Recombinant RNA Technology Research Articles

CRISPR-based engineering of RNA viruses.

CRISPR RNA-guided endonucleases have enabled precise editing of DNA. However, options for editing RNA remain limited. Here, we combine sequence-specific RNA cleavage by CRISPR ribonucleases with programmable RNA repair to make precise deletions and insertions in RNA. This work establishes a recombinant RNA technology with immediate applications for the facile engineering of RNA viruses.

Targeted mRNA Therapy for Ornithine Transcarbamylase Deficiency.

We describe a novel, two-nanoparticle mRNA delivery system and show that it is highly effective as a means of intracellular enzyme replacement therapy (i-ERT) using a murine model of ornithine transcarbamylase deficiency (OTCD). Our Hybrid mRNA Technology delivery system (HMT) comprises an inert lipid nanoparticle that protects the mRNA from nucleases in the blood as it distributes to the liver and a polymer micelle that targets hepatocytes and triggers endosomal release of mRNA. This results in high-level synthesis of the desired protein specifically in the liver. HMT delivery of human OTC mRNA normalizes plasma ammonia and urinary orotic acid levels, and leads to a prolonged survival benefit in the murine OTCD model. HMT represents a unique, non-viral mRNA delivery method that allows multi-dose, systemic administration for treatment of single-gene inherited metabolic diseases.

Advances in time course extracellular production of human pre-miR-29b from Rhodovulum sulfidophilum

The present study reports the successful production of human pre-miR-29b both intra- and extracellularly in the marine phototrophic bacterium Rhodovulum sulfidophilum using recombinant RNA technology. In a first stage, the optimal transformation conditions (0.025μg of plasmid DNA, with a heat-shock of 2min at 35°C) were established, in order to transfer the pre-miR-29b encoding plasmid to R. sulfidophilum host. Furthermore, the extracellular recovery of this RNA product from the culture medium was greatly improved, achieving quantities that are compatible with the majority of applications, namely for in vitro or in vivo studies. Using this system, the extracellular human pre-miR-29b concentration was approximately 182μg/L, after 40h of bacterial growth, and the total intracellular pre-miR-29b was of about 358μg/L, at 32h. At the end of the fermentation, it was verified that almost 87% of cells were viable, indicating that cell lysis is minimized and that the extracellular medium is not highly contaminated with the host intracellular ribonucleases (RNases) and endotoxins, which is a critical parameter to guarantee the microRNA (miRNA) integrity. These findings demonstrate that pre-miRNAs can be produced by recombinant RNA technology, offering novel clues for the production of natural pre-miRNA agents for functional studies and RNA interference (RNAi)-based therapeutics.

Abstract 541: Application of novel RNA aptamer-based RNase I activity assay for pancreatic cancer biomarker development

