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Natural antisense transcripts (NATs or AS transcripts) are frequently transcribed from many eukaryotic genes and post-transcriptionally regulate gene expression. The AS transcript is classified as noncoding RNA and acts as a regulatory RNA in concert with RNA-binding proteins that bind to cis-controlling elements on the mRNA, microRNAs, and drugs. The AS transcript that overlaps with mRNA regulates mRNA stability by interacting with mRNA, and the network of mRNAs, AS transcripts, microRNAs, and RNA-binding proteins finely tunes the output of gene regulation, i.e., mRNA levels. We found that single-stranded ‘sense’ oligonucleotides corresponding to an mRNA sequence decreased the mRNA levels by interfering with the mRNA-AS transcript interactions of several genes, such as inducible nitric oxide synthase (iNOS) and interferon-alpha1 (IFN-A1) genes. In contrast, AntagoNAT oligonucleotides, which are complementary to AS transcripts, are sense oligonucleotides when they overlap with mRNA, but they increase the levels of specific mRNAs. Collectively, the sense oligonucleotide is a powerful tool for decreasing or increasing mRNA levels. The natural antisense transcript-targeted regulation (NATRE) technology using sense oligonucleotides is a method with a unique modality for modulating cytosolic mRNA levels and may be used to treat human diseases in which AS transcripts are involved.
Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University, Japan
Tetsuya Okuyama
Department of Surgery, Kansai Medical University, Japan
Richi Nakatake
Department of Surgery, Kansai Medical University, Japan
*Address all correspondence to: nishizaw@sk.ritsumei.ac.jp
1. Introduction
The transcripts whose sequences are complementary to those of mRNA have been reported in many genes regardless of species, from bacteria to mammals. Because protein is encoded by mRNA, whose sequence is the same as the sense strand of a gene, i.e., double-stranded DNA, the transcript has been called a natural antisense transcript (NAT or AS transcript) [1, 2]. The AS transcripts do not code for proteins or only short peptides and are classified as one class of noncoding RNA (ncRNA). Accumulating genome-wide transcriptome analyses have demonstrated that natural antisense transcripts are transcribed from many eukaryotic genes [3]. HUGO proposed the nomenclature of human gene symbols for natural antisense transcripts AS (suffix) [4]. In this chapter, we use ‘AS transcript’ as a natural antisense transcript.
In contrast, classical types of ncRNA species, such as ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA), are well known and have definite functions in gene expression. These classical ncRNAs do not overlap mRNAs. Among other ncRNA species, microRNA (miRNA or miR), which is 20–23 nucleotides (nt) in the length, was found in nematodes and mammals. This very short ncRNA species, which is complementary to the 3′-untranslated region (3′UTR) of several mRNAs, inhibits translation and induces mRNA degradation [5]. Therefore, microRNA hybridizes with mRNA to regulate its functions.
Furthermore, long ncRNAs (lncRNAs), which are more than 200 nt long [6], were found. At first, their functions were unclear, but it has gradually been revealed that these lncRNAs are involved in gene expression [7]. To date, huge number of lncRNA sequences have been reported by RNA-seq analysis and deposited in public databases, such as LNCipedia 5 (human lncRNA transcripts) [8]. Nowadays, ncRNA species, including miRNA, and lncRNAs, are known as regulatory RNAs [9].
Many studies have demonstrated that the AS transcript, one class of lncRNA, is involved in various steps during gene expression [7]. When focusing on the AS transcript that overlaps with an mRNA, this type of AS transcript interacts with mRNA and plays an important role in gene expression, especially at post-transcriptional levels [1, 2]. Interestingly, most AS transcripts are transcribed at low levels [1, 2]. The analyses and application of AS transcripts are summarized by the reviews, for example, see [1, 2, 10].
During our functional analyses of AS transcripts, we found the mRNA-AS transcript interactions that regulate mRNA stability (described later). Although conventional methods, i.e., antisense and short interference RNA (siRNA) technologies [11, 12] were available for the analyses of AS transcript functions [13], we first used synthetic sense oligonucleotides that are complementary to the AS transcript. We found that sense oligonucleotides resulted in decreases in cytoplasmic mRNA levels, which may be applied to ‘knockdown of mRNA.’
Here, our method to regulate mRNA levels based on the mRNA-AS transcript interactions is described, and the application of this technology to treat disease is discussed.
