Development of a 3'-amino linker with high conjugation activity and its application to conveniently cross-link blunt ends of a duplex.

The 2-aminoethyl carbamate linker (ssH linker) exhibits high activity in modifying the 5'-termini of oligonucleotides; however, the ssH linker is not appropriate for 3'-terminal modification because it undergoes intramolecular trans-acylation under heat-aqueous ammonia conditions. We developed an N-(2-aminoethyl)carbamate linker (revH linker), in which the carbamate is oriented in the reverse direction relative to that in 2-aminoethyl carbamate. The revH linker was tolerant to heat-alkaline conditions and retained its high reactivity in conjugation with exogenous molecules. The 3'-revH linker was efficiently linked with the 5'-ssH linker at the termini of complementary double strands with a bifunctional molecule, producing a synthetic loop structure. An anti-microRNA oligonucleotide (AMO) was prepared from the chemical ligation of three-stranded 2'-O-methyl RNAs, and the AMO with two alkyl loops exhibited high inhibition activity toward miRNA function. The revH linker is not only useful for 3'-terminal modification of oligonucleotides but also expands the utility range in combination with the 5'-ssH linker.