Chimeric RNase H-competent oligonucleotides directed to the HIV-1 Rev response element.
Chimeric oligo-2'-O-methylribonucleotides containing centrally located patches of contiguous 2'-deoxyribonucleotides and terminating in a nuclease resistant 3'-methylphosphonate internucleotide linkage were prepared. The oligonucleotides were targeted to the 3'-side of HIV Rev response element (RRE) stem-loop IIB RNA, which is adjacent to the high affinity Rev protein binding site and is critical to virus function. Thermal denaturation experiments showed that chimeric oligonucleotides form very stable duplexes with a complementary single-stranded RNA, and gel electrophoretic mobility shift assays (EMSA) showed that they bind with high affinity and specificity to RRE stem-loop II RNA (K(D) approximately 200 nM). The chimeric oligonucleotides promote RNase H-mediated hydrolysis of RRE stem-loop II RNA and have half-lives exceeding 24h when incubated in cell culture medium containing 10% fetal calf serum. One of the chimeric oligonucleotides inhibited RRE mediated expression of chloramphenicol acetyl transferase (CAT) approximately 60% at a concentration of 300 nM in HEK 293T cells co-transfected with p-RRE/CAT and p-Rev mammalian expression vectors.
Prater
Chrissy E
CE
Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205, USA.
Saleh
Anthony D
AD
Wear
Maggie P
MP
Miller
Paul S
PS
D000818:Animals; D002417:Cattle; D002460:Cell Line; D002942:Circular Dichroism; D016373:DNA, Antisense / Q000235:genetics; D015870:Gene Expression; D015694:Gene Products, rev / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D005818:Genetic Engineering; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D006868:Hydrolysis; D008745:Methylation; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics*; D016914:Ribonuclease H / Q000378:metabolism*; D044967:Serum; D044366:Transition Temperature; D054321:rev Gene Products, Human Immunodeficiency Virus
D016373:DNA, Antisense; D015694:Gene Products, rev; D009841:Oligonucleotides; D012367:RNA, Viral; D054321:rev Gene Products, Human Immunodeficiency Virus; D016914:Ribonuclease H