2. Natural antisense transcripts that overlap with mRNAs
2.1 Structures of natural antisense transcripts
The AS transcript is frequently transcribed from inducible genes [14]. Our previous studies showed that an AS transcript harbors an overlapping sequence with 3′UTR of an mRNA [14]. Such a 3′UTR possesses a few AU-rich elements (AREs), which may be involved in mRNA stability because AREs may be the targets of miRNAs and RNA-binding proteins [15, 16]. Interestingly, the location of AREs in the 3′UTR of iNOS mRNA is conserved among species (rat, mouse, and human) [17, 18].
The sizes of AS transcripts are variable and show a smear pattern or discrete bands in Northern blot analysis. The former example is AS transcripts that are transcribed from rat inducible nitric oxide synthase (iNOS, NOS2) gene, and the size is ranging from 600 to 1000 nt (Figure 1) [19]. iNOS catalyzes the production of the inflammatory mediator nitric oxide (NO). iNOS is induced by various inflammatory stimuli; interleukin (IL)-1beta to hepatocytes and bacterial lipopolysaccharide (LPS) to macrophages [17, 18].
Figure 1.
mRNA and AS transcripts transcribed from the inducible nitric oxide synthase (iNOS) gene. The rat iNOS gene is schematically depicted. The iNOS gene consists of 27 exons, and the AS transcript overlaps with the exon 27, which includes the 3′UTR (white box) of the mRNA. The iNOS mRNA harbors AREs in its 3′UTR. The iNOS AS transcript starts at the end of exon 27 and stops at various sites, resulting in various sizes of the transcripts, which do not harbor a poly(A) tail. Figure reproduced with modification from [19] with permission.
The latter examples are about 4-kilobase (kb) AS transcript that is transcribed from the human interferon-alpha1 (IFN-A1) gene (Figure 2) [20]. IFN-A1 gene encodes the cytokine IFN-alpha1 and is one subset of human IFN-A genes, which consist of 13 subsets [22]. Viral infection induces IFN-alpha1, a member of the type I interferon family, which is a main innate immunity response. AS transcripts are also transcribed from other subtypes of IFN-A genes [23].
Figure 2.
mRNA and AS transcripts transcribed from interferon alpha1 (IFN-A1) gene. The human IFN-A1 gene consists of a single exon, which encodes IFN-alpha1 [20]. The coding sequence (from ATG to TAA) gives conserved stem-loop structures in the IFN-A1 mRNA, i.e., stem-loops (SL) and bulged-stem loop (BSL) [21]. These structures are responsible for chromosome region maintenance 1 (CRM1)-dependent nuclear export of IFN-A1 mRNA [21]. The IFN-A1 mRNA harbors AREs in its 3′UTR. Two AS transcripts are shown, both of which are spliced and harbor poly(A) tails. Nucleotides are numbered from the transcription initiation site. Figure reproduced with modification from [20] with permission.
Another example is the AS transcript from the rat tumor necrosis factor-alpha (Tnf) gene, which is about 2.5-kb long [24]. AS transcripts are transcribed from many genes that are involved in inflammation, such as mRNAs that encode alpha subunit (p19) of IL-23 (IL-23A), chemokine (C-C motif) ligand 2 (CCL2), chemokine (C-X3-C) motif ligand 1 (CX3CL1), p65 and p50 subunits of nuclear factor kappaB (NF-kappaB) [14]. Additionally, AS transcript is transcribed from the human gene encoding ephrin type A receptor 2 (EPHA2), which is a receptor tyrosine kinase whose over-expression is observed in various cancers [25, 26].
2.2 mRNA-AS transcript interactions and mRNA stability
2.2.1 iNOS mRNA-AS transcript interaction
When the AS transcript overlaps with the relevant mRNA, the interaction between the AS transcript and mRNA is expected. Indeed, the AS transcript is transcribed from the rat iNOS gene and interacts with and stabilizes the iNOS mRNA [19]. Further analyses demonstrated that the iNOS AS transcript interacts with the iNOS mRNA at the single-stranded loop or bulge of the overlapping region between the mRNA and AS transcript (Figure 3) [19].
Figure 3.
