U.S. patent application number 10/370819 was filed with the patent office on 2003-08-14 for screen employing fluorescence anisotropy to identify compounds with affinity for nucleic acids.
Invention is credited to Arenas, Jaime E., Cload, Sharon T., Laing, Lance G., Lillie, James W., Pakula, Andrew A..
Application Number | 20030152992 10/370819 |
Document ID | / |
Family ID | 21908003 |
Filed Date | 2003-08-14 |
United States Patent
Application |
20030152992 |
Kind Code |
A1 |
Laing, Lance G. ; et
al. |
August 14, 2003 |
Screen employing fluorescence anisotropy to identify compounds with
affinity for nucleic acids
Abstract
The present invention provides methods for screening for
bioactive compounds, in particular those that bind to RNA sequences
involved in the pathogenesis of disease or in regulation of a
physiological function. The methods involve assessing the stability
and/or the conformation of an RNA target in the presence and
absence of test ligands, and identifying as a ligand any test
ligand that causes a measurable change in target RNA stability
and/or conformation. In a preferred embodiment, the effect of a
ligand on target RNA stability and/or conformation is assessed by
measuring the fluorescence polarization of a fluorescently labeled
probe.
Inventors: |
Laing, Lance G.; (Belmont,
MA) ; Arenas, Jaime E.; (Lexington, MA) ;
Cload, Sharon T.; (Cambridge, MA) ; Lillie, James
W.; (Natick, MA) ; Pakula, Andrew A.;
(Lexington, MA) |
Correspondence
Address: |
DAVID R PRESTON & ASSOCIATES
12625 HIGH BLUFF DRIVE
SUITE 205
SAN DIEGO
CA
92130
US
|
Family ID: |
21908003 |
Appl. No.: |
10/370819 |
Filed: |
February 20, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10370819 |
Feb 20, 2003 |
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09961008 |
Sep 20, 2001 |
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6569628 |
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09961008 |
Sep 20, 2001 |
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09060449 |
Mar 5, 1998 |
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6331392 |
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60039911 |
Mar 5, 1997 |
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Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
C40B 40/06 20130101;
C40B 50/06 20130101; C12Q 1/6811 20130101; C12Q 1/6816 20130101;
C40B 30/10 20130101; C12Q 1/6813 20130101; C12Q 2561/119 20130101;
C12N 15/1048 20130101; C12Q 1/6811 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Claims
What is claimed is:
1. A method for identifying a ligand that binds a predetermined
target RNA sequence, which comprises: (a) selecting as test ligands
a plurality of compounds not known to bind to the target RNA
sequence; (b) incubating the target RNA sequence with one or more
fluorescently labeled probes in the presence of a test ligand to
produce a test combination; (c) incubating the target RNA sequence
with said one or more probes in the absence of a test ligand to
produce a control combination; (d) measuring one or more
fluorescence properties of said probe; and (e) selecting as a
ligand any test ligand that causes a measurable change in at least
one of the fluorescence properties measured in step (d) in the test
combination relative to the control combination, wherein said
property reflects the stability and/or conformation of said target
RNA.
2. A method as defined in claim 1, further comprising (f) repeating
steps (b)-(e) with a plurality of said test ligands until a ligand
that binds to the target RNA sequence is identified.
3. A method as defined in claim 1, wherein the fluorescence
property measured in step (d) is fluorescence polarization.
4. A method as defined in claim 3, further comprising subjecting
the test and control combinations to conditions that denature a
detectable fraction of the target RNA sequence in the control
combination.
5. A method as defined in claim 4, wherein the subjecting comprises
at least one of altering the temperature, altering the salt
concentration, adding denaturing compounds, and combinations
thereof.
6. A method as defined in claim 3, wherein the target RNA is from
about 5 to about 500 nucleotides in length.
7. A method as defined in claim 3, wherein the probe comprises an
oligonucleotide that preferentially hybridizes to a predetermined
conformation of the target RNA.
8. A method as defined in claim 3, wherein said probe comprises two
or more different fluorescent moieties.
9. A method as defined in claim 3, wherein in step (b), said target
RNA sequence is incubated with two or more different probes.
Description
FIELD OF INVENTION
[0001] This invention pertains to novel methods for screening for
pharmaceutical compounds, in particular those that bind to RNA
sequences involved in the pathogenesis of disease or in regulation
of a physiological function.
