[go: up one dir, main page]

WO2001062981A1 - Plate-forme destinee a la decouverte de genes bacteriens impliques dans la modification d'arn - Google Patents

Plate-forme destinee a la decouverte de genes bacteriens impliques dans la modification d'arn Download PDF

Info

Publication number
WO2001062981A1
WO2001062981A1 PCT/US2001/005920 US0105920W WO0162981A1 WO 2001062981 A1 WO2001062981 A1 WO 2001062981A1 US 0105920 W US0105920 W US 0105920W WO 0162981 A1 WO0162981 A1 WO 0162981A1
Authority
WO
WIPO (PCT)
Prior art keywords
molecule
sentinel
rna
rme
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2001/005920
Other languages
English (en)
Inventor
T. Guy Roberts
Wayne Mitchell
Kenneth Beckman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
MONTCLAIR GROUP
Original Assignee
MONTCLAIR GROUP
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by MONTCLAIR GROUP filed Critical MONTCLAIR GROUP
Priority to CA002401018A priority Critical patent/CA2401018A1/fr
Priority to AU2001239856A priority patent/AU2001239856A1/en
Priority to EP01914470A priority patent/EP1263986A4/fr
Publication of WO2001062981A1 publication Critical patent/WO2001062981A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1079—Screening libraries by altering the phenotype or phenotypic trait of the host

