Note: Descriptions are shown in the official language in which they were submitted.
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PLATFORM FOR THE DISCOVERY OF THE BACTERIAL GENES
INVOLVED IN RNA MODIFICATION
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is related to USSN 60/185,071, filed February 25, 2000,
USSN 60/185,000, also filed February 25, 2000; USSN 60/225,505, filed August
15,
2000; and USSN 60/225,506, also filed August 15, 2000. The present application
claims
priority to, and benefit of, these applications pursuant to 35 U. S. C.
~119(e).
COPYRIGHT NOTIFICATION
Pursuant to 37 C.F.R. 1.71(e), Applicants note that a portion of this
disclosure contains material which is subject to copyright protection. The
copyright owner
has no objection to the facsimile reproduction by anyone of the patent
document or patent
disclosure, as it appears in the Patent and Trademark Office patent file or
records, but
otherwise reserves all copyright rights whatsoever.
BACKGROUND OF THE INVENTION
The development of 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. However, 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.
SUMMARY OF THE INVENTION
Discovery of therapeutic agents is facilitated by an understanding of the
function of genes whose gene products represent safe and effective drug
targets.
Assigning an enzymatic activity to a gene product, protein or RNA, can be
difficult
without a lengthy investigation into the substrates of the catalyzed reaction.
Although it
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can be possible to assign a gene's product to a particular class of enzymes by
computational and comparative means, the specific activity often remains
unknown
without biochemical or genetic studies. Moreover, screening procedures that
seek to
identify an organism's essential genes can not reveal the function of these
genes. The
methods of the present invention differ from other drug target screening
procedures in that
these methods can be used to identify the genes directly responsible for a
particular
biosynthetic activity, for example, RNA modification activity. The expression
of a
candidate 'test' gene is modulated within an organism and the product of the
activity of
interest is analyzed for similar modulation. 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. Where the members of the chosen class of substrates,
herein
termed "sentinel molecules," can be modified by any of a number of catalytic
mechanisms, the assay is not limited to a specific enzymatic activity. When
performed in
a multi-well format, the methods employing these sentinel molecules can be
performed in
a high throughput fashion. Thus, it is an object of the invention to provide a
platform of
methods upon which modifications of a sentinel molecule, such as one or more
transfer
RNA (tRNA) molecules, can be examined and optionally correlated to the
presence,
absence, or expression of a particular gene. 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.
Within the assay solution, 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 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
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chromatography, HPLC, capillary electrophoresis, NMR spectroscopy, X-ray
crystallography, infrared spectroscopy, or cryo-electron microscopic analysis.
Accordingly, 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-occurnng small molecule substrates, and synthetic small molecule
substrates.
The assay solution is optionally a cellular extract, derived from either
bacterial sources or
eukaryotic sources. Alternatively, 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.
Depending
on the design of the assay, either 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 presence and extent of
these
modifications can be determined by one or more of a variety of analytical
techniques, such
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as mass spectrometry, thin layer chromatography, HPLC, capillary
electrophoresis, NMR
spectroscopy, X-ray crystallography, or cryo-electron microscopic analysis.
In addition, 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.
Furthermore, 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
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gene product (and by association, the test gene) participates in an RNA
modification
process. By 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. Using the methods of the present invention, 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.
BRIEF DESCRIPTION OF THE DRAWING
Figure 1 illustrates various methodologies for the analysis of gene function.
Figure 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.
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DETAILED DISCUSSION OF THE INVENTION
Definitions
Before describing the present invention in detail, it is to be understood that
this invention is not limited to particular compositions or biological
systems, which can, of
course, vary. It is also to be understood that the terminology used herein is
for the purpose
of describing particular embodiments only, and is not intended to be limiting.
As used in
this specification and the appended claims, the singular forms "a", "an" and
"the" include
plural referents unless the content clearly dictates otherwise. Thus, for
example, reference
to "a cell" includes a combination of two or more cells, reference to "a
sentinel molecule"
includes mixtures of sentinel molecules, and the like.
Unless defined otherwise, all technical and scientific terms used herein
have the same meaning as commonly understood by one of ordinary skill in the
art to
which the invention pertains. Although any methods and materials similar or
equivalent to
those described herein can be used in the practice for testing of the present
invention, the
preferred materials and methods are described herein. In describing and
claiming the
present invention, the following terminology will be used in accordance with
the
definitions set out below.
As used herein, 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.
