Properties of exonuclease-resistant, psoralen-conjugated oligodeoxyribonucleotides in vitro and in cell culture.
We have prepared oligodeoxyribonucleotides that are modified at the 3'-terminal with N4-(4-aminobutyl)deoxycytidine and derivatized at the 5'-end with a 4'-([N-(aminoethyl)amino]methyl)-4,5',8-trimethylpsoralen, (ae)AMT, and whose sequences are complementary to vesicular stomatitis virus (VSV), N-protein mRNA, (ae)AMT-II, or VSV M-protein mRNA, (ae)AMT-III. (ae)AMT-II cross-links exclusively to VSV N-mRNA when a mixture of the oligomer and poly(A+) RNA from VSV-infected cells is irradiated in vitro with long wavelength UV light at either 20 degrees or 37 degrees C. N4-(4-Aminobutyl)deoxycytidine at the 3'-end of (ae)AMT-II does not appear to affect the binding or cross-linking of the oligomer to its target RNA. Oligomer (ae)AMT-II is completely resistant to hydrolysis by the 3'-5'-exonuclease activity found in fetal calf serum whereas a similar oligomer, (ae)AMT-I, which contains a 3'-terminal deoxycytidine, is hydrolyzed within 30 min when incubated at 37 degrees C. Intact (ae)AMT-II was found in both the cell lysate and cell culture medium after 12 hr of incubation with mouse L-cells along with d-(ae)AMTpT, which appears to result from endonuclease degradation of the oligomer. In contrast no intact (ae)AMT-I was found in either the cell lysate or the culture medium after 1 hr incubation. Although 10 microM (ae)AMT-II had no effect on VSV-protein synthesis in either unirradiated or UV-irradiated VSV-infected mouse L-cells, 10 microM (ae)AMT-III inhibited VSV protein synthesis 30% in irradiated cells. These results show that introduction of a N4-(4-aminobutyl)deoxycytidine at the 3'-end of an oligodeoxyribonucleotide significantly increases the resistance of the oligomer to degradation by 3'-5'-exonucleases but does not interfere with its ability to bind selectively to complementary RNA. Further derivatization with psoralen creates an oligomer that can be triggered to cross-link with RNA in a sequence-specific manner, is taken up intact by mammalian cells in culture, and exhibits biological activity. In combination, these two modifications endow the oligodeoxyribonucleotide with novel properties that could be exploited in the design of antisense or antigene reagents for use in controlling gene expression in mammalian cells.
Levis
J T
JT
Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205.
Miller
P S
PS
D000818:Animals; D002460:Cell Line; D003432:Cross-Linking Reagents; D003470:Culture Media; D004355:Drug Stability; D005092:Exonucleases; D005363:Ficusin / Q000737:chemistry*; D051379:Mice; D016376:Oligonucleotides, Antisense / Q000737:chemistry* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics*
D003432:Cross-Linking Reagents; D003470:Culture Media; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D012367:RNA, Viral; D005092:Exonucleases; D005363:Ficusin