Koralewska N et al. (2024), Short 2'-O-methyl/LNA oligomers as highly-selec...

XB-ART-60715

Nucleic Acids Res 2024 Apr 27; doi: 10.1093/nar/gkae284.

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Short 2'-O-methyl/LNA oligomers as highly-selective inhibitors of miRNA production in vitro and in vivo.

Koralewska N , Corradi E , Milewski MC , Masante L , Szczepanska A , Kierzek R , Figlerowicz M , Baudet ML , Kurzynska-Kokorniak A .

Abstract
MicroRNAs (miRNAs) that share identical or near-identical sequences constitute miRNA families and are predicted to act redundantly. Yet recent evidence suggests that members of the same miRNA family with high sequence similarity might have different roles and that this functional divergence might be rooted in their precursors' sequence. Current knock-down strategies such as antisense oligonucleotides (ASOs) or miRNA sponges cannot distinguish between identical or near identical miRNAs originating from different precursors to allow exploring unique functions of these miRNAs. We here develop a novel strategy based on short 2'-OMe/LNA-modified oligonucleotides to selectively target specific precursor molecules and ablate the production of individual members of miRNA families in vitro and in vivo. Leveraging the highly conserved Xenopus miR-181a family as proof-of-concept, we demonstrate that 2'-OMe/LNA-ASOs targeting the apical region of pre-miRNAs achieve precursor-selective inhibition of mature miRNA-5p production. Furthermore, we extend the applicability of our approach to the human miR-16 family, illustrating its universality in targeting precursors generating identical miRNAs. Overall, our strategy enables efficient manipulation of miRNA expression, offering a powerful tool to dissect the functions of identical or highly similar miRNAs derived from different precursors within miRNA families.

