Controlled sulfur-based engineering confers mouldability to phosphorothioate antisense oligonucleotides

Phosphorothioates (PS) have proven their effectiveness in the area of therapeutic oligonucleotides with applications spanning from cancer treatment to neurodegenerative disorders. Initially, PS substitution was introduced for the antisense oligonucleotides (PS ASOs) because it confers an increased nuclease resistance meanwhile ameliorates cellular uptake and in-vivo bioavailability. Thus, PS oligonucleotides have been elevated to a fundamental asset in the realm of gene silencing therapeutic methodologies. But, despite their wide use, little is known on the possibly different structural changes PS-substitutions may provoke in DNA·RNA hybrids. Additionally, scarce information and significant controversy exists on the role of phosphorothioate chirality in modulating PS properties. Here, through comprehensive computational investigations and experimental measurements, we shed light on the impact of PS chirality in DNA-based antisense oligonucleotides; how the different phosphorothioate diastereomers impact DNA topology, stability and flexibility to ultimately disclose pro-Sp S and pro-Rp S roles at the catalytic core of DNA Exonuclease and Human Ribonuclease H; two major obstacles in ASOs-based therapies. Altogether, our results provide full-atom and mechanistic insights on the structural aberrations PS-substitutions provoke and explain the origin of nuclease resistance PS-linkages confer to DNA·RNA hybrids; crucial information to improve current ASOs-based therapies.© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research

Psychometric Validation of the Sensory Experiences Questionnaire

We evaluated the psychometric properties of the Sensory Experiences Questionnaire (Version 1; Baranek, David, Poe, Stone, & Watson 2006), a brief caregiver questionnaire for young children with autismand developmental delays used to identify sensory processing patterns in the context of daily activities

Synthesis and characterisation of ruthenium complexes containing a pendent catechol ring

A series of [Ru(bipy)₂L]⁺ and [Ru(phen)₂L]⁺ complexes where L is 2-[5-(3,4-dimethoxyphenyl)-4H-1,2,4-triazol-3-yl]pyridine (HL1) and 4-(5-pyridin-2-yl-4H-1,2,4-triazol-3-yl)benzene-1,2-diol (HL2) are reported. The compounds obtained have been characterised using X-ray crystallography, NMR, UV/Vis and emission spectroscopies. Partial deuteriation is used to determine the nature of the emitting state and to simplify the NMR spectra. The acid-base properties of the compounds are also investigated. The electronic structures of [Ru(bipy)₂L1]⁺ and Ru(bipy)₂HL1]²⁺ are examined using ZINDO. Electro and spectroelectrochemical studies on [Ru(bipy)₂(L2)]⁺ suggest that proton transfer between the catechol and triazole moieties on L2 takes place upon oxidation of the L2 ligand

Rigid spin-labeled nucleoside Ç: a nonperturbing EPR probe of nucleic acid conformation

Rigid spin-labeled nucleoside Ç, an analog of deoxycytidine that base-pairs with deoxyguanosine, was incorporated into DNA oligomers by chemical synthesis. Thermal denaturation experiments and circular dichroism (CD) measurements showed that Ç has a negligible effect on DNA duplex stability and conformation. Nucleoside Ç was incorporated into several positions within single-stranded DNA oligomers that can adopt two hairpin conformations of similar energy, each of which contains a four-base loop. The relative mobility of nucleotides in the alternating C/G hairpin loops, 5′-d(GCGC) and 5′-d(CGCG), was determined by electron paramagnetic resonance (EPR) spectroscopy. The most mobile nucleotide in the loop is the second one from the 5′-end, followed by the third, first and fourth nucleotides, consistent with previous NMR studies of DNA hairpin loops of different sequences. The EPR hairpin data were also corroborated by fluorescence spectroscopy using oligomers containing reduced Ç (Çf), which is fluorescent. Furthermore, EPR spectra of duplex DNAs that contained Ç at the end of the helix showed features that indicated dipolar coupling between two spins. These data are consistent with end-to-end duplex stacking in solution, which was only observed when G was paired to Ç, but not when Ç was paired with A, C or T

