Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 140
Filter
1.
Mol Biotechnol ; 62(4): 219-227, 2020 Apr.
Article in English | MEDLINE | ID: mdl-32103426

ABSTRACT

The ribosome is an essential organelle in charge of the translational processes in all kinds of cells. Currently, the scenario of its function has been significantly expanded from the classic machine for protein synthesis to a regulatory platform for quality control to maintain the protein homeostasis in a living cell. The ribosome is much more than a mechanical device with a static structure: it is inherently dynamic in structure and function, especially in response to the environmental fluctuations. Considerable effort has been made to regulate its structure and physiological function by engineering the components of a ribosome. The findings of the pioneering studies significantly deepened our understanding of a ribosome and exemplified how a ribosome could be engineered for biotechnology purposes in the era of synthetic biology. The engineering of ribosome offered highly accessible methods capable of comprehensively optimizing the performance of strains of industrial importance. In this article, the relevant recent advances were systematically reviewed.


Subject(s)
Amino Acids/chemistry , Biotechnology/methods , Protein Biosynthesis , Ribosomes/chemistry , Ribosomes/enzymology , Synthetic Biology/methods , Amino Acids/chemical synthesis , Amino Acids/metabolism , Codon, Nonsense/chemistry , Codon, Nonsense/genetics , Drug Resistance, Bacterial/genetics , Metabolic Engineering/methods , Protein Engineering/methods , RNA, Catalytic/biosynthesis , RNA, Catalytic/chemistry , RNA, Catalytic/genetics , RNA, Ribosomal/chemistry , RNA, Transfer/chemistry , RNA, Transfer/genetics , Ribosomes/metabolism
2.
Methods ; 161: 41-45, 2019 05 15.
Article in English | MEDLINE | ID: mdl-30738128

ABSTRACT

A large number of catalytic RNAs, or ribozymes, have been identified in the genomes of various organisms and viruses. Ribozymes are involved in biological processes such as regulation of gene expression and viral replication, but biological roles of many ribozymes still remain unknown. Ribozymes have also inspired researchers to engineer synthetic ribozymes that function as sensors or gene switches. To gain deeper understanding of the sequence-function relationship of ribozymes and to efficiently engineer synthetic ribozymes, a large number of ribozyme variants need to be examined which was limited to hundreds of sequences by Sanger sequencing. The advent of high-throughput sequencing technologies, however, has allowed us to sequence millions of ribozyme sequences at low cost. This review focuses on the recent applications of high-throughput sequencing to both characterize and engineer ribozymes, to highlight how the large-scale sequence data can advance ribozyme research and engineering.


Subject(s)
Genetic Engineering/methods , High-Throughput Nucleotide Sequencing/methods , RNA, Catalytic/biosynthesis , RNA, Catalytic/genetics , Animals , Genetic Engineering/trends , High-Throughput Nucleotide Sequencing/trends , Humans , Mutation/physiology
3.
Methods ; 161: 35-40, 2019 05 15.
Article in English | MEDLINE | ID: mdl-30639182

ABSTRACT

Hammerhead ribozyme-based RNA switches have been proven to be powerful tools for conditional gene regulation in various organisms. We present neomycin-dependent hammerhead ribozymes (HHR) that influence gene expression in a ligand- and dose-dependent manner in S. cerevisiae. We utilized a novel design of fusing the aptamer domain to the HHR enabling for the first time the identification of genetic ON- and OFF-switches within the same library. For this purpose a neomycin aptamer was fused to stem 1 of a type 3 hammerhead ribozyme via an addressable three-way junction that shows high flexibility at the connection site. An in vivo screening approach identified sequences that allow to induce or repress gene expression 2- to 3-fold in response to neomycin addition. The ribozyme switches operate at neomycin concentrations that show no toxic effect on cell growth and pose powerful genetic tools to study and modulate cellular function in yeast.


