Phosphorothioate Research Articles

Endosomal Escape of ASOs Internalized by Stabilin Receptors Is Regulated by Rab5C and EEA1.

One of the major drug classes that has been gaining traction in the pharmacological world has been the emergence of Antisense oligonucleotides (ASOs). Increased modifications of these drugs have led to major breakthroughs leading to a boost in this drug class being adapted to clinical trials. Second generation (Gen 2) Antisense oligonucleotides show increased nuclease stability and affinity for their RNA targets which has translated to improved potency and therapeutic index in the clinic. Gen 2 ASOs are typically modified using the phosphorothioate (PS) backbone modification which enhances ASO interactions with plasma, cell surface and intracellular proteins. This facilitates ASO distribution to peripheral tissues and also promotes cellular uptake after injection into animals. Recent work of ours has shown that a group of class H Stabilin receptors specifically internalize PS‐ASOs in the sinusoidal cells of the liver and the spleen. This work identifies a breakthrough candidate for the determination of a molecular pathway for these drugs to reach their desired target in the cell. Further exploration of the endocytotic uptake by these proteins has led to a rudimentary understanding of this pathway. By modulating expression of specific proteins involved in the trafficking and maturation of the endo‐lysosomal compartments, we show that the proteins Rab5C and EEA1 play a direct role in the early endosomal pathway. These proteins, along with Rab7A and LBPA (lyso‐lipid) in the late endosomal pathway, are important for trafficking of PS‐ASOs and facilitate their escape from endo‐lysosomal compartments after Stabilin‐mediated internalization. In conclusion, this research identifies key interacting partners involved in ASO‐containing endosomal maturation, as well as late endosomal partners involved in PS‐ASO endosomal release. This work also identifies the limiting proteins in the pathway for PS‐ASO translocation and escape from the endosome.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structural Determinants for the Interactions of Chemically Modified Nucleic Acids with the Stabilin-2 Clearance Receptor.

The Stabilin receptors are systemic clearance receptors for some classes of chemically modified nucleic acid therapeutics. In this study, the recombinant human secreted ecto-domain of the small isoform of Stabilin-2 (s190) was purified from cell culture and evaluated for direct binding with a multitude of antisense oligonucleotides (ASOs) using a fluorescence polarization-based assay. The tested ASOs varied in their backbone composition, modification of the ribose 2′ position, overall length of the oligo, and sequence of the nucleotide bases. A fully phosphorothioate (PS) ASO with a 5–10–5 pattern of flanking 2′-O-methoxyethyl modifications was then used to test the effects of pH and salt concentration on receptor binding. These tests concluded that the PS backbone was the primary determinant for ASO binding and that decreasing pH and increasing salt generally increased the rate of ligand dissociation and fit within the biological parameters expected of a constitutive recycling receptor. These results will be useful in the rational design of therapeutic oligonucleotides for enhancing their affinity or avoidance of the Stabilin receptors.

Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides.

Chemically modified antisense oligonucleotides (ASOs) with phosphorothioate (PS) linkages have been extensively studied as research and therapeutic agents. PS-ASOs can enter the cell and trigger cleavage of complementary RNA by RNase H1 even in the absence of transfection reagent. A number of cell surface proteins have been identified that bind PS-ASOs and mediate their cellular uptake; however, the mechanisms that lead to productive internalization of PS-ASOs are not well understood. Here, we characterized the interaction between PS-ASOs and epidermal growth factor receptor (EGFR). We found that PS-ASOs trafficked together with EGF and EGFR into clathrin-coated pit structures. Their co-localization was also observed at early endosomes and inside enlarged late endosomes. Reduction of EGFR decreased PS-ASO activity without affecting EGF-mediated signaling pathways and overexpression of EGFR increased PS-ASO activity in cells. Furthermore, reduction of EGFR delays PS-ASO trafficking from early to late endosomes. Thus, EGFR binds to PS-ASOs at the cell surface and mediates essential steps for active (productive) cellular uptake of PS-ASOs through its cargo-dependent trafficking processes which migrate PS-ASOs from early to late endosomes. This EGFR-mediated process can also serve as an additional model to better understand the mechanism of intracellular uptake and endosomal release of PS-ASOs.

Electrochemical Biosensor Using DNA Embedded Phosphorothioate Modified RNA for Mercury Ion Determination.