The term "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. As used herein, a
test
compound is meant to encompass both a single compound, as well as a group, or
"library,"
of compounds.
The term "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.
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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). In addition, the
test gene can
be one or more components of a library of sequences.
The term "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.
Discovery of Therapeutic Targets and Novel Dru~pounds
Pharmaceutical companies are pursuing new antimicrobial drug targets by
several methods, including the random screening of microbial genes for
lethality upon
disruption, for gene expression, or for activity upon exposure to compound
libraries. An
alternative approach to the identification of cellular targets having
therapeutic value (for
example, targets that demonstrate a sensitivity to antibiotics) is to place
the focus of the
screens on the products of the reactions catalyzed by the gene products and
assay for the
effect of modulation of "test genes" on the presence or abundance of the
covalent
modification on the "sentinel molecule." Often, these modifications (and the
gene
products that generate them) will differ among viral, prokaryotic and
eukaryotic systems,
or among members of these groups (for example, among different types of
prokaryotes),
providing some selectivity of the therapeutic target.
The present invention provides methods for screening test compounds, as
well as methods for screening compounds for antibiotic activity. In addition,
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. In the in vitro and in vivo methods, 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.
Alternatively, in
the screening methods, 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.
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. These methods, and the sentinel
molecules employed in these methods, are described in further detail below.
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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. Once the phenotype is known, the gene is then isolated,
and the
resulting gene product is shown to be capable of converting a defined
substrate into an
expected product. In the methods of the present invention, 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). 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) is expressed in either a
cell-based or in
vitro system, and the expression system is then modulated. If the expression
is down-
regulated, and the phenotype or the desired modification of the seminal
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.
Optionally, the gene expression is up-regulated and the cellular lysate is
combined with
synthetic tRNA substrates (i.e. the unmodified seminal molecule), and
optionally one or
more small molecule substrates. The resulting products (the modified tRNA
molecules)
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.
Sentinel Molecules
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
CA 02401018 2002-08-20
WO 01/62981 PCT/USO1/05920
molecule that is added to the system for the propose 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.
Examples of general classes of sentinel molecules 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. Examples of 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 seminal
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 seminal molecules
can also be
employed in the methods of the present invention; carbohydrate-based seminal
molecules
include, but are not limited to, carbohydrates, glycoproteins, glycolipids,
lipopolysaccharides, peptidoglycans, fucoidans, and the like.
Cells and Cell Lines
The cells used in the methods of the present invention include either
bacterial cells as well as eukaryotic cells. Examples of 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,
Helicobacter, Heliobacterium, Haemophillus, Methanobacterium, Methanococcus,
Mycobacterium, Mycoplasma, Pyrococcus, Rickettsia, Synechocystis, and
Treponema
(See, for example, the lists of microorganism genera provided by DSMZ-Deutsche
~ Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany,
at
http://www.dsmz.de/species). Alternatively, eukaryotic cell lines, including
mammalian
(for example, murine, rodent, guinea pig, rabbit, canine, feline, primate or
human cells),
amphibian, reptile, fish, nematode, fungal, and plant cells, can be employed.
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In addition to clinical and/or environmental samples, 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).
Generally, one of skill in the art is fully able to culture and transfect
cells
from animals, plants, fungi, bacteria and other cells using available
techniques. A variety
of cell culture media are described in The Handbook of Microbiological Media,
Atlas and
Parks (eds.) (1993, CRC Press, Boca Raton, FL). References describing the
techniques
involved in bacterial and animal cell culture include Sambrook et al.,
Molecular Cloning
A Laboratory Manual (2nd Ed.), Vol. 1-3 (1989, Cold Spring Harbor Laboratory,
Cold
Spring Harbor, New York); Current Protocols in Molecular Biolo~y, F.M. Ausubel
et al.,
eds., Current Protocols, (a joint venture between Greene Publishing
Associates, Inc. and
John Wiley & Sons, Inc., supplemented through 2000); Freshney, Culture of
Animal Cells,
a Manual of Basic Technique, third edition (1994, Wiley-Liss, New York) and
the
references cited therein; Humason, Animal Tissue Techniques, fourth edition
(1979, W.H.
Freeman and Company, New York); and Ricciardelli, et al., In Vitro Cell Dev.
Biol.
