Phosphorothioate Research Articles

Fast Electron Detachment Dissociation of Oligonucleotides in Electron-Nitrogen Plasma Stored in Magneto Radio-Frequency Ion Traps.

We report "plasma" electron detachment dissociation (EDD), a novel electron-activated dissociation (EAD) method for the fast sequencing of oligonucleotides with a high sequence coverage. To reduce the repulsive Coulombic force between the deprotonated oligonucleotides and the electron beam, we performed EDD in a neutral electron-nitrogen (N2+) plasma stored in a magneto radio-frequency ion trap. We confirmed that plasma EDD accomplished a high sequence coverage (100%) of RNA with 40 mers in the reaction time of 10 ms using the electron beam kinetic energy of 35 eV. This new technique was applied to various modifications in oligonucleotide therapeutics (ONTs). Phosphorothioate (PS) positions showed an extremely high dissociation efficiency, i.e., 100 times higher than the standard phosphate (PO) in DNA. Locked nucleotides did not show intensive dissociation in EDD; however, collision-induced dissociation (CID) helped sequence these portions. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a ZenoTOF mass spectrometer equipped with the plasma EDD technique successfully identified impurities in degraded samples.

Low Energy Photoionization of Phosphorothioate DNA-Oligomers and Ensuing Hole Transfer.

Phosphorothioate (PS) modified oligonucleotides (S-DNA) naturally exist in bacteria and archaea genome and are widely used as an antisense strategy in gene therapy. However, the introduction of PS as a redox active site may trigger distinct UV photoreactions. Herein, by time-resolved spectroscopy, we observe that 266 nm excitation of S-DNA d(Aps)20 and d(ApsA)10 leads to direct photoionization on the PS moiety to form hemi-bonded -P-S∴S-P- radicals, in addition to A base ionization to produce A+•/A(-H)•. Fluorescence spectroscopy and global analysis indicate that an unusual charge transfer state (CT) between the A and PS moiety might populate in competition with the common CT state among bases as key intermediate states responsible for S-DNA photoionization. Significantly, the photoionization bifurcating to PS and A moieties of S-DNA is discovered, suggesting that the PS moiety could capture the oxidized site and protect the remaining base against ionization lesion, shedding light on the understanding of its existence in living organisms.

Analysis of the Selective Flotation of Elemental Gold from Pyrite Using Diisobutyl Monothiophosphate

The gold contained in copper ores is an important resource for the gold industry. In some cases, elemental gold is present and can be recovered by selective flotation. It has been reported that the gold grade and recovery can be increased, without sacrificing the copper recovery, by replacing AERO 3477 (diisobutyl dithiophosphate (DTP)) with AERO 7249 (mixture of diisobutyl monothiophosphate (MTP) and diisobutyl dithiophosphate (DTP)) as the main collector. The fundamental understanding of the improvement in selectivity with the addition of MTP in the flotation of elemental gold from pyrite is limited and is the subject of this paper. In this regard, the hydrophobicity and selectivity of DTP and MTP in the flotation of pyrite and gold are compared and discussed. Density functional theory (DFT) was used to examine the electron density, reactivity, highest occupied molecular orbital (HOMO), and lowest unoccupied molecular orbital (LUMO) of the MTP and DTP collectors. The interaction energies for the adsorption of MTP and DTP from fresh pyrite, oxidized pyrite and gold surfaces were calculated and discussed with respect to the experimental results reported in the literature. Molecular dynamics simulation (MDS) was used to examine the adsorption state of MTP and DTP on the pyrite (100) and Au (111) surfaces.

A Sensitive Aptamer Fluorescence Anisotropy Sensor for Cd2+ Using Affinity-Enhanced Aptamers with Phosphorothioate Modification.

