Controlling Gene Expression in Living Cells Through Small Molecule-RNA Interactions

Werstuck, Geoff H.; Green, Michael R.

doi:10.1126/science.282.5387.296

Cited by 401 publications

(310 citation statements)

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“…4,5 Such knowledge is necessary to advance the understanding of RNA-small molecule recognition and for the continued development of small organic molecules as modulators of cellular processes at the RNA level. 6,7 The hammerhead ribozyme (HH) is one of the best characterized RNA enzymes. 8À10 A wealth of information regarding its conserved base requirements, kinetics, mechanisms and structure is available.…”

Section: Introductionmentioning

confidence: 99%

2-Aminopurine as a real-time probe of enzymatic cleavage and inhibition of hammerhead ribozymes

Kirk

2001

Bioorganic &Amp; Medicinal Chemistry

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Abstract-The design, synthesis and study of internally fluorescent hammerhead (HH) ribozymes, where changes in fluorescence parameters directly reflect the progress of the ribozyme's cleavage chemistry, are described. The approach relies on a HH substrate modified at position 1.1, proximal to the cleavage site, with 2-aminopurine (2AP), an intensely fluorescent adenosine isoster. The incorporation of 2AP, an unnatural nucleoside, does not interfere with the ribozyme folding and catalysis. Since 2AP is highly sensitive to environmental changes, its fluorescence is dramatically altered upon ribozyme-mediated cleavage of the substrate. This generates a measurable signal that directly reflects the progress of the ribozyme's reaction in real time. Identical pseudo first order rate constants are obtained for HH constructs using both continuous fluorescence monitoring and radioactive labeling. This rapid and real-time monitoring facilitates the study of ribozyme activity under different conditions (e.g., ionic strength, pH, etc.), and provides a useful assay to rapidly screen potential inhibitors. Three hitherto unknown HH inhibitors are presented and compared to neomycin B and chlortetracycline, two previously studied HH inhibitors. All three new small molecules, neo-acridine, guanidinoneomycin B, and [Á-(Eilatin)Ru(bpy) 2 ] 2+ , prove to be better inhibitors than neomycin B or chlortetracycline. Investigating HH inhibition under different ionic strengths reveals that the binding of neo-acridine, [Á-(Eilatin)Ru(bpy) 2 ] 2+ , and chlortetracycline to the HH involves hydrophobic interactions as their RNA affinities are largely unaffected by increasing salt concentrations. In contrast, neomycin B loses more than 50-fold of its inhibitory ability as the NaCl concentration is increased from 50 to 500 mM. #

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Section: Introductionmentioning

confidence: 99%

2-Aminopurine as a real-time probe of enzymatic cleavage and inhibition of hammerhead ribozymes

Kirk

2001

Bioorganic &Amp; Medicinal Chemistry

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“…An additional appeal is that chemicalfootprinting techniques, which are currently more effective for nucleic acids than proteins, allow for determining the precise site of binding of a small molecule on an RNA target by using routine sequencing methods on small quantities of material (17-19). We demonstrate here that such a chemical-footprinting technique can be used to identify a drug-like, RNA-binding molecule that is selective for a physiologically relevant site in mRNA and induces a change in the synthesis rate of the encoded protein.There have been a limited number of reported ligand-RNA interactions that affect protein synthesis (20)(21)(22). Werstuck and Green (20) described such a system in which RNA aptamers selected for aminoglycoside antibiotics were inserted within the 5Ј untranslated region (UTR) of a gene; subsequent translation in the presence of the selected antibiotic showed a dosedependent inhibitory effect on translation of protein both in vitro and in living cells.…”

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confidence: 99%

The up-regulation of ferritin expression using a small-molecule ligand to the native mRNA