(1989) vol. 25, pp.1016-1024. Information regarding plant cell culture can be
found in
Plant Cell and Tissue Culture in Liquid Systems, by Payne et al. (1992, John
Wiley &
Sons, Inc. New York, NY);Plant Cell, Tissue and Orgzan Culture: Fundamental
Methods
by Gamborg and Phillips, eds. (1995, Springer Lab Manual, Springer-Verlag,
Berlin ), and
is also available in commercial literature such as the Life Science Research
Cell Culture
Catalo;.~,ue (1998) from Sigma- Aldrich, Inc (St Louis, MO) (Sigma-LSRCCC) and
the
Plant Culture Catalogue and supplement (1997) also from Sigma-Aldrich, Inc (St
Louis,
MO) (Sigma-PCCS).
Genes, Gene Expression, and Gene Products
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
CA 02401018 2002-08-20
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Incyte Genomics (Palo Alto, CA). Alternatively, 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 polymerise mediated, ligation-mediated and
combination
ligation/ polymerise 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. Preferably, 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.
Methods of introducing genetic material into cells, including plant and
animal cells, e.g., for cloning and sequencing and/or for expression and
selection of
encoded molecules are generally available, as are methods of expressing
proteins encoded
by such nucleic acids (see, for example, Ausubel, supra). Transformation
methodologies
include, but are not limited to, bacterial-mediated transformation, phage
transduction,
s
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
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Technigues, Methods in Enzymolog-y 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 Enz~gy (1994) vol.
235, pp.385-404; Kleckner et al. Methods in Enzymolo~y (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.
Modifications to Sentinel Molecules
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,
farnsylation, and the like. Alternatively, modifications to seminal 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.
Known modifications of RNA molecules can be found, for example, in
Genes VI, Chapter 9 ("Interpreting the Genetic Code"), Lewis, ed. (1997,
Oxford
University Press, New York), and Modification and Editing of RNA, Grosjean and
Benne,
eds. (1998, ASM Press, Washington DC). Modified RNA components include the
following: 2'-O-methylcytidine; N4-methylcytidine; N4-2'-O-dimethylcytidine;
N4-
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; 5-methyl-2-thiouridine; 5-hydroxyuridine; 5-methoxyuridine;
uridine
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5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 5-
carboxymethyluridine; 5-
methoxycarbonylmethyluridine; 5-methoxycarbonylmethyl-2'-O-methyluridine; 5-
methoxycarbonylmethyl-2'-thiouridine; 5-carbamoylmethyluridine; 5-
carbamoylmethyl-
2'-O-methyluridine; 5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)
uridinemethyl ester; 5-aminomethyl-2-thiouridine; 5-methylaminomethyluridine;
5
methylaminomethyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5
carboxymethylaminomethyluridine; 5-carboxymethylaminomethyl-2'-O-
methyluridine; 5-
carboxymethylaminomethyl-2thiouridine; dihydrouridine; dihydroribosylthymine;
2'-O-
methyladenosine; 2-methyladenosine; N6N-methyladenosine; N~, N6-
dimethyladenosine;
N6,2'-O-trimethyladenosine; 2-methylthio-N6 N6-isopentenyladenosine; N6-(cis-
hydroxyisopentenyl)-adenosine; 2-methylthio-N6-(cis-hydroxyisopentenyl)-
adenosine; N6-
glycinylcarbamoyl)adenosine; N6-threonylcarbamoyl adenosine; N6-methyl-N6-
threonylcarbamoyl adenosine; 2-methylthio-N~-methyl-N~-threonylcarbamoyl
adenosine;
N6-hydroxynorvalylcarbamoyl adenosine; 2-methylthio- N6-
hydroxnorvalylcarbamoyl
adenosine; 2'-O-ribosyladenosine (phosphate); inosine; 2'-O-methyl inosine; 1-
methyl
inosine; 1;2'-O-dimethyl inosine; 2'-O-methyl guanosine; 1-methyl guanosine;
N2-methyl
guanosine; NZ,NZ-dimethyl guanosine; N2, 2'-O-dimethyl guanosine; N2, N2, 2'-O-
trimethyl guanosine; 2'-O-ribosyl guanosine (phosphate); 7-methyl guanosine;
N2;7-
dimethyl guanosine; NZ; NZ°'-trimethyl guanosine; wyosine;
methylwyosine; under-
modified hydroxywybutosine; wybutosine; hydroxywybutosine; peroxywybutosine;
queuosine; epoxyqueuosine; galactosyl-queuosine; mannosyl-queuosine; 7-cyano-7-
deazaguanosine; arachaeosine [also called 7-formamido-7-deazaguanosine]; and 7-
aminomethyl-7-deazaguanosine. The methods of the present invention or others
in the art
can be used to identify additional modified RNA molecules.