Rapid and sensitive detection of heavy metal cadmium ions (Cd2+) is of great significance to food safety and environmental monitoring, as Cd2+ contamination and exposure cause serious health risk. In this study we demonstrated an aptamer-based fluorescence anisotropy (FA) sensor for Cd2+ with a single tetramethylrhodamine (TMR)-labeled 15-mer Cd2+ binding aptamer (CBA15), integrating the strengths of aptamers as affinity recognition elements for preparation, stability, and modification, and the advantages of FA for signaling in terms of sensitivity, simplicity, reproducibility, and high throughput. In this sensor, the Cd2+-binding-induced aptamer structure change provoked significant alteration of FA responses. To acquire better sensing performance, we further introduced single phosphorothioate (PS) modification of CBA15 at a specific phosphate backbone position, to enhance aptamer affinity by possible strong interaction between sulfur and Cd2+. The aptamer with PS modification at the third guanine (G) nucleotide (CBA15-G3S) had four times higher affinity than CBA15. Using as an aptamer probe CBA15-G3S with a TMR label at the 12th T, we achieved sensitive selective FA detection of Cd2+, with a detection limit of 6.1 nM Cd2+. This aptamer-based FA sensor works in a direct format for detection without need for labeling Cd2+, overcoming the limitations of traditional competitive immuno-FA assay using antibodies and fluorescently labeled Cd2+. This FA method enabled the detection of Cd2+ in real water samples, showing broad application potential.

P.193 Phosphoryl guanidine-containing backbone modifications enhance exon skipping, dystrophin restoration and survival in a severe mouse model for DMD

Although recent regulatory approval of splice-switching oligonucleotides (SSOs) for the treatment of Duchenne muscular dystrophy (DMD) has been an advance for the field, current SSO chemistries have shown limited clinical benefit due to poor pharmacology. There also remains substantial unmet clinical need for approaches that reach cardiac and respiratory muscles, where restoration of dystrophin may be essential to improve survival. To overcome limitations of existing technologies, we engineered stereopure oligonucleotides with phosphorothioate (PS), phosphodiester (PO), and phosphoryl guanidine-containing (PN) backbones. In survival studies, bi-weekly doses of the PS/PO/PN SSO prevented premature death and improved median survival from 49 days to at least 280 days in the double utrophin/dystrophin knockout mouse, a model with an aggressive dystrophic phenotype. These profound survival benefits were achieved at doses anticipated to be clinically relevant. Importantly, the PS/PO/PN SSO increased dystrophin levels across several muscle types (skeletal, respiratory, and cardiac) and improved both respiratory and skeletal muscle function. Treatment also corresponded with decreased serum biomarkers of muscle damage and improved weight gain. These data demonstrate that chemical optimization can profoundly impact oligonucleotide pharmacology and highlight the therapeutic potential of stereopure PN-containing SSOs in DMD. Further, these data help to support the advancement of WVE-N531, an investigational compound currently in clinical testing (NCT04906460) that incorporates this technology, for the treatment of patients with DMD amenable to exon 53 skipping.

Functional Downregulation of PD-L1 and PD-L2 by CpG and non-CpG Oligonucleotides in Melanoma Cells.

Simple SummaryAlthough metastatic melanoma is still not a curable disease, targeting of immunologically relevant checkpoints represents a turning point in the treatment. Particularly, targeting the interaction between PD-L1 and its referring receptor PD-1 with antibodies has been shown to activate T-cell function abrogating the evasion of tumor cells from immune recognition. Here, we present another approach that interferes with this system by showing that treatment of melanoma cells with oligonucleotides reduces the expression of PD-L1 (and PD-L2) on tumor cells. Specifically, non-CpG-6-PTO, an ODN that forms superstructures known as G-quartets, has been found to inhibit the interferon-γ-induced signaling cascade which fosters PD-L1 expression. These findings suggest a new therapeutic strategy to interfere with one of the most important immune checkpoints.The clinical application of immune checkpoint inhibitors represents a breakthrough progress in the treatment of metastasized melanoma and other tumor entities. In the present study, it was hypothesized that oligonucleotides (ODNs), known as modulators of the immune response, have an impact on the endogenous expression of checkpoint molecules, namely PD-L1 and PD-L2 (PD-L1/2). IFNγ-stimulated melanoma cells (A375, SK-Mel-28) were treated with different synthetically manufactured oligonucleotides which differed in sequence, length and backbone composition. It was found that a variety of different ODN sequences significantly suppressed PD-L1/2 expression. This effect was dependent on length and phosphorothioate (PTO) backbone. In particular, a sequence containing solely guanines (nCpG-6-PTO) was highly effective in downregulating PD-L1/2 at the protein, mRNA and promoter levels. Mechanistically, we gave evidence that ODNs with G-quartet-forming motifs suppress the interferon signaling axis (JAK/STAT/IRF1). Our findings identify a subset of ODNs as interesting pharmacological compounds that could expand the arsenal of targeted therapies to combat the immunological escape of tumor cells.

