In vitro optimization of antisense oligodeoxynucleotide design: an example using the connexin gene family.
The completion of the human and mouse genomes has identified at least 20 connexin isomers in this family of intercellular channel proteins. However, there are no specific gap junction blockers or channel-blocking mimetic peptides available for the study of specific connexins. We designed antisense oligodeoxynucleotides that functionally reduce targeted connexin protein expression and can be used to reveal the biological function of individual connexins in vivo. Connexin mRNA was firstly exposed in vitro to deoxyribozymes complementing the sense coding sequence. Those that cleaved the target connexin mRNA in defined regions were used as the basis to design oligodeoxynucleotides to the accessible sites, thus taking into account tertiary mRNA configurations rather than relying on computed predictions. Antisense oligodeoxynucleotides designed to bind to accessible mRNA sites selectively reduced connexin26 and -43 mRNA expression in a corneal epithelium ex vivo model. Connexin43 protein levels were reduced correlating with the knockdown in mRNA and the protein's rapid turnover; protein levels of connexin26 did not alter, supporting lower turnover rates reported for that protein. We show, for the first time, an inexpensive and empirical approach to the preparation of specific and functional antisense oligodeoxynucleotides against known gene targets in the post-genomic era.
Law
Lee Yong
LY
Department of Developmental and Cell Biology, University of California, Irvine, CA, USA.
Zhang
Wei V
WV
Stott
N Susan
NS
Becker
David L
DL
Green
Colin R
CR
D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D000072259:Connexin 26; D018031:Connexin 43 / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017630:Connexins / Q000032:analysis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003315:Cornea / Q000166:cytology; D021881:DNA, Catalytic / Q000737:chemistry / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D007150:Immunohistochemistry; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology*; D009924:Organ Culture Techniques; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats
D018031:Connexin 43; D017630:Connexins; D021881:DNA, Catalytic; C000607042:GJB2 protein, human; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D000072259:Connexin 26