-bending force, and these results are supplemented by expulsion time measurements with 18 participants, as well as injections in a porcine model. Based on the expulsion time measurements, where 80 N injection force was found to be difficult for most people, we consider the maximum acceptable injection force to be 40 N, and recommend targeting no more than 20 N, especially if the configuration may be used in an autoinjector or similar device. The injectability of antisense oligonucleotide solutions was assessed to determine optimal dosing materials (among those evaluated) for a variety of solution viscosities. Dosing materials varied in syringe inner diameter, needle inner diameter, needle length, and needle wall thickness: standard-wall vs. thin-wall. In general, short (6–8 mm) thin-wall needles are recommended as a way to improve patient perception and comfort during subcutaneous dose administration.

Optically Triggered Melting of DNA on Individual Semiconducting Carbon Nanotubes

AbstractOptical excitation of nanostructures is known to induce local heating, a phenomenon that has been intensely exploited for drug release, gene delivery, cancer thermotherapy, and energy harvesting. However, the effect is typically small requiring collective heating of a large concentration or aggregates of particles. Herein, we show that optical excitation of individual semiconducting single‐walled carbon nanotubes triggers strongly localized heating adequate to melt non‐covalently attached double‐stranded oligonucleotides in solution. In contrast to conventional thermal dehybridization, this optically triggered DNA melting occurs at a solution temperature that is 22 °C lower than the DNA melting temperature. This unexpectedly large localized optical heating effect provides important new insights to design selective optical nanoheaters at the single particle level.

Optically Triggered Melting of DNA on Individual Semiconducting Carbon Nanotubes.

Optical excitation of nanostructures is known to induce local heating, a phenomenon that has been intensely exploited for drug release, gene delivery, cancer thermotherapy, and energy harvesting. However, the effect is typically small requiring collective heating of a large concentration or aggregates of particles. Herein, we show that optical excitation of individual semiconducting single-walled carbon nanotubes triggers strongly localized heating adequate to melt non-covalently attached double-stranded oligonucleotides in solution. In contrast to conventional thermal dehybridization, this optically triggered DNA melting occurs at a solution temperature that is 22 °C lower than the DNA melting temperature. This unexpectedly large localized optical heating effect provides important new insights to design selective optical nanoheaters at the single particle level.

Dual-Phase Iontophoresis for the Delivery of Antisense Oligonucleotides.

In support of ongoing research in the study of corneal and skin wound healing, we sought to improve on previously published results by using iontophoresis to deliver RNA interference-based oligonucleotides. By using a electromechanics-based approach, we were able to devise a two-phase solution that separated the buffering solution from the antisense oligonucleotide (ASO) solution. The separation was obtained by making the drug solution a higher density than the buffer, leading it to sink directly onto the tissue surface. This change immediately decreased the distance that the ASO would have to travel before delivery. The changes enabled delivery into ex vivo skin and corneas in 10 or fewer minutes and into in vivo corneas in 5 min. In vivo studies demonstrated short-term bioavailability of at least 24 h, a lack of chemical or thermal injury, a lack of interference in the healing of a corneal injury, and an antisense effect till at least day 7, but not day 14. The only side-effect observed was postdelivery edema that was not present when the vehicle alone was iontophoresed. This suggests that electro-osmotic flow from the delivery chamber was not the mechanism, but that the delivered solute likely increased the tissue's osmolarity. These results support the continued development and utilization of this ASO delivery approach in research-grade oligonucleotides to probe molecular biological pathways and in support of testing therapeutic ASOs in the skin and cornea.

Oxyluciferin Derivatives: A Toolbox of Environment-Sensitive Fluorescence Probes for Molecular and Cellular Applications.

In this work, we used firefly oxyluciferin (OxyLH2) and its polarity-dependent fluorescence mechanism as a sensitive tool to monitor biomolecular interactions. The chromophores, OxyLH2, and its two analogues, 4-MeOxyLH and 4,6'-DMeOxyL, were modified trough carboxylic functionalization and then coupled to the N-terminus part of Tat and NCp7 peptides of human immunodeficiency virus type-1 (HIV-1). The photophysical properties of the labeled peptides were studied in live cells as well as in complex with different oligonucleotides in solution. By monitoring the emission properties of these derivatives we were able, for the first time, to study in vitro biomolecular interactions using oxyluciferin as a sensor. As an additional application, cyclopropyl-oxyluciferin (5,5-Cpr-OxyLH) was site-specifically conjugated to the thiol group (Cys-232) of the human protein α-1 antytripsin to investigate its interaction with porcine pancreatic elastase. Our data demonstrate that OxyLH2 and its derivatives can be used as fluorescence reporters for monitoring biomolecular interactions.

Osmolality of antisense oligonucleotide parenteral formulations: Implications on counterion dissociation and recommended osmometry techniques

The intrinsic osmolality of aqueous solutions of sodium salt antisense oligonucleotides (ASOs) has been studied to inform formulation practices, understand the molecular basis underlying the difference between theoretical and empirical results, and determine suitable measurement methods. It was found that regardless of nucleotide sequence, ASO concentration of ∼140mg/mL has isotonic osmolality of ∼290mOsm/kg water (SI unit: mmol osmotically-active particles/kg water), such that lower concentration formulations require excipients for tonicity adjustment. The range of osmolality values at a given active ingredient concentration can be ascribed to drug substance lot-to-lot purity differences impacting total oligonucleotide content (i.e., including oligonucleotide-related impurities). Empirical osmolality measurements were found to be ∼70% of theoretical values, which corresponds to an osmotic coefficient value of ∼0.7, thus inferring incomplete counterion dissociation. When comparing theoretical (ideal) osmolality of multiple sequences with various nucleotide compositions and chemistries at the same w/v concentration, the “average osmolar mass” (molar mass of the oligonucleotide, including the sodium counterions, divided by the ideal Van’t Hoff factor, iid) appears to be the strongest factor governing theoretical osmolality values. Other factors examined were the sequence length, backbone chemistry, 2′ sugar chemistry, and nucleotide composition. A head-to-head comparison between two osmolality techniques showed that vapor pressure osmometry is generally more suitable than freezing point osmometry for oligonucleotide solutions greater than ∼150mg/mL due to viscosity effects, but the two techniques are comparable otherwise.

Liquid Crystal Ordering and Isotropic Gelation in Solutions of Four-Base-Long DNA Oligomers.

Liquid crystal ordering is reported in aqueous solutions of the oligomer 5'-ATTAp-3' and of the oligomer 5'-GCCGp-3'. In both systems, we quantitatively interpret ordering as stemming from the chaining of molecules via a "running-bond" type of pairing, a self-assembly process distinct from the duplex aggregation previously reported for longer oligonucleotides. While concentrated solutions of 5'-ATTAp-3' show only a columnar liquid crystal phase, solutions of 5'-GCCGp-3' display a rich phase diagram, featuring a chiral nematic phase analogous to those observed in solutions of longer oligonucleotides and two unconventional phases, a columnar crystal and, at high concentration, an isotropic amorphous gel. The appearance of these phases, which can be interpreted on the basis of features of 5'-GCCGp-3'molecular structure, suggests distinctive assembly motifs specific to ultrashort oligonucleotides.

Developments in the large-scale preparation of oligonucleotides in solution

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