Yeast genetic selections to optimize RNA decoys for transcription factor NF-κB

Abstract

In vitro-selected RNA aptamers are potential inhibitors of diseaserelated proteins. Our laboratory previously isolated an RNA aptamer that binds with high affinity to human transcription factor NF-B. This RNA aptamer competitively inhibits DNA binding by NF-B in vitro and is recognized by its target protein in vivo in a yeast three-hybrid system. In the present study, yeast genetic selections were used to optimize the RNA aptamer for binding to NF-B in the eukaryotic nucleus. Selection for improved binding to NF-B from RNA libraries encoding (i) degenerate aptamer variants and (ii) sequences present at round 8 of 14 total rounds of in vitro selection yielded RNA aptamers with dramatically improved in vivo activity. Furthermore, we show that an in vivo-optimized RNA aptamer exhibits specific ''decoy'' activity, inhibiting transcriptional activation by its NF-B target protein in a yeast one-hybrid assay. This decoy activity is enhanced by the expression of a bivalent aptamer. The combination of in vitro and in vivo genetic selections was crucial for obtaining RNA aptamers with in vivo decoy activity.T he artificial modulation of gene expression is a major goal of modern disease therapeutics. Recently, RNA aptamers have been investigated as inhibitors of disease-related proteins (reviewed in ref. 1). In vitro selections using large combinatorial RNA libraries have made trivial the identification of RNA sequences that bind with high affinities to target macromolecules. However, it is not intuitive that an RNA molecule selected for binding in vitro likewise will bind and inhibit the target protein within the complex environment of a living eukaryotic cell. Despite this caveat, some RNA aptamers identified through in vitro selection have been shown to perform the desired inhibitory function when expressed in cells. For example, Famulok and colleagues (2) used in vitro selection to identify an RNA aptamer that inhibits the intracellular domain of the ␤2 integrin LFA-1. A second example of an in vitro-selected RNA aptamer that subsequently was shown to function in vivo is an inhibitory aptamer identified by Lis and colleagues (3) against the Drosophila B52 splicing protein.We have been intrigued by the possibility of identifying an RNA ''decoy'' for a model transcription factor, human NF-B. Such a decoy molecule might fold so as to bind at or near the DNA-binding site of the transcription factor, sequestering NF-B and reducing its ability to activate target genes. A member of the rel homology protooncogene family of proteins, NF-B exists in cells primarily as a homodimer composed of two p50 subunits [(p50) 2 ] or a heterodimer composed of one p50 and one p65 subunit (p50͞p65). Both forms of NF-B bind similar sites in duplex DNA [e.g., 5Ј-GGGACTTTCC (4)] but appear to activate transcription through distinct mechanisms because, unlike p65, p50 lacks a potent carboxyl-terminal transcription activation domain (5). Inhibition of NF-B activity by the controlled expression of an RNA decoy potentially could have important thera...

Combining SELEX and the yeast three-hybrid system for in vivo selection and classification of RNA aptamers

“…Combination of SELEX and the three-hybrid system might become particularly attractive for drug discovery, RNA diagnostics, and therapeutics (Cassiday and Maher 2003). Since both techniques can be automated (Cox et al 2002;Eulberg et al 2005;Stelzl et al 2005), high-throughput applications also are feasible.…”

“…This has been demonstrated by combining SELEX with the three-hybrid system to identify an RNA aptamer in vivo that functions as an RNA decoy for the transcription factor NF-kB Maher 2001, 2003). Aptamers with improved in vivo activity in the three-hybrid system were selected by testing mutagenized versions of the original RNA aptamer or assaying binding candidates after a few cycles of SELEX under increased selection pressure for HIS3 expression (using higher 3-AT concentrations) (Cassiday and Maher 2003). In the latter case an additional in vivo binder could be identified that might have been lost after excessive in vitro cycles.…”

Selection and characterization of anti-NF-κB p65 RNA aptamers

“…The NF-kB aptamers presented herein also provide us with the exciting prospect of optimizing p65 2 binding within a eukaryotic organism, such as Saccharomyces cerevisiae, as we have previously reported for anti-p50 (Cassiday and Maher 2001;Cassiday and Maher 2003). Another potential strategy involves selection of optimal RNA linker sequences to combine the p50 and p65 aptamers to form a heterodimeric aptamer that is competent for simultaneous binding of both subunits of NF-kB heterodimers.…”

Die chemische Biologie von Aptameren

“…[165,[169][170][171][172] Die Analyse und Verbesserung der In-vivoAktivität von in vitro selektierten Aptameren durch eine auf dem Hefe-3-Hybrid-System beruhende Selektion ist kürzlich beschrieben worden. [173][174][175][176][177] Lipid-basierte Transfektionen, die für die Transmembranlokalisation von Plasmiden und siRNA-Molekülen eingesetzt werden, lassen sich ebenfalls für das Einbringen von Aptameren in das Cytosol nutzen (Abbildung 6). [178] In einer bahnbrechenden Studie von Lis et al werden andere Methoden wie Mikroinjektion, Rezeptor-vermittelte Internalisierung und die Entwicklung transgener Tiere, die das Aptamer unter Kontrolle eines konditionalen Promotors exprimieren, vorgestellt.…”