Efficient and specific inhibition of plant microRNA function by anti-microRNA oligonucleotides (AMOs) in vitro and in vivo

Key message

Anti-microRNA oligonucleotides (AMOs) are efficient and sequence-specific inhibitors of plant miRNA function both in vitro and in vivo.

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in developmental and physiological processes in plants and animals. Although miRNA knockdown by chemically modified antisense oligonucleotides prevails in animal and therapeutic studies, no such application has ever been reported in plants. Here, we show that sucrose-mediated delivery of 2′-O-methyl (2′-O-Me) anti-miRNA oligonucleotides (AMOs) is an efficient and sequence-specific way of inhibiting plant miRNA activity both in vitro and in vivo. Administration of AMOs to rice protoplasts and intact leaves resulted in efficient inhibition of miRNAs with concurrent de-repression of their target genes. AMOs caused simultaneous inhibition of miRNAs from the same family but exerted negligible effects on miRNAs from different families. In rice seedlings, a single-dose AMO treatment conferred long-lasting miRNA inhibition for at least 7 days. Although simultaneous dysregulation of multiple miRNAs by an AMO-and-miRNA-mimic mixture resulted in severe root defects, the phenotypic effects of individual AMOs and miRNA mimics were negligible, suggesting that those miRNAs function together in regulatory networks to ensure homeostasis. Our results validate the utility of AMOs as an efficient tool for plant miRNA loss-of-function studies in vivo, and this approach may prove to be a highly promising general method for unraveling miRNA-mediated gene-regulatory networks.

     

Sequence-dependent off-target inhibition of TLR7/8 sensing by synthetic microRNA inhibitors

Anti-microRNA (miRNA) oligonucleotides (AMOs) with 2′-O-Methyl (2′OMe) residues are commonly used to study miRNA function and can achieve high potency, with low cytotoxicity. Not withstanding this, we demonstrate the sequence-dependent capacity of 2′OMe AMOs to inhibit Toll-like receptor (TLR) 7 and 8 sensing of immunostimulatory RNA, independent of their miRNA-targeting function. Through a screen of 29 AMOs targeting common miRNAs, we found a subset of sequences highly inhibitory to TLR7 sensing in mouse macrophages. Interspecies conservation of this inhibitory activity was confirmed on TLR7/8 activity in human peripheral blood mononuclear cells. Significantly, we identified a core motif governing the inhibitory activity of these AMOs, which is present in more than 50 AMOs targeted to human miRNAs in miRBaseV20. DNA/locked nucleic acids (LNA) AMOs synthesized with a phosphorothioate backbone also inhibited TLR7 sensing in a sequence-dependent manner, demonstrating that the off-target effects of AMOs are not restricted to 2′OMe modification. Taken together, our work establishes the potential for off-target effects of AMOs on TLR7/8 function, which should be taken into account in their therapeutic development and in vivo application.     

Effective delivery of anti-miRNA DNA oligonucleotides by functionalized gold nanoparticles

MicroRNAs (miRNAs) are gaining recognition as essential regulators involved in many biological processes, and they are emerging as therapeutic targets for treating disease. Here, we introduce a method for effective delivery of anti-miRNA oligonucleotides (AMOs) using functionalized gold nanoparticles (AuNPs). To demonstrate the ability of AMOs to silence miRNA, we selected miR-29b, which is known to downregulate myeloid cell leukemia-1 (MCL-1), a factor responsible for promoting cell survival. We first generated AuNPs coated with cargo DNA, which was then coupled to complementary DNA linked to an antisense miR-29b sequence. When the AuNPs were delivered into HeLa cells, MCL-1 protein and mRNA levels were increased significantly. Furthermore, apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was inhibited, proving that AMOs targeting miR-29b were effectively delivered by our innovative AuNP. In addition, we provided evidence that AuNP could deliver other AMOs against miR-21 into two independent cell lines, KGN and 293T, suggesting that the AuNP conjugates can be versatile for any AMO and cell type.     