Mercury (Hg) and its compounds, originating from a variety of natural and anthropogenic sources, are ubiquitous in the natural environment, and cause severe environmental contamination and pose irreversible harm to human health. A fast and accurate sensing approach is of significant importance for mercury detection. Here, a label-free biosensor using Hg(II)-induced cleavage of phosphorothioate (PS) modified RNA was exploited. We designed a specific single-stranded DNA embedded with four PS-modified RNA (Hg-DPR) to improve the cleavage reaction yield, and then Hg-DPR was covalently linked with single-walled carbon nanotube field effect transistor (SWNTs/FET) via a peptide bond. The Hg-DPR can be efficiently cleaved after exposure to Hg(II), which further causes the conductivity of the SWNTs to change. Using the relative resistance change, the Hg-DPR/SWNTs/FET successfully detected Hg(II) levels as low as 10 pM, and the calibration curves were linear in the range of 50 pM to 100 nM and 100 nM to 10 μM. Additionally, Hg-DPR/SWNTs/FET exhibited excellent sensitivity, portability, and low-cost for Hg(II) detection.

Receptor-Mediated Uptake of Phosphorothioate Antisense Oligonucleotides in Different Cell Types of the Liver.

Oligonucleotide therapeutics have emerged as a third distinct platform for drug discovery within the pharmaceutical industry. Five oligonucleotide-based drugs have been approved by the US FDA and over 100 oligonucleotides drugs are currently at different stages of human trials. Several of these oligonucleotide drugs are modified using the phosphorothioate (PS) backbone modification where one of the nonbridging oxygen atoms of the phosphodiester linkage is replaced with sulfur. In this review, we summarize our knowledge on receptor-mediated uptake of PS antisense oligonucleotides (ASOs) within different cell types of the liver-a privileged organ for the discovery of oligonucleotide-based therapeutics.

Acute hepatotoxicity of 2' fluoro-modified 5-10-5 gapmer phosphorothioate oligonucleotides in mice correlates with intracellular protein binding and the loss of DBHS proteins.

We reported previously that a 2′ fluoro-modified (2′ F) phosphorothioate (PS) antisense oligonucleotides (ASOs) with 5–10–5 gapmer configuration interacted with proteins from Drosophila behavior/human splicing (DBHS) family with higher affinity than PS-ASOs modified with 2′-O-(2-methoxyethyl) (2′ MOE) or 2′,4′-constrained 2′-O-ethyl (cEt) did. Rapid degradation of these proteins and cytotoxicity were observed in cells treated with 2′ F PS-ASO. Here, we report that 2′ F gapmer PS-ASOs of different sequences caused reduction in levels of DBHS proteins and hepatotoxicity in mice. 2′ F PS-ASOs induced activation of the P53 pathway and downregulation of metabolic pathways. Altered levels of RNA and protein markers for hepatotoxicity, liver necrosis, and apoptosis were observed as early as 24 to 48 hours after a single administration of the 2′ F PS-ASO. The observed effects were not likely due to the hybridization-dependent RNase H1 cleavage of on- or potential off-target RNAs, or due to potential toxicity of 2′ F nucleoside metabolites. Instead, we found that 2′ F PS-ASO associated with more intra-cellular proteins including proteins from DBHS family. Our results suggest that protein-binding correlates positively with the 2′ F modification-dependent loss of DBHS proteins and the toxicity of gapmer 2′ F PS-ASO in vivo.

Optimizing Molecular Beacons for Intracellular Analysis of RNA.

Conventional molecular beacons (MBs) have been used extensively for imaging specific endogenous RNAs in living cells, but their tendency to generate false-positive signals as a result of nuclease degradation and/or nonspecific binding limits sensitive and accurate imaging of intracellular RNAs. In an attempt to overcome this limitation, MBs have been synthesized with various chemically modified oligonucleotide backbones to confer greater biostability. We have recently developed a new MB architecture composed of 2'-O-methyl RNA (2Me), a fully phosphorothioate (PS) modified loop domain and a phosphodiester stem (2Me/PSLOOP MB). We showed that this new MB exhibits a marginal level of false-positive signals and enables accurate single-molecule imaging of target RNA in living cells. In this chapter, we describe detailed methods that led us to conclude that, among various PS-modified configurations, the 2Me/PSLOOP MB is an optimal design for intracellular RNA analysis.

Double-stranded phosphodiester cytosine-guanine oligodeoxynucleotide complexed with calcium phosphate as a potent vaccine adjuvant for activating cellular and Th1-type humoral immunities.