A putative mechanism of the mRNA-AS transcript interaction and mRNA degradation by the introduction of a sense oligonucleotide (iNOS gene). (A) Stable complex on the iNOS mRNA. When the iNOS AS transcript is expressed, it partially hybridizes with a single-stranded loop in the iNOS mRNA that harbors a cap structure (open circle) and a poly(A) tail. By recruiting an RNA-binding protein, it forms an mRNA-AS transcript-protein complex to stabilize the iNOS mRNA in the cytoplasm. (B) Interference with the mRNA-AS transcript interaction. Because a sense oligonucleotide to the iNOS mRNA harbors the same sequence as the mRNA, it competitively inhibits the hybridization of iNOS mRNA with the iNOS AS transcript, leading to interference with the mRNA-AS transcript interaction and then the mRNA degradation. This mechanism confers to the basis of the NATRE technology.
Because the orientation of RNA is 5′-to-3′, base complementarity indicates that the secondary structure of AS transcript is a mirror image of that of mRNA. This means that stem-loop structures in the AS transcript are formed at the complementary sites in the corresponding mRNA, leading to loop-loop hybridization between the mRNA and AS transcript [1, 2]. This loop-loop hybridization forms a short RNA:RNA duplex (usually <10 base pairs), which is thermodynamically unstable due to the low melting temperature of the duplex. Then, RNA-binding proteins (e.g., HuR) bind to the iNOS mRNA and/or AS transcript to stabilize the mRNA-AS transcript-protein complex, which protects from the degradation of RNAs and facilitates translation in the cell [19].
2.2.2 INF-A1 mRNA-AS transcript interaction
As another putative mechanism, the human IFN-A1 AS transcript interacts with and stabilizes IFN-A1 mRNA by blocking the microRNA binding to IFN-A1 mRNA [20]. IFN-A1 AS transcript is expressed at a low level. After the AS transcript transiently interacts with IFN-A1 mRNA, it moves on and targets the next mRNA molecule in a ‘hit-and-run’ fashion [1, 20].
Sense oligonucleotides to BSL of IFN-A1 mRNA (see Figure 2) decreased IFN-A1 mRNA levels. Because a potent binding site of a microRNA (miR-1270) is present in BSL, miR-1270 may bind to BSL of IFN-A1 mRNA. A microRNA-binding site is also called a microRNA-responsive element (MRE). Next, a short AS oligoribonucleotide (asORN), which is the part of IFN-A1 AS transcript corresponding to BSL, was introduced into the cells (This is not NATRE technology.). IFN-A1 asORN increased IFN-A1 mRNA levels, but it did not alter IFN-A1 AS transcript levels [20]. These data imply that IFN-A1 asORN stabilizes IFN-A1 mRNA by simulating the AS transcript.
When the levels of transcripts were measured, miR-1270 was much more excess to IFN-A1 AS transcript [23]. Although IFN-A1 AS transcripts include several MREs, they are stoichiometrically unable to sponge all miR-1270 molecules. Further study indicated another mechanism.
When microRNAs are shared by mRNAs and AS transcripts, the transcripts function as competing endogenous RNAs (ceRNAs). Several AS transcripts are transcribed from the specific subsets of IFN-A gene family (IFN-A1, A7, A8, A10, and A14 genes) and harbor common MREs for miR-1270 [23]. MiR-1270 can bind to both mRNA and AS transcript from IFN-A gene, as well as mRNAs from the specific subsets of IFN-A gene family (IFN-A8, A10, A14, and A17 genes). Because the mRNAs and AS transcripts share MREs for miR-1270, they sponge and sequester the miR-1270 molecules. Collectively, IFN-A mRNAs and AS transcripts from IFN-A gene family form a ceRNA network to antagonize miR-1270 [23, 27]. This network finely tunes IFN-A1 mRNA levels by common MREs that are present in the mRNAs and AS transcripts at the post-transcriptional level. Possible interactions among transcripts are depicted in Figure 4. See detailed discussion in [28].
Figure 4.
A model of interactions among transcripts to regulate mRNA levels. mRNA1 has a site of interaction (loop) with AS transcript1 and an MRE for microRNA3. AS transcript1 stabilizes mRNA1, whereas microRNA destroys mRNA1 through its MRE. AS transcript2 transcribed from another gene has common MREs. AS transcript2 sponges microRNA3, resulting in the sequestration of microRNA3. Therefore, AS transcript2 competes with microRNA3 and functions as a ceRNA. A typical example is seen among IFN-A1 mRNA and AS transcripts from the specific subsets of IFN-A gene family. See details in the text.
