Design and Application of Mini‐libraries of miRNA Probes for an Efficient and Versatile miRNA‐mRNA Cross‐linking

Malinowska, A.; Łaski, Artur; Hall, Jonathan

doi:10.1002/chem.202101171

Cited by 6 publications

(2 citation statements)

References 51 publications

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“…It has been reported that cross-linkers modifying the base of nucleotides are positioned in the major groove, while those modifying the ribose are positioned in the minor groove of a miRNA−target duplex. 25 Considering that the modified diazirine at C-9 of the phenoxazine nucleoside is positioned in the major groove, we wanted to compare differences in the impact of cross-linker position in the major groove versus the minor groove on photo-cross-linking efficiency. For this purpose, we tethered different diazirines (R 2 and R 3 ) to the 2′-O-position of phenoxazine nucleoside ribose (structure 3 in Scheme 1C) using a 1,2,3-triazole linker.…”

Section: ■ Resultsmentioning

confidence: 99%

G-Clamp Heterocycle Modification Containing Interstrand Photo-Cross-Linker to Capture Intracellular MicroRNA Targets

Shen,

Hou,

et al. 2024

J. Am. Chem. Soc.

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MicroRNAs (miRNAs) play indispensable roles in posttranscriptional gene regulation. The identification of target mRNAs is essential for dissecting the recognition basis, dynamics, and regulatory mechanism of miRNA−mRNA interactions. However, the lack of an unbiased method for detecting weak miRNA−mRNA interactions remains a long-standing obstacle for miRNA research. Here, we develop and provide proof-of-concept evidence demonstrating a chemical G-clampenhanced photo-cross-linking strategy for covalent capture of intracellular miRNA targets in different cell lines. This approach relies on an aryldiazirine-G-clamp-modified-nucleoside (ARAGON) miRNA probe containing an alkynyl group that improves the thermal stability of miRNA− target mRNA duplex molecules and can rapidly cross-link with the complementary strand upon UV 365 nm activation, enhancing the transient capture of mRNA targets. After validating the accuracy and binding properties of ARAGON-based miRNA probes through the successful enrichment for the known targets of miR-106a, miR-21, and miR-101, we then extend ARAGON's application to screen for previously unknown targets of different miRNAs in various cell lines. Ultimately, results in this study uncover GAB1 as a target of miR-101 in H1299 lung cancer cells and show that miR-101 silencing of GAB1 can promote apoptosis in H1299 cells, suggesting an oncogenic mechanism of GAB1. This study thus provides a powerful and versatile tool for enhanced screening of global miRNA targets in cells to facilitate investigations of miRNA functions in fundamental cellular processes and disease pathogenesis.

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Section: ■ Resultsmentioning

confidence: 99%

G-Clamp Heterocycle Modification Containing Interstrand Photo-Cross-Linker to Capture Intracellular MicroRNA Targets

Shen,

Hou,

et al. 2024

J. Am. Chem. Soc.

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“…It is recognized that miRNAs interact with the downstream mRNAs by base pairing with mRNA 3′UTRs [Malinowska et al, 2021]. Through bioinformatic analysis and experiments, we found SETD1A was targeted by The survival fraction was determined in MKN-45 and AGS cells introduced with sh-NC, sh-FOXD2-AS1#1, or sh-FOXD2-AS1#1 + pcDNA3.1/SETD1A.…”

Section: Discussionmentioning

confidence: 95%

Downregulation of lncRNA FOXD2-AS1 Confers Radiosensitivity to Gastric Cancer Cells via miR-1913/SETD1A Axis

Guo

Zhang

2022

Cytogenet Genome Res

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Long noncoding RNA FOXD2 adjacent opposite strand RNA1 (FOXD2-AS1) plays an oncogenic role in various cancers, including gastric cancer (GC). However, the function of FOXD2-AS1 in regulating radiosensitivity of GC cells and its underlying molecular mechanisms have not been elucidated. This study aimed to figure out the potential mechanisms of FOXD2-AS1 in regulating GC cell radiosensitivity. RT-qPCR revealed upregulation of FOXD2-AS1 in GC cells exposed to radiation. Subcellular fractionation assay was used to localize FOXD2-AS1 in GC cells. Colony formation, MTT, EdU, and flow cytometry assays were performed to investigate the role of FOXD2-AS1 in regulating cell proliferation, cell cycle progression, and cell apoptosis. Western blotting was used to assess protein levels of apoptosis-associated markers and SET domain containing 1A (SETD1A). Homologous recombination reporter assay was conducted to explore the effect of FOXD2-AS1 on DNA damage repair. The downstream molecules of FOXD2-AS1 were identified with RNA pulldown, luciferase reporter, and RNA immunoprecipitation assays. The results showed that FOXD2-AS1 knockdown suppressed cell proliferation and cell cycle progression and promoted cell apoptosis and radiosensitivity of GC. FOXD2-AS1 could bind with miR-1913 in GC cells. In addition, miR-1913 targeted SETD1A, which was highly expressed in GC cells. Overexpression of SETD1A reversed FOXD2-AS1 silencing-induced effects on proliferation, apoptosis, and radiosensitivity of GC cells. In conclusion, knocking down FOXD2-AS1 enhances the radiosensitivity of GC cells by sponging miR-1913 to upregulate SETD1A expression.

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Uracil‐Selective Cross‐Linking in RNA and Inhibition of miRNA Function by 2‐Amino‐6‐vinyl‐7‐deazapurine Deoxynucleosides

Soemawisastra,

Okamura,

Abdelhady

et al. 2024

ChemBioChem

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MicroRNAs (miRNAs) regulate gene expression through RNA interference. Consequently, miRNA inhibitors, such as anti‐miRNA oligonucleotides (AMOs), have attracted attention for treating miRNA overexpression. To achieve efficient inhibition, we developed 2‐amino‐6‐vinylpurine (AVP) nucleosides that form covalent bonds with uridine counterparts in RNA. We demonstrated that mRNA cross‐linked with AVP‐conjugated antisense oligonucleotides with AVP were protected from gene silencing by exogenous miRNA. However, endogenous miRNA function could not be inhibited in cells, probably because of slow cross‐linking kinetics. We recently developed ADpVP, an AVP derivative bearing a 7‐propynyl group—which boasts faster reaction rate than the original AVP. Here, we synthesized dADpVP—a deoxy analog of ADpVP—through a simplified synthesis protocol. Evaluation of the cross‐linking reaction revealed that the reaction kinetics of dADpVP were comparable to those of ADpVP. In addition, structural analysis of the cross‐linked adduct discovered N3 linkage against uridine. Incorporating dADpVP into two types of miRNA inhibitors revealed a marginal impact on AMO efficacy yet improved the performance of target site blockers. These results indicate the potential of cross‐linking nucleosides for indirect miRNA function inhibition.

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Design and Application of Mini‐libraries of miRNA Probes for an Efficient and Versatile miRNA‐mRNA Cross‐linking

Cited by 6 publications

References 51 publications

G-Clamp Heterocycle Modification Containing Interstrand Photo-Cross-Linker to Capture Intracellular MicroRNA Targets

G-Clamp Heterocycle Modification Containing Interstrand Photo-Cross-Linker to Capture Intracellular MicroRNA Targets

Downregulation of lncRNA FOXD2-AS1 Confers Radiosensitivity to Gastric Cancer Cells via miR-1913/SETD1A Axis

Uracil‐Selective Cross‐Linking in RNA and Inhibition of miRNA Function by 2‐Amino‐6‐vinyl‐7‐deazapurine Deoxynucleosides

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