Synthesis of 4′-C-aminoalkyl-2′-O-methyl modified RNA and their biological properties

Koizumi, Kana; Maeda, Yusuke; Kano, Toshifumi; Yoshida, Hisae; Sakamoto, Taiichi; Yamagishi, Kazumasa; Ueno, Yoshihito

doi:10.1016/j.bmc.2018.05.025

Cited by 32 publications

(48 citation statements)

References 43 publications

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“…Pinpointing the origin(s) of the slight loss in stability seen with Ufo, Ufme and uo relative to Uf may be quite challenging. Finally, the recently evaluated 4′- Cα -aminoalkyl-2′- O -methyl modifications appear to be more destabilizing in the context of RNA duplexes than the 4′- Cα -OMe and 4′- Cα -Me substituted uridines examined here as the former resulted in T m losses of between –2.0 and –2.3°C per incorporated nucleotide ( 38 ).…”

Section: Discussionmentioning

confidence: 80%

Structural basis for the synergy of 4′- and 2′-modifications on siRNA nuclease resistance, thermal stability and RNAi activity

Harp

Guenther

Bisbe

et al. 2018

Nucleic Acids Research

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Chemical modification is a prerequisite of oligonucleotide therapeutics for improved metabolic stability, uptake and activity, irrespective of their mode of action, i.e. antisense, RNAi or aptamer. Phosphate moiety and ribose C2′/O2′ atoms are the most common sites for modification. Compared to 2′-O-substituents, ribose 4′-C-substituents lie in proximity of both the 3′- and 5′-adjacent phosphates. To investigate potentially beneficial effects on nuclease resistance we combined 2′-F and 2′-OMe with 4′-Cα- and 4′-Cβ-OMe, and 2′-F with 4′-Cα-methyl modification. The α- and β-epimers of 4′-C-OMe-uridine and the α-epimer of 4′-C-Me-uridine monomers were synthesized and incorporated into siRNAs. The 4′α-epimers affect thermal stability only minimally and show increased nuclease stability irrespective of the 2′-substituent (H, F, OMe). The 4′β-epimers are strongly destabilizing, but afford complete resistance against an exonuclease with the phosphate or phosphorothioate backbones. Crystal structures of RNA octamers containing 2′-F,4′-Cα-OMe-U, 2′-F,4′-Cβ-OMe-U, 2′-OMe,4′-Cα-OMe-U, 2′-OMe,4′-Cβ-OMe-U or 2′-F,4′-Cα-Me-U help rationalize these observations and point to steric and electrostatic origins of the unprecedented nuclease resistance seen with the chain-inverted 4′β-U epimer. We used structural models of human Argonaute 2 in complex with guide siRNA featuring 2′-F,4′-Cα-OMe-U or 2′-F,4′-Cβ-OMe-U at various sites in the seed region to interpret in vitro activities of siRNAs with the corresponding 2′-/4′-C-modifications.

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Section: Discussionmentioning

confidence: 80%

Structural basis for the synergy of 4′- and 2′-modifications on siRNA nuclease resistance, thermal stability and RNAi activity

Harp

Guenther

Bisbe

et al. 2018

Nucleic Acids Research

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“…Previous studies showed that PS-type modification reduces the avidity of the synthetic miRNA with the target mRNA, and the AE-type modification reduces its avidity with the complementary strand. 38,39,[45][46][47][48][49] Therefore, in this study, we aimed to improve the avidity of the synthetic miRNAs with the target mRNA with 2 0 -OMe and 2 0 -F modifications of the 2 0 nucleotide region. Consequently, the synthetic miR-214 5AE reduced COP1 protein expression similar to that mediated by naturally occurring mature miR-214 and manifested a stronger anti-tumour activity than mature miR-214.…”

Section: Discussionmentioning

confidence: 99%

“…In the oligospermine method, spermine consisting of a polyamine that includes four nitrogen atoms was bound to the 3 0 end of the sense (passenger) strand. [38][39][40] The sequences of the synthetic miR-214s are shown in Figure 1. The synthetic miR-214s were synthesized using methods previously described.…”

Section: Mature Mir-214 and Synthetic Mir-214mentioning

confidence: 99%

“…In previous studies, siRNA modified with 4 0 -aminoethyl nucleoside showed enhanced RNase resistance and became hard to lyse. 38,39 Furthermore, it is known that nuclease resistance is improved by introducing siRNA-type modification and PS-type modification into siRNA. [45][46][47][48][49] We examined whether these chemically modified miR-214 showed nuclease resistance in this study (results are shown in Figure 7).…”

Section: Synthetic Mir-214s Were More Stable Than Mature Mir-214 Inmentioning

confidence: 99%

“…AE-type and OS-type modifications have been reported to increase the intracellular uptake of siRNA in previous studies. [38][39][40] We speculated that cells transfected with these chemically modified synthetic F I G U R E 4 Apoptotic cell count by Annexin V/PI double staining. All of synthetic miR-214s induced the early and late phases of apoptosis.…”

Section: Chemical Modification Improved the Quantity Of Mir-214 In/mentioning

confidence: 99%

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

Yoshikawa

Heishima

Ueno

et al. 2020

Vet Comparative Oncology

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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 0-aminoethyl-2 0-fluoro, 2 0-fluoro, 2 0-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.

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

Katsuzaki

Tsukimura

Chandela

et al. 2022

Chemistry & Biodiversity

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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.

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Synthesis of 4′-C-aminoalkyl-2′-O-methyl modified RNA and their biological properties

Cited by 32 publications

References 43 publications

Structural basis for the synergy of 4′- and 2′-modifications on siRNA nuclease resistance, thermal stability and RNAi activity

Structural basis for the synergy of 4′- and 2′-modifications on siRNA nuclease resistance, thermal stability and RNAi activity

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

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

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