Definitions

  • antibiotic resistance is a natural process in bacteria, commonly achieved through the acquisition of mechanisms to expel the antibiotic from the cellular system, or to modify it into a less toxic form.
  • the prevalent and often casual use of currently-available antibiotics has accelerated this process, leading to multiply-resistant bacterial strains.
  • This rapid development of antibiotic resistance can be delayed by a more selective approach to the application of antibiotics, but is unlikely to be avoided entirely. Therefore, the identification and development of new antibiotics will continue to be necessary.
  • the present invention meets these and other needs by providing new antibiotic targets, and antibiotic and antibiotic target discovery platforms.
  • Assays based on detection of the products of a reaction whether enzymatic modification of a biomolecule or general detection of substrate-to-product conversion, necessarily links the gene of interest (e.g., the test gene) to a particular function or activity. Furthermore, if this activity is vital to the pathogenicity of an organism or a disease, then the identification of the responsible enzyme and its gene in the above manner serves to characterize a useful drug target.
  • the present invention systemizes this process of drug target identification by providing methods for correlating molecular and cellular structures to their causative genes. Furthermore, this invention provides methods for the simultaneous discovery of classes of enzymes that share at least one substrate in common.
  • sentinel molecules can be modified by any of a number of catalytic mechanisms
  • the assay is not limited to a specific enzymatic activity.
  • the methods employing these sentinel molecules can be performed in a high throughput fashion.
  • tRNA transfer RNA
  • This platform can be used, for example, to identify whether the sentinel molecule is modified, the type of modification present, the genes involved in the modification, the gene products which execute the modification, and one or more test compounds which affect the occurrence and/or extent of the modification.
  • the sentinel molecule can be modified by the gene product in a number of ways, including, but not limited to, methylation, alkylation, acetylation, esterification, ubiquitination, lysinylation, phosphorylation, sulfation, glycosylation, or a combination thereof.
  • the present invention also provides methods for screening a test compound for activity, comprising the steps of preparing an assay solution having a gene product (for example, an enzyme or a catalytic RNA) capable of modifying a sentinel molecule; incubating the assay solution with the sentinel molecule and a test compound; and determining whether the sentinel molecule was modified by the gene product in the presence of the test compound, thereby screening the test compound for activity.
  • the test compound can be, for example, an antibiotic compound.
  • the sentinel molecule can be any of a number of cellular components, including, but not limited to, various RNA molecules (for example, tRNA, rRNA, mRNA, guide RNA, snRNA molecules, snoRNA molecules, and hnRNA molecules), DNA molecules, peptides, proteins, carbohydrates, lipids, naturally-occurring small molecule substrates, and synthetic small molecule substrates.
  • the assay solution is optionally a cellular extract, derived from either bacterial sources or eukaryotic sources.
  • the assay solution is a solution containing a purified enzyme and one or more substrates (in, for example, purified or synthetic forms, or in crude mixtures, i.e., processed cellular lysates).
  • the present invention also provides methods for screening a compound for antibiotic activity.
  • the antibiotic screening method includes the steps of providing a cell line comprising a sentinel RNA molecule that is normally modified in a prokaryotic system, but not modified in a eukaryotic system; treating the cell line with the compound to be screened; and monitoring the sentinel RNA molecule for modification.
  • a single compound or multiple compounds can be screened for antibiotic activity using the methods provided.
  • the gene to be tested can be expressed in a cell line or tissue of interest, or the gene can be genetically engineered into tissues or cell lines of eukaryotic, archae or eubacterial origin.
  • the gene product can be assayed in vitro or in vivo.
  • the sentinel molecule can be any type of molecule, but is described here as a tRNA of natural or synthetic source.
  • the assay solution can be either a cellular extract or a solution of defined components, within which the sentinel RNA molecule can be modified in a number of ways, including, but not limited to, methylation, alkylation, acetylation, esterification, ubiquitination, lysinylation, phosphorylation, sulfation, glycosylation, or a combination thereof.
  • the present invention provides in vitro methods for identifying one or more gene products involved in RNA modification.
  • the in vitro methods include the steps of providing at least one cell having one or more test genes that encode at least one gene product; preparing a cellular extract from the cell, such that the cellular extract contains the gene product; incubating the cellular extract with a sentinel RNA molecule; and determining whether the sentinel RNA molecule has been modified by the gene product, thereby determining whether the gene product (and by association, the test gene) participates in an RNA modification process.
  • the cellular extract can be derived from, for example, a bacterial cell or a eukaryotic cell.
  • the test gene can be a part of the cellular genome, or it can be introduced into the cell by a number of techniques, for example, via an expression vector.
  • the expression level of the gene product can be altered for use in the method of the present invention, particularly if generation of a particular genotype, or particular phenotype, is desired.
  • the expression level can, for example, be induced, increased, reduced, or even eliminated.
  • the sentinel RNA molecule can be any of a number of RNA molecules, including, but not limited to, tRNA, rRNA, mRNA, guide RNA, snRNA molecules, snoRNA molecules and hnRNA molecules.
  • the assay solution is optionally a cellular extract, within which the sentinel molecule can be modified in a number of ways, including, but not limited to, methylation, alkylation, acetylation, esterification, ubiquitination, lysinylation, phosphorylation, sulfation, glycosylation, or a combination thereof.
  • the presence and extent of these modifications can be determined by one or more of a variety of analytical techniques, such as mass spectrometry, thin layer chromatography, HPLC, capillary electrophoresis, NMR spectroscopy, X-ray crystallography, or cryo-electron microscopic analysis.
  • the methods can further include the step of identifying the test gene or genes that encode the gene product that performed the modification.
  • the present invention provides in vivo methods for identifying one or more gene products involved in RNA modification.
  • the in vivo methods include the steps of providing a cell having at least one sentinel RNA molecule, and one or more test genes of interest; manipulating the test gene (e.g., altering the expression of the gene product); and monitoring the sentinel RNA molecule for modification by the one or more gene products encoded by the one or more test genes, thereby determining whether the gene product (and by association, the test gene) participates in an RNA modification process.
  • manipulating the test gene present in the in vivo tissue expression of the gene product can be induced, increased, reduced, or eliminated.
  • the in vivo method can further include monitoring whether the gene product encoded by the test gene is increased, reduced or eliminated.
  • the sentinel RNA molecule can be any of a number of RNA molecules, such as tRNA, rRNA, mRNA, guide RNA, snRNA molecules, snoRNA molecules, and hnRNA molecules.
  • the presence and extent of the modifications to the sentinel RNA molecule can be determined by one or more of a variety of analytical techniques, such as HPLC, mass spectrometry, liquid chromatography/mass spectrometry (LC/MS), thin layer chromatography, capillary electrophoresis, NMR spectroscopy, X-ray crystallography, or cryo-electron microscopic analysis.
  • the methods can further include the step of identifying the test gene or genes that encode the gene product.