Abstract Background: Ribonuclease I (RNase I) is highly expressed in pancreas and also seen in serum. Previous reports have noted its differential activity and association to a number of cancers including pancreatic adenocarcinoma (PDAC). In search of clinically relevant biomarkers for PDAC detection, in this study, we have pursued classification characterization by a novel plasma RNase I activity assay for discriminating PDAC cases from benign conditions and other malignancies. Methods: We have developed a new assay for accurate and precise quantification of RNase activities based on a specific and strong fluorescence intensity of malachite green (MG) bound to an RNA aptamer (MGA). The MGA chimeras were produced in E. coli using a novel optional noncoding RNA scaffold (OnRS) based recombinant RNA technology established in our lab. MGA chimeras were thus purified to a high degree of homogeneity (>98% pure) with an anion-exchange fast protein liquid chromatograph (FPLC) method and utilized in our assays. The cleavage of MGA by RNases (dominantly by RNase I) results in a dose- and time-dependent decrease in the fluorescence intensity at 630/650 nm (excitation/emission) for MGA-bound MG, which could be abolished by RNase inhibitor and utilized to determine RNase activities in any given sample. We applied this assay to quantitate the serum RNase activity levels in a set of prospectively collected serum from our IRB-approved Pancreas Registry including PDAC, benign cases (including chronic pancreatitis) and other (non-PDAC) malignancies, and normalized to that of a pooled serum sample from healthy subjects. Data analysis was performed for group comparison of the RNase activity levels and significance evaluated by Mann-Whitney U test (P<0.05 significant). Results: A total of 140 patients’ serum specimen was analyzed. 60 PDAC cases (stage I (n = 8), stage II (n = 25), stage III/IV (n = 27)), 60 benign conditions (chronic pancreatitis (n = 15), acute pancreatitis (n = 3), benign cysts (n = 17), normal pancreas (n = 25)), and 20 non-PDAC malignancies (Pancreatic neuroendocrine tumor (n = 11), CholangioCA(n = 2), Ampullary CA(n = 3), Hepatoma (n = 2), GIST (n = 1), lymphoma (n = 1)) Group comparison of RNase activity: PDAC vs benign: Median PDAC = 1.454; median benign group = 1.181 (P-value, <0.0001) Resectable-stage PDAC (stages I & II) vs benign: Median PDAC = 1.616; median benign group = 1.181 (P-value, <0.0001) PDAC vs other Malignancies: Median PDAC = 1.454; median other Malignancies = 1.242 (P-value = 0.0748) Conclusion: RNase activity level successfully discriminates PDAC cases from the benign conditions including chronic pancreatitis. Specifically, early-stage PDAC group is significantly separated from the benign group. Further testing is being performed for evaluating the test as a potential early detection diagnostic modality. Citation Format: Shiro Urayama, Aiming M. Yu. Application of novel RNA aptamer-based RNase I activity assay for pancreatic cancer biomarker development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 541. doi:10.1158/1538-7445.AM2015-541

Recombinant messenger RNA technology and its application in cancer immunotherapy, transcript replacement therapies, pluripotent stem cell induction, and beyond.

In recent years, the interest in using messenger RNA (mRNA) as a therapeutic means to tackle different diseases has enormously increased. This holds true not only for numerous preclinical studies, but mRNA has also entered the clinic to fight cancer. The advantages of using mRNA compared to DNA were recognized very early on, e.g., the lack of risk for genomic integration, or the expression of the encoded protein in the cytoplasm without the need to cross the nuclear membrane. However, it was generally assumed that mRNA is just not stable enough to give rise to sufficient expression of the encoded protein. Yet, an initially small group of mRNA aficionados could demonstrate that the stability of mRNA and the efficiency, by which the encoded protein is translated, can be significantly increased by selecting the right set of cis-acting structural elements (including the 5'-cap, 5'- and 3'-untranslated regions, poly(A)-tail, and modified building blocks). In parallel, significant advances in RNA packaging and delivery have been made, extending the potential for this molecule. This paved the way for further work to prove mRNA as a promising therapeutic for multiple diseases. Here, we review the developments to optimize mRNA regarding stability, translational efficiency, and immune-modulating properties to enhance its functionality and efficacy as a therapeutic. Furthermore, we summarize the current status of preclinical and clinical studies that use mRNA for cancer immunotherapy, for the expression of functional proteins as so-called transcript (or protein) replacement therapy, as well as for induction of pluripotent stem cells.

Chimeric MicroRNA-1291 Biosynthesized Efficiently in Escherichia coli Is Effective to Reduce Target Gene Expression in Human Carcinoma Cells and Improve Chemosensitivity.

In contrast to the growing interests in studying noncoding RNAs (ncRNAs) such as microRNA (miRNA or miR) pharmacoepigenetics, there is a lack of efficient means to cost effectively produce large quantities of natural miRNA agents. Our recent efforts led to a successful production of chimeric pre-miR-27b in bacteria using a transfer RNA (tRNA)-based recombinant RNA technology, but at very low expression levels. Herein, we present a high-yield expression of chimeric pre-miR-1291 in common Escherichia coli strains using the same tRNA scaffold. The tRNA fusion pre-miR-1291 (tRNA/mir-1291) was then purified to high homogeneity using affinity chromatography, whose primary sequence and post-transcriptional modifications were directly characterized by mass spectrometric analyses. Chimeric tRNA/mir-1291 was readily processed to mature miR-1291 in human carcinoma MCF-7 and PANC-1 cells. Consequently, recombinant tRNA/mir-1291 reduced the protein levels of miR-1291 target genes, including ABCC1, FOXA2, and MeCP2, as compared with cells transfected with the same doses of control methionyl-tRNA scaffold with a sephadex aptamer (tRNA/MSA). In addition, tRNA-carried pre-miR-1291 suppressed the growth of MCF-7 and PANC-1 cells in a dose-dependent manner, and significantly enhanced the sensitivity of ABCC1-overexpressing PANC-1 cells to doxorubicin. These results indicate that recombinant miR-1291 agent is effective in the modulation of target gene expression and chemosensitivity, which may provide insights into high-yield bioengineering of new ncRNA agents for pharmacoepigenetics research.

