Yusuke Maeda

Synthesis and properties of cationic oligopeptides with different side chain lengths that bind to RNA duplexes

A series of artificial peptides bearing cationic functional groups with different side chain lengths were designed, and their ability to increase the thermal stability of nucleic acid duplexes was investigated. The peptides with amino groups selectively increased the stability of RNA/RNA duplexes, and a relationship between the side chain length and the melting temperature (Tm) of the peptide-RNA complexes was observed. On the other hand, while peptides with guanidino groups exhibited a similar tendency with respect to the peptide structure and thermal stability of RNA/RNA duplexes, those with longer side chain lengths, such as L-2-amino-4-guanidinobutyric acid (Agb) or L-arginine (Arg) oligomers, stabilized both RNA/RNA and DNA/DNA duplexes, and those with shorter side chain lengths exhibited a higher ability to selectively stabilize RNA/RNA duplexes. In addition, peptides were designed with different levels of flexibility by introducing glycine (Gly) residues into the L-2-amino-3-guanidinopropionic acid (Agp) oligomers. It was found that insertion of Gly did not affect the thermal stability of the peptide-RNA complexes, but an alternate arrangement of Gly and Agp apparently decreased the thermal stability. Therefore, in the Agp oligomer, consecutive Agp sequences are essential for increasing the stability of RNA/RNA duplexes.

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.

Artificial cationic oligosaccharides for heteroduplex oligonucleotide-type drugs

Heteroduplex oligonucleotides (HDOs), composed of a DNA/LNA gapmer and its complementary RNA, are a novel, promising candidates for antisense drugs. We previously reported oligodiaminogalactoses (ODAGals), designed to bind to A-type nucleic acid duplexes such as DNA/RNA and RNA/RNA duplexes. In this paper, we report oligodiguanidinogalactoses (ODGGals) as novel A-type duplex binding molecules. We aimed to study in detail applicability of ODAGals and ODGGals for additives to HDOs as an antisense drug. The effect of ODAGal4 (ODAGal 4mer) and ODGGal3 (ODGGal 3mer) on an HDO were evaluated by UV melting analyses, RNA degradation study by ribonuclease A (RNase A), and ribonuclease H (RNase H). Cleavage of a 13mer HDO by RNase A, which is considered to be the main cause of RNA degradation in serum, was effectively inhibited by the addition of only one equivalent of ODAGal4 and ODGGal3. In contrast, RNase H activity, which involves the cleavage of target RNAs by an antisense mechanism, was only slightly affected by the presence of the cationic oligosaccharides. These results suggest that ODAGal4 and ODGGal3 are useful because they could both stabilize the HDO and maintain RNase H activity of the gapmer.

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.

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 1u202fnM 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 48u202fh of incubation. Thus, modification of siRNAs by the 4-C-aminoethyl-2-fluoro analog would be useful for the development of therapeutic siRNA molecules.

Solid-Phase Synthesis of Oligopeptides Containing Sterically Hindered Amino Acids on Nonswellable Resin Using 3-Nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphonium Hexafluorophosphate (PyNTP) as the Condensing Reagent

Peptides are still difficult to synthesize when they contain sterically hindered amino acids, such as α,α-disubstituted amino acids and N-substituted amino acids. In this study, solid-phase syntheses of oligopeptides containing multiple α-aminoisobutyric acid (Aib) residues were performed in high yields by using a nonswellable resin as the solid-support and 3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyNTP) as the condensing reagent.

Benzene-glycol nucleic acid (BGNA)–DNA chimeras: synthesis, binding properties, and ability to elicit human RNase H activity

This paper describes the synthesis and properties of benzene-glycol nucleic acid (BGNA)–DNA chimeras containing four nucleoside analogs – thymidine, cytidine, adenosine, and guanosine – with a base-benzene-glycol structure. We found that the BGNA–DNA chimeras are able to form thermally and thermodynamically stable duplexes with complementary RNAs, and have base-discriminating abilities. The BGNA–DNA chimeras were 20-fold more stable in a buffer containing 30% bovine serum than unmodified DNA. Furthermore, BGNA–DNA chimera/RNA duplexes were found to be good substrates for human RNase H. Thus, BGNA–DNA chimeras are good candidates for the development of therapeutic antisense molecules.

Synthesis of the Phosphoramidite Units for Benzene‐Glycol Nucleic Acid

Benzene‐glycol nucleic acid‐DNA chimeras form thermally and thermodynamically stable duplexes with complementary RNAs, and have base‐discriminating abilities. This unit describes the synthesis of four nucleoside analogs, an adenine, cytosine, thymine, and guanine analogs with base‐benzene‐glycol structure. The synthesis starts with conversion of (S)‐mandelic acid in arylboronic acid derivative, common intermediate. Nucleobase coupling of the intermediate and phosphitylation afford to phosphoroamidite units.

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.