  • the present invention also provides methods of identifying a gene encoding a desired gene product.
  • the methods include providing a library of nucleic acids and expressing the library to provide a plurality of gene products for analysis.
  • the plurality of gene products are incubated with one or more sentinel molecules and the resulting products are analyzed for the presence or absence of one or more modifications to one or more of the sentinel molecules.
  • it can be determined whether the plurality of gene products includes one or more desired gene products, and the gene encoding the desired gene product is identified.
  • the desired gene product can be any of a number of proteins, enzymes, RNA molecules, and the like.
  • the present invention also provides the modified sentinel molecules generated during the methods of the present invention, the gene products which perform these modifications, the genes that encode these gene products, and the test compounds and/or antibiotics which, for example, enhance or inhibit the modification of the sentinel molecules.
  • Figure 1 illustrates various methodologies for the analysis of gene function.
  • FIG. 2 is a flowchart depicting one embodiment of the methods of the present invention.
  • Figure 3 depicts an in vitro assay as performed by the methods of the present invention.
  • the term "sentinel molecule” refers to one or more molecules that can be monitored for change in structure (e.g., by the addition or removal of one or more atoms) or change in one or more physical properties (e.g., the ability to bind an enzyme), usually within the context of a cellular system.
  • the sentinel molecule can be a molecule that naturally occurs in the biological system being examined, or it can be a molecule that is added to the system for the purpose of monitoring, for example, a specific enzymatic activity.
  • test compound refers to a compound which is being added to an assay system to assess the effect that the compound has upon the assay system.
  • the test compound can be a synthetic compound (i.e. prepared by chemical synthesis or chemical modification), or it can be a naturally-occurring compound.
  • a test compound is meant to encompass both a single compound, as well as a group, or "library,” of compounds.
  • test gene refers to one or more nucleic acid sequences that encode one or more gene products.
  • the test gene can be a portion of a cellular genome, an isolated DNA sequence, or a synthetically prepared or artificially manipulated sequence.
  • the test gene can be part of the cellular genome, or it can be external to the cellular genome (for example, a component of an expression vector).
  • the test gene can be one or more components of a library of sequences.
  • gene product refers to product encoded by one or more genes.
  • the gene product can be a protein, an RNA molecule, or a DNA molecule.
  • the present invention provides methods for screening test compounds, as well as methods for screening compounds for antibiotic activity.
  • the present invention provides both in vitro as well as in vivo methods for identifying gene products involved in RNA modification.
  • a common component to these methods is the use of a "sentinel molecule" for the purpose of, for example, monitoring the activity of one or more gene products.
  • the sentinel molecule is used to identify gene products of interest (i.e., therapeutic targets), by determining whether the sentinel molecule has been modified in the presence of the gene product.
  • the assay systems are examined for a change (for example, an enhancement or inhibition) in the modification of the sentinel molecule, thus indicating whether a test compound or a putative antibiotic has an effect on the cellular metabolism.
  • a change for example, an enhancement or inhibition
  • These assays can be performed in a high-throughput manner, such that libraries of compounds, or collections of gene sequences, can be tested for involvement in microbial metabolism, and optionally RNA modification.
  • Figure 1 depicts various methodologies for the analysis of gene function, according to one class of embodiments of the present invention. For example, as depicted in the upper panel, gene function is commonly determined by looking at a phenotype of the cell or organism.
  • the gene is then isolated, and the resulting gene product is shown to be capable of converting a defined substrate into an expected product.
  • gene function is typically determined without the intervening step of analysis of the gene product and without the need for phenotype information.
  • the gene is simply expressed (e.g., in an expression library format) and the cells or cellular components are directly assayed for the ability of the expressed gene product to convert a known substrate (such as an unmodified RNA) to a particular product (e.g., a modified tRNA molecule).
  • a known substrate such as an unmodified RNA
  • a particular product e.g., a modified tRNA molecule.
  • Expression of the gene can occur in vitro (e.g., using a transcription-translation faormat) or in vivo (e.g., in cells of a library of interest).
  • Figure 2 depicts one embodiment of the methods for identifying a gene encoding an RNA modification enzyme as provided by the present invention.
  • a gene of interest e.g., a member of a nucleic acid library
  • the expression system is then modulated. If the expression is down- regulated, and the phenotype or the desired modification of the sentinal molecule is altered (e.g., if the RNA modification does or does not occur, and the cell gains or loses enzymatic functionality), then the function of the gene as an RNA modification enzyme is identified and the gene or gene product is potentially of interest as a drug target.
  • the gene expression is up-regulated and the cellular lysate is combined with synthetic tRNA substrates (i.e. the unmodified sentinal molecule), and optionally one or more small molecule substrates.
  • synthetic tRNA substrates i.e. the unmodified sentinal molecule
  • small molecule substrates optionally one or more small molecule substrates.
  • the resulting products are digested to component nucleosides (or nucleotides), which are then analyzed by LC/MS for the presence or absence of one or more modifications.
  • the presence of an RNA modification indicates that the gene being over-expressed functions as an RNA modification enzyme, and is a potential drug target.
  • a sentinel molecule is any molecule that can be monitored for change in structure (e.g., by the addition or removal of one or more atoms) or change in one or more physical properties (e.g., the ability to bind an enzyme).
  • the sentinel molecule can be a molecule that naturally occurs in the biological system being examined, or it can be a molecule that is added to the system for the purpose of monitoring, for example, an enzymatic activity.
  • Assays employing sentinel molecules can use a single type of sentinel molecule, a set of structurally similar sentinel molecules, or a group of structurally diverse sentinel molecules; the term "sentinel molecule" is intended to cover all of these possibilities.
  • sentinel molecules examples include, but are not limited to, RNA molecules, DNA molecules, peptides, proteins, carbohydrates, lipids, naturally-occurring small molecule substrates, and synthetic small molecule substrates.
  • One preferred class of sentinel molecule employed in the methods of the present invention includes sentinel RNA molecules.
  • RNA molecules that can act as sentinel molecules include, but are not limited to, transfer RNA (tRNA) molecules, ribosomal RNA (rRNA) molecules, messenger RNA (mRNA) molecules, guide RNA molecules, heterogeneous nuclear RNA (hnRNA) molecules, small nuclear RNA (snRNA) molecules, small nucleolar RNA (snoRNA) molecules, and the like.
  • Another class of sentinal molecules employed in the methods of the present invention includes lipid-based molecules, including, but not limited to, glycolipids, phospholipids, triglycerides, lipopolysaccharides, lipoproteins, mycolic acids, teichoic acids, teichuronic acids, lipoteichoic acids, and the like.
  • Carbohydrate-containing sentinal molecules can also be employed in the methods of the present invention; carbohydrate-based sentinal molecules include, but are not limited to, carbohydrates, glycoproteins, glycolipids, lipopolysaccharides, peptidoglycans, fucoidans, and the like.
  • the cells used in the methods of the present invention include either bacterial cells as well as eukaryotic cells.