A single anti-microRNA antisense oligodeoxyribonucleotide (AMO) targeting multiple microRNAs offers an improved approach for microRNA interference

Anti-miRNA antisense inhibitors (AMOs) have demonstrated their utility in miRNA research and potential in miRNA therapy. Here we report a modified AMO approach in which multiple antisense units are engineered into a single unit that is able to simultaneously silence multiple-target miRNAs, the multiple-target AMO or MTg-AMO. We validated the technique with two separate MTg-AMOs: anti-miR-21/anti-miR-155/anti-miR-17-5p and anti-miR-1/anti-miR-133. We first verified the ability of the MTg-AMOs to antagonize the repressive actions of their target miRNAs using luciferase reporter activity assays and to specifically knock down the levels of their target miRNAs using real-time RT-PCR methods. We then used the MTg-AMO approach to identify several tumor suppressors—TGFBI, APC and BCL2L11 as the target genes for oncogenic miR-21, miR-155 and miR-17-5p, respectively, and two cardiac ion channel genes HCN2 (encoding a subunit of cardiac pacemaker channel) and CACNA1C (encoding the α-subunit of cardiac L-type Ca²⁺ channel) for the muscle-specific miR-1 and miR-133. We further demonstrated that the MTg-AMO targeting miR-21, miR-155 and miR-17-5p produced a greater inhibitory effect on cancer cell growth, compared with the regular single-target AMOs. Moreover, while using the regular single-target AMOs excluded HCN2 as a target gene for either miR-1 or miR-133, the MTg-AMO approach is able to reveal HCN2 as the target for both miR-1 and miR-133. Our findings suggest the MTg-AMO as an improved approach for miRNA target finding and for studying function of miRNAs. This approach may find its broad application for exploring biological processes involving multiple miRNAs and multiple genes.     

Potent inhibition of microRNA in vivo without degradation

Chemically modified antisense oligonucleotides (ASOs) are widely used as a tool to functionalize microRNAs (miRNAs). Reduction of miRNA level after ASO inhibition is commonly reported to show efficacy. Whether this is the most relevant endpoint for measuring miRNA inhibition has not been adequately addressed in the field although it has important implications for evaluating miRNA targeting studies. Using a novel approach to quantitate miRNA levels in the presence of excess ASO, we have discovered that the outcome of miRNA inhibition can vary depending on the chemical modification of the ASO. Although some miRNA inhibitors cause a decrease in mature miRNA levels, we have identified a novel 2′-fluoro/2′-methoxyethyl modified ASO motif with dramatically improved in vivo potency which does not. These studies show there are multiple mechanisms of miRNA inhibition by ASOs and that evaluation of secondary endpoints is crucial for interpreting miRNA inhibition studies.     

Search for oligonucleotides selectively binding oncogenic miR-21

In the pathogenesis of malignancies, an active regulatory role belongs to small noncoding RNAs, miRNA (miR). miRNA expression profiles are often associated with the prognosis and therapeutic outcome of different oncological diseases. It is well known that in comparison with normal tissues cancer cells are characterized by hyperexpression of oncogenic miRNAs which leads to oncogenic transformation, carcinogenesis and metastasis progression. From this point of view, selective down-regulation of miRNA expression by specific agents, such as antisense oligonucleotides that recognize particular sequences, therefore, can be an effective tool to regulate the amount of miRNA in cancer cells and decrease tumor malignancy. In this paper, we have designed a series of antisense oligonucleotides addressed to the oncogenic miR-21 with a view to its selective binding and studied patterns of interaction of miR-21 with these oligonucleotides in vitro. The series included linear and hairpin oligonucleotides with the length of antisense fragment of 10–16 nucleotides (nt) complementary to the 5'- or the 3'-end of miRNA target. Hairpin oligonucleotides consist of a sequence complementary to miR-21 and a hairpin containing a four-nucleotide loop and stem of 6–9 bp necessary for stabilizing the complex with miR-21. It has been shown that inclusion of the hairpin with the stem of 6 bp to the oligonucleotide structure leads to a 1.6-fold increase in binding efficiency with miR-21 in comparison with a linear oligonucleotide and elongation of the stem from six to nine bp does not increase binding efficiency. Hairpin oligonucleotides with an antisense sequence of 14 nt effectively hybridize with miR-21 and are not inferior to 16-mer linear and hairpin oligonucleotides in the efficiency of complex formation. Thus, we have shown that hairpin oligonucleotides with antisense fragment of 14 nt and a hairpin, including the stem of 6 bp, are optimal for selective and effective sequestering of mature miR-21.     

