H1 RNA polymerase III promoter-driven expression of an RNA aptamer leads to high-level inhibition of intracellular protein activity.
Aptamers offer advantages over other oligonucleotide-based approaches that artificially interfere with target gene function due to their ability to bind protein products of these genes with high affinity and specificity. However, RNA aptamers are limited in their ability to target intracellular proteins since even nuclease-resistant aptamers do not efficiently enter the intracellular compartments. Moreover, attempts at expressing RNA aptamers within mammalian cells through vector-based approaches have been hampered by the presence of additional flanking sequences in expressed RNA aptamers, which may alter their functional conformation. In this report, we successfully expressed a 'pure' RNA aptamer specific for NF-kappaB p50 protein (A-p50) utilizing an adenoviral vector employing the H1 RNA polymerase III promoter. Binding of the expressed aptamer to its target and subsequent inhibition of NF-kappaB mediated intracellular events were demonstrated in human lung adenocarcinoma cells (A549), murine mammary carcinoma cells (4T1) as well as a human tumor xenograft model. This success highlights the promise of RNA aptamers to effectively target intracellular proteins for in vitro discovery and in vivo applications.
Mi
Jing
J
Department of Surgery, Duke University Medical Center Durham, NC, USA.
Zhang
Xiuwu
X
Rabbani
Zahid N
ZN
Liu
Yingmiao
Y
Su
Zhen
Z
Vujaskovic
Zeljko
Z
Kontos
Christopher D
CD
Sullenger
Bruce A
BA
Clary
Bryan M
BM
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D052157:Aptamers, Nucleotide / Q000096:biosynthesis / Q000235:genetics*; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D052002:NF-kappa B p50 Subunit / Q000037:antagonists & inhibitors*; D009369:Neoplasms / Q000378:metabolism / Q000473:pathology; D011401:Promoter Regions, Genetic; D012320:RNA Polymerase III / Q000378:metabolism*; D015533:Transcriptional Activation; D023041:Xenograft Model Antitumor Assays
D052157:Aptamers, Nucleotide; D052002:NF-kappa B p50 Subunit; D012320:RNA Polymerase III