PubMed ID: 38676942
PMC ID: PMC11162791
Article link: Nucleic Acids Res
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	Graphical Abstract.
	Figure 1. Designing ASOs targeting the apical region of pre-miRNA. (A) Schematic representation of the inhibitors design workflow. (B, C) Predicted secondary structures of pre-miR-181a-1 (left) and pre-miR-181a-2 (right). Scissors indicate Dicer cleavage sites. (B) Mature miRNA sequences are coloured and grey boxes indicate the target sequence used in the ASOs design process. (C) Grey circles mark regions targeted by the selected oligomers (AL1-4), asterisks indicate the position of LNA within the AL-ASO sequence. (D) Schematic representation of the control oligomers design workflow. (E) Native gel analysis of the interaction between 5′-32P-labeled pre-miRNA and tested oligomers (Ctrl, AL1-4). Abbreviations: AL, apical loop; Ctrl, control oligomer; C–, pre-miRNA with no other oligomer added.
	Figure 2. Characterization of the pre-miRNA cleavage pattern generated in cytosolic extracts. (A) Schematic of the experimental paradigm for cytosolic extracts from stage 44 X. laevis embryos. (B) Schematic representation of domain composition of human and X. laevis Dicer proteins. A cross-mark indicates amino-acid substitutions in the catalytic core of X. laevis Dicer S. (C) Representative gels showing 5′-32P-labeled pre-miRNA cleavage patterns upon incubation with Xenopus cytosolic extracts or human Dicer with or without EDTA. The ladder was created based on pre-miR-181a-1 and pre-miR-181a-2 cleavage patterns generated by RNase T1. Red and blue arrowheads indicate bands accepted as representing miR-181a-5p isoforms generated in X. laevis extracts and by human Dicer, respectively. Gray arrowheads point to the bands marked as non-Dicer products. Reproducible results were obtained using different extract batches. (D) Schematic representation of the cleavage pattern of pre-miR-181a-1 and pre-miR-181a-2 established based on the PAGE results. Arrowheads as in (C); orange stars indicate 5′-32P-labeling. (E) Results of Northern blot analysis of the cleavage pattern of pre-miR-181a-1 and pre-miR-181a-2 in X. laevis extracts using probes targeting the 5′ or 3′ arm of the precursor. Pre-miRNA regions targeted by the probes are indicated and schematic representation of the pre-miRNA, AL-ASO and the cleavage products is given on left to the blots. Abbreviations: Xla, stage 44 X. laevis cytosolic extracts; hDicer, human Dicer; no prot, no protein control; DUF283, domain of unknown function 283; PAZ, Piwi/Argonaute/Zwille domain; dsRBD, dsRNA binding domain. (F) Schematic representation of the tertiary structure of Dicer in a complex with pre-miRNA. Prepared based on cryo-EM structure obtained for human Dicer by Liu et al. (93). Scissors indicate the sites of pre-miRNA cleavage by RNase III domains. Figure adapted from (94). Color code as in (B). Abbreviations: DUF283, domain of unknown function 283; PAZ, Piwi/Argonaute/Zwille domain; dsRBD, dsRNA binding domain.
	Figure 3. In vitro validation of 2′-OMe/LNA ASOs targeting miR-181a-5p precursors. (A, B) Results of the inhibition assay assessing the effect of tested oligomers (AL1-4, Ctrl) on the cleavage of 5′-32P-labeled pre-miR-181a-1 (A) or 5′-32P-labeled pre-miR-181a-2 (B) in Xenopus cytosolic extracts. Triangles indicate increasing concentration of a given oligomer (0.1, 1, 10 μM). Graphs show miRNA production efficiency normalized to the level of miRNA generated in C+for pre-miR-181a-1 (A) and pre-miR-181a-2 (B). Data information: Values are mean ± SD. Statistics: n = 5 independent experiments, each data point represents a single assay, unpaired t-test. Abbreviations: ns, not significant; C–, pre-miRNA incubated with no extract nor oligomer added; C+, pre-miRNA incubated with extract but no oligomer added; T1, RNase T1 ladder.
	Figure 4. In vivo validation of 2′-OMe/LNA ASOs targeting miR-181a-5p precursors using microinjection. (A) Schematic of the experimental paradigm. Quantity used: 0.5 ng pCS2-eGFP and 1 ng AL1 and AL3 (anti-181 mix) or 1 ng of the control oligomer (Ctrl). (B) Survival embryos rate after microinjection of oligomers (AL1 + 3, Ctrl) or PBS in comparison to non-injected embryos (WT). (C) miRNAs expression levels quantified using the 2−ΔCt method and U6 as normalizer. Data are normalized to PBS control. Data information: Values are mean ± SEM. Statistics: n = 5 independent experiments, each data point represents a single RT–qPCR, unpaired t-test. Abbreviations: ns, not significant.
	Figure 5. In vivo validation of 2′-OMe/LNA ASOs targeting miR-181a-5p precursors using electroporation. (A, E) Schematic of the experimental paradigm. Concentrations used: 125 μM anti-181 mix (AL1 + AL3) or 125 μM control oligomer (Ctrl), 0.5 μg/μl pCS2-GFP. (B) Snapshot of representative electroporated embryos stage 40 in all the experimental conditions (WT, AL1 + 3 and Ctrl). Bottom left, schematic of the ALs inhibitors used for in vivo experiments. (C, D) Eye area (C) and diameter (D) of stage 40 wild type, AL1 + 3 or Ctrl electroporated embryos. (F) miR-181a-5p expression levels quantified using the 2−ΔCt method and U6 as normalizer. Data are normalized to PBS control. Each data point represents a single RT-qPCR. Data information: Values are mean ± SEM (C, D, F). Statistics: n = 3 independent experiments, one-way ANOVA, Tukey's Multiple Comparison Test (C, D); n = 4 independent experiments, unpaired t-test (F). Scale bars: 500 μm (B left, embryo), 200 μm (B right, eye). Abbreviations: ns, not significant.
	Figure 6. In vivo validation of 2′-OMe/LNA ASOs targeting miR-181a-5p precursors within axons. (A) Schematics of the experimental paradigm. Concentrations used: 2 μM AL1 + AL3 or 2 μM Ctrl, 200 ng/ml (Sema3A). (B) Representative RGC culture before and after explant manual removal. Severing axons do not cause collapse per se (arrowheads indicate not-collapsed growth cones). (C) Frequency (in percentage) of collapsed growth cones after 10 min Sema3A bath application. The number of counted growth cones is reported in each column. Data information: Values are mean ± SEM. Statistics: each data point corresponds to one independent experiment, n = 3 independent experiments, two-way ANOVA, Sidak's multiple comparison test. Scale bars: 50 μm. Abbreviations: ns, not significant.
	Figure 7. In vitro validation of MO ASOs inhibition potential and target-specificity. (A) Predicted secondary structures of pre-miR-181a-1 (left) and pre-miR-181a-2 (right). Scissors mark Dicer cleavage sites. Grey circles indicate the regions targeted by MOs. (B, C) Results of the inhibition assay assessing the effect of MOs on the cleavage of 5′-32P-labeled pre-miR-181a-1 (B) or 5′-32P-labeled pre-miR-181a-2 (C) in Xenopus cytosolic extracts. Triangles indicate increasing concentration of a given MO (0.1, 1, 10 μM). Schematic representation of the pre-miRNA hairpin and MO (yellow line) is given above each triangle. (D, E) Comparison of the inhibitory potential of AL1-4 and tested MOs calculated as miR-181a-1-5p production efficiency normalized to the level of miR-181a-1-5p generated in C+ for pre-miR-181a-1 (D) and pre-miR-181a-2 (E). Data derived from Figure 3 and Supplementary Figure S8 for AL1-4, and (B, C) for MOs. Abbreviations: MO, morpholino; C–, pre-miRNA incubated with no extract nor MO added; C+, pre-miRNA incubated with extract but no MO added; T1, RNase T1 ladder.
	Figure 8. In vitro validation of AL-ASOs targeting human miR-16-1 precursors. (A) Predicted secondary structures of pre-miR-16-1 (left) and pre-miR-16-2 (right). Scissors indicate Dicer cleavage sites. Mature miRNA sequences are coloured in red and blue/purple, regions targeted by the selected oligomers (hAL16-1 and hAL16-2) are in grey. Asterisks mark the position of LNA within the AL-ASO sequence. (B) Native gel analysis of the interaction between 5′-32P-labeled pre-miRNA and tested oligomers (hCtrl, hAL16-1, hAL16-2). (C, D) Results of the inhibition assay assessing the effect of tested oligomers on the cleavage of 5′-32P-labeled pre-miR-16-1 (C) or 5′-32P-labeled pre-miR-16-2 (D) in HEK293T cytosolic extracts. Triangles indicate increasing concentration of a given oligomer (0.1, 1, 10 μM). Graphs show miRNA production efficiency normalized to the level of miRNA generated in C+. Data information: Values are mean ± SD. Statistics: n = 3 independent experiments, unpaired t-test. Abbreviations: AL, apical loop; ns, not significant; C–, pre-miRNA incubated with no extract nor oligomer added; C+, pre-miRNA incubated with extract but no oligomer added; C++, pre-miRNA incubated with recombinant human Dicer; T1, RNase T1 ladder.

References [+] :

Abbott, The let-7 MicroRNA family members mir-48, mir-84, and mir-241 function together to regulate developmental timing in Caenorhabditis elegans. 2005, Pubmed