  • bacterial cell lines which can be used in the methods of the present invention include, but are not limited to, those from the genuses Aquifex, Archaeoglobus, Bacillus, Borrelia, Chlamydia, Escherichia,
  • Mycobacterium Mycoplasma, Pyrococcus, Rickettsia, Synechocystis, and Treponema
  • cells for use in the methods of the present invention are available from cell repositories such as the American Type Culture Collection (www.atcc.org), the World Data Center on Microorganisms (http://wdcm.nig.ac.jp), European Collection of Animal Cell Culture (www.ecacc.org) and the Japanese Cancer Research Resources Bank (http://cellbank.nihs.go.jp), and companies such as Clonetics Corporation (www.clonetics.com).
  • Genes that encode gene products employed in methods of the present invention can be part of the cellular genome, or they can be added to the cells, for example, in the form of expression vectors. As such, the identity of the genes and the functions of their respective gene products may or may not be defined.
  • the genes can be derived from a library of genomic fragments, such as those publicly or commercially available from a number of sources, for example, Gorilla Genomics (Alameda, CA) or Incyte Genomics (Palo Alto, CA).
  • nucleic acid sequences for use as genes can be amplified in vitro, synthesized de novo and/or assembled using techniques known to those in the art, such as polymerase mediated, ligation-mediated and combination ligation/ polymerase mediated assembly methods.
  • Custom-synthesized nucleic acid sequences can be ordered from any of a variety of commercial sources, such as The Midland Certified Reagent Company (mcrc@oligos.com), The Great American Gene Company (http://www.genco.com), ExpressGen Inc. (www.expressgen.com), Operon Technologies Inc. (Alameda, CA) and the like.
  • a variety of expression vectors can be employed to deliver the genes of interest and control their expression in bacterial and/or eukaryotic cells, including, but not limited to, viruses, plasmids, episomes, transposons ,phages, artificial chromosomes (such as a bacterial or yeast artificial chromosome), and the like.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the test gene.
  • Exemplary promoters for use in the methods of the present invention include, but are not limited to, the E. coli lac or trp promoter, SV40 promoter, phage lambda PL promoter, and CMV promoter. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available.
  • Transformation methodologies include, but are not limited to, bacterial-mediated transformation, phage transduction, conjugation, transfection, liposome-mediated transformation, protoplast fusion techniques, particle bombardment, electroporation, and the like.
  • Several of the methods of the present invention involve alteration of the test gene expression levels. This can be accomplished by a number of mechanisms known to those in the art, such as commercially available expression cassettes, expression vectors, and other transcription regulatory systems.
  • the resulting level of gene product expressed in the cells is increased, reduced, or eliminated, depending upon the treatment performed; upon lysing the cells, the resulting assay solution contains an altered amount of gene product as compared to untreated cells.
  • references describing nucleic acid manipulation techniques are known in the art, and include, for example, Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 (Academic Press, Inc., San Diego, CA); PCR Protocols A Guide to Methods and Applications (Innis et al. eds.) (1990, Academic Press Inc., San Diego, CA); De Lorenzo and Timis Methods in Enzymology (1994) vol. 235, pp.385-404; Kleckner et al. Methods in Enzymology (1991) vol. 204, chapter 7; as well as Sambrook and Ausubel, both supra.
  • These and other references cited herein describe cell culture techniques and recombinant nucleic acid methodologies appropriate for use in the methods of the present invention.
  • the sentinel molecule is modified by one or more gene products, leading to a modified sentinel molecule.
  • the modifications orchestrated by the gene products can cover a range of potential changes to the structure of the sentinel molecule.
  • Modifications to the sentinel molecule can include, but are not limited to, methylation, alkylation, acetylation, esterification, ubiquitination (ubiquitinulation), lysinylation, phosphorylation, sulfation, sulfonation, glycosylation, famsylation, and the like.
  • modifications to sentinal molecules can be the result of the activities of various transferases, synthases, isomerases, dehydrogenases, and the like.
  • a preferred class of sentinel molecule employed in the methods of the present invention includes sentinel RNA molecules.
  • Sentinel RNA molecules encompass, but are not limited to, tRNA molecules, rRNA molecules, mRNA molecules, guide RNA molecules, snRNA molecules, snoRNA molecules, and hnRNA molecules.
  • One or more component nucleotides of the sentinel RNA molecules can be modified to generate a modified sentinel molecule.
  • RNA molecules can be found, for example, in Genes VI, Chapter 9 ("Interpreting the Genetic Code"), Lewis, ed. (1997, Oxford
  • RNA components include the following: 2'-O-methylcytidine; N 4 -methylcytidine; N 4 -2'-O-dimethylcytidine; N 4 - acetylcytidine; 5-methylcytidine; 5,2'-O-dimethylcytidine; 5-hydroxymethylcytidine; 5- formylcytidine; 2'-O-methyl-5-formaylcytidine; 3-methylcytidine; 2-thiocytidine; lysidine; 2'-O-methyluridine; 2-thiouridine; 2-thio-2'-O-methyluridine; 3,2'-O- dimethyluridine; 3-(3-amino-3-carboxypropyl)uridine; 4-thiouridine; ribosylthymine; 5,2'- O-dimethyluridine
  • RNA molecules can be used to identify additional modified RNA molecules.
  • Another preferred sentinal molecule used in the methods of the present invention is a carbohydrate-based or lipid based molecule.
  • Exemplary sentinal molecules include, but are not limited to, phospholipids, triglycerides, lipopolysaccharides, glycolipids, glycoproteins, peptidoglycans, lipoproteins, mycolic acids, teichoic acids, teichuronic acids, lipoteichoic acids, and the like.
  • RNA-based sentinal molecules As with the RNA-based sentinal molecules described in the previous paragraph, a number of modifications can be made to the carbohydrate-based or lipid based sentinel molecule, such as phosphorylation or sulfation; methylation, alkylation, or acetylation; esterification; and addition of relatively large moieties such as amino acids (e.g., lysine), various carbohydrate structures, and proteins such as ubiquitin.
  • modifications to sentinal molecules can be the result of the activities of various transferases, synthases, isomerases, dehydrogenases, and the like.
  • Some preferred analytical techniques for use in determining whether the sentinel molecule has been modified, the extent of modification, and/or the type of modification include, but are not limited to, mass spectrometry, thin layer chromatography (TLC), high pressure liquid chromatography (HPLC), capillary electrophoresis (CE), NMR spectroscopy, X-ray crystallography, cryo-electron microscopic analysis, or a combination thereof.
  • RNA molecules have been processed using thin layer chromatographic techniques on radioactive substrates. References exist which address this traditional analytical methodology. More recently, mass spectrometry (MS) has been used by several academic groups to assess the modification states of RNA molecules.
  • MS mass spectrometry
  • Mass spectrometry is a particularly versatile analytical tool, and includes techniques and/or instrumentation such as electron ionization, fast atom/ion bombardment, MALDI (matrix-assisted laser desorption/ionization), electrospray ionization, tandem mass spectrometry, and the like.
  • MALDI matrix-assisted laser desorption/ionization
  • electrospray ionization tandem mass spectrometry, and the like.
  • the assay solutions (containing the newly modified sentinel molecules) are prepared for mass spectrometry and then transferring the resulting yield of sentinel molecules into a suitable solvent system.
  • Analysis by mass spectrometry yields a spectrogram from which both the mass and composition of the sentinel molecule can be determined, in both the modified or unmodified states.
