Inhibition of restriction endonuclease cleavage by triple helix formation with RNA and 2'-O-methyl RNA oligonucleotides containing 8-oxo-adenosine in place of cytidine.
The ability of homopyrimidine oligoribonucleotides (RNA) and oligo-2'-O-methyl-ribonucleotides (2'-O-methyl RNA) containing 8-oxo-adenosine (AOH) and 8-oxo-2'-O-methyl (AmOH) adenosine to form stable, triple-helical structures with sequences containing the recognition site for the class II-S restriction enzyme, Ksp632-I, was studied as a function of pH. The AOH- and AmOH-substituted RNA and 2'-O-methyl RNA oligonucleotides were shown to bind within the physiological pH range in a pH-independent fashion, without a compromise in specificity. The substitutions of three cytidine residues with AOH showed higher endonuclease inhibition than the substitution of either one or two cytidine residues with AOH. In particular, the 2'-O-methyl RNA oligonucleotide with only one cytidine substituted with AmOH showed higher endonuclease inhibition than the homopyrimidine RNA and 2'-O-methyl RNA oligonucleotides and the RNA oligonucleotides containing either one or two AOH moieties. Furthermore, the AmOH-substituted 2'-O-methyl RNA oligonucleotides were stable (53%) after an incubation in 10% fetal bovine serum for 8 h, whereas the RNA oligonucleotides were completely degraded. Increased resistance to nucleases is observed with the introduction of 2'-O-methylnucleosides. This stabilization should help us to design much more efficient third strand homopyrimidine oligomer and antisense nucleic acid-based antiviral therapies, which could be used as tools in cellular biology.
Ushijima
K
K
Department of Industrial Chemistry, Chiba Institute of Technology, Japan.
Ishibashi
T
T
Yamakawa
H
H
Tsukahara
S
S
Takai
K
K
Maruyama
T
T
Takaku
H
H
D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D003562:Cytidine / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004279:DNA, Viral / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D005092:Exonucleases / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D009843:Oligoribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D011743:Pyrimidines / Q000737:chemistry / Q000378:metabolism; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics; D013873:Thionucleotides / Q000138:chemical synthesis / Q000378:metabolism
D004279:DNA, Viral; D004791:Enzyme Inhibitors; D009843:Oligoribonucleotides; D011743:Pyrimidines; D013873:Thionucleotides; C051802:triplex DNA; C036009:8-oxoadenosine; D003562:Cytidine; D012313:RNA; D004247:DNA; D005092:Exonucleases; C022650:spleen exonuclease; C057073:endodeoxyribonuclease Ksp632I; D015252:Deoxyribonucleases, Type II Site-Specific; D000241:Adenosine