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Viroids are small circular, noncoding, highly base-paired RNAs able to infect higher plants. Recently, it has been shown that viroids can be used as very stable scaffolds to produce recombinant RNA in Escherichia coli. Coexpression of an RNA precursor consisting of a viroid monomer, in which the RNA of interest is inserted, flanked by domains of the viroid hammerhead ribozyme, along with a host plant tRNA ligase, the enzyme that catalyzes viroid circularization in infected plants, allows for accumulation of large amounts of the chimeric viroid-RNA of interest in E. coli. Since viroids do not replicate in E. coli, high accumulation most probably results from viroid scaffold stability, resistance to exonucleases due to circularity, and accumulation as a ribonucleoprotein complex with tRNA ligase. Purification of the recombinant RNA from total E. coli RNA is also facilitated by the circular structure of the product.

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

Viruses have been engineered into useful biotechnological tools for gene therapy or to induce the synthesis of products of interest, such as therapeutic proteins and vaccines, in animal and fungal cells, bacteria or plants. Viroids are a particular class of infectious agents of higher plants that exclusively consist of a small non-protein-coding circular RNA molecule. In the same way as viruses have been transformed into useful biotechnological devices, can viroids be converted into beneficial tools? We show herein that, by expressing Eggplant latent viroid (ELVd) derived RNAs in Escherichia coli together with the eggplant tRNA ligase, this being the enzyme involved in viroid circularization in the infected plant, RNAs of interest like aptamers, extended hairpins, or other structured RNAs are produced in amounts of tens of milligrams per liter of culture. Although ELVd fails to replicate in E. coli, ELVd precursors self-cleave through the embedded hammerhead ribozymes and the resulting monomers are, in part, circularized by the co-expressed enzyme. The mature viroid forms and the protein likely form a ribonucleoprotein complex that transitorily accumulates in E. coli cells at extraordinarily amounts.

The regulation of gene expression, triggered by conformational changes in RNA molecules, is widely observed in cellular systems. Here, we examine this mode of control by means of a model-based design and construction of a fully synthetic riboregulatory device. We present a theoretical framework that rests on a simple energy model to predict the dynamic response of such a system. Following an equilibrium description, our framework integrates thermodynamic properties—anticipated with an RNA physicochemical model—with a detailed description of the intermolecular interaction. The theoretical calculations are confirmed with an experimental characterization of the action of the riboregulatory device within living cells. This illustrates, more broadly, the predictability of genetic robustness on synthetic systems, and the faculty to engineer gene expression programs from a minimal set of first principles., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Small RNAs (sRNAs) can operate as regulatory agents to control protein expression by interaction with the 5' untranslated region of the mRNA. We have developed a physicochemical framework, relying on base pair interaction energies, to design multi-state sRNA devices by solving an optimization problem with an objective function accounting for the stability of the transition and final intermolecular states. Contrary to the analysis of the reaction kinetics of an ensemble of sRNAs, we solve the inverse problem of finding sequences satisfying targeted reactions. We show here that our objective function correlates well with measured riboregulatory activity of a set of mutants. This has enabled the application of the methodology for an extended design of RNA devices with specified behavior, assuming different molecular interaction models based on Watson-Crick interaction. We designed several YES, NOT, AND, and OR logic gates, including the design of combinatorial riboregulators. In sum, our de novo approach provides a new paradigm in synthetic biology to design molecular interaction mechanisms facilitating future high-throughput functional sRNA design.

Analysis of the population of cherry small circular RNAs (cscRNAs) from trees affected by cherry chlorotic rusty spot (CCRS) showed two groups of variants with similar sequence but differing in size (394-415 and 372-377 nt for cscRNA1 and cscRNA2, respectively) because of the presence or absence of a 27-nt fragment folding into a hairpin in their predicted quasi-rod-like secondary structures. These structures were preserved by co-variations and compensatory mutations, as well as by additional complex rearrangements. The variability also preserved the central conserved core and the stability of the helices of the plus and minus hammerhead ribozymes, supporting their role in replication of cscRNAs. The smaller variants most likely derive from the larger through recombination events. Possible functional relationships between cscRNAs and certain mycoviral-like double-stranded RNAs, also associated with CCRS, are discussed.