Structures of annexin A2-PS DNA complexes show dominance of hydrophobic interactions in phosphorothioate binding.

The introduction of phosphorothioate (PS) linkages to the backbone of therapeutic nucleic acids substantially increases their stability and potency. It also affects their interactions with cellular proteins, but the molecular mechanisms that underlie this effect are poorly understood. Here, we report structural and biochemical studies of interactions between annexin A2, a protein that does not possess any known canonical DNA binding domains, and phosphorothioate-modified antisense oligonucleotides. We show that a unique mode of hydrophobic interactions between a sulfur atom of the phosphorothioate group and lysine and arginine residues account for the enhanced affinity of modified nucleic acid for the protein. Our results demonstrate that this mechanism of interaction is observed not only for nucleic acid-binding proteins but can also account for the association of PS oligonucleotides with other proteins. Using the anomalous diffraction of sulfur, we showed that preference for phosphorothioate stereoisomers is determined by the hydrophobic environment around the PS linkage that comes not only from protein but also from additional structural features within the ASO such as 5-Me groups on cytosine nucleobases.

Simultaneous Targeting of Multiple oncomiRs with Phosphorothioate or PNA-Based Anti-miRs in Lymphoma Cell Lines.

MicroRNAs (miRNAs) are short (~ 22 nts) RNAs that regulate gene expression via binding to mRNA. MiRNAs promoting cancer are known as oncomiRs. Targeting oncomiRs is an emerging area of cancer therapy. OncomiR-21 and oncomiR-155 are highly upregulated in lymphoma cells, which are dependent on these oncomiRs for survival. Targeting specific miRNAs and determining their effect on cancer cell progression and metastasis have been the focus of various studies. Inhibiting a single miRNA can have a limited effect, as there may be other overexpressed miRNAs present that may promote tumor proliferation. Herein, we target miR-21 and miR-155 simultaneously using nanoparticles delivered two different classes of antimiRs: phosphorothioates (PS) and peptide nucleic acids (PNAs) and compared their efficacy in lymphoma cell lines. Poly-Lactic-co-Glycolic acid (PLGA) nanoparticles (NPs) containing PS and PNA-based antimiR-21 and -155 were formulated, and comprehensive NP characterizations: morphology (scanning electron microscopy), size (differential light scattering), and surface charge (zeta potential) were performed. Cellular uptake analysis was performed using a confocal microscope and flow cytometry analysis. The oncomiR knockdown and the effect on downstream targets were confirmed by gene expression (real time-polymerase chain reaction) assay. We demonstrated that simultaneous targeting with NP delivered PS and PNA-based antimiRs resulted in significant knockdown of miR-21 and miR-155, as well as their downstream target genes followed by reduced cell viability ex vivo. This project demonstrated that targeting miRNA-155 and miR-21 simultaneously using nanotechnology and a diverse class of antisense oligomers can be used as an effective approach for lymphoma therapy.

Dual-Signaling Photoelectrochemical Biosensor Based on Biocatalysis-Induced Vulcanization of Bi2MoO6 Nanosheets.