Another preferred seminal molecule used in the methods of the present
invention is a carbohydrate-based or lipid based molecule. Exemplary seminal
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. As with the RNA-based
seminal
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
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proteins such as ubiquitin. Such modifications to seminal molecules can be the
result of
the activities of various transferases, synthases, isomerases, dehydrogenases,
and the like.
Analysis of Modified Sentinel Molecules
Methods and techniques for analyzing reaction components and products
are varied and well known in the art. 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.
Traditionally, analysis of modifications of sentinel molecules such as RNA
molecules has been performed 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.
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. A brief review of mass spectrometry
techniques
commonly used in biotechnology can be found, for example, in Mass Spectrometry
for
Biotechnolo~y by G. Siuzdak (1996, Academic Press, San Diego).
In the methods of the present invention, the assay solutions (containing the
newly modified sentinel molecules) are prepared for mass spectrometry and then
transfernng 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. By direct comparison of these spectrograms, 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.
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Alternatively, 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. Suitable deuterated solvent solutions include,
but are not
limited to D20 (deuterium oxide), CDCl3, DMSO-d6, acetone-d6, and the like
(available
from Cambridge Isotope Labs, Andover, MA; www.isotope.com). Optionally, the
samples can be analyzed using LC-NMR spectroscopy. Analysis by these
methodologies
can provide information related to both the presence of one or more
modifications, as well
as the type or identity of the modification (see, for example, NMR of
Macromolecules: A
Practical Approach, G.C.K. Roberts, ed., 1993, Oxford University Press, New
York).
Screening Test Compounds using Cellular Assay Solutions
One embodiment of the methods of the present invention provides methods
for screening a test compound for activity. In these methods, 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.
In preparing the assay solution, 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. For
example, 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. Thus,
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.
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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. Optionally, 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.
Within the assay solution, the gene product is allowed to interact with the
sentinel molecule. After incubation, 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.
Methods of Screening a Compound for Antibiotic Activity
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). In one embodiment, the targeted organism includes prokaryotic cells,
while the
non-targeted organism are eukaryotic cells. In another embodiment, the
targeted organism
includes pathogenic eukaryotic cells (for example, yeast and fungi).
Alternatively, 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. Thus,
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
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products needed to screen for the antibiotic compounds (for example, the cell
line can be
transformed with expression vectors that encode specific enzymatic
activities).
During the methods of the present invention, 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. Preferably, the sentinel molecule is
an RNA
molecule, such as a tRNA, rRNA, mRNA, guide RNA, snoRNA, snRNA, hnRNA, and the
like. Alternatively, the seminal 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.
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In Vitro Methods for Identifying Gene Products involved in RNA
Modification
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.
Alternatively, 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.
In addition, the expression level of the gene products) can be altered or
manipulated. In the methods wherein augmentation of the test gene activity is
desired,
over-expression constructs can be used to produce elevated levels of the test
gene activity
and increased expression of the gene product(s). Alternatively, in protocols
wherein the
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.
A cellular extract is prepared from the cell or cells, such that the extract
contains the gene products) 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).
Examples
of 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. Optionally,
the
sentinel RNA molecules are tRNA molecules. It should be noted that either a
single type
of sentinel RNA molecule, or a collection of sentinel RNA molecules, can be
employed in
the present invention.
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After incubation, 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 seminal
molecules
and DMAPP (dimethylallyl diphosphate). After the substrate adenosines had been
modified, the seminal molecules were digested to component nucleosides and
analyzed by
LC/MS (using reverse-phase column chromatography on a C18 column (Higgins
Analytical, Mountain View CA), and triple quadropole mass detection (Quattro
IITM,
Micromass Inc, Beverly, MA) equipped with a Z SprayTM electrospray ionization
source
operated in positive ion mode (single ion recording mode), monitoring a mass
to charge
ratio range of about 100-450 daltons. Induction of transferase activity and
concomitant
increases in product i6A levels as measured by mass spectrometry data are
shown in
Figure 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).
In Vivo Methods for Identifying Gene Products involved in RNA
M~dificatinn
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
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CA 02401018 2002-08-20
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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.
Alternatively, 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.
In the in vivo methods for identifying one or more gene products involved
in RNA modification, a cell (or group of cells) is provided that 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. Optionally, 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.
The 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 products) 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.