2.2.3 Tnf mRNA-AS transcript interaction
Different from the iNOS and IFN-A1 mRNA cases, AS transcripts sometimes downregulate gene expression. The stability of Tnf mRNA is modulated by RNA-binding proteins that bind to the AREs in its 3′UTR: human homolog R of embryonic lethal-abnormal visual protein (HuR), which stabilizes the mRNA; and tristetraprolin (TTP), which destabilizes the mRNA [24]. A putative mechanism of mRNA destabilization is schematically shown in Figure 5 and discussed in [1, 2]. Possible involvement of microRNA in the Tnf mRNA-AS transcript interaction is also mentioned [24].
Figure 5.
A putative mechanism of the increase in mRNA levels by the introduction of a sense oligonucleotide (Tnf gene). (A) The Tnf mRNA that forms with a destabilizing RNA-binding protein. The Tnf AS transcript partially hybridizes with a single-stranded loop in the Tnf mRNA. An RNA-binding protein forms an mRNA-AS transcript-protein complex. (B) Interference with the mRNA-AS transcript interaction. A sense oligonucleotide to the Tnf mRNA competitively inhibits the hybridization of Tnf mRNA with the Tnf AS transcript, releasing a destabilizing RNA-binding protein. Finally, the Tnf mRNA becomes stable in the cytoplasm.
Other than these mechanisms, there are several AS transcript-mediated mechanisms that regulate gene expression [7]. For example, AS transcripts may epigenetically repress transcription at the chromatin level.
3.1 Natural antisense transcript-targeted regulation technology using sense oligonucleotides
If the mRNA-AS transcript interactions are inhibited, it is speculated that an mRNA-AS transcript-protein complex is not formed and that the mRNA becomes unstable [1, 2]. According to this hypothesis, we used single-stranded sense oligonucleotides to block the mRNA-AS transcript interactions at a post-transcriptional level. The sense oligonucleotide harbors the same sequence as that of the relevant mRNA.
We first applied the sense oligonucleotide corresponding to the iNOS mRNA sequence to decrease the iNOS mRNA levels in hepatocytes [19]. In a search of the literature to date, there are not any other reports on applying sense oligonucleotides to knockdown AS transcript(s) and finally mRNA. After refinement of this method and confirmation of its versatility, we established this method as a natural antisense transcript-targeted regulation (NATRE) technology using sense oligonucleotides [14, 18]. The introduction of sense oligonucleotides may regulate the mRNA levels of AS transcript-expressing genes. Similarly, the sense oligonucleotides to other mRNAs reduced the levels of IFN-A1 and other mRNAs [14, 20]. Therefore, the introduction of sense oligonucleotides may be used as loss-of-function experiments of AS transcripts.
In the absence of an AS transcript, a sense oligonucleotide cannot hybridize with the relevant mRNA and does not affect mRNA stability. Therefore, the presence of AS transcript and mRNA-AS transcript interactions are essential for the NATRE technology using sense oligonucleotides.
3.2 Design of sense oligonucleotides
3.2.1 Prediction of secondary structure
A sense oligonucleotide should harbor an overlapping sequence of an mRNA-AS transcript interaction, i.e., a single-stranded loop or bulge [18, 20]. The single-stranded loops are the potential sites of mRNA-AS transcript interactions. To seek the single-stranded regions of an mRNA where the relevant AS transcript interacts with, secondary structures of mRNA (especially, 3′UTR) were predicted using the mfold program [29]. Other prediction programs can be used. Regions conserved among predicted mRNA (especially 3′UTR) structures are selected, and candidates of several sense oligonucleotides are designed from the stem-loop regions.
Generally, it is unnecessary to predict the secondary structures of AS transcripts. As mentioned above, the overlapping sequence of an AS transcript is complementary to that of the corresponding mRNA. Therefore, the secondary structures of the AS transcript are a mirror image of the mRNA. The stems and loops of the AS transcript are formed at the same positions as the mRNA.