Rapid production of novel pre‐microRNA agent hsa‐mir‐27b in Escherichia coli using recombinant RNA technology for functional studies in mammalian cells

MicroRNAs (miRNAs or miRs) are a large family of noncoding RNAs that have been identified as critical epigenetic factors in the regulation of various cellular processes including drug metabolism and disposition. However, the study on miRNA functions is limited to the use of synthetic RNA and recombinant DNA agents. Herein, we show that novel pre‐miRNA‐27b (mir‐27b) agents could be biosynthesized in Escherichia coli using tRNA‐based recombinant RNA technology, and recombinant tRNA/mir‐27b chimera was readily purified to a high degree of homogeneity (> 95%) using anion‐exchange fast protein liquid chromatography. The tRNA‐fusion mir‐27b was revealed to be processed to mature miRNA miR‐27b in human carcinoma LS‐180 cells in a dose and time dependent manner. Moreover, recombinant tRNA/mir‐27b agents were biologically active in reducing the mRNA and protein expression levels of cytochrome P450 3A4 (CYP3A4) which consequently led to a lower midazolam 1'‐hydroxylase activity. These findings demonstrate that human pre‐miRNAs may be produced in Escherichia coli by recombinant RNA technology as novel ncRNA agents for functional studies in drug metabolism.

Let the cells do the work: Production of novel recombinant pre‐miRNAs in E. coli as therapeutic agents

Noncoding microRNAs (miRNAs or miRs) have been revealed as critical epigenetic factors in the control of various cellular processes including cell proliferation, survival and disease progression. However, research on miRNA functions and development of miRNA‐based therapy is limited to the use of synthetic RNA and recombinant DNA agents. Recently we have demonstrated that novel human pre‐miRNA agents can be expressed in Escherichia coli on large scale using tRNA‐based recombinant RNA technology. Milligrams of recombinant tRNA/pre‐miRNA chimeras were readily purified to high degrees of homogeneity (> 95%) using chromatography. We further showed that tRNA‐carried pre‐miRNAs were selectively processed to mature miRNAs in human carcinoma cells, which consequently suppressed the expression of miRNA target genes and modulate cell proliferation, apoptosis and chemosensistivity. Furthermore, recombinant miRNAs were active in controlling tumor progression in xenograft tumor mouse models and well tolerated in animal models. Herein we use hsa‐mir‐34a as an example to present our findings on recombinant pre‐miRNA agents, which shall serve as novel tools for the study on miRNA functions and development of RNA‐based therapeutics.

A general approach to high-yield biosynthesis of chimeric RNAs bearing various types of functional small RNAs for broad applications.

RNA research and therapy relies primarily on synthetic RNAs. We employed recombinant RNA technology toward large-scale production of pre-miRNA agents in bacteria, but found the majority of target RNAs were not or negligibly expressed. We thus developed a novel strategy to achieve consistent high-yield biosynthesis of chimeric RNAs carrying various small RNAs (e.g. miRNAs, siRNAs and RNA aptamers), which was based upon an optimal noncoding RNA scaffold (OnRS) derived from tRNA fusion pre-miR-34a (tRNA/mir-34a). Multi-milligrams of chimeric RNAs (e.g. OnRS/miR-124, OnRS/GFP-siRNA, OnRS/Neg (scrambled RNA) and OnRS/MGA (malachite green aptamer)) were readily obtained from 1 l bacterial culture. Deep sequencing analyses revealed that mature miR-124 and target GFP-siRNA were selectively released from chimeric RNAs in human cells. Consequently, OnRS/miR-124 was active in suppressing miR-124 target gene expression and controlling cellular processes, and OnRS/GFP-siRNA was effective in knocking down GFP mRNA levels and fluorescent intensity in ES-2/GFP cells and GFP-transgenic mice. Furthermore, the OnRS/MGA sensor offered a specific strong fluorescence upon binding MG, which was utilized as label-free substrate to accurately determine serum RNase activities in pancreatic cancer patients. These results demonstrate that OnRS-based bioengineering is a common, robust and versatile strategy to assemble various types of small RNAs for broad applications.