Subject(s)
Gene Expression Regulation, Fungal , Neomycin/pharmacology , RNA, Catalytic/biosynthesis , RNA, Catalytic/genetics , Saccharomyces cerevisiae Proteins/biosynthesis , Saccharomyces cerevisiae Proteins/genetics , Anti-Bacterial Agents/pharmacology , Gene Expression Regulation, Fungal/drug effects , Saccharomyces cerevisiae
4.
Proc Natl Acad Sci U S A ; 115(36): E8547-E8556, 2018 09 04.
Article in English | MEDLINE | ID: mdl-30127005

ABSTRACT

Inherited retinal degenerations are caused by mutations in >250 genes that affect photoreceptor cells or the retinal pigment epithelium and result in vision loss. For autosomal recessive and X-linked retinal degenerations, significant progress has been achieved in the field of gene therapy as evidenced by the growing number of clinical trials and the recent commercialization of the first gene therapy for a form of congenital blindness. However, despite significant efforts to develop a treatment for the most common form of autosomal dominant retinitis pigmentosa (adRP) caused by >150 mutations in the rhodopsin (RHO) gene, translation to the clinic has stalled. Here, we identified a highly efficient shRNA that targets human (and canine) RHO in a mutation-independent manner. In a single adeno-associated viral (AAV) vector we combined this shRNA with a human RHO replacement cDNA made resistant to RNA interference and tested this construct in a naturally occurring canine model of RHO-adRP. Subretinal vector injections led to nearly complete suppression of endogenous canine RHO RNA, while the human RHO replacement cDNA resulted in up to 30% of normal RHO protein levels. Noninvasive retinal imaging showed photoreceptors in treated areas were completely protected from retinal degeneration. Histopathology confirmed retention of normal photoreceptor structure and RHO expression in rod outer segments. Long-term (>8 mo) follow-up by retinal imaging and electroretinography indicated stable structural and functional preservation. The efficacy of this gene therapy in a clinically relevant large-animal model paves the way for treating patients with RHO-adRP.


Subject(s)
Dependovirus , Gene Knock-In Techniques/methods , Gene Knockdown Techniques/methods , Genetic Therapy/methods , Genetic Vectors , RNA, Catalytic , Retinal Rod Photoreceptor Cells/metabolism , Retinitis Pigmentosa , Rhodopsin , Animals , Dogs , HEK293 Cells , Humans , RNA, Catalytic/biosynthesis , RNA, Catalytic/genetics , Retinal Rod Photoreceptor Cells/pathology , Retinitis Pigmentosa/genetics , Retinitis Pigmentosa/metabolism , Retinitis Pigmentosa/pathology , Rhodopsin/biosynthesis , Rhodopsin/genetics
5.
J Biochem ; 161(5): 451-462, 2017 May 01.
Article in English | MEDLINE | ID: mdl-28096453

ABSTRACT

RNA is a biopolymer that is attractive for constructing nano-scale objects with complex structures. Three-dimensional (3D) structures of naturally occurring RNAs often have modular architectures. The 3D structure of a group I (GI) ribozyme from Tetrahymena has a typical modular architecture, which can be separated into two structural modules (ΔP5 and P5abc). The fully active ribozyme can be reconstructed by assembling the two separately prepared modules through highly specific and strong assembly between ΔP5 ribozyme and P5abc RNA. Such non-covalent assembly of the two modules allows the design of polygonal RNA nano-structures. Through rational redesign of the parent GI ribozyme, we constructed variant GI ribozymes as unit RNAs for polygonal-shaped (closed) oligomers with catalytic activity. Programmed trimerization and tetramerization of the unit RNAs afforded catalytically active nano-sized RNA triangles and squares, the structures of which were directly observed by atomic force microscopy (AFM).


Subject(s)
RNA, Catalytic/chemistry , RNA, Catalytic/metabolism , Tetrahymena/enzymology , Electrophoretic Mobility Shift Assay , Microscopy, Atomic Force , Nucleic Acid Conformation , RNA/chemistry , RNA/metabolism , RNA, Catalytic/biosynthesis
6.
Molecules ; 21(11)2016 Oct 31.
Article in English | MEDLINE | ID: mdl-27809244

ABSTRACT

Group I introns in nuclear ribosomal RNA of eukaryotic microorganisms are processed by splicing or circularization. The latter results in formation of full-length circular introns without ligation of the exons and has been proposed to be active in intron mobility. We applied qRT-PCR to estimate the copy number of circular intron RNA from the myxomycete Didymium iridis. In exponentially growing amoebae, the circular introns are nuclear and found in 70 copies per cell. During heat-shock, the circular form is up-regulated to more than 500 copies per cell. The intron harbours two ribozymes that have the potential to linearize the circle. To understand the structural features that maintain circle integrity, we performed chemical and enzymatic probing of the splicing ribozyme combined with molecular modeling to arrive at models of the inactive circular form and its active linear counterpart. We show that the two forms have the same overall structure but differ in key parts, including the catalytic core element P7 and the junctions at which reactions take place. These differences explain the relative stability of the circular species, demonstrate how it is prone to react with a target molecule for circle integration and thus supports the notion that the circular form is a biologically significant molecule possibly with a role in intron mobility.