A magnetic-assisted photoelectrochemical (PEC) and colorimetric (CL) dual-modal biosensing platform with high precision was established to monitor prostate-specific antigen (PSA) based on Bi2MoO6 nanosheets (BMO) by coupling the aptamer-guided hybridization chain reaction (HCR) with the hydrolysate-induced vulcanization reaction of Bi2MoO6 nanosheets. Upon addition of PSA, trigger DNA (tDNA) was released by the interaction between the target analyte and the aptamer and then further hybridized with anchor DNA (aDNA) conjugated on magnetic beads (MBs). The as-released tDNA initiated the target-assisted HCR in the presence of two alternating hairpin sequences (Bio-H1 and Bio-H2) to produce nicked long double-stranded DNA on the surface of MBs, where numerous alkaline phosphatase (ALP) enzymes could assemble with MBs through the biotin-avidin reaction, resulting in the hydrolysis of sodium thiophosphate (TP) to H2S. The as-produced H2S reacted with BMO to form vulcanized BMO (BMO-S), thus leading to obvious enhanced PEC performance under visible light with the color change from light yellow to brown. Having optimized the test conditions, the magnetic-assisted biosensing system holds a good quantitative diagnosis sensitivity area in a range of 5.0 pg mL-1-100 ng mL-1 with a calculated detection limit down to 3.5 pg mL-1. Meanwhile, a visual colorimetric assay on basis of the change in the color of the materials was also realized. Given the exceptional performance of the constructed biosensor, it may possess great promise as an advanced bioanalytical tool for practical applications.

The Combination of Mesyl-Phosphoramidate Inter-Nucleotide Linkages and 2'-O-Methyl in Selected Positions in the Antisense Oligonucleotide Enhances the Performance of RNaseH1 Active PS-ASOs.

Antisense oligonucleotides (ASOs) that mediate RNA target degradation by RNase H1 are used as drugs to treat various diseases. Previously we found that introduction of a single 2′-O-methyl (2′-OMe) modification in position 2 of the central deoxynucleotide region of a gapmer phosphorothioate (PS) ASO, in which several residues at the termini are 2′-methoxyethyl, 2′ constrained ethyl, or locked nucleic acid, dramatically reduced cytotoxicity with only modest effects on potency. More recently, we demonstrated that replacement of the PS linkage at position 2 or 3 in the gap with a mesyl-phosphoramidate (MsPA) linkage also significantly reduced toxicity without meaningful loss of potency and increased the elimination half-life of the ASOs. In this study, we evaluated the effects of the combination of MsPA linkages and 2′-OMe nucleotides on PS ASO performance. We found that two MsPA modifications at the 5′ end of the gap or in the 3′-wing of a Gap 2′-OMe PS ASO substantially increased the activity of ASOs with OMe at position 2 of the gap without altering the safety profile. Such effects were observed with multiple sequences in cells and animals. Thus, the MsPA modification improves the RNase H1 cleavage rate of PS ASOs with a 2′-OMe in the gap, significantly reduces binding of proteins involved in cytotoxicity, and prolongs elimination half-lives.

Microfluidics-Assisted Fluorescence Mapping of DNA Phosphorothioation.

As the key player of a new restriction modification system, DNA phosphorothioate (PT) modification, which swaps oxygen for sulfur on the DNA backbone, protects the bacterial host from foreign DNA invasion. The identification of PT sites helps us understand its physiological defense mechanisms, but accurately quantifying this dynamic modification remains a challenge. Herein, we report a simple quantitative analysis method for optical mapping of PT sites in the single bacterial genome. DNA molecules are fully stretched and immobilized in a microfluidic chip by capillary flow and electrostatic interactions, improving the labeling efficiency by maximizing exposure of PT sites on DNA while avoiding DNA loss and damage. After screening 116 candidates, we identified a bifunctional chemical compound, iodoacetyl-polyethylene glycol-biotin, that can noninvasively and selectively biotinylate PT sites, enabling further labeling with streptavidin fluorescent nanoprobes. With this method, PT sites in PT+ DNA can be easily detected by fluorescence, while almost no detectable ones were found in PT- DNA, achieving real-time visualization of PT sites on a single DNA molecule. Collectively, this facile genome-wide PT site detection method directly characterizes the distribution and frequency of DNA modification, facilitating a better understanding of its modification mechanism that can be potentially extended to label DNAs in different species.