Conventional class B cytosine-guanine (CpG) (CpG-B) oligodeoxynucleotide (ODNs) consisting of a single-stranded (ss) phosphorothioate (PT) backbone (ss CpG-B-PT) is converted from a proinflammatory cytokine inducer to a type-I interferon (IFN) inducer when complexed with cationic materials. In this study, we designed ss CpG-B and double-stranded (ds) CpG-B ODNs with a phosphodiester (PD) backbone (ss CpG-B-PD and ds CpG-B-PD, respectively) that became type-I IFN inducers upon complexation with Lipofectamine 2000 (Lipo), a cationic liposome. The ds CpG-B-PD complex induced higher IFN-β expression in mouse macrophage-like RAW264 cells than ss CpG-B-PD and ss CpG-B-PT complexes. The fold induction of IFN-β increased with the number of CpG motifs in ds CpG-B-PD, and a complex of ds CpG-B-PD consisting of 72 base pairs with nine CpG motifs (ds CpG-B72-PD) and Lipo showed the highest capacity to induce IFN-β. The materials and method used for complexation influenced the degree of IFN-β induction: ds CpG-B72-PD entrapped by calcium phosphate (CaP) (ds CpG-B72-PD/CaP) showed a higher induction capacity than ds CpG-B72-PD adsorbed onto the CaP surface. Entrapment of ds CpG-B72-PD by CaP also enhanced the induction of the proinflammatory cytokine interleukin-12. Vaccinating mice with ds CpG-B72-PD/CaP in conjunction with ovalbumin (OVA) increased the ratios of OVA-specific CD8+ T cells to total CD8+ T cells in peripheral blood and of OVA-specific IgG2a associated with helper T (Th)1 cells to OVA-specific IgG1 associated with Th2 cells. These results indicate that ds CpG-B72-PD/CaP is an effective vaccine adjuvant that can activate both cellular and Th1-type humoral immune responses.

Chemical Changes in Layered Ferroelectric Semiconductors Induced by Helium Ion Beam

Multi-material systems interfaced with 2D materials, or entirely new 3D heterostructures can lead to the next generation multi-functional device architectures. Physical and chemical control at the nanoscale is also necessary tailor these materials as functional structures approach physical limit. 2D transition metal thiophosphates (TPS), with a general formulae Cu1−xIn1+x/3P2S6, have shown ferroelectric polarization behavior with a Tc above the room temperature, making them attractive candidates for designing both: chemical and physical properties. Our previous studies have demonstrated that ferroic order persists on the surface, and that spinoidal decomposition of ferroelectric and paraelectric phases occurs in non-stoichiometric Cu/In ratio formulations. Here, we discuss the chemical changes induced by helium ion irradiation. We explore the TPS compound library with varying Cu/In ratio, using Helium Ion Microscopy, Atomic Force Microscopy (AFM), and Time of Flight-Secondary Ion Mass Spectrometry (ToF-SIMS). We correlate physical nano- and micro- structures to the helium ion dose, as well as chemical signatures of copper, oxygen and sulfur. Our ToF-SIMS results show that He ion irradiation leads to oxygen penetration into the irradiated areas, and diffuses along the Cu-rich domains to the extent of the stopping distance of the helium ions.

Adsorption and degradation of methyl parathion (MP), a toxic organophosphorus pesticide, using NaY/Mn0.5Zn0.5Fe2O4 nanocomposite

In this research, the applicability of NaY/Mn0.5Zn0.5Fe2O4 as a new synthesized nanocomposite adsorbent for the adsorption and degradation of methyl parathion (MP, O,O-dimethyl O-p-nitrophenyl phosphorothioate), an organophosphorus pesticide, is investigated. IThe prepared samples were characterized via SEM-EDAX, TEM, XRD, FTIR, VSM and N2-BET techniques. The role of several experimental factors such as contact time, adsorbent dose and initial concentration of methyl parathion on the removal efficiency of this pesticide were considered and evaluated via 31P nuclear magnetic resonance (31PNMR) spectroscopy. 31PNMR spectroscopy showed that methyl parathion was desirably removed by the NaY/Mn0.5Zn0.5Fe2O4 nanocomposite with a yield of more than 90% under certain optimized conditions. Factors including contact time (80 min), adsorbent dose (0.5 g), and initial pesticide concentration (15 mg/L) were studied and optimized for the reaction. Reaction kinetic status was surveyed using a first order model. The values of the half-life (T 1/2) and rate constant (k) were 54.6 min and 0.0127 min−1, respectively. The products of the degradation reaction between methyl parathion and NaY/Mn0.5Zn0.5Fe2O4 were dimethyl phosphorothioic acid (DMPA) and p-nitrophenol (PNP), which are significantly less toxic than the primary pesticide.