A protocol is described for general application for cloning small circular RNAs which requires only minimal amounts of template (approximately 50 ng) of unknown sequence. Both cDNA strands are synthesized with a 26-mer primer whose six 3'-terminal positions are totally degenerate in two consecutive reactions catalyzed by reverse transcriptase and DNA polymerase, respectively. The cDNAs are then PCR-amplified, using a 20-mer primer with the non-degenerate sequence of the previous primer, cloned and sequenced. This information permits the synthesis of one or more pairs of specific and adjacent primers for obtaining full-length cDNA clones by a protocol which is also described.

Analysis by a PAGE approach for detecting small circular RNAs showed the existence of one such molecular species (RNA 1) accumulating at high levels in cherimoya. Sequencing of cDNA clones of RNA 1 revealed a size of 281 nt and a sequence identical to the 3'-terminal region of the 494-nt tRNALeu(UAA) group I intron from cherimoya. Northern blot hybridizations with a probe complementary to RNA 1 showed that this RNA coexists in vivo with its corresponding linear form, with the presumed full-length intron, and with minor amounts of two additional small circular species (RNAs 2 and 3). RNAs 2 and 3 had sizes of 216 and 156 nt, respectively, and sequences identical to different moieties of the 3'-terminal region of the tRNALeu(UAA) intron. The three cyclization sites giving rise to RNAs 1, 2, and 3, located within loop 8, are preceded by CUU or UUU trinucleotides and followed by sequences capable of forming base pairing interactions with the internal guide sequence characteristic of group I introns. The good correlation observed between the stabilities of these interactions and the in vivo accumulation levels of the corresponding cherimoya circular RNAs support the hypothesis that they emerge through a common mechanism similar to that advanced previously for the generation of circular RNAs derived from other group I introns. The lack of interactions of similar stabilities in tobacco, in which no circular RNAs derived from the tRNALeu(UAA) intron were detected, is consistent with this proposal, although other factors are also probably important in the synthesis and accumulation of the small circular RNAs in cherimoya.

Two PCR-based methods are described for obtaining clones of small circular RNAs of unknown sequence and for which only minute amounts are available. To avoid introducing any assumption about the RNA sequence, synthesis of the cDNAs is initiated with random primers. The cDNA population is then PCR-amplified using a primer whose sequence is present at both sides of the cDNAs, since they have been obtained with random hexamers and then a linker with the sequence of the PCR primer has been ligated to their termini, or because the cDNAs have been synthesized with an oligonucleotide that contains the sequence of the PCR primer at its 5' end and six randomized positions at its 3' end. The procedures need only approximately 50 ng of purified RNA template. The reasons for the emergence of cloning artifacts and precautions to avoid them are discussed.

Northern blot hybridizations with a probe complementary to a 275-nt circular RNA isolated from carnation plants revealed that this RNA co-exists in vivo with minor amounts of other small circular RNAs. Sequencing of cDNA clones of nine RNA species demonstrated deletions and duplications of the predominant 275-nt RNA. Minor sequence heterogeneities were observed at the crossover sites. Deletions mapped to three arms of the cruciform structure of lowest free energy obtained previously for the parental 275-nt RNA, whereas repeats encompassed mostly regions of the arm where deletions were not found. Some of the deleted and duplicated regions corresponded to sequences forming part of the two hammerhead structures involved in the in vitro self-cleavage of the plus and minus strands of the 275-nt RNA. A copy choice model is proposed for the emergence of deletions and duplications, where the RNA polymerase with the nascent strand dissociates from the template at regions rich in secondary and possibly tertiary structures, and reinitiates synthesis at different upstream and downstream positions.

The sequence of a circular RNA from carnation has been determined and found to consist of 275 nucleotide residues adopting a branched secondary structure of minimum free energy. Both plus and minus strands of this RNA can form the hammerhead structures proposed to mediate the in vitro self-cleavage of a number of small infectious plant RNAs and the transcript of satellite 2 DNA from the newt. Minus full- and partial-length transcripts of the carnation circular RNA including the hammerhead structure showed self-cleavage during transcription and after purification, indicating the involvement of a single-hammerhead structure in the self-cleavage reaction. In the case of the plus transcripts only a dimeric RNA, but not a monomeric one, self-cleaved efficiently during transcription and after purification, strongly supporting the implication in this process of a double-hammerhead structure theoretically more stable than the corresponding single cleavage domain. However, a plus monomeric transcript self-cleaved after purification at a slow rate in a concentration-independent reaction which most probably occurs through an intramolecular mechanism. Comparative sequence analysis has revealed that the circular RNA from carnation shares similarities with some representative members of the viroid and viroid-like satellites RNAs from plants, suggesting that it is a new member of either these two groups of small pathogenic RNAs.