Subject(s)
Heat-Shock Response/physiology , Introns , Myxomycetes/metabolism , RNA, Catalytic/biosynthesis , Myxomycetes/genetics , RNA, Catalytic/genetics
7.
Proc Natl Acad Sci U S A ; 113(35): 9786-91, 2016 08 30.
Article in English | MEDLINE | ID: mdl-27528667

ABSTRACT

In all extant life, genetic information is stored in nucleic acids that are replicated by polymerase proteins. In the hypothesized RNA world, before the evolution of genetically encoded proteins, ancestral organisms contained RNA genes that were replicated by an RNA polymerase ribozyme. In an effort toward reconstructing RNA-based life in the laboratory, in vitro evolution was used to improve dramatically the activity and generality of an RNA polymerase ribozyme by selecting variants that can synthesize functional RNA molecules from an RNA template. The improved polymerase ribozyme is able to synthesize a variety of complex structured RNAs, including aptamers, ribozymes, and, in low yield, even tRNA. Furthermore, the polymerase can replicate nucleic acids, amplifying short RNA templates by more than 10,000-fold in an RNA-catalyzed form of the PCR. Thus, the two prerequisites of Darwinian life-the replication of genetic information and its conversion into functional molecules-can now be accomplished with RNA in the complete absence of proteins.


Subject(s)
DNA-Directed RNA Polymerases/biosynthesis , Directed Molecular Evolution , Polymerase Chain Reaction/methods , RNA, Catalytic/biosynthesis , RNA/genetics , Aptamers, Nucleotide/biosynthesis , Aptamers, Nucleotide/genetics , Base Pairing , DNA-Directed RNA Polymerases/genetics , Nucleic Acid Conformation , RNA/chemistry , RNA/metabolism , RNA, Catalytic/genetics , Templates, Genetic
8.
Cytokine ; 82: 80-6, 2016 06.
Article in English | MEDLINE | ID: mdl-26841929

ABSTRACT

Normal cell proliferation is controlled by a balance between signals that promote or halt cell proliferation. Micro RNAs are emerging as key elements in providing fine signal balance in different physiological situations. Here we report that STAT5 signaling induces the miRNAs miR-19 and miR-155, which potentially antagonize the tumor suppressor axis composed by the STAT5 target gene SOCS1 (suppressor of cytokine signaling-1) and its downstream effector p53. MiRNA sponges against miR-19 or miR-155 inhibit the functions of these miRNAs and potentiate the induction of SOCS1 and p53 in mouse leukemia cells and in human myeloma cells. Adding a catalytic RNA motif of the hammerhead type within miRNA sponges against miR-155 leads to decreased miR-155 levels and increased their ability of inhibiting cell growth and cell migration in myeloma cells. The results indicate that antagonizing miRNA activity can reactivate tumor suppressor pathways downstream cytokine stimulation in tumor cells.


Subject(s)
Leukemia/metabolism , MicroRNAs/metabolism , Multiple Myeloma/metabolism , RNA, Catalytic/biosynthesis , RNA, Neoplasm/metabolism , Suppressor of Cytokine Signaling 1 Protein/metabolism , Tumor Suppressor Protein p53/metabolism , Animals , Humans , Leukemia/genetics , Mice , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , Multiple Myeloma/genetics , RAW 264.7 Cells , RNA, Catalytic/genetics , RNA, Neoplasm/antagonists & inhibitors , RNA, Neoplasm/genetics , Suppressor of Cytokine Signaling 1 Protein/genetics , Tumor Suppressor Protein p53/genetics
9.
J Biosci Bioeng ; 121(4): 471-6, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26386750