3.2.2 Design of sense oligonucleotides
The sense oligonucleotide consists of either synthetic oligodeoxyribonucleotide (DNA) or synthetic oligoribonucleotides (RNA) with modifications of the oligonucleotide backbone, sugars, bases, and the 5′-phosphate (described later). The sequences of sense oligonucleotides (about 20 nt long in our cases) designed according to the mRNA sequence included at least one single-stranded loop in the conserved region [14, 19]. From the sequences of sense oligonucleotides, the elements that may provoke innate immunity responses through Toll-like receptors (TLR3, 7, 8, and 9) should be eliminated, such as GU-rich motifs (e.g., 5′-GUGU-3′), CG, GGG, GGGG, and CCCC [14, 19, 30]. It is possible that some oligonucleotides show off-target effects, even after the exclusion of these motifs. To attain the specificity of a target mRNA and avoid off-target effects, homology search in the DDBJ/EMBL/GenBank databases should be performed. Trials and errors are necessary to select effective sense oligonucleotides among several candidates.
Note that not all the candidate sense oligonucleotides reduce the levels of specific mRNA species, whereas some oligonucleotides increase the mRNA levels. As above-mentioned, AS transcripts modulate the expression of each gene either positively or negatively; the AS transcript stabilizes mRNA (Figures 3 and 4) or destabilizes mRNA (Figure 5).
Changes in mRNA levels also depend on the region of the mRNA-AS transcript interactions [14, 24]. Six sense oligonucleotides to the 3′UTR of the rat Tnf mRNA were designed to the regions whose secondary structures were conserved [24]. Among them, only one sense oligonucleotide decreased Tnf mRNA levels, four increased, and one did not alter. RNA-binding proteins, e.g., HuR and TTP, may change the effect of each sense oligonucleotide.
Additionally, both several RNA-binding proteins and microRNAs control the mRNA stability (see Section 2.2). Therefore, it is difficult to predict whether knockdown of AS transcript using a sense oligonucleotide causes either an increase or decrease in mRNA levels (see also Section 3.3).
3.2.3 Negative controls of sense oligonucleotides
Several types of oligonucleotides are frequently used as negative controls. A negative control that is suitable for your experiments should be selected because not all the negative controls work well in the experiments.
3.2.3.1 Sense oligonucleotides to stems
A sense oligonucleotide to stem regions is used as a negative control. A stem region consists of double-stranded RNA:RNA hybrid and is not involved in the interactions with an AS transcript [20].
3.2.3.2 Mismatch oligonucleotides or mutated oligonucleotides
Mismatch oligonucleotides are designed by introducing mutated bases at the site of mRNA-AS transcript interactions [20, 31].
3.2.3.3 Random oligonucleotides
Random oligonucleotides (20 nt) harbor random sequences, i.e., 5′-N20-3′ (N = A, C, G, or T) with phosphorothioate bonds [18].
3.2.3.4 Scrambled oligonucleotides and mock transfection
A scrambled oligonucleotide is designed by base shuffling without changing the base composition of the relevant sense oligonucleotide [31]. Homology search screened by the BLAST program must eliminate candidates harboring unexpected homology to other mRNAs in the DDBJ/EMBL/GenBank databases.
When a sense oligonucleotide is introduced into cells using a transfection reagent, mock transfection is also necessary as a negative control of transfection. The mock transfection requires a transfection reagent alone, and an oligonucleotide is not introduced to the cells [18, 20].
3.2.4 Modification of sense oligonucleotides
A variety of nucleases are present in the cells and blood, such as exonuclease, endonuclease, and ribonuclease (RNase) H1. RNase activity is very high in various cell lines, as well as blood and cells in many organs, including the liver. To protect sense oligonucleotides from these nucleases, phosphorothioate bonds and modified nucleic acids are commonly introduced to replace the phosphodiester bonds and (deoxy)ribose rings of native nucleotides, respectively [32, 33]. Indeed, iNOS sense oligonucleotides without modification did not reduce iNOS mRNA levels [18]. Locked nucleic acid (LNA) [34] and 2′-O-methyl nucleic acid (OmeNA) are frequently used as modified nucleic acids. In our cases, iNOS sense oligonucleotides were substituted with partial phosphorothioate bonds and LNAs or OmeNAs reduced the levels of iNOS mRNA and iNOS protein in hepatocytes [18, 19]. This is a critical point at which modifications are included to obtain effective sense oligonucleotides.
