Properties and synergistic effects of a nonionic backbone and aminoalkyl modified nucleosides in RNAs.

In this study, we report the synthesis of two formacetal (FA)-linked dimer building blocks, namely 2'-O-methyluridyl-2'-O-methyluridine and 2'-O-methyluridyl-2'-O-aminoethyluridine. We utilize the former dimer in combination with (S)-5'-C-aminopropyl-2'-O-methylnucleosides (5'-APs) as a neutral trimer unit, and the latter dimer as a cationic unit. Double-stranded RNA containing the neutral trimer unit exhibits greater stability compared to the cationic unit and maintains nuclease stability in a serum-containing buffer. Furthermore, this unit appears to establish additional hydrogen bonds with complementary bases, as supported by modeling simulations and mismatch melting temperature assays. Importantly, siRNAs modified with this unit enhance RNA interference activity in cultured cells. These findings suggest that the trimer unit holds promise for therapeutic siRNAs.

Non-terminal conjugation of small interfering RNAs with spermine improves duplex binding and serum stability with position-specific incorporation.

The conjugation of small interfering RNAs (siRNAs) has been studied using lipid and ligand conjugates for efficient delivery. However, most conjugates have been inserted at the terminal position; very few have been inserted at non-terminal positions. Herein, we synthesized a 4'-C-propyllevulinate-2'-O-methyluridine analog for non-terminal conjugation of spermine into the passenger strand of siRNA. Solid-phase oligonucleotide synthesis using this analog was successful, with the conjugation of one or two spermine molecules. The siRNAs conjugated with spermine displayed improved thermodynamic stability and resistance against nucleases, which depended on the site of conjugation in each case. Circular dichroism spectroscopy revealed that the A-type helical structure of the RNA duplex was not altered by these modifications. However, the gene-silencing activity of conjugated siRNAs was reduced and further decreased when the number of spermine molecules was increased. Hence, this work supplies valuable information and provides scope for the further development of drug-delivery systems through non-terminal conjugation.

Synthesis and characterization of novel (S)-5'-C-aminopropyl-2'-fluorouridine modified oligonucleotides as therapeutic siRNAs.

The lack of stability of natural nucleosides limits their application in small interfering RNA (siRNA)-mediated RNA interference (RNAi). Various chemical modifications have been reported to improve their pharmacokinetic behavior; however, the development of potential candidates is still underway. In this study, we designed and synthesized (S)-5'-C-aminopropyl-2'-fluorouridine (5'-AP-2'-FU) and evaluated the properties of siRNAs containing this analog. A comparative thermodynamic study revealed the enhanced thermal stability of double-stranded RNAs (dsRNAs) containing 5'-AP-2'-FU in a position-specific manner, whereas (S)-5'-C-aminopropyl-2'-O-methyluridine (5'-AP-2'-MoU)-modified dsRNAs exhibited lower melting temperatures. This improved thermal stability of RNA duplexes is attributed to favorable entropy loss, which induces the duplex into an N-type (C3'-endo) conformation and enhances duplex　binding in this case. The 5'-AP-2'-FU analog was also suitable for incorporation into the passenger strand to induce gene-silencing activity. Gene knockdown efficacy was comparable to that of unmodified siRNAs, and the best response was observed by introducing 5'-AP-2'-FU near the 3'-terminal end of the passenger strand. In addition, the single-stranded RNAs (ssRNAs) modified with 5'-AP-2'-FU showed strong resistance against decomposition by nucleases when treated with buffer containing bovine serum, which was similar to 5'-AP-2'-MoU.

Antisense Gapmers with LNA-Wings and (S)-5'-C-Aminopropyl-2'-arabinofluoro-nucleosides Could Efficiently Suppress the Expression of KNTC2.

