Endogenous Ribonucleases: Therapeutic Targeting of the Transcriptome Through Oligonucleotide-Triggered RNA Inactivation

Figures

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  Figure 1. Eukaryotic RNase H1 and its role in target RNA degradation. (a) Domain organization of RNase H1. (b) Schematic representation of RNase H1 domain interactions with the RNA-DNA heteroduplex modified from [45]. (c) Scheme of phosphodiester bond cleavage reaction catalyzed by RNase H enzymes. (d) Degradation of target RNA in the complex with antisense oligonucleotide (ASO) via recruitment of RNase H1. The time required for phosphorothioate-ASO distribution, pre-hybridization, and enzyme recruitment stages after ASO transfection into cells indicated in parentheses are according to [46].

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  Figure 2. Chemical modifications compatible with RNase H1 activity and structure of gapmer-type antisense oligonucleotides. (a) Examples of chemical modifications that, as part of ASOs, promoting heteroduplex formation with target RNA recognizable by RNase H1. Modifications of the phosphodiester backbone are highlighted in red, sugar moiety modifications in green, and modifications of nucleobases and sugar in blue. (b) Structure of gapmer ASO, consisting of a central region recognized by RNase H1, and flanking regions containing modified oligonucleotides (orange frame) enhancing target-binding efficiency and/or nuclease resistance. The structure of FDA-approved gapmer ASOs, such as mipomersen, inotersen, volanesorsen and tofersen, is characterized by a fully PS-modified backbone with five 2′-O-MOE modifications flanking each terminus. Cytosines within these molecules are typically methylated at the 5′-position, enhancing their stability and affinity for target RNA.

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  Figure 3. Structural and functional characteristics of RNase P-mediated cleavage of target RNA using external guide sequences (EGS). (a) Structural organization of human RNase P, consisting of catalytic RNA component (H1 RNA) (green line) and ten auxiliary protein subunits (green circles). (b) Structure of precursor tRNA (pre-tRNA), a natural substrate of RNase P. (c) Complex formed by EGS and target mRNA, structurally mimicking pre-tRNA and specifically recognized and cleaved by RNase P.

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  Figure 4. Domain organization and functional structure of human AGO family proteins. (a) Schematic representation of AGO/PIWI protein domain organization with description of their main functions. (b) Model of AGO protein interaction with the small RNA and target RNA heteroduplex. (c) Functional regions of the small RNA guide strand upon itsincorporation into AGO protein.

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  Figure 5. AGO-mediated inhibition of target RNAs using synthetic single-stranded oligonucleotides. (a) Scheme of target RNA suppression by single-stranded small interfering RNA (ss-siRNA) involving AGO2 protein. (b) Generalized optimized structures of modified ss-siRNAs used for gene suppression as reported in [161,162,164,165,166,167,168] (1), [169] (2), [170] (3). (c) Chemical structures of modifications used in the design of the single-stranded oligonucleotides. (d) Scheme of target RNA suppression by single-stranded microRNA (ss-miRNA) mimic involving AGO2 protein. (e) Chemically modified structures of ss-miRNA mimics used for gene suppression in [163] (1) and [171] (2). (f) Scheme of target RNA suppression by transfer RNA-derived small RNA (tsRNA) involving AGO protein. RISC—RNA-induced silencing complex; 5′-(E)-VP—5′-(E)-vinylphosphonate; 2′-F—2′-fluororibose; 2′-OMe—2′-O-methyl; PS—phosphorothioate; 2′-O-MOE—2′-O-methoxyethyl; LNA—locked nucleic acid; 3dT—3′-deoxythymidine; (C16)−lipophilic linear carbon chain; UNA—unlocked nucleic acid; ribo—ribonucleotide; deoxy—deoxyribonucleotide.

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  Figure 6. Biogenesis of transfer RNA-derived small RNAs (tsRNA). Schematic representation of tsRNA generation from mature tRNA. This process produces distinct tsRNA classes, including transfer RNA-derived fragments (5′-tRFs, 3′-tRFs), tRNA-derived stress-induced RNAs (5′-tiRNA, 3′-tiRNA) and internal fragments (i-tRF and tRF-2).

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  Figure 7. piRNA biogenesis and compartment-specific functions of PIWI family proteins in mammals. (a). piRNA biogenesis and processing pathway. piRNA production occurs through two main pathways: (1) primary processing begins with transcription of piRNA clusters followed by PLD6 endonuclease cleavage with MOV10L1 helicase assistance, generating 5′-monophosphorylated fragments that are loaded onto PIWI proteins. These fragments are then 3′-trimmed by PNLDC1 exonuclease and 2′-O-methylated by HENMT1, yielding mature primary piRNAs that mediate both transcriptional and post-transcriptional gene silencing; (2) secondary biogenesis occurs via the ping-pong amplification cycle, primarily active in germline cells and absent or reduced in most somatic tissues. In this pathway, PIWI proteins (PIWIL2 and PIWIL4) guided by piRNAs cleave target transcripts, generating new 5′-monophosphorylated piRNA precursors that are processed and loaded onto another PIWI protein for reciprocal targeting, establishing a self-amplifying piRNA production system and creating mature secondary piRNAs for functional gene regulation [195,196]. (b). Subcellular functional specialization of PIWI proteins. Compartmentalization…

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  Figure 8. Clinical translation progress of ribonuclease-recruiting oligonucleotide therapies. The chronological scheme shows years of initial application and current development stages illustrated by coloring from preclinical research (light) to clinical application (dark). ASO—antisense oligonucleotides; EGS—external guide sequences; siRNA—small interfering RNA; ds and ss—double-stranded and single-stranded; tsRNA—tRNA-derived small RNA; piRNA—PIWI-interacting RNA.