Assessing single-stranded oligonucleotide drug-induced effects in vitro reveals key risk factors for thrombocytopenia.

Single-stranded oligonucleotides (ON) comprise a promising therapeutic platform that enables selective modulation of currently undruggable targets. The development of novel ON drug candidates has demonstrated excellent efficacy, but in certain cases also some safety liabilities were reported. Among them are events of thrombocytopenia, which have recently been evident in late stage trials with ON drugs. The underlying mechanisms are poorly understood and the risk for ON candidates causing such events cannot be sufficiently assessed pre-clinically. We investigated potential thrombocytopenia risk factors of ONs and implemented a set of in vitro assays to assess these risks. Our findings support previous observations that phosphorothioate (PS)-ONs can bind to platelet proteins such as platelet collagen receptor glycoprotein VI (GPVI) and activate human platelets in vitro to various extents. We also show that these PS-ONs can bind to platelet factor 4 (PF4). Binding to platelet proteins and subsequent activation correlates with ON length and connected to this, the number of PS in the backbone of the molecule. Moreover, we demonstrate that locked nucleic acid (LNA) ribosyl modifications in the wings of the PS-ONs strongly suppress binding to GPVI and PF4, paralleled by markedly reduced platelet activation. In addition, we provide evidence that PS-ONs do not directly affect hematopoietic cell differentiation in culture but at higher concentrations show a pro-inflammatory potential, which might contribute to platelet activation. Overall, our data confirm that certain molecular attributes of ONs are associated with a higher risk for thrombocytopenia. We propose that applying the in vitro assays discussed here during the lead optimization phase may aid in deprioritizing ONs with a potential to induce thrombocytopenia.

Reconstruction of Toll-like receptor 9-mediated responses in HEK-Blue hTLR9 cells by transfection of human macrophage scavenger receptor 1 gene

We used human Toll-like receptor 9 (hTLR9)-expressing HEK-Blue hTLR9 cells, which release secreted embryonic alkaline phosphatase (SEAP) upon response to CpG DNA, to evaluate the immunological properties of nucleic acid drug candidates. Our preliminary studies showed that phosphodiester CpG DNA hardly induced any SEAP secretion in HEK-Blue hTLR9 cells. In the current study, therefore, we developed HEK-Blue hTLR9 cells transduced with human macrophage scavenger receptor-1 (hMSR1), a cell-surface DNA receptor, and determined whether HEK-Blue hTLR9/hMSR1 cells respond to phosphorothioate (PS) CpG DNA and phosphodiester (PO) CpG DNA. We selected PS CpG2006, a single-stranded PO CpG DNA (ssCpG), and a tetrapod-like structured DNA (tetrapodna) containing ssCpG (tetraCpG) as model TLR9 ligands. Alexa Fluor 488-labeled ligands were used for flow cytometry. Unlike the mock-transfected HEK-Blue hTLR9 cells, the HEK-Blue hTLR9/hMSR1 cells efficiently took up all three CpG DNAs. SEAP release was almost proportional to the uptake. Treatment of HEK-Blue hTLR9/hMSR1 cells with an anti-hMSR1 antibody significantly reduced the uptake of ssCpG and tetraCpG. Collectively, reconstruction of TLR9-mediated responses to CpG DNA in HEK-Blue hTLR9 cells can be used to evaluate the toxicity of nucleic acid drug candidates with diverse physicochemical properties.

Chromatographic approaches for the characterization and quality control of therapeutic oligonucleotide impurities

Phosphorothioate (PS) oligonucleotides are a rapidly rising class of drugs with significant therapeutic applications. However, owing to their complex structure and multistep synthesis and purification processes, generation of low-level impurities and degradation products are common. Therefore, they require significant investment in quality control and impurity identification. This requires the development of advanced methods for analysis, characterization and quantitation. In addition, the presence of the PS linkage leads to the formation of chiral centers which can affect their biological properties and therapeutic efficiency. In this review, the different types of oligonucleotide impurities and degradation products, with an emphasis on their origin, mechanism of formation and methods to reduce, prevent or even eliminate their production, will be extensively discussed. This review will focus mainly on the application of chromatographic techniques to determine these impurities but will also discuss other approaches such as mass spectrometry, capillary electrophoresis and nuclear magnetic resonance spectroscopy. Finally, the chirality and formation of diastereomer mixtures of PS oligonucleotides will be covered as well as approaches used for their characterization and the application for the development of stereochemically-controlled PS oligonucleotides.

