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Structurally constrained phosphonate internucleotide linkage impacts oligonucleotide-enzyme interaction, and modulates siRNA activity and allele specificity.

ABSTRACT: Oligonucleotides is an emerging class of chemically-distinct therapeutic modalities, where extensive chemical modifications are fundamental for their clinical applications. Inter-nucleotide backbones are critical to the behaviour of therapeutic oligonucleotides, but clinically explored backbone analogues are, effectively, limited to phosphorothioates. Here, we describe the synthesis and bio-functional characterization of an internucleotide (E)-vinylphosphonate (iE-VP) backbone, where bridging oxygen is substituted with carbon in a locked stereo-conformation. After optimizing synthetic pathways for iE-VP-linked dimer phosphoramidites in different sugar contexts, we systematically evaluated the impact of the iE-VP backbone on oligonucleotide interactions with a variety of cellular proteins. Furthermore, we systematically evaluated the impact of iE-VP on RNA-Induced Silencing Complex (RISC) activity, where backbone stereo-constraining has profound position-specific effects. Using Huntingtin (HTT) gene causative of Huntington's disease as an example, iE-VP at position 6 significantly enhanced the single mismatch discrimination ability of the RISC without negative impact on silencing of targeting wild type htt gene. These findings suggest that the iE-VP backbone can be used to modulate the activity and specificity of RISC. Our study provides (i) a new chemical tool to alter oligonucleotide-enzyme interactions and metabolic stability, (ii) insight into RISC dynamics and (iii) a new strategy for highly selective SNP-discriminating siRNAs.

SUBMITTER: Yamada K

PROVIDER: S-EPMC8643693 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Spatial variation in connectivity is an integral aspect of the brain's architecture. In the absence of this variability, the brain may act as a single homogenous entity without regional specialization. In this study, we investigate the variability in functional links categorized on the basis of the presence of direct structural paths (primary) or indirect paths mediated by one (secondary) or more (tertiary) brain regions ascertained by diffusion tensor imaging. We quantified the variability in functional connectivity using an unbiased estimate of unpredictability (functional connectivity entropy) in a neuropsychiatric disorder where structure-function relationship is considered to be abnormal; 34 patients with schizophrenia and 32 healthy controls underwent DTI and resting state functional MRI scans. Less than one-third (27.4% in patients, 27.85% in controls) of functional links between brain regions were regarded as direct primary links on the basis of DTI tractography, while the rest were secondary or tertiary. The most significant changes in the distribution of functional connectivity in schizophrenia occur in indirect tertiary paths with no direct axonal linkage in both early (P=0.0002, d=1.46) and late (P=1×10(-17), d=4.66) stages of schizophrenia, and are not altered by the severity of symptoms, suggesting that this is an invariant feature of this illness. Unlike those with early stage illness, patients with chronic illness show some additional reduction in the distribution of connectivity among functional links that have direct structural paths (P=0.08, d=0.44). Our findings address a critical gap in the literature linking structure and function in schizophrenia, and demonstrate for the first time that the abnormal state of functional connectivity preferentially affects structurally unconstrained links in schizophrenia. It also raises the question of a continuum of dysconnectivity ranging from less direct (structurally unconstrained) to more direct (structurally constrained) brain pathways underlying the progressive clinical staging and persistence of schizophrenia.

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Structurally constrained phosphonate internucleotide linkage impacts oligonucleotide-enzyme interaction, and modulates siRNA activity and allele specificity.

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