Nucleic Acids Research, Vol 26, Issue 24 5670-5675, Copyright © 1998 by Oxford University Press
GJ Veal, S Agrawal and RA Byrn
We have used a ribonuclease protection assay to investigate RNase H
cleavage of HIV-1 mRNA mediated by phosphorothioate antisense
oligonucleotides complementary to the gag region of the HIV-1 genome in
vitro. Cell lysate experiments in H9 and U937 cells chronically infected
with HIV-1 IIIB showed RNase H cleavage of unspliced gag message but no
cleavage of spliced message which did not contain the target gag region.
RNase H cleavage products were detected at oligonucleotide concentrations
as low as 0.01 microM and the RNase H activity was seen to be concentration
dependent. Similar experiments with 1-, 3- and 5-mismatch oligonucleotides
demonstrated sequence specificity at low concentrations, with cleavage of
gag mRNA correlating with the predicted activities of the parent and
mismatch oligonucleotides based on their hybridization melting
temperatures. Experiments in living cells suggested that RNase H-specific
antisense activity was largely determined by the amount of oligonucleotide
taken up by the different cell lines studied. RNase H cleavage products
were detected in antisense oligonucleotide treated MT-4 cells acutely
infected with HIV-1 IIIB, but not in infected H9 cells treated with
oligonucleotide under the same conditions. The data presented demonstrate
potent and specific RNase H cleavage of HIV-1 mRNA mediated by an antisense
oligonucleotide targeted to HIV-1 gag mRNA, and are in agreement with
previous reports that the major obstacle to demonstrating antisense
activity in living cells remains the lack of penetration of these agents
into the desired cellular compartment.
ARTICLES
Sequence-specific RNase H cleavage of gag mRNA from HIV-1 infected cells by an antisense oligonucleotide in vitro
Divisions of Hematology, Oncology and Experimental Medicine, Beth Israel Deaconess Medical Center,Harvard Medical School, Boston, MA 02215, USA. g.j.veal@nc1.ac.uk
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