Rational Design of Short Locked Nucleic Acid-Modified 2'-O-Methyl Antisense Oligonucleotides for Efficient Exon-Skipping InVitro.

Locked nucleic acid is a prominent nucleic acid analog with unprecedented target binding affinity to cDNA and RNA oligonucleotides and shows remarkable stability against nuclease degradation. Incorporation of locked nucleic acid nucleotides into an antisense oligonucleotide (AO) sequence can reduce the length required without compromising the efficacy. In this study, we synthesized a series of systematically truncated locked nucleic acid-modified 2′-O-methyl AOs on a phosphorothioate (PS) backbone that were designed to induce skipping exon 23 from the dystrophin transcript in H-2Kb-tsA58 mdx mouse myotubes in vitro. The results clearly demonstrated that shorter AOs (16- to 14-mer) containing locked nucleic acid nucleotides efficiently induced dystrophin exon 23 skipping compared with the corresponding 2′-O-methyl AOs. Our remarkable findings contribute significantly to the existing knowledge about the designing of short LNA-modified oligonucleotides for exon-skipping applications, which will help reduce the cost of exon-skipping AOs and potential toxicities, particularly the 2′-OMe-based oligos, by further reducing the length of AOs.

Control of phosphorothioate stereochemistry substantially increases the efficacy of antisense oligonucleotides.

Whereas stereochemical purity in drugs has become the standard for small molecules, stereoisomeric mixtures containing as many as a half million components persist in antisense oligonucleotide (ASO) therapeutics because it has been feasible neither to separate the individual stereoisomers, nor to synthesize stereochemically pure ASOs. Here we report the development of a scalable synthetic process that yields therapeutic ASOs having high stereochemical and chemical purity. Using this method, we synthesized rationally designed stereopure components of mipomersen, a drug comprising 524,288 stereoisomers. We demonstrate that phosphorothioate (PS) stereochemistry substantially affects the pharmacologic properties of ASOs. We report that Sp-configured PS linkages are stabilized relative to Rp, providing stereochemical protection from pharmacologic inactivation of the drug. Further, we elucidated a triplet stereochemical code in the stereopure ASOs, 3'-SpSpRp, that promotes target RNA cleavage by RNase H1 in vitro and provides a more durable response in mice than stereorandom ASOs.

Two Completely Different Mechanisms for Highly Specific Na+ Recognition by DNAzymes

Our view of the interaction between Na+ and nucleic acids was changed by a few recently discovered Na+ -specific RNA-cleaving DNAzymes. In addition to nonspecific electrostatic interactions, highly specific recognition is also possible. Herein, two such DNAzymes, named EtNa and Ce13d, are compared to elucidate their mechanisms of Na+ binding. Mutation studies indicate that they have different sequence requirements. Phosphorothioate (PS) substitution at the scissile phosphate drops the activity of EtNa 140-fold, and it cannot be rescued by thiophilic Cd2+ or Mn2+ , whereas the activity of PS-modified Ce13d can be rescued. Na+ -dependent activity assays indicate that two Na+ ions bind cooperatively in EtNa, and each Na+ likely interacts with a nonbridging oxygen atom in the scissile phosphate, whereas Ce13d binds only one Na+ ion in a well-defined Na+ aptamer, and this Na+ ion does not directly interact with the scissile phosphate. Both DNAzymes display a normal pH-rate profile, with a single deprotonation reaction required for catalysis. For EtNa, Na+ fails to protect the conserved nucleotides from dimethyl sulfate attack, and no specific Na+ binding is detected by 2-aminopurine fluorescence, both of which are different from those observed for Ce13d. This work suggests that EtNa binds Na+ mainly through its scissile phosphate without significant involvement of the nucleotides in the enzyme strand, whereas Ce13d has a well-defined aptamer for Na+ binding. Therefore, DNA has at least two distinct ways to achieve highly selective Na+ binding.