BACKGROUND OF THE INVENTION
[0002] Pharmaceuticals can be developed from lead compounds that
are identified through a random screening process directed towards
a target, such as a nucleic acid or a protein receptor. Large scale
screening approaches can be complicated by a number of factors.
First, many assays are laborious or expensive to perform. Assays
may involve experimental animals, cell lines, or tissue cultures
that are difficult or expensive to acquire or maintain. These
considerations often place practical limitations on the number of
compounds that reasonably can be screened. Thus, those employing
random screening methods are frequently forced to limit their
search to those compounds for which some prior knowledge suggests
that the compounds are likely to be effective. This strategy limits
the range of compounds tested, and many useful drugs may be
overlooked.
[0003] Furthermore, the specificity of many biochemical assays may
exclude a wide variety of useful chemical compounds, because the
interactions between the ligand and the target are outside the
scope of the assay. With such a specific assay, many potential
pharmaceuticals may not be detected.
[0004] Finally, in most existing biochemical screening approaches
to drug discovery, the system in question must be
well-characterized before screening can begin. Consequently,
biochemical screening for therapeutic drugs directed against many
targets must await detailed biochemical characterization, a process
that generally requires extensive research.
[0005] The present invention pertains specifically to the use of
RNA targets in high-throughput screening methods for identification
of useful ligands. The invention takes advantage of the existence
of higher-order structures in naturally-occurring and synthetic RNA
molecules. For example, RNA exists in both single stranded and
helical duplex forms. These duplexes may be distorted by loops,
bulges, base triples and junctions between helices. The structural
diversity of RNA is far greater than that of DNA, and similar to
that of proteins, making RNA a likely target for unique binding of
small molecules (reviewed in Wyatt and Tinoco, 1993).
[0006] Small molecules can bind RNA with high affinity and
specificity and can block essential functions of the bound RNA. The
best example of such molecules are antibiotics such as erythromycin
and aminoglycosides. The first suggestion that some antibiotic
translation inhibitors interact specifically with RNA was the
genetic mapping of resistance to kanamycin and gentamicin to the
methylation of 16S RNA (Thompson et al., Mol. Gen. Genet. 201:168,
1985). Erythromycin binds to bacterial RNA and releases
peptidyl-tRNA and mRNA (Menninger et al., Mol. Gen. Genet. 243:225,
1994). 2-DOS-containing aminoglycosides bind specifically to the
structures of HIV RNA known as the RRE, block binding of the HIV
Rev protein to this RNA, and thereby inhibit HIV replication in
tissue culture cells (Zapp et al., Cell 74:969, 1993). In addition,
although aminoglycosides have long been developed as translation
inhibitors, they were only recently shown to bind to rRNA in the
absence of proteins (Purohit and Stern, Nature 370:659, 1994;
Fourmy et al., Science 274:1367, 1996). Hygromycin B inhibits
coronaviral RNA synthesis and is thought to do so by binding to the
viral RNA and blocking specifically the translation of viral RNA
(Macintyre et al., Antimicrob. Agents Chemother. 35:2630,
1991).
[0007] Existing assays for ligands of nucleic acids, such as, for
example, methods that use equilibrium dialysis, differential
scanning calorimetry, viscometric analyses, UV measurement of
hyperchromic effect, or fluorescence, are often unfeasible for
high-throughput applications, because of insufficient
characterization, unavailability of components, expense, and/or
complexity. (See, for example, Murakami et al., Nuc.Acids Res.
19:4097, 1991; Walker et al., Nuc.Acids Res. 24:348, 1996). Thus,
prior to the present invention, random screening approaches for
non-oligonucleotide ligands of RNA were limited to compounds for
which some prior knowledge suggested that they might be effective.
This strategy has been successful (Zapp et al., 1993), but is
limited in the range of compounds that can be tested on a practical
scale.
[0008] U.S. Pat. No. 5,270,163 describes the SELEX system for the
identification of oligonucleotides that bind specific targets. In
this system, random oligonucleotides are affinity-selected and
amplified, followed by several cycles of re-selection and
amplification. This method, however, is limited to screening for
oligonucleotide ligands and cannot be applied in reverse, i.e., to
search for non-oligonucleotide ligands that bind to nucleic
acids.
[0009] U.S. Pat. No. 5,306,619 discloses a screening method to
identify compounds that bind particular DNA target sequences. In
this method, a test nucleic acid is constructed in which the target
sequence is placed adjacent to a known protein-binding DNA
sequence. The effect of test compounds on the binding of the
cognate protein to the protein-binding DNA sequence is then
measured. This method requires conditions in which melting of DNA
hybrids and unfolding of DNA structure do not occur. RNA, by
contrast, can undergo much more dramatic variations in patterns of
base-pairing and overall conformation.