  • the modification state of the sentinel molecule is revealed in a computationally straightforward manner. The presence or absence of modifications on these sentinel molecules determines the relevance of the manipulated test gene to the enzymatic pathway that produces the modifications. Direct analysis of the sentinel molecules is also possible, though this requires a complete analysis of the composition of the unmodified sentinel molecule.
  • the assay solutions containing the newly modified sentinel molecules are prepared for NMR spectroscopy by removal of the original solvent solution (for example, by lyophilization), and re-dissolution into a stable-isotope solvent, such as a deuterated solvent solution.
  • a deuterated solvent solution include, but are not limited to D 2 O (deuterium oxide), CDC1 3 , DMSO-d6, acetone-d6, and the like (available from Cambridge Isotope Labs, Andover, MA; www.isotope.com).
  • the samples can be analyzed using LC-NMR spectroscopy.
  • an assay solution is prepared, containing a gene product that is capable of modifying a sentinel molecule.
  • the assay solution can be a cellular extract, prepared from a single cell or a plurality of cells (i.e. a cell line or an in vivo tissue).
  • the gene product can be a protein (for example, an enzyme), a ribonucleic acid sequence (such as a ribozyme), or a deoxyribonucleic acid (for example, a cDNA prepared by reverse transcription, or a PCR product).
  • the gene that encodes the gene product used in the methods can be a gene present in the cellular genome, or it can be a gene present in a structure external to the cellular genome, such as a virus, a plasmid, an expression vector and the like.
  • the cells can be treated such that the expression level of the gene product is altered.
  • Manipulation of the expression of the gene product can be performed at the level of the gene or at the level of the gene product.
  • the expression of gene product can be controlled at the gene level through stimulation or inhibition of various transcription activities, alteration of promoters, generation of temperature-sensitive mutations and the like.
  • Genes can be manipulated through knock-in, knock-down, and/or knock-out techniques. Production of the gene product can be influenced by the levels of translation factors'available, by the presence of transcript-specific ribozymes, or using anti-sense technology.
  • the activity of the gene product can be directly affected by addition of inhibitors or enhancers.
  • the method used to manipulate the gene product can vary from assay to assay, depending upon the compound to be assayed and the gene product employed.
  • the assay solution is incubated with the sentinel molecule, and one or more test compounds to be screening for activity.
  • the test compound can be a single compound, or a library of compounds to be screened for activity. Sources for test compounds include, but are not limited to, chemical catalogs such as those available from Sigma or Aldrich, and commercial libraries of compounds.
  • the test compound can be one or more antibiotic compounds.
  • the addition of the test compound to the assay solution can increase the extent of modification or alter the type of modification that the sentinel molecule undergoes; alternatively, the compound being tested can inhibit or interfere with the modification of the compound.
  • the gene product is allowed to interact with the sentinel molecule.
  • the sentinel molecule is examined for modification by one or more analytical techniques.
  • a change in the modification state of the sentinel molecule indicates that the compound or compounds being screened have activity with respect to the gene product and sentinel molecule used in the method.
  • Another embodiment of the methods of the present invention provides methods for screening a compound for antibiotic activity.
  • the screening capitalizes on differences in cellular metabolism between targeted and non-targeted organisms, to identify compounds which detrimentally affect the targeted organism (generally, a pathogenic organism) without affecting or harming other, non-targeted cells (e.g., a host organism, or nonpathogenic cells existing in the same environment as the pathogenic cells).
  • the targeted organism includes prokaryotic cells, while the non-targeted organism are eukaryotic cells.
  • the targeted organism includes pathogenic eukaryotic cells (for example, yeast and fungi).
  • this screening procedure can be used to identify antibiotics that, for example, affect the metabolism of certain classes of prokaryotes or microbes but not other classes.
  • compounds with antibiotic activity can be identified that affect certain aspects of cellular metabolism, for example, the generation and recognition of modified tRNA molecules, which may differ among organisms.
  • These methods start with providing a cell line that has a sentinel molecule that is normally modified in the targeted cell, but not modified in the non-targeted cell (e.g., the host).
  • the cell line can be modified, if necessary, to express one or more gene products needed to screen for the antibiotic compounds (for example, the cell line can be transformed with expression vectors that encode specific enzymatic activities).
  • the cell line is optionally lysed to produce a cellular extract.
  • the sentinel molecule can be expressed in the cell line, or it can subsequently be added to the cells or cellular extract.
  • Sentinel molecules which can be used in these methods include, but are not limited to, RNA molecules, DNA molecules, peptides, proteins, carbohydrates, lipids, naturally-occurring small molecule substrates, and synthetic small molecule substrates.
  • the sentinel molecule is an RNA molecule, such as a tRNA, rRNA, mRNA, guide RNA, snoRNA, snRNA, hnRNA, and the like.
  • the sentinal molecule is a carbohydrate-based or lipid based molecule (e.g., phospholipids, triglycerides, lipopolysaccharides, glycolipids, glycoproteins, peptidoglycans, lipoproteins, mycolic acids, teichoic acids, teichuronic acids, lipoteichoic acids, and the like).
  • the sentinel molecule can be a single type of molecule, or it can be a mixture of molecules. Using a mixture of sentinel molecules increases the number of gene products participating in the screening assay, and thus improves the likelihood of finding a test compound with activity.
  • the assay solution is incubated with the sentinel molecule, and one or more compounds to be screening for antibiotic activity.
  • the compound to be screened can be a single compound, or a library of compounds. Sources for such test compounds include, but are not limited to, chemical catalogs such as those available from Sigma or Aldrich, and commercial libraries of compounds.
  • the addition of the test compound to the assay solution can increase the extent of modification or alter the type of modification that the sentinel molecule undergoes; alternatively, the compound being tested can inhibit or interfere with the modification of the compound.
  • To screen the compound (or a mixture of compounds) for antibiotic activity the compound is added to the cell line. After treating the cell line, the sentinel molecule is monitored for modification, thus determining whether the compound has an antibiotic activity.
  • the monitoring for modification can be performed by any of the techniques as described previously, alone or in combination, or as otherwise known in the art. In Vitro Methods for Identifying Gene Products involved in RNA
  • Yet another embodiment of the methods of the present invention provides in vitro methods for identifying one or more gene products involved in RNA modification, as well as the genes that encode these gene products.
  • the in vitro methods can include protocols in which the test gene activity is augmented, as well as protocols in which there is a reduction of the test gene activity. Both protocols are described below.
  • At least one cell having one or more test genes of interest is used in these in vitro methods.
  • Current micro-manipulation technology and sensitive analytical techniques have made it possible to perform experiments on single cells.
  • a group of cells or a cell line can be used.
  • the cells can be prokaryotic cells or eukaryotic cells.
  • the test gene or genes being examined for whether they encode one or more gene products involved in RNA modification can be part of the cellular genome, or they can be added to the cells, for example, in the form of expression vectors. In this manner, large numbers of nucleic acid segments, or libraries of genomic fragments, can be analyzed.
  • the expression level of the gene product(s) can be altered or manipulated.