Fatty Acid-Modified Gapmer Antisense Oligonucleotide and Serum Albumin Constructs for Pharmacokinetic Modulation

Delivery technologies are required for realizing the clinical potential of molecular medicines. This work presents an alternative technology to preformulated delivery systems by harnessing the natural transport properties of serum albumin using endogenous binding of gapmer antisense oligonucleotides (ASOs)/albumin constructs. We show by an electrophoretic mobility assay that fatty acid-modified gapmer and human serum albumin (HSA) can self-assemble into constructs that offer favorable pharmacokinetics. The interaction was dependent on fatty acid type (either palmitic or myristic acid), number, and position within the gapmer ASO sequence, as well as phosphorothioate (PS) backbone modifications. Binding correlated with increased blood circulation in mice (t1/2 increased from 23 to 49min for phosphodiester [PO] gapmer ASOs and from 28 to 66min for PS gapmer ASOs with 2× palmitic acid modification). Furthermore, a shift toward a broader biodistribution was detected for PS compared with PO gapmer ASOs. Inclusion of 2× palmitoyl to the ASOsshifted the biodistribution to resemble that of natural albumin. This work, therefore, presents a novel strategy based on the proposed endogenous assembly of gapmer ASOs/albumin constructs for increased circulatory half-life and modulation of the biodistribution of gapmer ASOs that offers tunable pharmacokinetics based on the gapmer modification design.

DNA Backbone Sulfur-Modification Expands Microbial Growth Range under Multiple Stresses by its anti-oxidation function

DNA phosphorothioate (PT) modification is a sulfur modification on the backbone of DNA introduced by the proteins DndA-E. It has been detected within many bacteria isolates and metagenomic datasets, including human pathogens, and is considered to be widely distributed in nature. However, little is known about the physiological function of this modification, and thus its evolutionary significance and application potential remains largely a mystery. In this study, we focused on the advantages of DNA PT modification to bacterial cells coping with environmental stresses. We show that the mesophile Escherichia coli and the extremophile Shewanella piezotolerans both expanded their growth ranges following exposure to extreme temperature, salinity, pH, pressure, UV, X-ray and heavy metals as a result of DNA phophorothioation. The phophorothioated DNA reacted to both H2O2 and hydroxyl radicals in vivo, and protected genomic DNA as well as sensitive enzymes from intracellular oxidative damage. We further demonstrate that this process has evolved separate from its associated role in DNA restriction and modification. These findings provide a physiological role for a covalent modification widespread in nature and suggest possible applications in biotechnology and biomedicine.

Oxidation of phosphorothioate DNA modifications leads to lethal genomic instability.

Genomic modification with sulfur as phosphorothioate (PT) is widespread among prokaryotes, including human pathogens. Apart from its physiological functions, the redox and nucleophilic properties of PT sulfur suggest effects on bacterial fitness in stressful environments. Here we show that PTs are dynamic and labile DNA modifications that cause genomic instability during oxidative stress. Using coupled isotopic labeling-mass spectrometry, we observed sulfur replacement in PTs at a rate of ~2%/h in unstressed Escherichia coli and Salmonella enterica. While PT levels were unaffected by exposure to hydrogen peroxide (H2O2) or hypochlorous acid (HOCl), PT turnover increased to 3.8–10%/h for HOCl and was unchanged for H2O2, consistent with repair of HOCl-induced sulfur damage. PT-dependent HOCl sensitivity extended to cytotoxicity and DNA strand-breaks, which occurred at orders-of-magnitude lower doses of HOCl than H2O2. The genotoxicity of HOCl in PT-containing bacteria suggests reduced fitness in competition with HOCl-producing organisms and during human infections.

Electronic Structures of LNA Phosphorothioate Oligonucleotides.

Important oligonucleotides in anti-sense research have been investigated in silico and experimentally. This involves quantum mechanical (QM) calculations and chromatography experiments on locked nucleic acid (LNA) phosphorothioate (PS) oligonucleotides. iso-potential electrostatic surfaces are essential in this study and have been calculated from the wave functions derived from the QM calculations that provide binding information and other properties of these molecules. The QM calculations give details of the electronic structures in terms of e.g., energy and bonding, which make them distinguish or differentiate between the individual PS diastereoisomers determined by the position of sulfur atoms. Rules are derived from the electronic calculations of these molecules and include the effects of the phosphorothioate chirality and formation of electrostatic potential surfaces. Physical and electrochemical descriptors of the PS oligonucleotides are compared to the experiments in which chiral states on these molecules can be distinguished. The calculations demonstrate that electronic structure, electrostatic potential, and topology are highly sensitive to single PS configuration changes and can give a lead to understanding the activity of the molecules.