Modified oligodeoxynucleotide primers for reverse-transcription of target RNAs that can discriminate among length variants at the 3'-terminus.

To discriminate among miRNA length variants, we synthesized conformationally restricted or unrestricted oligonucleotides containing a cyclohexyl phosphate residue. These oligonucleotides formed duplexes with length-matched complementary miRNAs more tightly than with length variants. The use of one of these modified oligodeoxynucleotides as a reverse transcription primer enabled a novel RT-PCR that can discriminate among miRNA length variants.

Short-RNA selective binding of oligonucleotides modified using adenosine and guanosine derivatives that possess cyclohexyl phosphates as substituents.

We have developed new artificial oligonucleotides which distinguish short RNA targets from long ones. The modification of the 5' termini of oligonucleotides by using adenosine derivatives that possess a bulky cyclohexyl phosphate moiety at their base moiety and a phosphate group at the position of their 5'-hydroxyl group maximized their short RNA selectivity. The 2'-O-methyl-RNA (5'-XC(m)A(m)A(m)C(m)C(m)U(m)A(m)C(m)U(m)) having these modifications exhibits ca.

Synthesis and properties of terminally modified oligonucleotides capable of short-RNA selective hybridization.

We have developed 2'-O-methyl-RNAs having phosphorylated cyclohexane at the 5' and/or 3'-terminal adenine. These 2'-O-methyl-RNAs formed less stable duplex with the longer target RNA than that with the short target RNA. We tried to improve the short-RNA selective hybridization property by the synthesis of 2'-O-methyl-RNAs having terminal guanine modifications.

Synthesis of terminally modified oligonucleotides and their hybridization dependence on the size of the target RNAs.

We have developed new artificial oligonucleotide probes that show selective recognition for short RNA targets over long RNA targets. Our results suggested that modification of the termini of the oligonucleotide probes by bulky substituents such as cyclohexyl and 4-(3,6,9-trioxaundecylenedioxy)phenyl (Bzcr) groups significantly improved the selectivity of the probes toward the short RNA targets. The selectivity was further improved by the addition of a phosphate group on the cyclohexane ring.

Synthesis of oligonucleotides terminally modified by bulky and negatively charged substituents and their hybridization with RNA targets.

2'-O-methyl RNAs having phosphorylated cyclohexane groups at the 5' and 3'-terminal bases were synthesized and their hybridization properties toward RNA targets were studied. It was proved that the duplex of the modified 2'-O-methyl RNAs and the target RNA longer than the 2'-O-methyl RNA was less stable than that with the short target. In addition, it was also clarified that the selectivity toward the short target was improved by the introduction of the phosphate group in comparison with that without the phosphate group.