PNA-based antisense oligonucleotides for micrornas inhibition in the absence of a transfection reagent

MicroRNAs (miRNAs) are noncoding RNAs approximately 22 nucleotides in length that play a major role in the regulation of important biological processes, including cellular development, differentiation, and apoptosis. Antisense oligonucleotides against miRNAs are useful tools for studying the biological mechanisms and therapeutic targets of miRNAs. Various antisense oligonucleotides chemistries, including peptide nucleic acids (PNAs), have been developed to enhance nuclease-resistance and affinity and specificity for miRNA targets. PNAs have a greater specificity and affinity for DNA and RNA than do natural nucleic acids, and they are resistant to nucleases-an essential property of an miRNA inhibitor that will be exposed to cellular nucleases. However, the main limiting factor in the use of PNAs is their reduced penetration into cells. Recently, several cell-penetrating peptides (CPPs) have been investigated as a means to overcome the limited penetration of PNAs. Here, we evaluated the ability of 11 CPPs to transport PNAs inside cells in the absence of transfection reagents and then investigated the ability of these CPPs to inhibit miRNAs. Of the 11 CPPs tested, Tat-modified-conjugated PNA showed the most effective penetration into cells in the absence of transfection reagents and most effectively inhibited miRNAs. Our data demonstrate that Tat-modified-conjugated CPP is the most suitable for supporting PNA-mediated miRNA inhibition.     

A highly effective and long-lasting inhibition of miRNAs with PNA-based antisense oligonucleotides

MiRNAs are non-coding RNAs that play a role in the regulation of major processes. The inhibition of miRNAs using antisense oligonucleotides (ASOs) is a unique and effective technique for the characterization and subsequent therapeutic targeting of miRNA function. Recent advances in ASO chemistry have been used to increase both the resistance to nucleases and the target affinity and specificity of these ASOs. Peptide nucleic acids (PNAs) are artificial oligonucleotides constructed on a peptide-like backbone. PNAs have a stronger affinity and greater specificity to DNA or RNA than natural nucleic acids and are resistant to nucleases, which is an essential characteristic for a miRNA inhibitor that will be exposed to serum and cellular nucleases. For increasing cell penetration, PNAs were conjugated with cell penetrating peptides (CPPs) at N-terminal. Among the tested CPPs, Tat-modified peptide-conjugated PNAs have most effective function for miRNA inhibition. PNA-based ASO was more effective miRNA inhibitor than other DNA-based ASOs and did not show cytotoxicity at concentration up to 1,000 nM. The effects of PNA-based ASOs were shown to persist for 9 days. Also, PNA-based ASOs showed considerable stability at storage temperature. These results suggest that PNA-based ASOs are more effective ASOs of miRNA than DNA-based ASOs and PNA-based ASO technology, compared with other technologies used to inhibit miRNA activity can be an effective tool for investigating miRNA functions.     

MicroRNA‐488 suppresses cell migration through modulation of the focal adhesion activity during chondrogenic differentiation of chick limb mesenchymal cells

miRNAs (microRNAs) have proven to play essential roles in diverse biological processes including early development, cell proliferation and cell death, and cell differentiation. However, there is only limited amount of information about their potential role in chondrogenesis. In the present study, we investigated the role of miRNA‐488 in the cellular condensation, which is essential initiation for chondrogenic differentiation. We found that miRNA‐488 expression is up‐regulated at the precondensation stage and then down‐regulated at the postcondensation stage. Blockade of miRNA‐488 via the use of PNA (peanut agglutinin)‐based ASOs (antisense oligonucleotides) decreased the protein level of integrins β1 and phosphorylated FAK (focal adhesion kinase) and resulted in the suppression of cell motility and migration. Moreover, in parallel with theses observation, treatment of anti‐miRNA‐488 oligonucleotides up‐regulated the level of MMP (matrix metalloprotease)‐2 activity, and co‐treatment with GM6001, an MMP inhibitor, induced recovery of cellular condensation inhibited by blockade of miRNA‐488. Collectively, our results suggest that miRNA‐488 is one of regulator in cell to ECM (extracellular matrix) interaction through modulation of focal adhesion activity by MMP‐2 during chondrogenesis of limb mesenchymal cells.     

Propionic and methylmalonic acidemia: antisense therapeutics for intronic variations causing aberrantly spliced messenger RNA