Improvement of chemo- and stereoselectivity for phosphorothioate oligonucleotides in capillary electrophoresis by addition of cyclodextrins

Phosphorothioate (PS) modification is one of the most widely used oligonucleotide chemical alterations in the oligonucleotide backbone. It has proven to be crucial in the field of therapeutic oligonucleotides regarding the optimization of their physicochemical and biological properties. In this study, a capillary electrophoresis (CE) method with an acidic background electrolyte (BGE) containing a combination of β- and γ-cyclodextrins derivatives as chiral selectors is proposed for the diastereomeric separation of 5-mer oligonucleotides containing 0, 1, 2, or 3 phosphorothioate linkages (5´-TCGTG-3´). The effects of the BGE pH, organic modifier addition, and type of cyclodextrin (CD) on chemo- and stereoselectivity and resolution were studied. A mixture of 25 mM (2-hydroxy-3-N,N,N-trimethylamino)propyl-γ-CD and 10 mM carboxymethyl-β-cyclodextrin in a pH 3 buffer was found to be the most appropriate system for the qualitative evaluation of the short oligonucleotides investigated. These phosphorothioate oligonucleotides were separated with high efficiency in less than 11 min with no capillary treatment. The suggested approach can be the basis for purity testing of this new generation of therapeutics.

A self-assembly amplification strategy for ultra-sensitive detection of microRNA based on phosphorothioated probes

Based on self-assembly amplification, we designed a novel microRNA (miRNA)-detection method with high specificity and sensitivity. Two unique DNA probes named Linker A and Linker B were modified with phosphorothioate (PS) at both ends. In the presence of the target miRNA, these two DNA probes were ligated together by T4 DNA ligase enzyme to form a dumbbell-shaped DNA. Then the dumbbell-shaped structure would be extended with Bst 2.0 DNA polymerase enzyme, triggering the strand displacement activity without using additional primers. These results revealed our method's ultralow detection limit (300 fM), excellent selectivity, simple operation, and capability to discriminate single-base mismatches. It is believed that this proposed approach would have great application potential in clinical diagnosis and other involved fields.

Developing predictive hybridization models for phosphorothioate oligonucleotides using high-resolution melting.

The ability to predict nucleic acid hybridization energies has been greatly enabling for many applications, but predictive models require painstaking experimentation, which may limit expansion to non-natural nucleic acid analogues and chemistries. We have assessed the utility of dye-based, high-resolution melting (HRM) as an alternative to UV-Vis determinations of hyperchromicity in order to more quickly acquire parameters for duplex stability prediction. The HRM-derived model for phosphodiester (PO) DNA can make comparable predictions to previously established models. Using HRM, it proved possible to develop predictive models for DNA duplexes containing phosphorothioate (PS) linkages, and we found that hybridization stability could be predicted as a function of sequence and backbone composition for a variety of duplexes, including PS:PS, PS:PO, and partially modified backbones. Individual phosphorothioate modifications destabilize helices by around 0.12 kcal/mol on average. Finally, we applied these models to the design of a catalytic hairpin assembly circuit, an enzyme-free amplification method used for nucleic acid-based molecular detection. Changes in PS circuit behavior were consistent with model predictions, further supporting the addition of HRM modeling and parameters for PS oligonucleotides to the rational design of nucleic acid hybridization.

The minor groove topology of nucleic acid ligands plays a key role in nuclease resistance and targeting of HMGB1