Previously reported (S)-5'-C-aminopropyl-2'-arabinofluoro-thymidine (5ara-T) and newly synthesized (S)-5'-C-aminopropyl-2'-arabinofluoro-5-methyl-cytidine (5ara-MeC) analogs were incorporated into a series of antisense gapmers containing multiple phosphorothioate (PS) linkages and locked nucleic acids (LNAs) in their wing regions. The functional properties of the gapmers were further evaluated in vitro. Compared with the positive control, for the LNA-wing full PS gapmer without 5ara modification, it was revealed that each gapmer could have a high affinity and be thermally stable under biological conditions. Although the cleavage pattern was obviously changed; gapmers with 5ara modification could still efficiently activate E. coli RNase H1. In addition, incorporating one 5ara modification into the two phosphodiester linkages could reverse the destabilization in enzymatic hydrolysis caused by fewer PS linkages. In vitro cellular experiments were also performed, and the Lipofectamine® 2000 (LFA)+ group showed relatively higher antisense activity than the LFA-free group. KN5ara-10, which contains fewer PS linkages, showed similar or slightly better antisense activity than the corresponding full PS-modified KN5ara-3. Hence, KN5ara-10 may be the most promising candidate for KNTC2-targeted cancer therapy.

Synthesis of 4'-C-(Aminoethyl)thymidine and 4'-C-[(N-Methyl)aminoethyl] Thymidine Nucleosides to Enhance DNA Stability.

Antisense oligonucleotide (ASO) therapeutics target the pathogenic mRNA directly and modulate protein expression. Novel chemical modifications help to improve the action of ASOs with better thermal stability and resistance against nucleases. Oligodeoxynucleotides (ODNs) containing 4'-C-(aminoethyl)thymidine modifications exhibit efficient and stable hybridization with complementary DNA as well as RNA strands showing remarkably improved resistance against nucleolytic hydrolysis, which makes them promising candidates for antisense therapeutics. This article describes the synthesis of a novel nucleoside analog, 4'-C-[(N-methyl)aminoethyl]-thymidine (4'-MAE-T), 3, and previously reported 4'-C-aminoethyl-thymidine (4'-AE-T), 2, through a newly designed synthetic route to obtain a high overall yield. This has been established by changing the starting material from thymidine to diacetone-D-glucofuranose and synthesizing the known 4-C-hydroxyethyl pentofuranose. Conversion of the hydroxy group to an azide functional group through Mitsunobu azidation and performing acetolysis, provide the common intermediate 4-C-(2-azidoethyl)-ribofuranose. Subsequent coupling of the thymine nucleobase with the common intermediate under Vorbrüggen glycosylation conditions provides the corresponding modified nucleoside in high yield. It was subjected for conversion of the azide to an amine by Staudinger reaction and 2'-deoxygenation using Barton-McCombie conditions. Debenzylation with Lewis acid and mono-dimethoxytritylation of the 5'-OH afforded a fully protected 3'-OH intermediate for phosphitylation to give the corresponding phosphoramidites. In the case of 4'-MAE-T, benzyloxymethyl protection of the N3 -position and methylation were carried out prior to debenzylation. These phosphoramidite monomers were suitable with conventional oligonucleotide synthesis, and imparted ameliorated nuclease resistance, and competent RNase H activity, suggesting its potential utilization in ASO drugs. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of 4-C-(2-azidoethyl)-ribofuranose (6) Basic Protocol 2: Synthesis of 4'-C-aminoethyl thymidine phosphoramidite (15) Basic Protocol 3: Synthesis of 4'-C-(N-methyl)aminoethyl thymidine phosphoramidite (20).

4'-C-Aminoethoxy-Modified DNAs Exhibit Increased Nuclease Resistance, Sustained RNase H Activity, and Inhibition of KRAS Gene Expression.

The linear synthesis of 4'-C-aminoethoxy thymidine (AEoT) nucleoside phosphoramidite was accomplished using deoxythymidine as the starting material. This analog was incorporated into several oligonucleotides, the applicability of which as antisense oligonucleotides (ASOs) was then evaluated. The AEoT-modified DNA/RNA duplex exhibited improved thermal stability compared to unmodified and 4'-C-aminoethyl thymidine (4'-AET) modified heteroduplexes. The serum stability of AEoT-modified DNA was notably increased by several-folds compared to that of unmodified DNA. Furthermore, RNase H-dependent cleavage of the modified-DNA/RNA hybrids was found to be sustained. In addition, the modified antisense and unmodified oligonucleotides also displayed relatively comparable inhibition of the KRAS gene in human lung cancer cells. This study strengthens our understanding of the potential application of 4'-C-aminoethoxy-modified nucleotides as ASO therapeutics.

