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.

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.

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.

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.

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.

(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.

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.

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.

Diazirine-containing tag-free RNA probes for efficient RISC-loading and photoaffinity labeling of microRNA targets.

We designed and synthesized a photo-reactive and tag-free RNA probe for the identification of microRNA (miRNA) targets. To synthesize the RNA probe, we designed a novel nucleoside analog 1-O-[3-ethynyl-5-(3-trifluoromethyl-3H-diazirine-3-yl)]benzyl-ß-d-ribofuranose containing aryl trifluoromethyl diazirine and ethynyl moieties. The RNA probe containing this analog was observed to form crosslinks with complementary RNA by UV irradiation and was rapidly tagged by Cu-catalyzed azide alkyne cycloaddition (CuAAC). In addition, the tag-free and photo-reactive miRNA-145 probe showed comparable gene silencing activity to that of unmodified miRNA-145. Therefore, miRNA probes containing the nucleoside analog are promising candidates for the identification of target mRNAs of miRNAs.

Synthesis and properties of 4'-C-aminoalkyl-2'-fluoro-modified RNA oligomers.

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.

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

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.

Ni-Pd Catalyzed Cyclization of Sulfanyl 1,6-Diynes: Synthesis of 1'-Homonucleoside Analogues.

The Ni-Pd catalyzed addition-cyclization of sulfanyl 1,6-diynes 2-9 with nucleobases is described. The reactions of N-tethered 1,6-diynes with N3-benzoylthymine, N4,N4-bis(Boc)cytosine, N3-benzoyluracil and N6,N6-bis(Boc)adenine exclusively afforded the pyrrolylmethyl and furylmethyl nucleotides in good yields. Deprotection of nucleobases was completed by treatment with acids or bases. Furthermore, the reactions of pyrroles and furans with nucleophiles such as alkoxides and amines underwent detosylation and conversion to the alkoxymethyl- and arylaminomethyl-pyrroles and furans in good yields.

Synthesis of antisense oligonucleotides containing acyclic alkynyl nucleoside analogs and their biophysical and biological properties.

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.

Design and synthesis of novel photoinduced electron transfer-based hybridization probes.

Photoinduced electron transfer (PeT)-based hybridization probe is a linear and quencher-free oligonucleotide (ON) probe for DNA or RNA detection. In this report, we designed and synthesized novel adenosine analogues for PeT-based hybridization probe. In particular, the analogue containing a piperazinomethyl moiety showed effective quenching property under physiological conditions. When the probe containing the analogue was hybridized with a complementary DNA or RNA, the fluorescence increased 3- or 4-fold, respectively, compared to the single-stranded state.

Synthesis of a fluorescence resonance energy transfer-based probe containing a tricyclic nucleoside analog for single nucleotide polymorphism typing.

Here, we report the synthesis of a fluorescence resonance energy transfer (FRET)-based probe for single nucleotide polymorphism (SNP) typing. The probe contains a fluorescent tricyclic base, 8-amino-3-(2,3-dihydroxypropyl)imidazo[4',5':5,6]pyrido[2,3-d]pyrimidine, as a donor molecule and 7-diethylaminocoumarin-3-carboxylic acid as an acceptor molecule. FRET was observed between the donor and acceptor molecules on the probe. The identity of the target bases on DNA and RNA strands could be determined using the probe.

Photoinduced Electron-Transfer-Based Hybridization Probes for Detection of DNA and RNA.

Here, we report the synthesis of a hybridization probe for detection of RNA and DNA based on photoinduced electron transfer (PeT). We designed and synthesized an oligonucleotide containing an adenosine analogue with a 9-(N,N-dimethylaminomethyl)anthracenyl moiety at its 6-position via an ethynylene linker as the hybridization probe. When the probe was hybridized with a complementary RNA or DNA, the fluorescence intensity increased 3-fold or 4.5-fold, respectively, compared to the single-stranded state.

Incorporation of an acyclic alkynyl nucleoside analog into siRNA improves silencing activity and nuclease resistance.

In order to improve the silencing activity and nuclease resistance of small interfering RNA (siRNA), we designed and synthesized an acyclic thymidine analog containing 4-pentyne-1,2-diol instead of d-ribofuranose. The incorporation of this analog into siRNAs at specific positions in the strands was found to enhance the silencing activity of siRNAs and to increase the resistance of the siRNA to hydrolytic degradation by a 3' exonuclease.

Diazirine-containing RNA photo-cross-linking probes for capturing microRNA targets.

Here, we report the applicability of diazirine-containing RNA photo-cross-linking probes for the identification of microRNA (miRNA) targets. The RNA cross-linking probes were synthesized by substituting the RNA nucleobases with nucleoside analogues such as 1-O-[3-(3-trifluoromethyl-3H-diazirin-3-yl)]benzyl-ß-d-ribofuranose or 1-O-[4-(3-trifluoromethyl-3H-diazirin-3-yl)]benzyl-ß-D-ribofuranose that carry aryl trifluoromethyl diazirine moieties. The probes were successfully cross-linked with synthetic RNAs containing the four natural nucleosides on the opposite site of the nucleoside analogues. Furthermore, it was found that miRNAs containing these analogues were effective in regulating the expression of their target genes. Thus, RNAs containing the nucleoside analogues are promising candidates as photo-cross-linking probes to identify the target mRNAs of miRNAs.

A potent 2'-O-methylated RNA-based microRNA inhibitor with unique secondary structures.

MicroRNAs (miRNAs) are involved in various biological processes and human diseases. The development of strong low-molecular weight inhibitors of specific miRNAs is thus expected to be useful in providing tools for basic research or in generating promising new therapeutic drugs. We have previously described the development of 'Tough Decoy (TuD) RNA' molecules, which achieve the long-term suppression of specific miRNA activity in mammalian cells when expressed from a lentivirus vector. In our current study, we describe new synthetic miRNA inhibitors, designated as S-TuD (Synthetic TuD), which are composed of two fully 2'-O-methylated RNA strands. Each of these strands includes a miRNA-binding site. Following the hybridization of paired strands, the resultant S-TuD forms a secondary structure with two stems, which resembles the corresponding TuD RNA molecule. By analyzing the effects of S-TuD against miR-21, miR-200c, miR-16 and miR-106b, we have elucidated the critical design features of S-TuD molecules that will provide optimum inhibitory effects following transfection into human cell lines. We further show that the inhibitory effects of a single transfection of S-TuD-miR200c are quite long-lasting (>7 days) and induce partial EMT, the full establishment of which requires 11 days when using a lentivirus vector that expresses TuD-miR200c continuously.