ABSTRACT
We report, herein, a new class of RNAi trigger molecules based on the unconventional parallel hybridization of two oligonucleotide chains. We have prepared and studied several parallel stranded (ps) duplexes, in which the parallel orientation is achieved through incorporation of isoguanine and isocytosine to form reverse Watson-Crick base pairs in ps-DNA:DNA, ps-DNA:RNA, ps-(DNA-2'F-ANA):RNA, and ps-DNA:2'F-RNA duplexes. The formation of these duplexes was confirmed by UV melting experiments, FRET and CD studies. In addition, NMR structural studies were conducted on a ps-DNA:RNA hybrid for the first time. Finally, we provide evidence for the unprecedented finding that ps-DNA:RNA and ps-DNA:2'F-RNA hybrids can engage the RNAi pathway to silence gene expression in vitro.
Subject(s)
DNA/chemistry , RNA Interference , RNA/chemistry , Base Pairing , HEK293 Cells , Humans , Oligonucleotides/chemistry , RNA, Small Interfering/chemistryABSTRACT
MicroRNAs (miRNAs) originate from stem-loop-containing precursors (pre-miRNAs, pri-miRNAs) and mature by means of the Drosha and Dicer endonucleases and their associated factors. The let-7 miRNAs have prominent roles in developmental differentiation and in regulating cell proliferation. In cancer, the tumor suppressor function of let-7 is abrogated by overexpression of Lin28, one of several RNA-binding proteins that regulate let-7 biogenesis by interacting with conserved motifs in let-7 precursors close to the Dicer cleavage site. Using in vitro assays, we have identified a binding site for short modified oligoribonucleotides ('looptomirs') overlapping that of Lin28 in pre-let-7a-2. These looptomirs selectively antagonize the docking of Lin28, but still permit processing of pre-let-7a-2 by Dicer. Looptomirs restored synthesis of mature let-7 and inhibited growth and clonogenic potential in Lin28 overexpressing hepatocarcinoma cells, thereby demonstrating a promising new means to rescue defective miRNA biogenesis in Lin28-dependent cancers.
Subject(s)
MicroRNAs/metabolism , Neoplasms/genetics , Oligoribonucleotides/metabolism , RNA Processing, Post-Transcriptional , RNA-Binding Proteins/antagonists & inhibitors , Binding Sites , Cell Line, Tumor , Cell Proliferation , HEK293 Cells , Humans , MicroRNAs/chemistry , Neoplasms/enzymology , Neoplasms/pathology , Oligoribonucleotides/chemistry , RNA Precursors/chemistry , RNA Precursors/metabolism , RNA-Binding Proteins/metabolism , Ribonuclease III/metabolismABSTRACT
Dendritic cell (DC) migration via lymphatic vessels to draining lymph nodes (dLNs) is crucial for the initiation of adaptive immunity. We imaged this process by intravital microscopy (IVM) in the ear skin of transgenic mice bearing red-fluorescent vasculature and yellow-fluorescent DCs. DCs within lymphatic capillaries were rarely transported by flow, but actively migrated within lymphatics and were significantly faster than in the interstitium. Pharmacologic blockade of the Rho-associated protein kinase (ROCK), which mediates nuclear contraction and de-adhesion from integrin ligands, significantly reduced DC migration from skin to dLNs in steady-state. IVM revealed that ROCK blockade strongly reduced the velocity of interstitial DC migration, but only marginally affected intralymphatic DC migration. By contrast, during tissue inflammation, ROCK blockade profoundly decreased both interstitial and intralymphatic DC migration. Inhibition of intralymphatic migration was paralleled by a strong up-regulation of ICAM-1 in lymphatic endothelium, suggesting that during inflammation ROCK mediates de-adhesion of DC-expressed integrins from lymphatic-expressed ICAM-1. Flow chamber assays confirmed an involvement of lymphatic-expressed ICAM-1 and DC-expressed ROCK in DC crawling on lymphatic endothelium. Overall, our findings further define the role of ROCK in DC migration to dLNs and reveal a differential requirement for ROCK in intralymphatic DC crawling during steady-state and inflammation.