  • over-expression constructs can be used to produce elevated levels of the test gene activity and increased expression of the gene product(s).
  • induction of a mechanism that reduces or eliminates the expression of the test gene is employed. If the test gene is involved in the modification of the sentinel RNA molecule, then the induced mechanism against the test gene will lead to a lessened state of modification on the sentinel RNA.
  • a cellular extract is prepared from the cell or cells, such that the extract contains the gene product(s) potentially having RNA modification activity.
  • One or more sentinel RNA molecules are incubated with the cellular extract and gene products, during which time the sentinel RNA molecule can be modified by the gene product(s).
  • sentinel RNA molecules which can be used in the present invention include, but are not limited to tRNA molecules, rRNA molecules, mRNA molecules, guide RNA molecules, snRNA molecules, snoRNA molecules, hnRNA molecules, and the like.
  • the sentinel RNA molecules are tRNA molecules.
  • sentinel RNA molecule either a single type of sentinel RNA molecule, or a collection of sentinel RNA molecules, can be employed in the present invention.
  • the sentinel RNA molecules are analyzed for one or more modifications. Determining whether the sentinel RNA molecule has been modified can be performed, e.g., by any of the techniques as described previously, alone or in combination, or as otherwise known in the art.
  • the methods of the present invention can identify modified RNA molecules that have not previously been described.
  • the in vitro method can further include the step of identifying the test gene or test genes that encode the gene products involved in RNA modification by methods, such as DNA sequencing, which are well known to one in the art. References describing nucleic acid sequencing techniques are known in the art, and include, for example, Berger and Kimmel, Innis, Sambrook and Ausubel, all supra.
  • Figure 3 depicts an in vitro assay as performed by the methods of the present invention.
  • E. coli cells were transformed to over-express the miaA gene product (a tRNA isopentenyl pyrophosphate transferase; Leung et al. (1997) J Biol. Chem. 272:13073-83).
  • the transferase catalyzes the addition of an isopentenyl group to certain adenosines (e.g., A37) adjacent to the anticodon region of some tRNA molecules, the first step in the biosynthesis of 2-methylthio-N6-(delta 2-isopentenyl)-adenosine.
  • Lysate was prepared from the cells, and incubated with synthetic cysteine tRNA sentinal molecules and DMAPP (dimethylallyl diphosphate). After the substrate adenosines had been modified, the sentinal molecules were digested to component nucleosides and analyzed by LC/MS (using reverse-phase column chromatography on a C18 column (Higgins
  • FIG. 3 panel A ("uninduced state”) and panel B ("induced state”). Relative peak areas of the i6A product are depicted in panel C.
  • the data demonstrates the viability of this in vitro method for identifying one or more gene products involved in RNA modification (in this case, the known tRNA transferase).
  • RNA modification in this case, the known tRNA transferase.
  • a further embodiment of the methods of the present invention provides in vivo methods for identifying one or more gene products involved in RNA modification, as well as the genes that encode these gene products.
  • the in vivo approach typically involves the induction of a mechanism that reduces or eliminates the expression of the test gene from the genome of the cell, leading to a reduction in the concentration of the gene product. If the test gene is involved in the modification of the sentinel RNA, then this chain of cellular events will lead to decreased modification of the sentinel RNA.
  • the expression of the test gene can be induced, or increased, such that the sentinel RNA molecules are newly modified, or modified to a greater extent, as compared to in the uninduced state.
  • a cell expresses one or more sentinel RNA molecules, and has one or more test genes of interest.
  • the cells can be bacterial cells, or they can be of eukaryotic origin.
  • Sentinel RNA molecules that can be found within, or introduced into, the cell include, but are not limited to, tRNA molecules, rRNA molecules, mRNA molecules, guide RNA molecules, hnRNA molecules, snRNA molecules, snoRNA molecules, and the like.
  • the sentinel RNA molecules are tRNA molecules. It should be noted that either a single type of sentinel RNA molecule, or a group of sentinel RNA molecules, can be employed in the present invention.
  • test gene or genes to be examined can be part of the cellular genome, or they can be added to the cellular environment in the form of expression vectors. Using such expression vectors, various nucleic acid segments of interest, or libraries of genomic fragments, can be analyzed. Alternatively, transcriptional elements and/or promoters can be inserted into the cell's genomic material, thus providing a means by which the expression of proximal nucleic acid sequences can be manipulated. Thus, the expression level of the gene product(s) encoded by either the introduced or transcriptionally-modified sequences can be altered or manipulated.
  • the one or more test genes of interest are manipulated within the cellular environment, such that expression of the test gene is altered.
  • the mechanism used to alter the expression of the test gene can include, but is not limited to, the following techniques: targeted destruction of the RNA transcripts by gene-specific ribozymes; generation of temperature-sensitive mutations in the test gene that allow for temperature-dependent expression of the gene product; anti-sense technology; gene knock-out, knock-in, or knock-down technologies; or any other method known to those skilled in the art.
  • over-expression constructs can be used to produce elevated levels of the test gene activity and increased expression of the gene product(s).
  • test gene activity is reduced
  • induction of a mechanism that reduces or eliminates the expression of the test gene is employed. If the test gene is involved in the modification of the sentinel RNA molecule, then the induced mechanism against the test gene will lead to a lessened state of modification on the sentinel RNA.
  • the sentinel RNA molecules are analyzed for modification.
  • the determining whether the sentinel RNA molecule has been modified can be performed by any of the techniques as described previously, alone or in combination, or as otherwise known in the art.
  • the methods of the present invention can identify modified RNA molecules that have not previously been described.
  • the in vivo method can also include the step of identifying the test gene or test genes that encode the gene products involved in RNA modification.
  • the in vitro methods and the in vivo methods for identifying gene products can further include the step of identifying the gene that encodes the gene product.
  • in vitro amplification methods can also be used to amplify and/or sequence the test genes of interest.
  • examples of in vitro amplification and sequencing techniques including the polymerase chain reaction (PCR) the ligase chain reaction (LCR), Q ⁇ -replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA) can be found in Berger, Sambrook, and Ausubel, id., as well as in Mullis et al., (1987) U.S.
  • Patent No. 4,683,202 PCR Protocols A Guide to Methods and Applications by Innis et al. eds. (1990, Academic Press Inc., San Diego, CA ); Arnheim & Levinson (October 1, 1990) C&EN 36-47; The Journal Of NTH Research (1991) 3, 81-94; Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin.
  • a putative RNA modification gene (the test gene) is cloned into an expression vector plasmid having an inducible promoter (e.g., the E. coli lac promoter).
  • the plasmid also includes an antibiotic resistance gene (for example, beat-lactamase, which confers ampicillin resistance, or aminoglycoside 3'-phosphotransferase, which confers kanamycin resistance) to allow selection of plasmid-carrying cells and confirmation of stable transformation.
  • the test gene is operatively linked to the inducible promoter.
  • the expression vector comprising the gene is transferred into a bacterial cell line (e.g., E. coli) by methods familiar to those skilled in the art. References describing the techniques involved include Sambrook, supra and Ausubel, supra. As a control, the expression vector (sans the test gene) is transfected into additional cells and used to determine background levels of tRNA modifications.