Synthesis, gene silencing activity, thermal stability, and serum stability of siRNA containing four (S)-5'-C-aminopropyl-2'-O-methylnucleosides (A, adenosine; U, uridine; G, guanosine; and C, cytidine).

Herein, we report the synthesis of (S)-5'-C-aminopropyl-2'-O-methyladenosine and (S)-5'-C-aminopropyl-2'-O-methylguanosine phosphoramidites and the properties of small interfering RNAs (siRNAs) containing four (S)-5'-C-aminopropyl-2'-O-methylnucleosides (A, adenosine; U, uridine; G, guanosine; and C, cytidine). The siRNAs containing (S)-5'-C-aminopropyl-nucleosides at the 3'- and 5'-regions of the passenger strand were well tolerated for RNA interference (RNAi) activity. Conversely, the (S)-5'-C-aminopropyl modification in the central region of the passenger strand decreased the RNAi activity. Furthermore, the siRNAs containing three or four consecutive (S)-5'-C-aminopropyl-2'-O-methylnucleosides at the 3'- and 5'-regions of the passenger strand exhibited RNAi activity similar to that of the corresponding 2'-O-methyl-modified siRNAs. Finally, it was observed that (S)-5'-C-aminopropyl modifications effectively improved the serum stability of the siRNAs, compared with 2'-O-methyl modifications. Therefore, (S)-5'-C-aminopropyl-2'-O-methylnucleosides would be useful for improving the serum stability of therapeutic siRNA molecules without affecting their RNAi activities.

Synthesis of 4'-C-(aminoethyl)thymidine and 4'-C-[(N-methyl)aminoethyl]thymidine by a new synthetic route and evaluation of the properties of the DNAs containing the nucleoside analogs.

A gapmer-type antisense oligonucleotide is an oligonucleotide therapeutic that targets pathogenic mRNA directly, and it is expected to be a next-generation therapeutic drug. In this study, we designed and synthesized 4'-C-[(N-methyl)aminoethyl]-thymidine (4'-MAE-T) as a novel nucleoside analog and compared its properties with those of 4'-C-aminoethyl-thymidine (4'-AE-T). Furthermore, we designed a new synthetic route for 4'-C-aminoethyl-modified nucleosides and accomplished the synthesis of 4'-AE-T via a novel pathway with high total yield. DNA containing 4'-MAE-T analogs decreased RNA affinity slightly more than unmodified DNA and DNA containing 4'-AE-T, but significantly improved nuclease resistance compared to unmodified DNA in a solution containing bovine serum. In addition, the impact of 4'-MAE-T on DNA stability was higher than that of 4'-AE-T. Also, DNA containing these analogs can activate Escherichia coli-derived RNase H. Thus, 4'-MAE-T has the potential to be used in gapmer-type antisense nucleic acids as a suitable candidate for the development of therapeutic antisense oligonucleotides.

Intraperitoneal administration of synthetic microRNA-214 elicits tumor suppression in an intraperitoneal dissemination mouse model of canine hemangiosarcoma.

Canine hemangiosarcoma (HSA) has an extremely poor prognosis, making it necessary to develop new systemic treatment methods. MicroRNA-214 (miR-214) is one of many microRNAs (miRNA) that can induce apoptosis in HSA cell lines. Synthetic miR-214 (miR-214/5AE), which showed higher cytotoxicity and greater nuclease resistance than mature miR-214, has been developed for clinical application. In this study, we evaluated the effects of miR-214/5AE on stage 2 HSA in a mouse model. Mice intraperitoneally administered with miR-214/5AE (5AE group) had significantly fewer intraperitoneal dissemination tumor foci (median number: 72.5 vs. 237.5; p < 0.05) and a lower median foci weight (0.26 g vs. 0.61 g; p < 0.05). Mice in the 5AE group had increased expression of p53 and cleaved caspase-3, and a significantly lower proportion of Ki-67-positive cells, than those in the non-specific miR group. Notably, no significant side effects were observed. These results indicate that intraperitoneal administration of miR-214/5AE exhibits antitumor effects in an intraperitoneal dissemination mouse model of HSA by inducing apoptosis and suppressing cell proliferation. These results provide a basis for future studies on the antitumor effect of miR-214/5AE for HSA.

