Engineering precision RNA molecular switches

Abstract

Ligand-specific molecular switches composed of RNA were created by coupling preexisting catalytic and receptor domains via structural bridges. Binding of ligand to the receptor triggers a conformational change within the bridge, and this structural reorganization dictates the activity of the adjoining ribozyme. The modular nature of these tripartite constructs makes possible the rapid construction of precision RNA molecular switches that trigger only in the presence of their corresponding ligand. By using similar enzyme engineering strategies, new RNA switches can be made to operate as designer molecular sensors or as a new class of genetic control elements.Mastery of the molecular forces that dictate biopolymer folding and function would allow molecular engineers to participate in the design of enzymes-a task that to date has been managed largely by the random processes of evolution. The reward for acquiring this capability is substantial, considering that many applications in medicine, industry, and biotechnology demand high-speed enzymes with precisely tailored catalytic functions. ''Modular rational design'' has proven to be an effective means for conferring additional chemical and kinetic complexity on existing protein (1-4) and RNA enzymes (5-9). This engineering strategy takes advantage of the modular nature of many protein (10) and RNA subdomains (11-13), which can be judiciously integrated to form new multifunctional constructs. The recent discoveries of new catalytic RNA motifs (14, 15) and new ligand-binding motifs (16, 17) have considerably expanded the opportunities for ribozyme engineering.We have used modular rational design to create several artificial ribozymes that are activated or deactivated by the binding of specific small organic molecules such as ATP (5, 8) and flavin mononucleotide (FMN) (unpublished data). Each of these allosteric † ribozymes is composed of two independent structural domains: one an RNA-cleaving ribozyme and the other a receptor (or ''aptamer'') for a specific ligand. The conformational changes that occur within an aptamer domain on introduction of the ligand, termed ''adaptive binding '' (22-25), can trigger kinetic modulation of the adjoining catalytic domain by several different mechanisms that ultimately influence ribozyme folding (8,9). In this report, we describe the combined application of modular rational design and in vitro selection techniques that provide an effective strategy for the rapid generation of precision molecular switches made of RNA. MATERIALS AND METHODSOligonucleotides. Synthetic DNA and the 14-nt substrate RNA were prepared by standard solid phase methods (Keck Biotechnology Resource Laboratory, Yale University) and purified by denaturing (8 M urea) PAGE as described (5).RNA substrate was 5Ј 32 P-labeled with T4 polynucleotide kinase and [␥-32 P]ATP, and repurified by PAGE. Doublestranded DNA templates for in vitro transcription using T7 RNA polymerase were generated by extension of primer A (5Ј-TAATACGACTCACTATAGGGCGACCCTGATGAG) on a D...

Artificial riboswitches for gene expression and replication control of DNA and RNA viruses

“…Clearly, the fact that the constitutively active ribozyme caused potent inhibition of virus replication in all cases indicates that the aptazyme approach should be universally functional with optimized aptazymes. Artificial aptazymes have been mostly developed in vitro and in bacteria, where aptazymes with high induction rates were generated by high-throughput selection (12,13,20,33). However, only a few aptazymes have been reported to be functional in mammalian cells.…”

“…Aptazymes have been extensively developed and studied in vitro, in bacteria, and in yeast (6,12,13,(19)(20)(21)(22). Their application in mammalian systems, however, has been challenging.…”

“…Furthermore, they can be customized for responsiveness to different small-molecule ligands by exchange of the aptamer domain (12,13). Importantly, due to their RNA-intrinsic mode of action they should be applicable in both DNA and RNA viruses.…”

Evolutionary patterns of non-coding RNAs

“…A theoretical study shows that RNAs exhibiting very different secondary structures with near-groundstate energy, i.e., potential riboswitches, are relatively frequent and easily accessible in 7SL/SRP 300-400 signal recognition particle ubiquitous [124] vault 80-100 part of vault particle vertebrata Y 80-100 part of Ro particle metazoa tmRNA 300-400 tags protein for proteolysis bacteria, chloroplasts, cyanoplasts [457] miRNA ∼22 post-transcriptional regulation multicellular organisms [131] gRNA 40-80 RNA editing kinetoplastids [156] evolution [108]. Artificial riboswitches have been explored for biotechnological applications [378,207,364] and it has been demonstrated that such constructs can be specifically triggered by means of small "modifier" RNAs [277,142].…”

Thermozymes

“…Synthetic RNA thermometers based on ribozyme activity 17 such aptazymes have been applied in vivo for gene-regulatory purposes only after the discovery of catalytically well-behaved, fast-cleaving variants of the hammerhead ribozyme comprising stem I/II interactions. [18][19][20] In our opinion, ribozyme-based devices have the advantage of almost universal applicability for controlling RNA functions.…”