Crystal structure, stability and in vitro RNAi activity of oligoribonucleotides containing the ribo-difluorotoluyl nucleotide: insights into substrate requirements by the human RISC Ago2 enzyme

Short interfering RNA (siRNA) duplexes are currently being evaluated as antisense agents for gene silencing. Chemical modification of siRNAs is widely expected to be required for therapeutic applications in order to improve delivery, biostability and pharmacokinetic properties. Beyond potential improvements in the efficacy of oligoribonucleotides, chemical modification may also provide insight into the mechanism of mRNA downregulation mediated by the RNA–protein effector complexes (RNA-induced silencing complex or RISC). We have studied the in vitro activity in HeLa cells of siRNA duplexes against firefly luciferase with substitutions in the guide strand of U for the apolar ribo-2,4-difluorotoluyl nucleotide (rF) [Xia, J. et al. (2006) ACS Chem. Biol., 1, 176–183] as well as of C for rF. Whereas an internal rF:A pair adjacent to the Ago2 (‘slicer’ enzyme) cleavage site did not affect silencing relative to the native siRNA duplex, the rF:G pair and other mismatches such as A:G or A:A were not tolerated. The crystal structure at atomic resolution determined for an RNA dodecamer duplex with rF opposite G manifests only minor deviations between the geometries of rF:G and the native U:G wobble pair. This is in contrast to the previously found, significant deviations between the geometries of rF:A and U:A pairs. Comparison between the structures of the RNA duplex containing rF:G and a new structure of an RNA with A:G mismatches with the structures of standard Watson–Crick pairs in canonical duplex RNA leads to the conclusion that local widening of the duplex formed by the siRNA guide strand and the targeted region of mRNA is the most likely reason for the intolerance of human Ago2 (hAgo2), the RISC endonuclease, toward internal mismatch pairs involving native or chemically modified RNA. Contrary to the influence of shape, the thermodynamic stabilities of siRNA duplexes with single rF:A, A:A, G:A or C:A (instead of U:A) or rF:G pairs (instead of C:G) show no obvious correlation with their activities. However, incorporation of three rF:A pairs into an siRNA duplex leads to loss of activity. Our structural and stability data also shed light on the role of organic fluorine as a hydrogen bond acceptor. Accordingly, UV melting (TM) data, osmotic stress measurements, X-ray crystallography at atomic resolution and the results of semi-empirical calculations are all consistent with the existence of weak hydrogen bonds between fluorine and the H-N1(G) amino group in rF:G pairs of the investigated RNA dodecamers

Sequence-Dependent Fluorescence of Cyanine Dyes on Microarrays

Cy3 and Cy5 are among the most commonly used oligonucleotide labeling molecules. Studies of nucleic acid structure and dynamics use these dyes, and they are ubiquitous in microarray experiments. They are sensitive to their environment and have higher quantum yield when bound to DNA. The fluorescent intensity of terminal cyanine dyes is also known to be significantly dependent on the base sequence of the oligonucleotide. We have developed a very precise and high-throughput method to evaluate the sequence dependence of oligonucleotide labeling dyes using microarrays and have applied the method to Cy3 and Cy5. We used light-directed in-situ synthesis of terminally-labeled microarrays to determine the fluorescence intensity of each dye on all 1024 possible 5′-labeled 5-mers. Their intensity is sensitive to all five bases. Their fluorescence is higher with 5′ guanines, and adenines in subsequent positions. Cytosine suppresses fluorescence. Intensity falls by half over the range of all 5-mers for Cy3, and two-thirds for Cy5. Labeling with 5′-biotin-streptavidin-Cy3/-Cy5 gives a completely different sequence dependence and greatly reduces fluorescence compared with direct terminal labeling

