Cis-acting Hammerhead Ribozymes Research Articles

An Adaptive Defect Weighted Sampling Algorithm to Design Pseudoknotted RNA Secondary Structures.

Computational design of RNA sequences that fold into targeted secondary structures has many applications in biomedicine, nanotechnology and synthetic biology. An RNA molecule is made of different types of secondary structure elements and an important RNA element named pseudoknot plays a key role in stabilizing the functional form of the molecule. However, due to the computational complexities associated with characterizing pseudoknotted RNA structures, most of the existing RNA sequence designer algorithms generally ignore this important structural element and therefore limit their applications. In this paper we present a new algorithm to design RNA sequences for pseudoknotted secondary structures. We use NUPACK as the folding algorithm to compute the equilibrium characteristics of the pseudoknotted RNAs, and describe a new adaptive defect weighted sampling algorithm named Enzymer to design low ensemble defect RNA sequences for targeted secondary structures including pseudoknots. We used a biological data set of 201 pseudoknotted structures from the Pseudobase library to benchmark the performance of our algorithm. We compared the quality characteristics of the RNA sequences we designed by Enzymer with the results obtained from the state of the art MODENA and antaRNA. Our results show our method succeeds more frequently than MODENA and antaRNA do, and generates sequences that have lower ensemble defect, lower probability defect and higher thermostability. Finally by using Enzymer and by constraining the design to a naturally occurring and highly conserved Hammerhead motif, we designed 8 sequences for a pseudoknotted cis-acting Hammerhead ribozyme. Enzymer is available for download at https://bitbucket.org/casraz/enzymer.

Production of Homogeneous Recombinant RNA Using a tRNA Scaffold and Hammerhead Ribozymes.

Bacterial overproduction of recombinant RNA using a tRNA scaffold yields large amounts of chimeric RNA. For structural and functional characterizations of the RNA it is often necessary to remove the scaffold. Here we describe an efficient and facile method to release the RNA of interest from the tRNA scaffold by selective cleavage using cis-acting hammerhead ribozymes. After cleavage, the RNA of interest is purified to homogeneity using standard chromatographic and electrophoretic methods. Up to 5 mg of highly pure end-product RNA can be obtained from a single liter of bacterial culture.

Fast production of homogeneous recombinant RNA—towards large-scale production of RNA

In the past decades, RNA molecules have emerged as important players in numerous cellular processes. To understand these processes at the molecular and atomic level, large amounts of homogeneous RNA are required for structural, biochemical and pharmacological investigations. Such RNAs are generally obtained from laborious and costly in vitro transcriptions or chemical synthesis. In 2007, a recombinant RNA technology has been described for the constitutive production of large amounts of recombinant RNA in Escherichia coli using a tRNA-scaffold approach. We demonstrate a general applicable extension to the described approach by introducing the following improvements: (i) enhanced transcription of large recombinant RNAs by T7 RNA polymerase (high transcription rates, versatile), (ii) efficient and facile excision of the RNA of interest from the tRNA-scaffold by dual cis-acting hammerhead ribozyme mediated cleavage and (iii) rapid purification of the RNA of interest employing anion-exchange chromatography or affinity chromatography followed by denaturing polyacrylamide gel electrophoresis. These improvements in the existing method pave the tRNA-scaffold approach further such that any (non-)structured product RNA of a defined length can cost-efficiently be obtained in (multi-)milligram quantities without in vitro enzymatic manipulations.

Construction, modification and evaluation of apolipoprotein A-I promoter-driven shRNA expression vectors against hTERT

Liver-specific gene knockdown cannot be achieved by short hairpin RNA (shRNA) generated by RNA polymerase III promoter. Here we constructed, modified and evaluated apolipoprotein A-I (ApoA-I) promoter-driven shRNA expression vectors against human telomerase reverse transcriptase (hTERT) in SMMC-7721 cells. The roles of the cis-acting hammerhead ribozyme and the specific pausing site MAZ, the liver-specific promoter ApoA-I, as well as SV40 and CMV enhancers were first individually evaluated, and then they were incorporated to construct a liver-specific shRNA expression plasmid against hTERT, and the inhibitory effects on hTERT were examined in SMMC-7721 cells. The results showed that the introduction of the cis-acting hammerhead ribozyme and the specific pausing site MAZ did not change gene knockdown efficiency significantly, but eliminated the off-target effect. Green fluorescent protein (GFP) expressing plasmid under the control of ApoA-I promoter can produce liver-specific GFP expression, but at a much lower level compared to the CMV promoter. The CMV or SV40 enhancer-modified ApoA-I promoter caused a four or two folds increase in mRNA expression of GFP relative to ApoA-I alone, respectively. The liver-specific shRNA expression plasmid against hTERT under the control of CMV enhancer-modified ApoA-I promoter with the sequences of the cis-acting hammerhead ribozyme and MAZ, induced significant inhibitory effect on hTERT at both mRNA and protein levels in SMMC-7721 cells. Therefore, liver-specific gene therapy is made possible by utilizing shRNA expression vector under the control of CMV enhancer-modified ApoA-I promoter.

