Characterization of modified antisense oligonucleotides in Xenopus laevis embryos.
A wide variety of modified oligonucleotides have been tested as antisense agents. Each chemical modification produces a distinct profile of potency, toxicity, and specificity. Novel cationic phosphoramidate-modified antisense oligonucleotides have been developed recently that have unique and interesting properties. We compared the relative potency and specificity of a variety of established antisense oligonucleotides, including phosphorothioates (PS), 2'-O-methyl (2'OMe) RNAs, locked nucleic acids (LNAs), and neutral methoxyethyl (MEA) phosphoramidates with new cationic N,N-dimethylethylenediamine (DMED) phosphoramidate-modified antisense oligonucleotides. A series of oligonucleotides was synthesized that targeted two sites in the Xenopus laevis survivin gene and were introduced into Xenopus embryos by microinjection. Effects on survivin gene expression were examined using quantitative real-time PCR. Of the various modified oligonucleotide designs tested, LNA/PS chimeras (which showed the highest melting temperature) and DMED/phosphodiester chimeras (which showed protection of neighboring phosphate bonds) were potent in reducing gene expression. At 40 nM, overall specificity was superior for the LNA/PS-modified compounds compared with the DMED-modified oligonucleotides. However, at 400 nM, both of these compounds led to significant degradation of survivin mRNA, even when up to three mismatches were present in the heteroduplex.
Integrated DNA Technologies, Coralville, IA 52241, USA.
Kim A
KA
Lennox
Jaime L
JL
Sabel
Maegan J
MJ
Johnson
Bernardo G
BG
Moreira
Cherisa A
CA
Fletcher
Scott D
SD
Rose
Mark A
MA
Behlke
Andrei L
AL
Laikhter
Joseph A
JA
Walder
John M
JM
Dagle
D000577:Amides / Q000737:chemistry; D000818:Animals; D020137:Base Pair Mismatch; D001483:Base Sequence; D003851:Deoxyribonucleases / Q000737:chemistry; D004625:Embryo, Nonmammalian / Q000187:drug effects; D005029:Ethylenediamines / Q000737:chemistry*; D015870:Gene Expression / Q000187:drug effects; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D010756:Phosphoric Acids / Q000737:chemistry; D012333:RNA, Messenger / Q000037:antagonists & inhibitors*; D000077022:Survivin; D013696:Temperature; D029867:Xenopus Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D014982:Xenopus laevis
D000577:Amides; D005029:Ethylenediamines; D016376:Oligonucleotides, Antisense; D010756:Phosphoric Acids; D012333:RNA, Messenger; D000077022:Survivin; D029867:Xenopus Proteins; C472712:birc5.2 protein, Xenopus; C027450:dimethylethylenediamine; C011067:phosphoramidic acid; D003851:Deoxyribonucleases