Rapid production of novel pre-microRNA agent hsa-mir-27b in Escherichia coli using recombinant RNA technology for functional studies in mammalian cells.

Noncoding microRNAs (miRNAs or miRs) have been revealed as critical epigenetic factors in the regulation of various cellular processes, including drug metabolism and disposition. However, research on miRNA functions is limited to the use of synthetic RNA and recombinant DNA agents. Herein, we show that novel pre-miRNA-27b (miR-27b) agents can be biosynthesized in Escherichia coli using recombinant RNA technology, and recombinant transfer RNA (tRNA)/mir-27b chimera was readily purified to a high degree of homogeneity (>95%) using anion-exchange fast protein liquid chromatography. The tRNA-fusion miR-27b was revealed to be processed to mature miRNA miR-27b in human carcinoma LS-180 cells in a dose- and time-dependent manner. Moreover, recombinant tRNA/miR-27b agents were biologically active in reducing the mRNA and protein expression levels of cytochrome P450 3A4 (CYP3A4), which consequently led to lower midazolam 1'-hydroxylase activity. These findings demonstrate that pre-miRNA agents can be produced by recombinant RNA technology for functional studies.

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.

Recombinant RNA technology: the tRNA scaffold

RNA has emerged as a major player in most cellular processes. Understanding these processes at the molecular level requires homogeneous RNA samples for structural, biochemical and pharmacological studies. So far, this has been a bottleneck, as the only methods for producing such pure RNA have been in vitro syntheses. Here we describe a generic approach for expressing and purifying structured RNA in Escherichia coli, using tools that parallel those available for recombinant proteins. Our system is based on a camouflage strategy, the 'tRNA scaffold', in which the recombinant RNA is disguised as a natural RNA and thus hijacks the host machinery, escaping cellular RNases. This opens the way to large-scale structural and molecular investigations of RNA function.

Foreign Complementary Sequences Facilitate Genetic RNA Recombination in Brome Mosaic Virus

We have demonstrated that local antisense sequences can mediate genetic recombination within the 3′ noncoding region among brome mosaic virus (BMV) RNAs (P. Nagy and J. J. Bujarski, 1993, Proc, Natl. Acad. Sci. USA 90, 6390-6394). Here we show that foreign complementary inserts can direct crossovers between BMV RNA3 components within an internal region. A 170-nt polynucleotide derived from the cowpea chlorotic mottle virus (CCMV) RNA3 was inserted just upstream of the initiation cordon of the BMV coat protein open reading frame in either sense or antisense orientations. The resulting respective mutants, BCC+ and BCC-, maintained unchanged CCMV inserts when inoculated separately on leaves of a local lesion host for BMV. In contrast, when a mixture containing both mutated RNAs3 was inoculated, a significant fraction of lesions accumulated the BMV RNA3 lacking the CCMV insert. The presence of a 3′ marker mutation confirmed that the BMV RNA3 progeny arose due to crossovers between BCC+ and BCC- within the complementary sequences. The highest frequency of recombinant appearance was observed when the RNA mixtures were annealed prior to inoculation on the host plants. Our results confirm a concept predicting the general nature of the heteroduplex-mediated recombination functioning in RNA viruses. Examples of possible applications of this approach in recombinant RNA technology are discussed.