ABSTRACT

A simple, inexpensive flow-focusing device has been developed to make uniform droplets for biochemical reactions, such as in vitro transcription and cell-free protein synthesis. The device was fabricated from commercially available components without special equipment. Using the emulsion droplets formed by the device, a class I ligase ribozyme, bcI 23, was successfully synthesized from DNA attached to magnetic microbeads by T7 RNA polymerase. It was also ligated with an RNA substrate on the same microbeads, and detected using flow cytometry with a fluorescent probe. In addition, a single-chain derivative of the lambda Cro protein was expressed using an Escherichia coli cell-free protein synthesis system in emulsion, which was prepared using the flow-focusing device. In both emulsified reactions, usage of the flow-focusing device was able to greatly reduce the coefficient of variation for the amount of RNA or protein displayed on the microbeads, demonstrating the device is advantageous for quantitative analysis in high-throughput screening.


Subject(s)
Lab-On-A-Chip Devices , Protein Biosynthesis , Transcription, Genetic , Cell-Free System , DNA/genetics , DNA/metabolism , DNA-Directed RNA Polymerases/metabolism , Emulsions , Escherichia coli/metabolism , Flow Cytometry , Fluorescence , In Vitro Techniques/economics , In Vitro Techniques/instrumentation , In Vitro Techniques/methods , Lab-On-A-Chip Devices/economics , Ligases/analysis , Ligases/biosynthesis , Ligases/genetics , Magnetics , Microspheres , RNA, Catalytic/analysis , RNA, Catalytic/biosynthesis , RNA, Catalytic/genetics , Repressor Proteins/analysis , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Viral Proteins/metabolism , Viral Regulatory and Accessory Proteins/analysis , Viral Regulatory and Accessory Proteins/biosynthesis , Viral Regulatory and Accessory Proteins/genetics
10.
PLoS One ; 9(10): e109987, 2014.
Article in English | MEDLINE | ID: mdl-25299454

ABSTRACT

The error threshold of replication limits the selectively maintainable genome size against recurrent deleterious mutations for most fitness landscapes. In the context of RNA replication a distinction between the genotypic and the phenotypic error threshold has been made; where the latter concerns the maintenance of secondary structure rather than sequence. RNA secondary structure is treated as a proxy for function. The phenotypic error threshold allows higher per digit mutation rates than its genotypic counterpart, and is known to increase with the frequency of neutral mutations in sequence space. Here we show that the degree of neutrality, i.e. the frequency of nearest-neighbour (one-step) neutral mutants is a remarkably accurate proxy for the overall frequency of such mutants in an experimentally verifiable formula for the phenotypic error threshold; this we achieve by the full numerical solution for the concentration of all sequences in mutation-selection balance up to length 16. We reinforce our previous result that currently known ribozymes could be selectively maintained by the accuracy known from the best available polymerase ribozymes. Furthermore, we show that in silico stabilizing selection can increase the mutational robustness of ribozymes due to the fact that they were produced by artificial directional selection in the first place. Our finding offers a better understanding of the error threshold and provides further insight into the plausibility of an ancient RNA world.


Subject(s)
Nucleic Acid Conformation , RNA, Catalytic/genetics , RNA/genetics , Selection, Genetic/genetics , Computer Simulation , Genetic Drift , Genetic Fitness , Models, Theoretical , Mutation , RNA/biosynthesis , RNA, Catalytic/biosynthesis , Sequence Analysis, RNA
11.
J Biol Chem ; 288(46): 33096-106, 2013 Nov 15.
Article in English | MEDLINE | ID: mdl-24089519

ABSTRACT

Understanding the evolution of functional RNA molecules is important for our molecular understanding of biology. Here we tested experimentally how two evolutionary parameters, selection pressure and recombination, influenced the evolution of an evolving RNA population. This was done using four parallel evolution experiments that employed low or gradually increasing selection pressure, and recombination events either at the end or dispersed throughout the evolution. As model system, a trans-splicing group I intron ribozyme was evolved in Escherichia coli cells over 12 rounds of selection and amplification, including mutagenesis and recombination. The low selection pressure resulted in higher efficiency of the evolved ribozyme populations, whereas differences in recombination did not have a strong effect. Five mutations were responsible for the highest efficiency. The first mutation swept quickly through all four evolving populations, whereas the remaining four mutations accumulated later and more efficiently under low selection pressure. To determine why low selection pressure aided this evolution, all evolutionary intermediates between the wild type and the 5-mutation variant were constructed, and their activities at three different selection pressures were determined. The resulting fitness profiles showed a high cooperativity among the four late mutations, which can explain why high selection pressure led to inefficient evolution. These results show experimentally how low selection pressure can benefit the evolution of cooperative mutations in functional RNAs.