Tibodeau

Fox²,

Ropp³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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The binding of small molecules to distinctive three-dimensional structures in mRNA provides a new dimension in RNA control, previously limited to the targeting of secondary structures with antisense and RNA interference; such targeting can modulate mRNA function and rates of protein biosynthesis. Small molecules that selectively bind the iron-responsive element (IRE), a specific three-dimensional structure in the noncoding region of the ferritin mRNA model that is recognized by the iron-regulatory protein repressor, were identified by using chemical footprinting. The assay used involved an oxoruthenium(IV) complex that oxidizes guanine bases in RNA sequences. Small molecules that blocked oxidation of guanines in the internal loop region were expected to selectively increase the rate of ferritin synthesis, because the internal loop region of the ferritin IRE is distinctive from those of other IREs. The natural product yohimbine was found (based on gel mobility shifts) to block cleavage of the internal loop RNA site by >50% and seemed to inhibit protein binding. In the presence of yohimbine, the rate of biosynthesis of ferritin in a cell-free expression system (rabbit reticulocyte lysate) increased by 40%. Assignment of the IRE-yohimbine interaction as the origin of this effect was supported by a similar increase in synthesis of luciferase protein in a chimera of the IRE and luciferase gene. The identification of a small, drug-like molecule that recognizes a naturally occurring three-dimensional mRNA structure and regulates protein biosynthesis rates raises the possibility that small molecules can regulate protein biosynthesis by selectively binding to mRNA.footprinting ͉ protein synthesis ͉ RNA structure ͉ RNA-binding drugs ͉ gene expression T he targeting of small molecules to three-dimensional structures in RNA has the potential to change the synthesis rate of the encoded proteins (1-4); an example is alteration of the control of reverse-transcription rate rates of HIV-1 RNA by trans-activation responsive region RNA, a target of many druglike molecules (5-9). A principal challenge in targeting mRNA is identifying three-dimensional RNA structures that are functionally relevant and exhibit structures amenable to selective binding in the presence of large quantities of other nucleic acids (5-7). Recent findings on the activation of bacterial riboswitches by metabolites provide incentive to pursue mRNA binding as a new avenue for pharmaceutical research (10), particularly given the intriguing possibility that such elements exist in eukaryotes (11,12). The advantages of using drug-like molecules to target three-dimensional RNA targets include the ability to escape biological systems that respond to targeting helical RNA structures (13), the ability to identify potentially useful RNA targets in genome sequences (14,15), and the possibility of synthesizing reasonably large quantities of RNA for screening by chemical methods (2,16). An additional appeal is that chemicalfootprinting techniques, which are currently more ...

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“…Prototype technologies for controlled protein secretion and mRNA translation have been reported (Rivera et al, 2000;Werstuck and Green, 1998). However, these systems have been designed for therapeutic use to modulate insulin secretion of transgenic cell lines and to block translation of viral transcripts by rather toxic molecules without the scope of improving biopharmaceutical manufacturing.…”

Section: Discussionmentioning

confidence: 99%

Novel CNBP‐ and La‐based translation control systems for mammalian cells

Schlatter

Fussenegger²

2002

Biotech & Bioengineering

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Throughout the development of Xenopus, production of ribosomal proteins (rp) is regulated at the translational level. Translation control is mediated by a terminal oligopyrimidine element (TOP) present in the 5Ј untranslated region (UTR) of rp-encoding mRNAs. TOP elements adopt a specific secondary structure that prevents ribosome-binding and translation-initiation of rpencoding mRNAs. However, binding of CNBP (cellular nucleic acid binding protein) or La proteins to the TOP hairpin structure abolishes the TOP-mediated transcription block and induces rp production. Based on the specific CNBP-TOP/La-TOP interactions we have designed a translation control system (TCS) for conditional as well as adjustable translation of desired transgene mRNAs in mammalian cells. The generic TCS configuration consists of a plasmid encoding CNBP or La under control of the tetracycline-responsive expression system (TET OFF ) and a target expression vector containing a TOP module between a constitutive P SV40 promoter and the human model product gene SEAP (human secreted alkaline phosphatase) (P SV40 -TOP-SEAP-pA). The TCS technology showed excellent SEAP regulation profiles in transgenic Chinese hamster ovary (CHO) cells. Alternatively to CNBP and La, TOP-mediated translation control can also be adjusted by artificial phosphorothioate anti-TOP oligodeoxynucleotides. Confocal laser-scanning microscopy demonstrated cellular uptake of FITC-labeled oligodeoxynucleotides and their localization in perinuclear organelles within 24 hours. Besides their TOP-based translation-controlling capacity, CNBP and La were also shown to increase cap-independent translation from polioviral internal ribosomal entry sites (IRES) and La alone to boost cap-dependent translation initiation. CNBP and La exemplify for the first time the potential of RNAbinding proteins to exert translation control of desired transgenes and to increase heterologous protein production in mammalian cells. We expect both of these assets to advance current gene therapy and biopharmaceutical manufacturing strategies.

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Controlling Gene Expression in Living Cells Through Small Molecule-RNA Interactions

Cited by 401 publications

References 13 publications

2-Aminopurine as a real-time probe of enzymatic cleavage and inhibition of hammerhead ribozymes

2-Aminopurine as a real-time probe of enzymatic cleavage and inhibition of hammerhead ribozymes

The up-regulation of ferritin expression using a small-molecule ligand to the native mRNA

Novel CNBP‐ and La‐based translation control systems for mammalian cells

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