Synthesis and Properties of Nucleobase-Sugar Dual Modified Nucleic Acids: 2'-OMe-RNA and scpBNA Bearing a 5-Hydroxycytosine Nucleobase.

Naturally occurring 5-hydroxycytosine (5-OHCyt), which is associated with DNA damage, was recently found to reduce the hepatotoxicity of antisense oligonucleotides (ASOs) without compromising its antisense activity when used as a replacement for cytosine (Cyt). Additionally, sugar-modified nucleic acids, such as 2'-O-methylribonucleic acid (2'-OMe-RNA) and 2'-O,4'-C-spirocyclopropylene-bridged nucleic acid (scpBNA), have emerged as useful antisense materials. Herein, we aimed to combine these two advantages by designing dual modified nucleic acids 2'-OMe-RNA-5-OHCyt and scpBNA-5-OHCyt bearing the 5-OHCyt nucleobase to develop efficient and safe ASOs. We describe the synthesis of 2'-OMe-RNA-5-OHCyt and scpBNA-5-OHCyt phosphoramidites and their incorporation into oligonucleotides (ONs). The duplex-forming ability and base discrimination properties of 2'-OMe-RNA-5-OHCyt- and scpBNA-5-OHCyt-modified ONs were similar to those of 2'-OMe-RNA-Cyt- and scpBNA-mCyt-modified ONs, respectively. We also synthesized two 2'-OMe-RNA-5-OHCyt-modified ASOs, and one of the two was found to exhibit reduced hepatotoxicity while retaining target mRNA knockdown activity in in vivo experiments.

Synthesis and duplex-forming ability of oligonucleotides modified with 4'-C,5'-C-methylene-bridged nucleic acid (4',5'-BNA).

We describe herein the design and synthesis of 4'-C,5'-C-methylene-bridged nucleic acid (4',5'-BNA), a novel artificial nucleic acid with the torsion angle γ in a non-canonical +ac range. The 4',5'-BNA phosphoramidite bearing a thymine nucleobase was synthesized from a commercially available thymidine analog in 11 steps and successfully incorporated into oligonucleotides. The resulting oligonucleotides were evaluated for their duplex-forming ability toward single-stranded DNA and RNA.

Synthesis and Properties of Oligonucleotides Having Ethynylphosphonate Linkages.

Ethynylphosphonate (EP)-linked thymidine dimers were synthesized via a palladium-catalyzed cross-coupling reaction and successfully incorporated into oligonucleotides. The oligonucleotides containing EP linkages appropriately formed a duplex with their complementary single-stranded RNA (ssRNA) and single-stranded DNA. The oligonucleotides containing both the EP linkages and 2'-O,4'-C-methylene-bridged nucleic acid/locked nucleic acid exhibited strong duplex-forming ability toward the complementary ssRNA. The EP-modified oligonucleotides exhibited higher exonuclease resistances than their natural counterparts. Moreover, one EP modification to a gapmer-type antisense oligonucleotide resulted in a switch of the cleavage site in the target ssRNA. Therefore, the EP modification can be applied for controlling the cleavage site in the RNase H-dependent mechanism.

Hybridization and Mismatch Discrimination Abilities of 2',4'-Bridged Nucleic Acids Bearing 2-Thiothymine or 2-Selenothymine Nucleobase.

Oligonucleotides modified with 2'- O,4'- C-spirocyclopropylene-bridged nucleic acid (scpBNA) exhibit excellent duplex-forming ability with their complementary single-stranded RNA (ssRNA). Here, we demonstrate that scpBNA bearing a 2-thiothymine (scpBNA-S2T) or 2-selenothymine (scpBNA-Se2T) nucleobase provides robust mismatch discrimination capabilities to oligonucleotides without compromising their high binding affinities toward the full complementary ssRNA. X-ray crystallographic analysis of a self-assembling oligonucleotide featuring 2',4'-BNA/LNA-2-thiothymine (2',4'-BNA/LNA-S2T, where 2',4'-BNA and LNA stand for "2'- O,4'- C-methylene-bridged nucleic acid" and "locked nucleic acid", respectively), a prototype of scpBNA-S2T, revealed that the 2-thiocarbonyl moiety plays a crucial role in the destabilization of thymine-guanine mismatched wobble base pairs.

Synthesis of scpBNA-mC, -A, and -G Monomers and Evaluation of the Binding Affinities of scpBNA-Modified Oligonucleotides toward Complementary ssRNA and ssDNA.

We previously reported the synthesis and evaluation of 2'-O,4'-C-spirocyclopropylene-bridged nucleic acid (scpBNA) bearing a thymine (T) nucleobase. Oligonucleotides (ONs) modified with scpBNA-T exhibited strong binding affinity to complementary single-stranded RNA (ssRNA) and high enzymatic stability. These biophysical properties suggest that scpBNAs are well suited for use in antisense strategies. Herein, we describe the synthesis of scpBNA monomers bearing 5-methylcytosine (mC), adenine (A), and guanine (G) nucleobases for use in a variety of sequences. The prepared scpBNA monomers were incorporated into ONs at various positions. The scpBNA-modified ONs exhibited excellent duplex-forming ability with the complementary ssRNA comparable to ONs modified with 2'-O,4'-C-methylene-bridged nucleic acid (2',4'-BNA/LNA). Moreover, ON modified with scpBNA-mC, -A, and -G showed higher enzymatic stability than the corresponding 2',4'-BNA/LNA-modified ON. These results demonstrated a promising role for the incorporation of scpBNA monomers into therapeutic antisense ONs.

Synthesis and properties of 2'-O,4'-C-spirocyclopropylene bridged nucleic acid (scpBNA), an analogue of 2',4'-BNA/LNA bearing a cyclopropane ring.

2'-O,4'-C-Spirocyclopropylene bridged nucleic acid (scpBNA), an analogue of 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA/LNA) bearing a cyclopropane ring at the 6'-position, was synthesized and successfully incorporated into oligonucleotides. The scpBNA-modified oligonucleotides showed excellent duplex-forming ability with complementary single-stranded RNA and exhibited increased enzymatic stability as compared to the corresponding natural and 2',4'-BNA/LNA-modified oligonucleotides. Our results demonstrate the potential of scpBNA for gene therapeutics, such as antisense technology.