Subject(s)
Directed Molecular Evolution , Escherichia coli/metabolism , Mutation , RNA, Catalytic/biosynthesis , RNA, Catalytic/genetics , Escherichia coli/genetics
12.
Cancer Gene Ther ; 20(5): 298-307, 2013 May.
Article in English | MEDLINE | ID: mdl-23618950

ABSTRACT

Stathmin is the founding member of a family of microtubule-destabilizing proteins that have a critical role in the regulation of mitosis. Stathmin is expressed at high levels in breast cancer and its overexpression is linked to disease progression. Although there is a large body of evidence to support a role for stathmin in breast cancer progression, the validity of stathmin as a viable therapeutic target for breast cancer has not been investigated. Here, we used a bicistronic adenoviral vector that co-expresses green fluorescent protein and a ribozyme that targets stathmin messenger RNA in preclinical breast cancer models with different estrogen receptor (ER) status. We examined the effects of anti-stathmin ribozyme on the malignant phenotype of breast cancer cells in vitro and in xenograft models in vivo both as a single agent and in combination with chemotherapeutic agents. Adenovirus-mediated gene transfer of anti-stathmin ribozyme resulted in a dose-dependent inhibition of proliferation and clonogenicity associated with a G2/M arrest and increase in apoptosis in both ER-positive and ER-negative breast cancer cell lines. This inhibition was markedly enhanced when stathmin-inhibited breast cancer cells were exposed to low concentrations of taxol, which resulted in virtually complete loss of the malignant phenotype. Interestingly, breast cancer xenografts treated with low doses of anti-stathmin therapy and taxol showed regression in a majority of tumors, while some tumors stopped growing completely. In contrast, combination of anti-stathmin ribozyme and adriamycin resulted in only a modest inhibition of growth in vitro and in breast cancer xenografts in vivo. Although inhibition of tumor growth was observed in both the combination treatment groups compared with groups treated with single agent alone, combination of anti-stathmin therapy and taxol had a more profound inhibition of tumorigenicity, as both agents target the microtubule pathway. Clinically, these findings are highly relevant because taxol is one of the most active chemotherapeutic agents in breast cancer. These studies provide the proof-of-principle that stathmin provides an attractive molecular target, which could serve as a primary focus of novel approaches to breast cancer.


Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/therapy , Genetic Therapy/methods , RNA, Catalytic/genetics , Stathmin/antagonists & inhibitors , Stathmin/genetics , Adenoviridae/genetics , Animals , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Cycle Checkpoints/genetics , Cell Line, Tumor , Combined Modality Therapy , Doxorubicin/pharmacology , Female , Genetic Vectors/genetics , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Mice , Mice, Nude , RNA, Catalytic/biosynthesis , RNA, Catalytic/metabolism , Stathmin/metabolism , Xenograft Model Antitumor Assays
13.
Nucleic Acids Res ; 41(2): e41, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23155065

ABSTRACT

Synthetic RNA control devices that use ribozymes as gene-regulatory components have been applied to controlling cellular behaviors in response to environmental signals. Quantitative measurement of the in vitro cleavage rate constants associated with ribozyme-based devices is essential for advancing the molecular design and optimization of this class of gene-regulatory devices. One of the key challenges encountered in ribozyme characterization is the efficient generation of full-length RNA from in vitro transcription reactions, where conditions generally lead to significant ribozyme cleavage. Current methods for generating full-length ribozyme-encoding RNA rely on a trans-blocking strategy, which requires a laborious gel separation and extraction step. Here, we develop a simple two-step gel-free process including cis-blocking and trans-activation steps to support scalable generation of functional full-length ribozyme-encoding RNA. We demonstrate our strategy on various types of natural ribozymes and synthetic ribozyme devices, and the cleavage rate constants obtained for the RNA generated from our strategy are comparable with those generated through traditional methods. We further develop a rapid, label-free ribozyme cleavage assay based on surface plasmon resonance, which allows continuous, real-time monitoring of ribozyme cleavage. The surface plasmon resonance-based characterization assay will complement the versatile cis-blocking and trans-activation strategy to broadly advance our ability to characterize and engineer ribozyme-based devices.