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In the methods wherein augmentation of the test gene activity is desired,
over-expression constructs can be used to produce elevated levels of the test
gene activity
and increased expression of the gene product(s). Alternatively, in protocols
wherein the
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.
After the one or more test genes have been manipulated, and the resulting
changes in gene product expression have occurred, 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.
Furthermore,
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.
Identification of Genes 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.
Techniques for identification of genes and/or nucleic acid sequences are known
in the art.
Sequencing and other standard recombinant techniques useful for the present
invention
can be found, for example, in Berger and Kimmel, Guide to Molecular Cloning
Techniques, Methods in EnzXmology volume 152 (Academic Press, Inc., San Diego,
CA);
Sambrook et al., Molecular Cloning - A Laboratory Manual (2nd Ed.), Vol. 1-3,
(1989,
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) and Current
Protocols in
Molecular Biolo~y, F.M. Ausubel et al., eds., Current Protocols, a joint
venture between
Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented
through
1999).
Cloning and expression techniques which can be utilized in this step of the
methods of the present invention have been described previously, with respect
to
expressing the gene product in the cell. These references also describe
additional
approaches which can be employed to determine the identity of the test genes
of the
present invention. In addition, in vitro amplification methods can also be
used to amplify
and/or sequence the test genes of interest. Examples of in vitro amplification
and
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sequencing techniques, including the polymerise chain reaction (PCR) the
ligase chain
reaction (LCR), Q(3-replicase amplification and other RNA polymerise 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
A~nlications by Innis et al. eds. (1990, Academic Press Inc., San Diego, CA );
Arnheim &
Levinson (October 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3,
81-94;
Kwoh et al. (1989) Proc. Natl. Acid. Sci. USA 86, 1173; Guatelli et al. (1990)
Proc. Natl.
Acid. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin. Chem 35, 1826;
Landegren et al.,
(1988) Science 241, 1077-1080; Van Brunt (1990) Biotechnolo~y 8, 291-294; Wu
and
Wallace, (1989) Gene 4, 560; Barringer et al. (1990) Gene 89, 117, and
Sooknanan and
Malek (1995) Biotechnolo~y 13: 563-564; Wallace et al., U.S. Pat. No.
5,426,039; Cheng
et al. (1994) Nature 369: 684-685, and the references therein.
The function of a gene can be determined via over-expression of the
encoded gene product in vivo using the methods of the present invention. 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). Optionally, 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. In
the plasmid, 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.
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
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as guanidinium thiocyanate, phenol and detergent and buffered to pH of between
5.5 and
6.5 (see, for example, Berger and Kimmel, supra). 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 Pl, 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.
Using the methods of the present invention, resolution of many nucleosides
is possible by HPLC alone using reversed phase chromatography on a C18 column.
In the
event that a particular modification of interest (i.e., a sentinel structure)
is not resolved
from other modifications, the use of LC-MS has been demonstrated to
effectively detect
modified nucleosides in mixed solutions (Pomerantz and McCloskey (1990)
Methods in
Enzymology 193:796-824). A measured increase in the amount of a modified
nucleoside
indicates that the gene is involved in the pathway of tRNA modification.
High Throughput Methodology
Optionally, 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 mufti-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 mufti-well plates. Mufti-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
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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.
Thus, by
using a multi-well format, 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.
Data Analysis
The step of detecting the presence or absence of one or modifications to the
seminal molecule can further include analyzing data generated during the
detection
process. For example, 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).
Furthermore, 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. For example, 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 seminal molecules; a device for analyzing the
signals
from the modified seminal 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 seminal 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). Standard desktop
applications
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WO 01/62981 PCT/USO1/05920
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. Optionally, the
computer or
computer readable medium can provide the examination results in the form of an
output
file.
Uses of the Methods, Devices and Compositions of the Present Invention
Modifications can be made to the method and materials as described above
without departing from the spirit or scope of the invention as claimed, and
the invention
can be put to a number of different uses, including:
The use of any method herein, to identify a gene encoding an RNA
modification enzyme.
The use of a method or an integrated system to identify a gene encoding an
RNA modification enzyme.
An assay, kit or system utilizing a use of any one of the selection
strategies,
materials, components, methods or substrates hereinbefore described. 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.
In an additional aspect, the present invention provides kits embodying the
methods and devices herein. Kits of the invention optionally comprise one or
more of the
following: (1) a library of nucleic acid sequences, optionally incorporated
into expression
vectors; (2) one or more seminal molecules, such as RNA seminal 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.
In a further aspect, 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.
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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.
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