Highly efficient catalytic RNA cleavage by the cooperative action of two Cu(II) complexes embodied within an antisense oligonucleotide.
Based on our recent studies of RNA cleavage by oligonucleotide-terpyridine.Cu(II) complex 5'- and/or 3'-conjugates, we designed 2'-O-methyloligonucleotides with two terpyridine-attached nucleosides at contiguous internal sites. To connect the 2'-terpyridine-modified uridine residue at the 5'-side to the 5'-O-terpyridyl nucleoside residue at the 3'-side, a dimethoxytrityl derivative of 5-hydroxypropyl-5'-O-terpyridyl-2'-deoxyuridine-3'-phosphoramidite was newly synthesized. Using this unit, we constructed two terpyridine conjugates, with either an unusual phophodiester bond or the bond extended by a propanediol(s)-containing linker. Cleavage reactions of the target RNA oligomer, under the conditions of conjugate excess in the presence of Cu(II), indicated that the conjugates precisely cleaved the RNA at the predetermined site and that one propanediol-containing linker was the most appropriate for inducing high cleavage activity. Furthermore, a comparison of the activity of the propanediol agent with those of the control conjugates with one complex confirmed that the two complexes are required for efficient RNA cleavage. The reaction of the novel cleaver revealed a bell-shaped pH-rate profile with a maximum at pH approximately 7.5, which is a result of the cooperative action of the complexes. In addition, we demonstrated that the agent catalytically cleaves an excess of the RNA, with the kinetic parameter kcat/K(m) = 0.118 nM(-1) x h(-1).
Sakamoto
Satoshi
S
Department of Applied and Bioapplied Chemistry, Graduate School of Engineering, Osaka City University, Sugimoto 3-3-138, Sumiyoshi-ku, Osaka 558-8585, Japan.
Tamura
Takashi
T
Furukawa
Takako
T
Komatsu
Yasuo
Y
Ohtsuka
Eiko
E
Kitamura
Masaya
M
Inoue
Hideo
H
D001483:Base Sequence; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015394:Molecular Structure; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors
D016376:Oligonucleotides, Antisense; D009943:Organophosphorus Compounds; D016337:RNA, Catalytic; C434331:phosphoramidite; D003300:Copper