Subject(s)
RNA, Catalytic/biosynthesis , DNA/chemistry , Electrophoresis, Polyacrylamide Gel , RNA Cleavage , RNA Stability , RNA, Catalytic/chemistry , RNA, Catalytic/metabolism , Surface Plasmon Resonance/methods , Synthetic Biology/methods , Transcription, Genetic
15.
Nature ; 491(7422): 72-7, 2012 Nov 01.
Article in English | MEDLINE | ID: mdl-23075853

ABSTRACT

The origins of life on Earth required the establishment of self-replicating chemical systems capable of maintaining and evolving biological information. In an RNA world, single self-replicating RNAs would have faced the extreme challenge of possessing a mutation rate low enough both to sustain their own information and to compete successfully against molecular parasites with limited evolvability. Thus theoretical analyses suggest that networks of interacting molecules were more likely to develop and sustain life-like behaviour. Here we show that mixtures of RNA fragments that self-assemble into self-replicating ribozymes spontaneously form cooperative catalytic cycles and networks. We find that a specific three-membered network has highly cooperative growth dynamics. When such cooperative networks are competed directly against selfish autocatalytic cycles, the former grow faster, indicating an intrinsic ability of RNA populations to evolve greater complexity through cooperation. We can observe the evolvability of networks through in vitro selection. Our experiments highlight the advantages of cooperative behaviour even at the molecular stages of nascent life.


Subject(s)
Biocatalysis , Evolution, Chemical , Models, Biological , Origin of Life , RNA, Catalytic/biosynthesis , RNA, Catalytic/metabolism , Azoarcus/enzymology , Azoarcus/genetics , Base Pairing , Base Sequence , Introns/genetics , Models, Genetic , Molecular Sequence Data , RNA, Catalytic/chemistry , RNA, Catalytic/genetics , Recombinases/biosynthesis , Recombinases/chemistry , Recombinases/genetics , Recombinases/metabolism
16.
J Am Chem Soc ; 134(19): 8050-3, 2012 May 16.
Article in English | MEDLINE | ID: mdl-22551009

ABSTRACT

A nuclease-resistant RNA enzyme, constructed entirely from L-ribonucleotides, was shown to undergo ligand-dependent, self-sustained replication with exponential growth. The catalytic motif is based on a previously described RNA ligase that can undergo either self- or cross-replication but had been limited in its application to ligand sensing due to its susceptibility to degradation by ribonucleases. The self-replicating RNA enzyme and its RNA substrates were prepared synthetically from either D- or L-nucleoside phosphoramidites. The D and L reaction systems undergo isothermal, ligand-dependent exponential amplification in the same manner, but only the l system is impervious to ribonucleases and can operate, for example, in the presence of human serum. This system has potential for the quantitative detection of various ligands that are present within biological or environmental samples. In addition, this work provides the first demonstration of the self-sustained exponential amplification of nonbiological molecules.


Subject(s)
RNA, Catalytic/biosynthesis , RNA, Catalytic/metabolism , Self-Sustained Sequence Replication , Base Sequence , Humans , Kinetics , Ligands , RNA, Catalytic/genetics , Serum/metabolism
17.
Biochimie ; 94(7): 1457-63, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22465106

ABSTRACT

The RNA-world-theory is one possible explanation of how life on earth has evolved. In this context it is of high interest to search for molecular systems, capable of self-organization into structures with increasing complexity. We have engineered a simple catalytic system in which two short RNA molecules can catalyze their own ligation to form a larger RNA construct. The system is based on the hairpin ribozyme using a 2',3'-cyclophosphate as activated species for ligation. 2',3'-cyclic phosphates can be easily formed and occur in many natural systems, thus being superior candidates for activated building blocks in RNA world scenarios.