Preparation of large RNA oligonucleotides with complementary isotope-labeled segments for NMR structural studies

RNA structure determination by solution NMR spectroscopy is often restricted to small RNAs (<15 kDa) owing to the problem of chemical shift degeneracy. A fruitful coupling of novel NMR techniques with segmental RNA labeling methodologies could be a powerful tool to overcome the molecular mass limitation of RNA NMR spectroscopy. Herein, we describe a time- and cost-effective procedure to prepare and purify segmentally labeled large RNAs. Two sets of RNA fragments with complementary labeling schemes, such as one fragment (13)C- and the other (15)N-labeled, are prepared by in vitro transcription from a single plasmid DNA. The desired RNA fragments are excised from the primary transcript by two cis-acting hammerhead ribozymes, yielding the required engineered ends for subsequent, complementary ligation. The resulting RNA oligonucleotides display NMR spectra with greatly reduced resonance overlap and thus enable NMR studies of smaller labeled RNA segments within the native context of a large RNA. The procedure is expected to take 3-4 weeks to implement.

Development of strategies for conditional RNA interference

RNA interference (RNAi) represents a powerful tool with which to undertake sequence-dependent suppression of gene expression. Synthesized double-stranded RNA (dsRNA) or dsRNA generated endogenously from plasmid or viral vectors can be used for RNAi. For the latter, polymerase III promoters which drive ubiquitous expression in all tissues have typically been adopted. Given that dsRNA molecules must contain few 5' and 3' over-hanging bases to maintain potency, employing polymerase II promoters to drive tissue-specific expression of RNAi may be problematic due to potential inclusion of nucleotides 5' and 3' of siRNA sequences. To circumvent this, polymerase II promoters in combination with cis-acting hammerhead ribozymes and short-hairpin RNA sequences have been explored as a means to generate potent dsRNA molecules in tissues defined by the promoter in use. The novel constructs evaluated in this study produced functional siRNA which suppressed the enhanced green fluorescent protein (eGFP) both in vitro and in vivo (in mice). Additionally, the constructs did not appear to elicit a significant type-1 interferon response compared to traditional H1-transcribed shRNA. Given the potential 'off-target' effects of dsRNAs, it would be preferable in many cases to limit expression of dsRNA to the tissue of interest and moreover would significantly augment the resolution of RNAi technologies. Notably, the system under evaluation in this study could readily be adapted to achieve this objective.

1019. Development of Tissue-Specific RNAi for Gene Therapy

RNA interference (RNAi) is a post-transcriptional, sequence- specific gene-silencing technology that utilises double stranded (dsRNA) molecules to degrade messenger RNA containing the same sequence as the dsRNA. Although a significant body of research has been undertaken in the field of suppression (utilising RNAi) in mammalian cells and animals, in the majority of these studies synthetic siRNAs or else polymerase III promoter-driven shRNAs have been utilised. Notably, neither of these methods allow for temporal or spatial control of suppression. Whilst there are some studies describing methods of conditional RNAi (e.g. using cre recombinase), the current study represents the first demonstration of a single plasmid with components that enable tissue specific suppression in vitro and in vivo. In essence, the approach combines tissue specific polymerase II promoters with cis-acting hammerhead ribozymes and short-hairpin RNA (shRNA) sequences to generate potent dsRNA molecules in tissues defined by the promoter in use. Results in cell culture and in mice demonstrating that the approach outlined above can be utilized to provide tissue specific RNAi will be presented. More specifically, potent suppression of EGFP expression has been demonstrated in mouse kidney and liver using a cmv promoter driven shRNA construct, whilst an albumin promoter driven shRNA construct has been shown to elicit suppression only in mouse liver. Tissue-specific RNAi has several advantages over traditional methods not least of which is the ability to minimise off-target effects of RNAi. Additionally, tissue-specific RNAi, in principle, greatly increases the resolution of RNAi technology facilitating the ability to silence genes in individual tissues which, if down-regulated in all tissue types, may be lethal.