Phosphodiesterase 4 Inhibition Reduces Innate Immunity and Improves Isoniazid Clearance of Mycobacterium tuberculosis in the Lungs of Infected Mice

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is one of the leading infectious disease causes of morbidity and mortality worldwide. Though current antibiotic regimens can cure the disease, treatment requires at least six months of drug therapy. One reason for the long duration of therapy is that the currently available TB drugs were selected for their ability to kill replicating organisms and are less effective against subpopulations of non-replicating persistent bacilli. Evidence from in vitro models of Mtb growth and mouse infection studies suggests that host immunity may provide some of the environmental cues that drive Mtb towards non-replicating persistence. We hypothesized that selective modulation of the host immune response to modify the environmental pressure on the bacilli may result in better bacterial clearance during TB treatment. For this proof of principal study, we compared bacillary clearance from the lungs of Mtb-infected mice treated with the anti-TB drug isoniazid (INH) in the presence and absence of an immunomodulatory phosphodiesterase 4 inhibitor (PDE4i), CC-3052. The effects of CC-3052 on host global gene expression, induction of cytokines, and T cell activation in the lungs of infected mice were evaluated. We show that CC-3052 modulates the innate immune response without causing generalized immune suppression. Immune modulation combined with INH treatment improved bacillary clearance and resulted in smaller granulomas and less lung pathology, compared to treatment with INH alone. This novel strategy of combining anti-TB drugs with an immune modulating molecule, if applied appropriately to patients, may shorten the duration of TB treatment and improve clinical outcome

Phosphodiesterase-4 Inhibition Alters Gene Expression and Improves Isoniazid – Mediated Clearance of Mycobacterium tuberculosis in Rabbit Lungs

Tuberculosis (TB) treatment is hampered by the long duration of antibiotic therapy required to achieve cure. This indolent response has been partly attributed to the ability of subpopulations of less metabolically active Mycobacterium tuberculosis (Mtb) to withstand killing by current anti-TB drugs. We have used immune modulation with a phosphodiesterase-4 (PDE4) inhibitor, CC-3052, that reduces tumor necrosis factor alpha (TNF-α) production by increasing intracellular cAMP in macrophages, to examine the crosstalk between host and pathogen in rabbits with pulmonary TB during treatment with isoniazid (INH). Based on DNA microarray, changes in host gene expression during CC-3052 treatment of Mtb infected rabbits support a link between PDE4 inhibition and specific down-regulation of the innate immune response. The overall pattern of host gene expression in the lungs of infected rabbits treated with CC-3052, compared to untreated rabbits, was similar to that described in vitro in resting Mtb infected macrophages, suggesting suboptimal macrophage activation. These alterations in host immunity were associated with corresponding down-regulation of a number of Mtb genes that have been associated with a metabolic shift towards dormancy. Moreover, treatment with CC-3052 and INH resulted in reduced expression of those genes associated with the bacterial response to INH. Importantly, CC-3052 treatment of infected rabbits was associated with reduced ability of Mtb to withstand INH killing, shown by improved bacillary clearance, from the lungs of co-treated animals compared to rabbits treated with INH alone. The results of our study suggest that changes in Mtb gene expression, in response to changes in the host immune response, can alter the responsiveness of the bacteria to antimicrobial agents. These findings provide a basis for exploring the potential use of adjunctive immune modulation with PDE4 inhibitors to enhance the efficacy of existing anti-TB treatment

The interaction of bacterial pathogens with platelets.

In recent years, the frequency of serious cardiovascular infections such as endocarditis has increased, particularly in association with nosocomially acquired antibiotic-resistant pathogens. Growing evidence suggests a crucial role for the interaction of bacteria with human platelets in the pathogenesis of cardiovascular infections. Here, we review the nature of the interactions between platelets and bacteria, and the role of these interactions in the pathogenesis of endocarditis and other cardiovascular diseases