We describe the use of antisense morpholino oligonucleotides (AMOs) to restore normal splicing caused by intronic molecular defects identified in methylmalonic acidemia (MMA) and propionic acidemia (PA). The three new point mutations described in deep intronic regions increase the splicing scores of pseudoexons or generate consensus binding motifs for splicing factors, such as SRp40, which favor the intronic inclusions in MUT (r.1957ins76), PCCA (r.1284ins84), or PCCB (r.654ins72) messenger RNAs (mRNAs). Experimental confirmation that these changes are pathogenic and cause the activation of the pseudoexons was obtained by use of minigenes. AMOs were targeted to the 5′ or 3′ cryptic splice sites to block access of the splicing machinery to the pseudoexonic regions in the pre-mRNA. Using this antisense therapeutics, we have obtained correctly spliced mRNA that was effectively translated, and propionyl coenzyme A (CoA) carboxylase (PCC) or methylmalonylCoA mutase (MCM) activities were rescued in patients’ fibroblasts. The effect of AMOs was sequence and dose dependent. In the affected patient with MUT mutation, close to 100% of MCM activity, measured by incorporation of ¹⁴C-propionate, was obtained after 48 h, and correctly spliced MUT mRNA was still detected 15 d after treatment. In the PCCA-mutated and PCCB-mutated cell lines, 100% of PCC activity was measured after 72 h of AMO delivery, and the presence of biotinylated PCCA protein was detected by western blot in treated PCCA-deficient cells. Our results demonstrate that the aberrant inclusions of the intronic sequences are disease-causing mutations in these patients. These findings provide a new therapeutic strategy in these genetic disorders, potentially applicable to a large number of cases with deep intronic changes that, at the moment, remain undetected by standard mutation-detection techniques.     

A PCR-Based Method to Construct Lentiviral Vector Expressing Double Tough Decoy for miRNA Inhibition

DNA vector-encoded Tough Decoy (TuD) miRNA inhibitor is attracting increased attention due to its high efficiency in miRNA suppression. The current methods used to construct TuD vectors are based on synthesizing long oligonucleotides (~90 mer), which have been costly and problematic because of mutations during synthesis. In this study, we report a PCR-based method for the generation of double Tough Decoy (dTuD) vector in which only two sets of shorter oligonucleotides (< 60 mer) were used. Different approaches were employed to test the inhibitory potency of dTuDs. We demonstrated that dTuD is the most efficient method in miRNA inhibition in vitro and in vivo. Using this method, a mini dTuD library against 88 human miRNAs was constructed and used for a high-throughput screening (HTS) of AP-1 pathway-related miRNAs. Seven miRNAs (miR-18b-5p, -101-3p, -148b-3p, -130b-3p, -186-3p, -187-3p and -1324) were identified as candidates involved in AP-1 pathway regulation. This novel method allows for an accurate and cost-effective generation of dTuD miRNA inhibitor, providing a powerful tool for efficient miRNA suppression in vitro and in vivo.     

LidNA, a novel miRNA inhibitor constructed with unmodified DNA

Many miRNA inhibitors have been developed and they are chemically modified oligonucleotides such as 2′-O-methylated RNA and locked nucleic acid (LNA). Unmodified DNA was not yet reported as a miRNA inhibitor because of the low affinity of DNA/miRNA compared to mRNA/miRNA. We designed a structured unmodified DNA that significantly inhibits miRNA function. The clue structure for activity is the miRNA binding site between double stranded regions which is responsible for the miRNA inhibitory activity and tight binding to miRNA. We developed the miRNA inhibitor constructed with unmodified DNA, and named it LidNA, DNA that puts a lid on miRNA function.     

Efficient inhibition of miR-155 function in vivo by peptide nucleic acids

MicroRNAs (miRNAs) play an important role in diverse physiological processes and are potential therapeutic agents. Synthetic oligonucleotides (ONs) of different chemistries have proven successful for blocking miRNA expression. However, their specificity and efficiency have not been fully evaluated. Here, we show that peptide nucleic acids (PNAs) efficiently block a key inducible miRNA expressed in the haematopoietic system, miR-155, in cultured B cells as well as in mice. Remarkably, miR-155 inhibition by PNA in primary B cells was achieved in the absence of any transfection agent. In mice, the high efficiency of the treatment was demonstrated by a strong overlap in global gene expression between B cells isolated from anti-miR-155 PNA-treated and miR-155-deficient mice. Interestingly, PNA also induced additional changes in gene expression. Our analysis provides a useful platform to aid the design of efficient and specific anti-miRNA ONs for in vivo use.     

Double-stranded regions are essential design components of potent inhibitors of RISC function

While microRNAs (miRNAs) are recognized as playing a critical role in regulating eukaryotic gene expression, both the mechanism by which these small, noncoding RNAs function and the genes they target remain elusive. Previous studies have shown that short, single-stranded 2'-O-methyl-modified oligonucleotides that are complementary to mature microRNA sequences can interact with the miRNA-RISC nucleoprotein complex and weakly inhibit miRNA function. Here we report the identification of secondary structural elements that enhance the potency of these molecules. Incorporation of highly structured, double-stranded flanking regions around the reverse complement core significantly increases inhibitor function and allows for multi-miRNA inhibition at subnanomolar concentrations. The improved functionality of these double-stranded miRNA inhibitors may provide insights into the miRNA mechanism by suggesting the possible importance of such structures in or near endogenous miRNA target sites.