Recombinant photoreactive tRNA molecules as probes for cross-linking studies

Photoreactive tRNA derivatives have been used extensively for investigating the interaction of tRNA molecules with their ligands and substrates. Recombinant RNA technology facilitates the construction of such tRNA probes through site-specific incorporation of photoreactive nucleosides. The general strategy involves preparation of suitable tRNA fragments and their ligation either to a photoreactive nucleotide or to each other. tRNA fragments can be prepared by site-specific cleavage of native tRNAs, or synthesized by enzymatic and chemical means. A number of photoreactive nucleosides suitable for incorporation into tRNA are presently available. Joining of tRNA fragments is accomplished either by RNA ligase or by DNA ligase in the presence of a DNA splint. The application of this methodology to the study of tRNA binding sites on the ribosome is discussed, and a model of the tRNA-ribosome complex is presented.

A 2-thiouridine derivative in tRNAGlu is a positive determinant for aminoacylation by Escherichia coli glutamyl-tRNA synthetase

Early investigations into the interaction between Escherichia coli glutamyl-tRNA synthetase (GluRS) and tRNAGlu have implicated the modified nucleoside 5-[(methylamino)methyl]-2-thiouridine in the first position of the anticodon as an important contact for efficient aminoacylation. However, the experimental methods employed were not sufficient to determine whether the interaction was dependent on the presence of the modification or simply involved other anticodon loop-nucleotides, now occluded from interaction with the synthetase. Unmodified E. coli tRNA(Glu), derived by in vitro transcription of the corresponding gene, is a poor substrate for GluRS, exhibiting a 100-fold reduction in its specificity constant (kcat/KM) compared to that of tRNA(Glu) prepared from an overproducing strain. Through the use of recombinant RNA technology, we created several hybrid tRNAs which combined sequences from the in vitro transcript with that of the native tRNA, resulting in tRNA molecules differing in modified base content. By in vitro aminoacylation of these hybrid tRNA molecules and of tRNAs with base substitutions at positions of nucleotide modification, we show conclusively that the modified uridine at position 34 in tRNA(Glu) is required for efficient aminoacylation by E. coli GluRS. This is only the second example of a tRNA modification acting as a positive determinant for interaction with its cognate aminoacyl-tRNA synthetase.

Recognition of bases in Escherichia coli tRNA(Gln) by glutaminyl-tRNA synthetase: a complete identity set.

The fidelity of protein biosynthesis rests largely on the correct aminoacylation of transfer RNAs by their cognate aminoacyl-tRNA synthetases. Previous studies have demonstrated that the interaction of Escherichia coli tRNA(Gln) with glutaminyl-tRNA synthetase (GlnRS) provides an excellent system for studying the basis of this highly specific recognition process. Correct aminoacylation depends on the set of nucleotides (identity elements) in tRNA(Gln) responsible for correct interaction with GlnRS. Specific contacts between tRNA(Gln) and GlnRS include the 2-amino group of guanosines. Therefore, we made a set of tRNA(Gln) variants in which specific guanosines were replaced by inosine using recombinant RNA technology. This resulted in a set of tRNAs that varied by single deletions of the amino group from guanine residues, thus allowing us to test the functional importance of these contacts. In addition, a number of mutants were made by transcription of mutated tRNA genes with base changes at position 10, 16 or 25. In vitro aminoacylation of these mutants showed decreases in the specificity constant (kcat/KM) of up to 300-fold, with kcat being the parameter most affected. These experiments reveal G10 as a new element of glutamine identity. In addition, the interaction of G2, G3 and G10 with GlnRS via the 2-amino group is significant for tRNA discrimination. Based on these results, and on earlier data, we propose a complete set of bases as identity elements for tRNA(Gln).

Site-directed protection of RNA during nuclease digestion

We report the use of oligodeoxynucleotides to block the nucleolytic hydrolysis of single-stranded regions of RNA. Using complementary oligomers, the hydrolysis of the CCA terminus of methionine initiator tRNA could be prevented. This method can be useful in the production of specific single-stranded fragments of RNA, which are necessary in recombinant RNA technology.

The birth of recombinant RNA technology.

Compte rendu des travaux de l'Institute of Cancer Research de l'universite de Colombia permettant d'obtenir de grandes quantites d'ARN grâce au systeme de replication de la replicase Qβ