  • a bacterial cell line e.g., E. coli
  • the bacteria are allowed to grow in media containing the selected antibiotic and an agent that induces expression of the test gene (for example, the inducing agent IPTG can be employed for the lac operon promoter).
  • the cells are cultured until the cell density indicates that the bacteria are in early log phase growth.
  • Expression of the test gene is induced and the gene product is allowed to accumulate in the bacteria for a specified period of time, e.g., between 2 hours and overnight.
  • the bacteria are then harvested (e.g., by centrifugation, filtration, or other means known in the art) and the cells are lysed.
  • Lysis can be performed, e.g., with a solution containing chaotropic salts, such as guanidinium thiocyanate, phenol and detergent and buffered to pH of between 5.5 and 6.5 (see, for example, Berger and Kimmel, supra).
  • chaotropic salts such as guanidinium thiocyanate, phenol and detergent
  • the RNA is extracted from this mixture by the addition of chloroform and removal of the aqueous phase.
  • the RNA is precipitated from the aqueous phase by the addition to this mixture of 2.5 volumes of neat ice cold ethanol followed by high speed centrifugation.
  • the purified RNA is then dissolved in a buffered saline solution.
  • the tRNA can then be purified from the other cellular RNAs by, for example, gel filtration chromatography. See, for example, Reed et al. (1988) Cell 53:1949- 1961.
  • the tRNA-containing fractions of the eluate are pooled together and concentrated by extraction with neat 1-butanol.
  • the tRNA is digested to nucleosides (or nucleotides) using nuclease PI, alkaline phosphatase and phosphonucleotide di-esterase (see, for example, Crain (1990) Methods in Enzymology 193:782-790 and Nishimura et al. (1997) Methods in Enzymology 155:373-379).
  • the resulting nucleosides can be analyzed for a particular modification by one of several methods, depending on the nucleoside modification in question.
  • one or more detection techniques that will allow for the preparation and rapid analysis of the sentinel molecules can be employed in the methods of the present invention.
  • Techniques for the growth of bacteria in multi-well plates and transformation of cells within multi-well plates have been described elsewhere These techniques can be employed in the methods of the present invention.
  • Sentinel molecules such as tRNA from bacterial cells and cell lysates can be prepared in a parallel fashion for mass spectroscopy, LC/MS, LC-NMR or other analytical instrumentation in a parallel fashion using multi-well plates.
  • Multi-well plates having 96, 384, 768 or 1536 wells are available from various suppliers such as VWR Scientific Products (West Chester, PA).
  • Instrumentation for autosampling from 96-well plates (or other formats) can be used to transfer samples from the multi-well plates to, for example, the mass spectrometer; this instrumentation is available from several sources.
  • the methods of the present invention can be performed in a parallel high-throughput format.
  • the described procedure allows for the discovery of the gene encoding the enzymatic activity responsible for lysidinylation of cytosine as found in the isoleucyl tRNA of bacteria.
  • the methodologies for structural analysis can be used to discover new covalent modifications in bacteria of both eubacterial and archeae origins and to further identify the genes in these organisms that encode the enzymes that produce the covalent modifications.
  • the step of detecting the presence or absence of one or modifications to the sentinal molecule can further include analyzing data generated during the detection process.
  • data generated during mass spectrometry analysis can be quantitated and compared between samples containing the test gene product and control samples, by methods known to one in the art. Additional examples of data analysis are provided in, for example, provisional application 60/225,506 (filed August 15, 2000) and copending application (Attorney Docket No. 16-000220US, filed February 23, 2001).
  • the methods of the present invention can be automated, for example, for generation and analysis of data collected using high throughput methodologies.
  • Instruction sets for analyzing the results generated by the methods of the present invention can be constructed by one of skill using a standard programming language such as C, C++, Visual Basic, Fortran, Basic, Java, or the like.
  • a system for use with the methods of the present invention can include one or more of the following: a device for providing and/or sorting the libraries of nucleic acids used in the methods; a device for incubating the assay compositions with the sentinal molecules; a device for analyzing the signals from the modified sentinal molecules; a computer or computer-readable medium; software for analyzing the presence or absence of one or more modifications; software for picking "hits" from any expression library (e.g., library members which encode enzymes that are relevant to sentinal molecule modification) and, optionally, for sequencing or otherwise analyzing the hits; and a user interface (e.g., a GUI in a standard operating system such as a Windows, Macintosh, UNIX, LINUX, and the like).
  • a user interface e.g.,
  • Standard desktop applications which can be employed with one or more of these devices includes, but is not limited to, word processing software (e.g., Microsoft WordTM or Corel WordPerfectTM), spreadsheet and/or database software (e.g., Microsoft ExcelTM, Corel Quattro ProTM, Microsoft AccessTM, ParadoxTM, Filemaker ProTM, OracleTM, SybaseTM, and InformixTM ) and the like, which can be adapted for these (and other) purposes.
  • the computer or computer readable medium can provide the examination results in the form of an output file.
  • Kits will optionally additionally comprise instructions for performing the methods or assays, packaging materials, one or more containers which contain assay, device or system components, or the like.
  • kits embodying the methods and devices herein optionally comprise one or more of the following: (1) a library of nucleic acid sequences, optionally incorporated into expression vectors; (2) one or more sentinal molecules, such as RNA sentinal molecules; (3) one or more assay components, including, but not limited to buffers, substrates, cofactors, inhibitors, selection agents, antibiotics, enzymes, and the like; (4) a computer or computer- readable medium for performing the methods of the present invention and/or for storing the assay results; (5) instructions for practicing the methods described herein; and, optionally, (6) packaging materials.
  • the present invention provides for the use of any component or kit herein, for the practice of any method or assay herein, and/or for the use of any apparatus or kit to practice any assay or method herein. While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, all the techniques and apparatus described above can be used in various combinations. All publications, patents, patent applications, and/or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and/or other document were individually indicated to be incorporated by reference for all purposes.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des procédés d'identification de gènes et des produits de gènes impliqués dans la modification d'ARN (comme montré Figure 2), ainsi que des méthodes destinées au criblage d'activité de composés test ou d'antibiotiques. Elle concerne, en outre, les produits de gènes identifiés dans les méthodes, les gènes qui codent pour ces produits, des molécules sentinelles modifiées produites par ces produits, ainsi que des composés ou des antibiotiques qui permettent d'inhiber le processus de modification.
PCT/US2001/005920 2000-02-25 2001-02-23 Plate-forme destinee a la decouverte de genes bacteriens impliques dans la modification d'arn Ceased WO2001062981A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CA002401018A CA2401018A1 (fr) 2000-02-25 2001-02-23 Plate-forme destinee a la decouverte de genes bacteriens impliques dans la modification d'arn
AU2001239856A AU2001239856A1 (en) 2000-02-25 2001-02-23 Platform for the discovery of the bacterial genes involved in rna modification
EP01914470A EP1263986A4 (fr) 2000-02-25 2001-02-23 Plate-forme destinee a la decouverte de genes bacteriens impliques dans la modification d'arn