The architectural DNA‐binding protein High Mobility Group B1 (HMGB1) binds distorted double stranded DNA (e.g. kinked, cruciform) in the nucleus with extremely high affinity to modulate nuclear homeostasis. HMGB1 is a multifunctional protein that also plays a pivotal role outside of the nucleus. It is now clear that HMGB1 can also function as a Damage Molecular Pattern Molecule. In this capacity, HMGB1 can act as a friend or foe to: i)bolster innate immunity to ward off pathogens or ii)exacerbate unwanted immune responses that worsen disease states (e.g. lupus, rheumatoid arthritis). Research shows that cruciform DNA [four‐way junctions (4WJs)] and single strand DNA (ssDNA) can effectively bind/sequester HMGB1. The 4WJs and ssDNA investigated contain natural and phosphorothioate (PS) bonds. PS bonds are introduced to enhance nuclease resistance. Our prior study shows that intramolecular 4WJs composed of natural and PS bonds possess enhanced resistance against the endo‐ and exonucleases. We hypothesize that the increase in nuclease resistance is due to a combination of three factors: i)restricting access to the helical termini (end‐capping), ii)the presence of PS bonds and iii)altered minor groove topology. The objective of this study is to more stringently investigate the potential connection between minor groove topology and nuclease binding. Here, three nucleases are used that cleave double and ssDNA: DNase I, Exonuclease III (Exo III) and bacteriophage T5 Exonuclease (T5 Exo). The nucleases bind within the minor groove to initiate hydrolysis via a non‐specific (DNase I) or specific manner (Exo III and T5 Exo). We hypothesize that nucleases do not bind strongly to nucleic acid substrates with altered (likely wider) minor groove dimensions. As a result, nucleic acids with wider minor grooves tend to have enhanced nuclease resistance. Circular dichroism (CD) studies are used to investigate the minor groove topology of intramolecular 4WJs and ssDNA. The minor groove binder 4’,6‐diamidino‐2‐phenylindole (DAPI) is used to estimate the gross minor groove topology of each nucleic acid ligand. The data clearly shows the DNA substrates with altered minor groove dimensions, as reflected by weakened DAPI binding interactions, possess enhanced nuclease resistance. DNA substrates with enhanced nuclease resistance will be investigated further to target the DNA‐binding cytokine, HMGB1.

Nanopore Sequencing for Detection and Characterization of Phosphorothioate Modifications in Native DNA Sequences.

Bacterial DNA is subject to various modifications involved in gene regulation and defense against bacteriophage attacks. Phosphorothioate (PT) modifications are protective modifications in which the non-bridging oxygen in the DNA phosphate backbone is replaced with a sulfur atom. Here, we expand third-generation sequencing techniques to allow for the sequence-specific mapping of DNA modifications by demonstrating the application of Oxford Nanopore Technologies (ONT) and the ELIGOS software package for site-specific detection and characterization of PT modifications. The ONT/ELIGOS platform accurately detected PT modifications in a plasmid carrying synthetic PT modifications. Subsequently, studies were extended to the genome-wide mapping of PT modifications in the Salmonella enterica genomes within the wild-type strain and strains lacking the PT regulatory gene dndB (ΔdndB) or the PT synthetic gene dndC (ΔdndC). PT site-specific signatures were observed in the established motifs of GAAC/GTTC. The PT site locations were in close agreement with PT sites previously identified using the Nick-seq technique. Compared to the wild-type strain, the number of PT modifications are 1.8-fold higher in ΔdndB and 25-fold lower in ΔdndC, again consistent with known regulation of the dnd operon. These results demonstrate the suitability of the ONT platform for accurate detection and identification of the unusual PT backbone modifications in native genome sequences.

Involvement of the DNA Phosphorothioation System in TorR Binding and Anaerobic TMAO Respiration in Salmonella enterica.

ABSTRACTAlthough the phosphorothioate (PT) modification, in which the nonbridging oxygen in the DNA sugar-phosphate backbone is replaced by sulfur, has been reported to play versatile roles in multiple cellular processes, very little data have been obtained to define the role of PT in epigenetic regulation. In this study, we report that the PT system in Salmonella enterica serovar Cerro 87 is involved in the transcriptional regulation of the torCAD operon encoding the trimethylamine N-oxide (TMAO) respiration machinery that enables the use of TMAO as a terminal electron acceptor for respiration when oxygen is not available. In vitro, PT enhanced the binding of the transcriptional activator of the torCAD operon, namely, TorR, to its DNA substrate (tor boxes). However, in vivo, the PT modification protein complex DndCDE downregulated torCAD transcription through competing with the binding of TorR to the tor boxes. The altered expression of torCAD caused by PT modification proteins affected cell growth that relied on TMAO respiration. To our knowledge, this is the first report supporting that PT proteins participate in transcriptional regulation, showing a new function of PT systems.

