Synthesis and properties of a novel modified nucleic acid, 2'-N-methanesulfonyl-2'-amino-locked nucleic acid.

2'-Amino-locked nucleic acid has a functionalizable nitrogen atom at the 2'-position of its furanose ring that can provide desired properties to a nucleic acid as a scaffold. In this study, we synthesized a novel nucleic acid, 2'-N-methanesulfonyl-2'-amino-locked nucleic acid (ALNA[Ms]) and conducted comparative studies on the physical and pharmacological properties of the ALNA[Ms] and on conventional nucleic acids, such as 2'-methylamino-LNA (ALNA[Me]), which is a classical 2'-amino-LNA derivative, and also on 2',4'-BNA/LNA (LNA). ALNA[Ms] oligomers exhibited binding affinities for the complementary RNA strand that are similar to those of conventional nucleic acids. Four types of ALNA[Ms] nucleosides exhibited no genotoxicity in bacterial reverse mutation assays. The knockdown abilities of Malat1 RNA using the Matat1 antisense oligonucleotide (ASO) containing ALNA[Ms] were higher than those of ALNA[Me] and were closer to those of LNA. Furthermore, the ASO containing ALNA[Ms] showed different tissue tropism from that containing LNA. ALNA[Ms] exhibited biological activities that were distinct from conventional constrained nucleic acids, suggesting the possibility that ALNA[Ms] can serve as novel modified nucleic acids in oligonucleotide therapeutics.

2'-N-Alkylaminocarbonyl-2'-amino-LNA: Synthesis, duplex stability, nuclease resistance, and in vitro anti-microRNA activity.

2'-Amino-LNA has the potential to acquire various functions through chemical modification at the 2'-nitrogen atom. This study focused on 2'-N-alkylaminocarbonyl 2'-amino-LNA, which is a derivative of 2'-amino-LNA. We evaluated its practical usefulness as a chemical modification of anti-miRNA oligonucleotide. The synthesis of phosphoramidites of 2'-N-alkylaminocarbonyl substituted 2'-amino-LNA bearing thymine and 5-methylcytosine proceeded in good yields. Incorporating the 2'-N-alkylaminocarbonyl-2'-amino-LNA monomers into oligonucleotides improved the duplex stability for complementary RNA strands and robust nuclease resistance. Moreover, 2'-N-alkylaminocarbonyl-2'-amino-LNA is a promising scaffold that significantly increases the potency of anti-miRNA oligonucleotides.

Parallel synthesis of oligonucleotides containing N-acyl amino-LNA and their therapeutic effects as anti-microRNAs.

2'-Amino-locked nucleic acid (ALNA), maintains excellent duplex stability, and the nitrogen at the 2'-position is an attractive scaffold for functionalization. Herein, a facile and efficient method for the synthesis of various 2'-N-acyl amino-LNA derivatives by direct acylation of the 2'-amino moiety contained in the synthesized oligonucleotides and its fundamental properties are described. The introduction of the acylated amino-LNA enhances the potency of the molecules as therapeutic anti-microRNA oligonucleotides.

Structure-Activity Relationships of Anti-microRNA Oligonucleotides Containing Cationic Guanidine-Modified Nucleic Acids.

Anti-microRNA oligonucleotides (AMOs) are valuable tools for the treatment of diseases caused by the dysregulation of microRNA expression. However, the correlation between chemical modifications in AMO sequences and the microRNA-inhibitory activity has not been fully elucidated. In this study, we synthesized a series of AMOs containing cationic guanidine-bridged nucleic acids (GuNA) and evaluated their activities using a dual luciferase assay. We also optimized the site of GuNA substitution and found an effective design for the inhibition of microRNA-21, which was partially different from that of conventional nucleic acid derivatives. This study showed that GuNA-substituted AMOs are effective in inhibiting the function of microRNA.

Synthesis and properties of GuNA purine/pyrimidine nucleosides and oligonucleotides.

We recently designed guanidine-bridged nucleic acids (GuNA), and GuNA bearing a thymine (T) nucleobase was synthesized and successfully incorporated into oligonucleotides. The GuNA-T-modified oligonucleotides possessed high duplex-forming ability towards their complementary single-stranded RNAs and were highly stable against 3'-exonuclease. Therefore, GuNA is a promissing artificial nucleic acid for therapeutic antisense oligonucleotides. We herein report the facile synthesis of GuNA phosphoramidites bearing adenine (A), guanine (G), and 5-methylcytosine (mC) nucleobases and a robust method for the preparation of GuNA-modified oligonucleotides, even with sequences having acid-sensitive purine nucleobases. Oligonucleotides modified with GuNA-A, -G, or -mC possessed high duplex-forming ability, similar to those modified with GuNA-T. Moreover, some of the GuNA-modified oligonucleotides were revealed to have high base discriminating ability compared with that of their natural counterparts. GuNA nucleosides exhibited no genotoxicity in bacterial reverse mutation assays. Thus, all GuNAs (GuNA-T, -A, -G, and -mC) are now available to be examined in therapeutic applications.