3.2.5 Conjugation of sense oligonucleotides
When modified, but non-conjugated oligonucleotides are introduced in the body, they are transferred to the liver and kidney. To improve in vivo delivery to an organ or a tissue, sense oligonucleotides are often conjugated at their ends with cell-penetrating arginine-rich peptides and cell-permeable hydrophobic molecules [32, 33]. Cell-penetrating arginine-rich peptides are derived from the Tat protein of human immunodeficiency virus (HIV)-1 [35] and synthetic arginine oligomer peptides (Arg6); and cell-permeable hydrophobic molecules are cholesterol [36] and C12 spacer.
The conjugation does not affect the potency of iNOS oligonucleotides to decrease iNOS mRNA expression [18]. When an iNOS sense oligonucleotide conjugated to these molecules was introduced into hepatocytes, all conjugated sense oligonucleotides were as effectively decreasing iNOS mRNA levels as the non-conjugated sense oligonucleotide [18]. Appropriate conjugation may facilitate the delivery of sense oligonucleotides to target tissues or organs (see Section 3.7).
3.3 Regulation of mRNA levels by sense oligonucleotides in culture cells
Because the modification of sense oligonucleotides is essential, modified sense oligonucleotides were used for the introduction to cells. The in vitro effects of NATRE technology (including AntagoNAT technology) on mRNA levels in the cells are summarized in Table 1.
Examples of the in vitro effects of NATRE technology on mRNA levels.
Genes were classified by the main effect of sense oligonucleotides on the mRNA levels.
3.4 Administration of sense oligonucleotides to animals
When NO is excessively produced by iNOS in hepatocytes and Kupffer cells (resident macrophages) of the liver, it leads to multiple organ failure [38]. Endotoxemia model rats with hepatic failure are often used to evaluate drugs. These model rats are prepared either by intravenous injection of D-galactosamine (GalN) and LPS [38, 39, 40], or by LPS injection after partial hepatectomy [38, 41]. These rats resemble the animals suffering from sepsis or septic shock.
After optimization of the sequence and modification of iNOS sense oligonucleotides in hepatocytes, the best sense oligonucleotide was administered into the endotoxemia model rats [38]. When the sense oligonucleotide was intravenously injected with GalN and LPS to rats, the survival rate was markedly increased, and apoptosis in the hepatocytes markedly decreased in the sense oligonucleotide-treated rats [38].
Because LNA is efficiently accumulated in the liver [34], the LNA-modified iNOS sense oligonucleotide may function in the liver where the iNOS gene is highly expressed. Taken together, NATRE technology using iNOS sense oligonucleotides may be applicable to treat sepsis and septic shock.
3.5 AntagoNAT technology
To increase mRNA levels by modulating the mRNA-AS transcript interactions, an AntagoNAT oligonucleotide has been used, which is an antagonist to an AS transcript (NAT) and defined as a single-stranded antisense oligonucleotide to a specific AS transcript [37]. Each AntagoNAT oligonucleotide contained a mixture of OmeNAs and LNAs.
When an AS transcript overlaps with its corresponding mRNA, the AntagoNAT is identical to a sense oligonucleotide. Therefore, AntagoNAT technique is very close to NATRE technology. Both technologies use sense oligonucleotides to knockdown AS transcript. However, NATRE technology has been applied to decrease mRNA levels, whereas AntagoNAT technology has been applied to increase mRNA levels.
It has been reported that AntagoNAT-mediated knockdown of brain-derived neurotrophic factor (Bdnf) and glial-derived neurotrophic factor (Gdnf) AS transcripts resulted in increased levels of Bdnf and Gdnf mRNAs in HEK293T cells [37]. The underlying mechanism may be similar to those indicated in Figure 5, or to other mechanisms that were previously mentioned [7].
AntagoNAT oligonucleotides can be administered to animals. When Bdnf-AntagoNAT was intracerebroventricularly delivered, the Bdnf mRNA levels increased in the mouse brain [37]. Collectively, the Bdnf-AntagoNATs functioned in vitro and in vivo, although it increased the Bdnf mRNA levels.
AntagomiR (antagomir), which is a synthetic oligonucleotide complementary to a microRNA, is used to sequester endogenous microRNA [42]. Each antagomir sequence is identical to a specific mRNA and similar to several mRNAs that share microRNA-binding sites (seed sequences). Therefore, antagomirs are another type of sense oligonucleotides. When microRNA is involved in the mRNA-AS transcript interactions, the antagomir technology may be applied to analyze these interactions. See an example in [23].