Applications Claiming Priority (9)

Application Number Priority Date Filing Date Title
US18507100P 2000-02-25 2000-02-25
US18500000P 2000-02-25 2000-02-25
US60/185,071 2000-02-25
US60/185,000 2000-02-25
US60/255,506 2000-02-25
US22550600P 2000-08-15 2000-08-15
US22550500P 2000-08-15 2000-08-15
US60/225,505 2000-08-15
US25550600P 2000-12-13 2000-12-13

Publications (1)

Publication Number Publication Date
WO2001062981A1 true WO2001062981A1 (fr) 2001-08-30

Family

ID=34069426

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2001/005920 Ceased WO2001062981A1 (fr) 2000-02-25 2001-02-23 Plate-forme destinee a la decouverte de genes bacteriens impliques dans la modification d'arn

Country Status (3)

Country Link
EP (2) EP1263986A4 (fr)
AU (1) AU2001239856A1 (fr)
WO (1) WO2001062981A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013033019A1 (fr) * 2011-08-31 2013-03-07 University Of Utah Research Foundation Procédés de détermination de l'intégrité d'un échantillon biologique

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108251517A (zh) * 2017-12-29 2018-07-06 武汉艾德士生物科技有限公司 一种分析体系内相似序列相对数量的方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5834184A (en) * 1995-05-17 1998-11-10 Harada; Kazuo In vivo selection of RNA-binding peptides
US5989814A (en) * 1997-04-01 1999-11-23 Reagents Of The University Of California Screening methods in eucaryotic cells
US6008034A (en) * 1996-04-15 1999-12-28 Givaudan-Roure (International) Sa Hydroperoxide lyases

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR9106486A (pt) * 1990-05-23 1993-05-25 Isis Pharmaceuticals Inc Composicao para modulacao da atividade de um rna e processo para tratamento de um organismo que tem uma doenca que se caracteriza para producao indesejada de uma proteina
US5270170A (en) * 1991-10-16 1993-12-14 Affymax Technologies N.V. Peptide library and screening method
US5593835A (en) * 1995-05-12 1997-01-14 President And Fellows Of Harvard College Methods and kits for RNA binding compounds
US6337183B1 (en) * 1995-09-08 2002-01-08 Scriptgen Pharmaceuticals, Inc. Screen for compounds with affinity for nucleic acids
US5871987A (en) * 1996-11-01 1999-02-16 Cubist Pharmaceuticals, Inc. Candida tyrosyl-tRNA synthetase proteins, nucleic acids and strains comprising same
FR2762014B1 (fr) * 1997-04-10 1999-06-04 Itzik Harosh Utilisation d'apobeg1 ou des proteines associees, ou des genes responsables de la maladie d'andersons comme cible de traitement de l'atherosclerose et de l'obesite et techniques associees de clonage et de criblage d'inhibiteurs
AU9336701A (en) * 2000-04-19 2001-10-30 Ribotargets Limited Assay for identification of a test compound

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5834184A (en) * 1995-05-17 1998-11-10 Harada; Kazuo In vivo selection of RNA-binding peptides
US6008034A (en) * 1996-04-15 1999-12-28 Givaudan-Roure (International) Sa Hydroperoxide lyases
US5989814A (en) * 1997-04-01 1999-11-23 Reagents Of The University Of California Screening methods in eucaryotic cells

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP1263986A4 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013033019A1 (fr) * 2011-08-31 2013-03-07 University Of Utah Research Foundation Procédés de détermination de l'intégrité d'un échantillon biologique

Also Published As

Publication number Publication date
AU2001239856A1 (en) 2001-09-03
EP1261734A1 (fr) 2002-12-04
EP1263986A4 (fr) 2005-02-23
EP1263986A1 (fr) 2002-12-11

Similar Documents

Publication Publication Date Title
Suzuki et al. A complete landscape of post-transcriptional modifications in mammalian mitochondrial tRNAs
Wang et al. Quantifying the RNA cap epitranscriptome reveals novel caps in cellular and viral RNA
Kuhle et al. tRNA renovatio: rebirth through fragmentation
Darnell et al. Pre-mRNA processing includes N6 methylation of adenosine residues that are retained in mRNA exons and the fallacy of “RNA epigenetics”
Bushkin et al. m6A modification of a 3′ UTR site reduces RME1 mRNA levels to promote meiosis
Cohen et al. Quantitative gene expression analysis in renal biopsies: a novel protocol for a high-throughput multicenter application
Nechay et al. High-throughput approaches to profile RNA-protein interactions
Belin et al. Purification of ribosomes from human cell lines
Subkhankulova et al. Comparative evaluation of linear and exponential amplification techniques for expression profiling at the single-cell level
Byrne et al. The polyadenylation site of Mimivirus transcripts obeys a stringent ‘hairpin rule’
Mathur et al. Quantitative analysis of m6A RNA modification by LC-MS
Waduge et al. Chemical probing for examining the structure of modified RNAs and ligand binding to RNA
Yadav et al. FFPE‐ATAC: A highly sensitive method for profiling chromatin accessibility in formalin‐fixed paraffin‐embedded samples
US20010049103A1 (en) Platform for the discovery of the bacterial genes involved in RNA modification
Marcellin et al. Saccharopolyspora erythraea’s genome is organised in high-order transcriptional regions mediated by targeted degradation at the metabolic switch
Welp et al. Chemical crosslinking extends and complements UV crosslinking in analysis of RNA/DNA nucleic acid–protein interaction sites by mass spectrometry
Gorini et al. OxiDIP-seq for genome-wide mapping of damaged DNA containing 8-oxo-2'-deoxyguanosine
EP3669193B1 (fr) Capture d'interactome arn améliorée
Khoddami et al. Experimental approaches for target profiling of RNA cytosine methyltransferases
Rang et al. Identification of a TetR family regulator and a polyketide synthase gene cluster involved in growth development and butenyl-spinosyn biosynthesis of Saccharopolyspora pogona
WO2001062981A1 (fr) Plate-forme destinee a la decouverte de genes bacteriens impliques dans la modification d'arn
Anvar et al. Combined genome-scale fitness and paralog synthetic lethality screens with just 44k clones: the IN4MER CRISPR/Cas12a multiplex knockout platform
CA2401018A1 (fr) Plate-forme destinee a la decouverte de genes bacteriens impliques dans la modification d'arn
Maor-Nof et al. An optimized ATAC-seq protocol for genome-wide mapping of active regulatory elements in primary mouse cortical neurons
Anastasakis et al. Proximity‐CLIP and expedited non‐radioactive library preparation of small RNA footprints for next‐generation sequencing

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2401018

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2001239856

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2001914470

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2001914470

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Ref document number: 2001914470

Country of ref document: EP