A functional DNA nanosensor for highly sensitive and selective imaging of ClO− in atherosclerotic plaques

Atherosclerosis (AS) is the primary cause of cardiovascular disorders, which lead to one-third of all death globally. However, the atherosclerosis process remains elusive due to the lack of appropriate analytical methods. Herein, we present the first nanoflare-based DNA sensor that could report the distribution of ClO− (a typical marker of AS) in the atherosclerotic plaques. ClO−-responsive phosphorothioate (PS) was inserted into DNA, which was then assembled with a gold nanoparticle (AuNP) core to form a ClO−-specific nanoflare probe. The hydrolysis between PS and ClO− triggered fluorescence turn-on of the probes, showing excellent sensitivity (as low as 8.51 nM) and specificity for ClO− detection. In addition, the nanoflare probes exhibit superior performance in tracing the variations of endogenous and exogenous ClO− in living RAW264.7 cells. After intravenous injection, the efficient accumulation of probes and fluorescence signal enhancement were observed in the atherosclerotic plaques of AS model mice. The nanoflare probes possess significant feasibility in the precise identification of atherosclerotic plaques, which holds tremendous implications for clinical diagnosis, mechanism study, and assessment of therapeutic outcomes associated with AS.

Molecular Structure of Single-Stranded DNA on the ZnS Surface of Quantum Dots.

DNA-based nanoparticle assemblies have emerged as leading candidates in the development of bioimaging materials, photonic devices, and computing materials. Here, we combine atomistic simulations and experiments to characterize the wrapping mechanism of chimeric single-stranded DNA (ssDNA) on CdSe-ZnS (core-shell) quantum dots (QDs) at different ratios of the phosphorothioate (PS) modification of the bases. We use an implicit solvent, all-atom ssDNA model to match the experimentally calculated ssDNA conformation at low salt concentrations. Through simulation, we find that 3-mercaptopropionic acid (MPA) induces electrostatic repulsion and O-(2-mercaptoethyl)-Ó-methyl-hexa (ethylene glycol) (mPEG) induces steric exclusion, and both reduce the binding affinity of ssDNA. In both simulation and experiment, we find that ssDNA is closer to the QD surface when the QD size is larger. The effect of the PS-base ratio on the conformation of ssDNA is also elaborated in this work. We found through MD simulations, and confirmed by transmission electron microscopy, that the maximum valence numbers are 1, 2, and 3 on QDs of 6, 9, and 14 nm in diameter, respectively. We conclude that the maximum ssDNA valence number is linearly related to the QD size, n ∝ R, and justify this finding through an electrostatic repulsion mechanism.

Tandem mass spectrometric sequence characterization of synthetic thymidine-rich oligonucleotides.

Tandem mass spectrometry (MS/MS) can provide direct and accurate sequence characterization of synthetic oligonucleotide drugs, including modified oligonucleotides. Multiple factors can affect oligonucleotide MS/MS sequencing, including the intrinsic properties of oligonucleotides (i.e., nucleotide composition and structural modifications) and instrument parameters associated with the ion activation for fragmentation. In this study, MS/MS sequencing of a thymidine (T)‐rich and phosphorothioate (PS)‐modified DNA oligonucleotide was investigated using two fragmentation techniques: trap‐type collision‐induced dissociation (“CID”) and beam‐type CID also termed as higher‐energy collisional dissociation (“HCD”), preceded by a hydrophilic interaction liquid chromatography (HILIC) separation. A low to moderate charge state (−4), which predominated under the optimized HILIC‐MS conditions, was selected as the precursor ion for MS/MS analysis. Comparison of the two distinctive ion activation mechanisms on the same precursor demonstrated that HCD was superior to CID in promoting higher sequence coverage and analytical sensitivity in sequence elucidation of T‐rich DNA oligonucleotides. Specifically, HCD provided more sequence‐defining fragments with higher fragment intensities than CID. Furthermore, the direct comparison between unmodified and PS‐modified DNA oligonucleotides demonstrated a loss of MS/MS fragmentation efficiency by PS modification in both CID and HCD approaches, and a resultant reduction in sequence coverage. The deficiency in PS DNA sequence coverage observed with single collision energy HCD, however, was partially recovered by applying HCD with multiple collision energies. Collectively, this work demonstrated that HCD is advantageous to MS/MS sequencing of T‐rich PS‐modified DNA oligonucleotides.