3.6 Comparison with other methods
The mechanisms of two conventional technologies, i.e., antisense and siRNA technologies [11], are schematically shown (Figure 6).
Figure 6.
Mechanisms of conventional mRNA knockdown technologies. (A) Antisense technology. The mRNA that hybridizes with an antisense oligonucleotide is digested by RNase H1. (B) siRNA technology. siRNA recruits Argonaut (Ago) proteins to form RISC, which destroys mRNA. See details in the text.
3.6.1 Antisense technology
A single-stranded antisense oligonucleotide hybridizes with an mRNA and forms a local DNA:RNA hybrid. RNase H1 recognizes DNA:RNA hybrids and selectively digests the RNA strand, leading to the degradation of the mRNA. Therefore, the antisense oligonucleotides should be DNA. The presence of an AS transcript is not essential for this method.
3.6.2 siRNA technology
siRNA is a synthetic double-stranded RNA, and one strand of the siRNA ( i.e., guide strand) is complementary to a target mRNA. Typical siRNA consists of 19 base pairs and 2-nt 3′ overhangs. siRNA interacts with Argonaut (Ago) proteins to form RNA-induced silencing complex (RISC) and then binds to the target mRNA. The guide strand of siRNA hybridizes with the mRNA (especially, 3′UTR) in the RISC, resulting in degradation of the target mRNA. The other RNA strand (i.e., passenger strand) is destroyed during the RISC formation. This mechanism mimics mRNA degradation by microRNA.
As mRNA knockdown methods, NATRE technology using sense oligonucleotides is compared with conventional methods, i.e., antisense technology and siRNA technology (Table 2). Other than these technologies, there are various oligonucleotide technologies that are applied to therapies of disease.
The backbone, sugars, bases, and the 5′-phosphate of the oligonucleotide are modified.
MicroRNAs may interfere with these interactions.
3.6.3 RNA aptamers
Aptamers are oligoribonucleotides that form 3D structures and function like proteins, such as ligands and antibodies [42]. For example, pegaptanib, which is an aptamer drug developed for the treatment of macular degeneration, blocks vascular endothelial growth factor (VEGF) by preventing its binding to VEGF receptors [42]. Although there are no reports about the mRNA knockdown using aptamers up to date, the aptamers that simulate RNA-binding proteins may be utilized to modulate mRNA levels by affecting the mRNA-AS transcript interactions.
3.7 Drug delivery system (DDS)
The introduction of oligonucleotides to cells requires transfection reagents using liposomes, e.g., Lipofectamine (Thermo Fisher Scientific Inc., Waltham, MA, USA) and using iron nanoparticles, e.g., MATra A reagent (IBA, Göttingen, Germany) or PolyMag Magnetofection reagent (OZ Biosciences, Marseille, France).
To improve the in vivo delivery of sense oligonucleotides, several techniques have been developed [32, 33]. Because LNA is efficiently accumulated in the liver [34], iNOS sense oligonucleotides were modified with LNAs [38].
Recently, N-acetylgalactosamine (GalNAc) has been conjugated at the ends of the oligonucleotides to efficiently deliver the oligonucleotides to the liver [11, 43]. Because the liver and kidney receive high blood flow and permeability of their capillaries is high, sense oligonucleotides conjugated with GalNAc will improve the delivery and accumulation in these organs. To cross the blood-brain barrier, Bdnf-AntagoNATs in liposomes were delivered by nasal approaches [44, 45]. Additionally, aptamers capable of entering the cells may facilitate the delivery of oligonucleotides into the cells [42].
Because guinea pigs maintain a functional MX1 gene for the IFN-alpha1 pathway, they were infected with influenza virus to verify that the AS transcript-mRNA regulatory axis exerts in vivo control of innate immunity [46]. When an AS oligoribonucleotide (asORN) to guinea pig IFN-A1 mRNA in poly (D,L-lactide-co-glycolide (PLGA) nanoparticles was pulmonary-administered, it inhibited in vivo viral proliferation by modulating IFN-A1 mRNA levels. Although this experiment is not NATRE technology, PLGA nanoparticles can be used to deliver sense oligonucleotides to animals. Various lipid nanoparticles (LNPs) have been used to deliver nucleic acids, including oligonucleotides, in the body [47]. New-generation LNPs can deliver long RNA, such as LNP-based mRNA vaccines for COVID-19 [47].
