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Antisense oligonucleotides (ASOs) have been developed as potential therapeutic agents for genetic disorders and various life‐threatening conditions. This class of therapeutics facilitates the treatment of the currently undruggable targets. However, their low stability and poor activity must be overcome by developing new analogs with improved functions. Therefore, in this study, we synthesized 4′‐C‐(S)‐ and 4′‐C‐(R)‐2‐aminopropoxy−thymidine and investigated the stability of the modified oligonucleotides containing these analogs. Interestingly, both analogs responded differently to thermal denaturation, with the former exhibiting good stability closer to that of the unmodified oligonucleotide and the latter exhibiting slight thermal destabilization. Notably, DNA/RNA heteroduplexes modified with these analogs elicited RNase H activity by degrading the heteroduplexes upon treatment. In addition, the serum stability of the modified oligodeoxynucleotides was significantly enhanced by the introduction of these analogs. Overall, our findings highlight the potential application of these analogs as ASO therapeutic candidates. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

[Display omitted] 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. [ABSTRACT FROM AUTHOR]

The synthesis and properties of oligonucleotides (ONs) containing 9-(2,3,4-trihydroxybutyl)adenine, A C2 and A C3 , are described. The ON containing A C2 involves the 3′ → 4′ and 3′ → 5′ phosphodiester linkages in the strand, whereas that containing A C3 possesses the 3′ → 4′ and 2′ → 5′ phosphodiester linkages. It was found that incorporation of the analogs, A C2 or A C3 , into ONs significantly reduces the thermal and thermodynamic stabilities of the ON/DNA duplexes, but does not largely decrease the thermal and thermodynamic stabilities of the ON/RNA duplexes as compared with the case of the ON/DNA duplexes. It was revealed that the base recognition ability of A C2 is greater than that of A C3 in the ON/RNA duplexes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: This Letter describes the synthesis and properties of double-stranded antisense oligonucleotides connected with a pentaerythritol linker. We found that double-stranded antisense oligonucleotides with aminomethyl residues have high affinity for single-stranded DNA or RNA in buffer solutions with and without MgCl2. Thus, these oligonucleotides would be useful as antisense oligonucleotides for targeting single-stranded RNA through triplex formation. [Copyright &y& Elsevier]

[Display omitted] • Synthesis of siRNAs containing a nonionic backbone and an aminoalkyl modified nucleoside is described. • The siRNA containing the nonionic backbone and an aminoalkyl modified nucleoside is more thermally stable than the corresponding an aminoalkyl-modified siRNA. • The nonionic backbone and an aminoalkyl modified RNA is more nuclease-resistant than a natural RNA. 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. [ABSTRACT FROM AUTHOR]

[Display omitted] 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. [ABSTRACT FROM AUTHOR]

The synthesis of oligonucleotide (ON) analogs, which can be used as antisense molecules, has recently gained much attention. Here, we report the synthesis and properties of an ON analog containing acyclic thymidine and cytidine analogs with a 4-pentyl-1,2-diol instead of the d -ribofuranose moiety. The incorporation of these analogs into the ON improved its nuclease resistance to 3′-exonucleases. Furthermore, it was found that the incorporation of the acyclic thymidine analog into a DNA/RNA duplex accelerates the RNA cleavage of a DNA/RNA duplex by Escherichia coli RNase H. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Synthesis and properties of double-stranded RNAs (dsRNAs) and small interfering RNAs (siRNAs) containing 4′- C -aminoethyl-2′-deoxy-2′-fluorouridine are described. Thermal denaturation studies showed that incorporation of 4′- C -aminoethyl-2′-fluoro analog improved the thermal stabilities of dsRNAs and siRNAs compared to the corresponding 4′- C -aminoethyl-2′- O -methyl analog. siRNA incorporating eight 4′-aminoethyl-2′-fluoro analogs in the passenger strand showed sufficient RNAi activity at 1 nM concentration, which was similar to that of the unmodified siRNA. Furthermore, the siRNA containing the 4′- C -aminoethyl-2′-fluoro analog exhibited high stability in a buffer containing 20% bovine serum. Forty-eight percent of the siRNA remained intact after 48 h of incubation. Thus, modification of siRNAs by the 4′- C -aminoethyl-2′-fluoro analog would be useful for the development of therapeutic siRNA molecules. [ABSTRACT FROM AUTHOR]

In this paper, we describe the synthesis of 4′- C -aminoalkyl-2′- O -methylnucleosides and the properties of RNAs containing these analogs. Phosphoramidites of 4′- C -aminoethyl and 4′- C -aminopropyl-2′- O -methyluridines were prepared using glucose as starting material, and RNAs containing the analogs were synthesized using the phosphoramidites. Thermal denaturation studies revealed that these nucleoside analogs decreased the thermal stabilities of double-stranded RNAs (dsRNAs). Results of NMR, molecular modeling, and CD spectra measurements suggested that 4′- C -aminoalkyl-2′- O -methyluridine adopts an C2′- endo sugar puckering in dsRNA. The 4′- C -aminoalkyl modifications in the passenger strand and the guide strand outside the seed region were well tolerated for RNAi activity of siRNAs. Single-stranded RNAs (ssRNAs) and siRNAs containing the 4′- C -aminoethyl and 4′- C -aminopropyl analogs showed high stability in buffer containing bovine serum. Thus, siRNAs containing the 4′- C -aminoethyl and 4′- C -aminopropyl analogs are good candidates for the development of therapeutic siRNA molecules. [ABSTRACT FROM AUTHOR]

4′- C -Aminomethyl-2′-deoxy-2′-fluoroarabinonucleoside increases the nuclease resistance of DNA without inhibiting its ability 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-β- 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. [ABSTRACT FROM AUTHOR]