(S)-5'-C-Aminopropyl-2'-O-methyl nucleosides enhance antisense activity in cultured cells and binding affinity to complementary single-stranded RNA.

Antisense oligonucleotides (ASOs) are a promising clinical tool that could be applied for unmet medical needs, but there are several limitations for their therapeutic application. Here, we designed and synthesized (S)-5'-C-aminopropyl-2'-O-methylcytidine, and oligonucleotides containing (S)-5'-C-aminopropyl-2'-O-methyluridine and -methylcytidine. We then investigated the properties of ASOs containing these nucleoside analogs. (S)-5'-C-Aminopropyl modifications enhanced the thermal stability of DNA/RNA duplexes when compared to other commercially available 2'-O-methyl modifications. This suggested that the terminal ammonium cation on the alkyl side chains neutralized the negative charge of the phosphates in the duplex. Additionally, the overall conformation of ASO/RNA duplexes was retained with the modified ASOs. Thus, these duplexes exhibited the ability to elicit RNase H activity. Furthermore, we found that ASOs containing the (S)-5'-C-aminopropyl modification exhibited higher antisense potency than those containing the 2'-O-methyl modification in cultured cells. Therefore, the (S)-5'-C-aminopropyl-2'-O-methyl nucleosides synthesized in this study are promising candidates for developing antisense therapeutics.

IIAEK Targets Intestinal Alkaline Phosphatase (IAP) to Improve Cholesterol Metabolism with a Specific Activation of IAP and Downregulation of ABCA1.

IIAEK (Ile-Ile-Ala-Glu-Lys, lactostatin) is a novel cholesterol-lowering pentapeptide derived from bovine milk ß-lactoglobulin. However, the molecular mechanisms underlying the IIAEK-mediated suppression of intestinal cholesterol absorption are unknown. Therefore, we evaluated the effects of IIAEK on intestinal cholesterol metabolism in a human intestinal model using Caco-2 cells. We found that IIAEK significantly reduced the expression of intestinal cholesterol metabolism-associated genes, particularly that of the ATP-binding cassette transporter A1 (ABCA1). Subsequently, we chemically synthesized a novel molecular probe, IIXEK, which can visualize a complex of target proteins interacting with photoaffinity-labeled IIAEK by fluorescent substances. Through photoaffinity labeling and MS analysis with IIXEK for the rat small intestinal mucosa and intestinal lipid raft fractions of Caco-2 cells, we identified intestinal alkaline phosphatase (IAP) as a specific molecule interacting with IIAEK and discovered the common IIAEK-binding amino acid sequence, GFYLFVEGGR. IIAEK significantly increased IAP mRNA and protein levels while decreasing ABCA1 mRNA and protein levels in Caco-2 cells. In conclusion, we found that IIAEK targets IAP to improve cholesterol metabolism via a novel signaling pathway involving the specific activation of IAP and downregulation of intestinal ABCA1.

4'-C-Aminomethyl-2'-deoxy-2'-fluoroarabinonucleoside increases the nuclease resistance of DNA without inhibiting the ability of a DNA/RNA duplex to activate RNase H.

An antisense oligonucleotide is expected as an innovative drug for cancer and hereditary diseases. In this paper, we designed and synthesized DNAs containing a novel nucleoside analog, 1-(4-C-aminomethyl-2-deoxy-2-fluoro-ß-d-arabinofuranosyl)thymine, and evaluated their properties. It was revealed that the analog slightly decreases the thermal stability of the DNA/RNA duplex but significantly increases the stability of DNA in a buffer containing bovine serum. Furthermore, it turned out that the DNA/RNA duplex containing the analog is a good substrate for Escherichia coli RNase H. Thus, DNAs containing the nucleoside analog would be good candidates for the development of therapeutic antisense oligonucleotides.

Development of synthetic microRNA-214 showing enhanced cytotoxicity and RNase resistance for treatment of canine hemangiosarcoma.