Administration of oligonucleotides, including NATRE technology, is a unique therapeutic modality. Because the sequence of an oligonucleotide specifies the gene, one mRNA is selectively downregulated or upregulated (Figure 7). For example, administration of an iNOS sense oligonucleotide to endotoxemia/sepsis model rats showed little effects on endothelial NOS (eNOS) and neuronal NOS (nNOS) (unpublished data). Enzyme inhibitors (e.g., NOS inhibitors) generally have a broader specificity. Furthermore, it is rare that antibodies against oligonucleotides are raised.
Figure 7.
The NATRE technology using sense oligonucleotides. The principle of the NATRE technology is schematically shown. Potential therapeutic application using this technology is also shown.
NATRE technology is a powerful method to modulate in vitro and in vivo gene expression. Note that sense oligonucleotides to the mRNAs can be designed to inducible genes and many other genes. iNOS AS transcript was successfully administered to endotoxemia/sepsis model rats and improved their survival rate [38]. As shown in Table 1, many genes involved in inflammation are candidates suitable for clinical uses in the future. When a gene that is involved in diseases is selected, sense oligonucleotides to this gene can be easily designed and examined. Instead of sense oligonucleotides, antisense oligoribonucleotides (asORN) may be used to increase mRNA levels. Indeed, IFN-A1 asORN inhibited the proliferation of Influenza virus in guinea pigs [46].
Sense oligonucleotides may apply to cancer, neurodegenerative disorders, and other diseases. For example, EPHA2 is over-expressed in various cancers, and the EPHA2AS transcript may be involved in a subtype of breast cancer [26]. It is possible that sense oligonucleotides inhibit cancer progression and proliferation of cancer cells. Furthermore, the administration of Bdnf- and Gdnf-AntagoNATs to mice [44, 45] may facilitate the regeneration of neurons and glial cells in the central nervous system. When a gene that is involved in diseases is found, sense oligonucleotides to this gene can be designed. Furthermore, sense oligonucleotides can be designed in the genome of a pathogenic virus to inhibit viral multiplication.
Drugs and some constituents in functional foods and crude drugs of Japanese Kampo medicine mimic sense oligonucleotides by modulating mRNA stability [1]. When sodium salicylate reduced iNOS mRNA levels in hepatocytes, decreased mRNA stability was speculated [48]. It was reported that acetyl salicylate (aspirin) interacts with RNA by intercalating with the RNA duplex and destabilized the helix, resulting in a conformational change of the stem-loop structure of the RNA [49]. Because the drug-RNA interaction may affect mRNA stability, drugs and constituents may interfere with iNOS mRNA-AS transcript interactions like sense oligonucleotides. Indeed, several drugs or constituents in functional foods and crude drugs decreased the levels of both iNOS mRNA and AS transcript: dexamethasone (anti-inflammatory drug) [50], cucurbitacin B (triterpenoid in the fruit of Momordica charantia) [51], sakuranetin (flavonoid in the bark of Prunus jamasakura) [52], and standardized oligomerized-polyphenol from Litchi chinensis fruit extract (OPLFE) [53]. The investigation of the drugs and constituents may clarify the mechanism of action of the sense oligonucleotide in future.
When AS transcript is transcribed from a gene, NATRE technology can be applied to any gene to down- or up-regulate mRNA levels. NATRE technology using sense oligonucleotides may be useful to specifically inhibit mRNA-AS transcript interactions. Therefore, this method may be applied to many genes and contribute to the treatment of various human diseases in the future.
We thank Drs. Tominori Kimura, Tadayoshi Okumura, and Saki Shirako (Ritsumeikan University) for their valuable advice and discussion and Ms. Noriko Kanazawa for her secretarial assistance. This work was supported in part by the Japan Society for the Promotion of Science KAKENHI Grant Nos. 21K08697, 21K09032, and 19KK0413 and by the Asia-Japan Research Institute, Ritsumeikan University (Ibaraki, Osaka, Japan).
M.N. is an inventor of a patent describing the use of iNOS sense oligonucleotides to regulate iNOS mRNA levels. The other authors declare no conflict of interest.
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Written By
Mikio Nishizawa, Tetsuya Okuyama and Richi Nakatake
Submitted: 14 September 2022Reviewed: 26 September 2022Published: 17 October 2022
Open access peer-reviewed chapter