A Self-Processing Ribozyme Cassette: Utility against Human Papillomavirus 11 E6/E7 mRNA and Hepatitis B Virus

We have been developing a self-processing triple-ribozyme cassette, which consists of two cis-acting hammerhead ribozymes flanking an internal, trans-acting hammerhead ribozyme (ITRz). Here, the single ITRz was replaced by two contiguous ITRz (dITRz), and a short poly(A) tail was designed onto the 3′ end of the liberated dITRz, to produce the “SNIPAA” cassette. Self-processing of the cassette appeared to proceed efficiently in cells: The only region of the cassette identified in cells was the liberated dITRz, with approximately 10–20% of the dITRz found within the nucleus. We tested this reagent against two therapeutically important targets, human papillomavirus 11 E6/E7 mRNA and hepatitis B virus (HBV). Library selection protocols were utilized to define accessible target sites, and ribozymes targeted to these sites were very active in vitro. Pairs of the selected ribozymes were then inserted into the SNIPAA cassette. SNIPAA constructs targeted to the E6/E7 mRNA were tested in cell culture using a cotransfection approach. Significant reductions were produced in E6/E7 target, with 80–90% reductions observed at 5 days following cotransfection. SNIPAA constructs targeted to HBV RNA were tested in vivo in a transgenic mouse model. SNIPAA constructs were packaged in liposomes, which were targeted to hepatocytes using asialofetuin, and administered ip. After 2 weeks, a >80% reduction in viral liver DNA was observed. Immunohistochemical staining for core antigen showed a similar decrease in the number of hepatocytes staining positively, compounded by a concomitant loss of residual staining intensity. These results demonstrate the in vivo utility of the self-processing SNIPAA cassette against HBV.

Role of Polyadenylation in Nucleocytoplasmic Transport of mRNA

To examine the role of polyadenylation in the nuclear export of mRNA, we have replaced the poly(A) signal in a Rev-responsive human immunodeficiency virus type 1-based reporter gene with a cis-acting hammerhead ribozyme. Transcripts from this gene thus acquire a 3' terminus by cis-ribozyme cleavage rather than by polyadenylation. The nuclear and cytoplasmic distribution of transcripts was investigated using transient gene expression and quantitative RNase protection assays. In the absence of Rev, a basal level of polyadenylated unspliced mRNA transcribed from a poly(A) signal-containing control reporter gene was detected in the cytoplasm of transfected COS7 cells. However, cytoplasmic ribozyme-cleaved unspliced RNA was only barely detectable. The nuclear/cytoplasmic (n/c) ratio of polyadenylated RNAs was 3.8, while the n/c ratio for ribozyme cis-cleaved RNAs was 33. The cytoplasmic localization of the polyadenylated unspliced mRNA was enhanced about 10-fold in the presence of Rev and the Rev-responsive element. In marked contrast to this, ribozyme cleaved RNA accumulated almost exclusively (n/c ratio of 28) in the nucleus in the presence of Rev. Actinomycin D time course analysis suggested that the low levels of the cytoplasmic ribozyme-cleaved RNAs in both the presence and absence of Rev were due to serve export deficiency of ribozyme-cleaved RNA. Finally, by inserting a 90-nucleotide poly(A) stretch directly upstream of the ribozyme cassette, we have demonstrated that a long stretch of poly(A) near the 3' end of a ribozyme-cleaved transcript is not sufficient for directing mRNA export. Taken together, these results suggest that polyadenylation is required for the nucleocytoplasmic transport of mRNA and that Rev interaction with the Rev-responsive element cannot bypass this requirement.

Crystallization of RNA-protein complexes I. Methods for the large-scale preparation of RNA suitable for crystallographic studies

In vitro transcription using bacteriophage RNA polymerases and linearised plasmid or oligodeoxynucleotide templates has been used extensively to produce RNA for biochemical studies. This method is, however, not ideal for generating RNA for crystallisation because efficient synthesis requires the RNA to have a purine rich sequence at the 5' terminus, also the subsequent RNA is heterogenous in length. We have developed two methods for the large scale production of homogeneous RNA of virtually any sequence for crystallization. In the first method RNA is transcribed together with two flanking intramolecularly-, (cis-), acting ribozymes which excise the desired RNA sequence from the primary transcript, eliminating the promoter sequence and heterogeneous 3' end generated by run-off transcription. We use a combination of two hammerhead ribozymes or a hammerhead and a hairpin ribozyme. The RNA-enzyme activity generates few sequence restrictions at the 3' terminus and none at the 5' terminus, a considerable improvement on current methodologies. In the second method the BsmAI restriction endonuclease is used to linearize plasmid template DNA thereby allowing the generation of RNA with any 3' end. In combination with a 5' cis-acting hammerhead ribozyme any sequence of RNA may be generated by in vitro transcription. This has proven to be extremely useful for the synthesis of short RNAs.