MicroRNA-214 (miR-214), a pivotal tumour-suppressive miRNA, is downregulated in canine hemangiosarcoma (HSA) cells. Although these tumour-suppressive miRNAs are potential therapeutic agents, their clinical efficacy may be limited because of their vulnerability to RNase-rich microenvironments and low in vivo transfection rates. We developed synthetic miR-214s with enhanced cytotoxicity, RNase resistance and quantity of miR-214 in/on cells. These synthetic miR-214s were synthesized by various chemical modifications (such as 4'-aminoethyl-2'-fluoro, 2'-fluoro, 2'-O-methyl, phosphorothioate and oligospermine modifications) of the wild-type mature miR-214 sequences. Transfection of HSA cells with synthetic miR-214 (miR-214 5AE) demonstrated significant growth suppressive effect and induced the strongest apoptotic response. Synthetic miR-214s (miR-214 5AE, miR-214 10AE and miR-214 OS) were much more stable than mature miR-214s in foetal bovine serum. Similar to mature miR-214, 5AE and OS suppressed the expression level of COP1 in HSA cells. The quantity of synthetic miR-214s in/on cells was higher than that of mature miR-214. In conclusion, we developed a clinically applicable, synthetic miR-214 5AE that regulates the COP1 protein expression similar to that mediated by mature miR-214. Additionally, miR-214 5AE confers better cytotoxicity, nuclease resistance and transfection rate than mature miR-214. Thus, miR-214 5AE could potentially be a novel miRNA-based chemotherapeutic agent that could improve the prognosis of HSA. Its in vivo effects on canine HSA need to be examined in future.

Synthesis and characterization of 4'-C-guanidinomethyl-2'-O-methyl-modified RNA oligomers.

This study investigated the synthesis and properties of 4'-C-guanidinomethyl-2'-O-methyluridine and RNAs containing the analog. Thermal and thermodynamic stabilities of double-stranded RNAs (dsRNAs) containing the nucleoside analog were examined. It was found that although the analog decreased the thermal and thermodynamic stabilities of dsRNA, it had base-discrimination ability. The 4'-C-guanidinomethyl modification increased stability of RNAs in a buffer containing serum. Furthermore, small interference RNAs incorporating one analog at the passenger strand still preserved their RNA interference activities. It was suggested that the 4'-guanidinomethyl modification significantly improved cell membrane permeability of RNA. Thus, 4'-C-guanidinomethyl-2'-O-methyl analogs may be useful in improving the properties of therapeutic siRNA molecules.

Synthesis of siRNAs incorporated with cationic peptides R8G7 and R8A7 and the effect of the modifications on siRNA properties.

Small interfering RNA (siRNA) can be used as an innovative next-generation drug. However, there are several challenges in the therapeutic application of siRNAs, including their low cell membrane permeability. In this study, we designed and synthesized siRNAs, incorporating the cationic peptides R8G7 and R8A7 to improve cell membrane permeability of siRNAs. Thermal denaturation studies revealed that R8G7 and R8A7 modifications increased the thermal stability of the siRNA duplexes. Incorporating these peptides at the 3'-ends of the siRNA passenger strands increased the stability of the siRNAs in a buffer containing bovine serum. Further, we found that the peptide-siRNA conjugates did not show sufficient RNA interference (RNAi) activity in the absence of the transfection reagent; however, when the transfection reagent was used, the peptide-siRNA conjugates preserved their RNAi activity.

Synthesis and evaluation of (S)-5'-C-aminopropyl and (S)-5'-C-aminopropyl-2'-arabinofluoro modified DNA oligomers for novel RNase H-dependent antisense oligonucleotides.

We designed and synthesized two novel thymidine analogs: (S)-5'-C-aminopropyl-thymidine and (S)-5'-C-aminopropyl-2'-ß-fluoro-thymidine. Then, DNA oligomers containing these analogs were synthesized, and their functional properties were evaluated. Compared with the naturally occurring thymidine, it was revealed that (S)-5'-C-aminopropyl-2'-arabinofluoro-thymidine was sufficiently thermally stable, while (S)-5'-C-aminopropyl-thymidine featured thermal destabilization. The difference in thermal stability resulted from a moderate change in the secondary structure of the DNA/RNA duplexes and a molecular fluctuation in monomers derived from the (S)-5'-C-aminopropyl side chain, as well as from a variation in sugar puckering derived from the 2'-arabinofluoro modification. Meanwhile, the incorporation of these analogs significantly enhanced the nuclease resistance of the DNA oligomers. Moreover, the (S)-5'-C-aminopropyl-2'-arabinofluoro-modified DNA/RNA duplexes showed a superior ability to activate RNase H-mediated cleavage of the RNA strand compared to the (S)-5'-C-aminopropyl-modified DNA/RNA duplexes.