Subject(s)
Oligoribonucleotides/metabolism , RNA, Catalytic/biosynthesis , RNA, Catalytic/metabolism , Base Sequence , Biocatalysis , Computational Biology , Nucleic Acid Conformation , Oligoribonucleotides/genetics , Phosphates/metabolism , Polymerization , RNA, Catalytic/chemistry , RNA, Catalytic/genetics , Temperature
18.
Methods Mol Biol ; 848: 159-84, 2012.
Article in English | MEDLINE | ID: mdl-22315069

ABSTRACT

Ribozymes and riboswitches are RNA motifs that accelerate biological reactions and regulate gene expression in response to metabolite recognition, respectively. These RNA molecules gain functionality via complex folding that cannot be predicted a priori, and thus requires high-resolution three-dimensional structure determination to locate key functional attributes. Herein, we present an overview of the methods used to determine small RNA structures with an emphasis on RNA preparation, crystallization, and structure refinement. We draw upon examples from our own research in the analysis of the leadzyme ribozyme, the hairpin ribozyme, a class I preQ(1) riboswitch, and variants of a larger class II preQ(1) riboswitch. The methods presented provide a guide for comparable investigations of noncoding RNA molecules including a 48-solution, "first choice" RNA crystal screen compiled from our prior successes with commercially available screens.


Subject(s)
Crystallography, X-Ray/methods , RNA, Catalytic/chemistry , Riboswitch , Chromatography, Gel , DNA/biosynthesis , DNA/metabolism , DNA Polymerase I/metabolism , Electrophoresis, Polyacrylamide Gel , Ion Exchange , Protein Biosynthesis , RNA, Catalytic/biosynthesis , RNA, Catalytic/genetics , RNA, Catalytic/isolation & purification , Robotics
19.
Nucleic Acids Res ; 39(19): 8559-71, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21742763

ABSTRACT

Obtaining homogeneous population of natively folded RNAs is a crippling problem encountered when preparing RNAs for structural or enzymatic studies. Most of the traditional methods that are employed to prepare large quantities of RNAs involve procedures that partially denature the RNA. Here, we present a simple strategy using 'click' chemistry to couple biotin to a 'caged' photocleavable (PC) guanosine monophosphate (GMP) in high yield. This biotin-PC GMP, accepted by T7 RNA polymerase, has been used to transcribe RNAs ranging in size from 27 to 527 nt. Furthermore we show, using an in-gel fluorescence assay, that natively prepared 160 and 175 kDa minimal group II intron ribozymes have enhanced catalytic activity over the same RNAs, purified via denaturing conditions and refolded. We conclude that large complex RNAs prepared by non-denaturing means form a homogeneous population and are catalytically more active than those prepared by denaturing methods and subsequent refolding; this facile approach for native RNA preparation should benefit synthesis of RNAs for biophysical and therapeutic applications.


Subject(s)
Guanosine Monophosphate/chemistry , RNA, Catalytic/biosynthesis , RNA/biosynthesis , Avidin/chemistry , Base Sequence , Biotinylation , Catalysis , Chromatography, Affinity , Click Chemistry , DNA-Directed RNA Polymerases/metabolism , Introns , Molecular Sequence Data , Photochemical Processes , RNA/chemistry , RNA/isolation & purification , RNA, Catalytic/metabolism , RNA, Ribosomal/metabolism , Transcription, Genetic , Viral Proteins/metabolism
20.
Mol Biosyst ; 7(8): 2419-27, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21603688

ABSTRACT

Synthetic regulatory devices are key components for the development of complex biological systems and the reprogramming of cellular functions and networks. Here we describe the selection of tetracycline inducible hammerhead ribozymes. A tetracycline aptamer was fused to the full-length hammerhead ribozyme via a variable linker region. 11 rounds of in vitro selection were applied to isolate linker sequences that mediate tetracycline dependent hammerhead cleavage. We identified allosteric ribozymes that cleave in the presence of 1 µM tetracycline as fast as the full-length ribozyme whereas cleavage is inhibited up to 333-fold in the absence of tetracycline. Reporter gene assays indicate that the allosteric ribozymes can be employed to control gene expression in yeast.


Subject(s)
Gene Expression Regulation, Fungal/drug effects , RNA, Catalytic/metabolism , Tetracycline/pharmacology , Yeasts/genetics , Animals , Aptamers, Nucleotide/genetics , Base Sequence , Enzyme Induction/drug effects , Genes, Reporter/drug effects , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Catalytic/biosynthesis , RNA, Catalytic/genetics , Saccharomyces cerevisiae/genetics , Schistosoma mansoni/genetics , Yeasts/drug effects , Yeasts/enzymology
SELECTION OF CITATIONS
SEARCH DETAIL
...