Synthesis and Biophysical Characterization of RNAs Containing ( R)- and ( S)-5'- C-Aminopropyl-2'- O-methyluridines.

We designed and synthesized ( R)-5'- C-aminopropyl-2'- O-methyluridine and ( S)-5'- C-aminopropyl-2'- O-methyluridine, which are applicable to small interfering RNAs (siRNAs). We have evaluated the properties of siRNAs containing ( R)-5'- C-aminopropyl-2'- O-methyl and ( S)-5'- C-aminopropyl-2'- O-methyl modifications and have compared them to that of the 4'- C-aminopropyl-2'- O-methyl modification. Although these modifications decreased the thermal stability of double-stranded RNAs (dsRNAs) and siRNAs, the dsRNA containing the ( S)-5'- C-aminopropyl-2'- O-methyl modification showed the highest melting temperature ( Tm) among them. Silencing activity of the modified siRNAs was assessed by a dual luciferase reporter assay using HeLa cells. Incorporation of the ( R)-5'- C-aminopropyl-2'- O-methyl and ( S)-5'- C-aminopropyl-2'- O-methyl modifications on a passenger and guide strand was found to be tolerated for the silencing activity of siRNAs except for in the seed region on the guide strand. Furthermore, these modifications significantly increased the stability of single-stranded RNAs (ssRNAs) and siRNAs in a buffer containing bovine serum.

Synthesis and characterization of small interfering RNAs with haloalkyl groups at their 3'-dangling ends.

With the aim to create a small interfering RNA (siRNA) with enhanced activity and resistance to nuclease degradation, we synthesized and evaluated the properties of the following siRNAs containing haloalkyl ß-d-ribofuranosides at their 3'-dangling ends: 2,2,2-trifluoroethyl ß-d-ribofuranoside, 2,2,2-trichloroethyl ß-d-ribofuranoside and 2,2,2-tribromoethyl ß-d-ribofuranoside. The gene silencing activities of the modified siRNAs were investigated through a dual luciferase reporter assay using HeLa cells. The highest silencing activity was observed for the trichloroethyl analog modified siRNA, which was closely followed by the trifluoroethyl and tribromoethyl analogs. The modified siRNAs were found to show increased binding affinity towards the Piwi-Argonaute-Zwille (PAZ) domain protein based on computational analysis and an experimental study. Furthermore, the RNAs modified with the analogs at their 3'-ends exhibited improved resistance to hydrolysis by a 3'-exonuclease.

Design, synthesis and evaluation of novel, branched trident small interfering RNA nanostructures for sequence-specific RNAi activity.

Small interfering RNAs (siRNAs) are potential candidates for gene regulation with efficient activity, but off-target effects and limited systemic delivery. Herein, we report the design and synthesis of the branched siRNA nanostructures with highly improved resistance against exonucleases. Also, these branched siRNAs showed suppression of off-target gene silencing through selection of the passenger strand as the branching unit. The physical characterization of branched siRNAs showed that they form a compact assembly with a hydrodynamic diameter of 6.9 nm against 2.8 nm of the duplex. We demonstrated that a branched siRNA synthesized with a trebling solid-support selectively exhibits RNAi activity and suppresses the off-target effect.

Enhanced Intercellular Delivery of cRGD-siRNA Conjugates by an Additional Oligospermine Modification.

Small interfering RNA (siRNA), consisting a 21-mer duplex molecule, is often modified by conjugation with specific ligands to enhance its capacity for tissue-specific delivery. However, these attempts are hampered by the low permeability of negatively charged RNA molecules to enter the cell membrane. In this study, we designed and synthesized siRNA conjugates modified with cationic oligospermine and cyclic RGD (cRGD) to overcome the low-membrane permeability of siRNA. The siRNA conjugate, which contains 15 spermines and a cRGD peptide, showed sufficient gene-silencing activity at 250 nM final concentration without a transfection reagent. Under these conditions, the cationic oligospermine and cRGD-siRNA conjugate did not show any cytotoxicity.