[0010] Thus, there is a need in the art for efficient and
cost-effective high-throughput methods for random screening of
large numbers of non-oligonucleotide small molecules for their
ability to bind physiologically, medically, or commercially
significant RNA molecules.
SUMMARY OF THE INVENTION
[0011] The present invention encompasses screening methods to
identify ligands that bind any predetermined target RNA. The
methods are carried out by the steps of: selecting as test ligands
a plurality of compounds not known to bind to the target RNA
sequence; incubating the target RNA sequence with one or more
fluorescently labeled probes in the presence of a test ligand to
produce a test combination, and in the absence of a test ligand to
produce a control combination; measuring one or more fluorescence
properties of the probe(s) to evaluate the stability and/or
conformation of the target RNA in each combination; and selecting
as a ligand any test ligand that causes a measurable change in at
least one of the fluorescence properties of the probes in the test
combination relative to the control combination. Preferably, the
incubating and measuring steps are repeated with a plurality of
said test ligands until a ligand that binds to the target RNA
sequence is identified. Furthermore, the test and control
combinations may be subjected to conditions that, in the absence of
ligands (i.e., in the control combination), denature a detectable
fraction of the target RNA.
[0012] In general, ligands identified by the methods of the present
invention are expected to stabilize, i.e., reduce .DELTA.G.degree.
of a particular conformation of the target RNA. Stabilization may
lead to a more folded conformation, a less folded conformation, or
a change from a first folded conformation to a second, alternative,
folded conformation. Preferably, ligands stabilize a particular
conformation which inhibits the normal function of the target
RNA.
[0013] Probes useful in practicing the invention include molecules
which comprise fluorescent moieties whose measurable fluoresence
properties, particularly polarization or anisotropy, are sensitive
to the stability and/or conformation of the target RNA as reflected
in the binding state of the probe. Preferably, the fluorescence
anisotropy of a sample containing the probe is compared between the
test and control combinations. Ligands may be identified as
compounds that cause the anisotropy to increase or decrease
relative to the anisotropy of a control solution.
[0014] In a preferred embodiment, the target RNA is hybridized with
a complementary oligonucleotide to which one or more fluorescent
probes have been attached, and the effect of test compounds on
fluorescence anisotropy of the probe is used to detect ligand
binding to the target RNA.
[0015] A "measurable change" in target RNA stability or
conformation as detected by any of the above or other methods is
one in which the difference in the measured parameter between the
test and control combinations is greater than that expected due to
random statistical variation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a schematic illustration of the equilibrium
equation for the hybridization to a target RNA of a fluorophore
attached to an oligonucleotide probe.
[0017] FIG. 2 is a schematic illustration of the effect on
fluorescence polarization of a fluorophore binding to two different
regions of a target RNA structure.
[0018] FIG. 3 shows a photograph of a 15% native polyacrylamide gel
showing the formation of hybrids between a fluorescent probe and a
target RNA.
[0019] FIG. 4 is a schematic illustration of a model target RNA
structure.
[0020] FIG. 5 is a graphic illustration of the time course of
fluorescence polarization of hybridization reactions between the
target RNA shown schematically in FIG. 4 and two different
fluorescently-labeled oligonucleotide probes. mP, millipolarization
units.
DETAILED DESCRIPTION OF THE INVENTION
[0021] All patent applications, patents, and literature references
cited in this specification are hereby incorporated by reference in
their entirety. In case of conflict, the present description,
including definitions, will prevail.
[0022] Definitions
[0023] As used herein, the term "ligand" refers to an agent that
binds a target RNA. The agent may bind the target RNA when the
target RNA is in a native or alternative conformation, or when it
is partially or totally unfolded or denatured. According to the
present invention, a ligand can be an agent that binds at any site
on the target RNA. Therefore, the ligands of the present invention
encompass agents that in and of themselves may have no apparent
biological function, beyond their ability to bind to the target
RNA.
[0024] As used herein, the term "test ligand" refers to an agent,
comprising a compound, molecule or complex, which is being tested
for its ability to bind to a target RNA. Test ligands can be
virtually any agent, including without limitation metals, peptides,
proteins, lipids, polysaccharides, small organic molecules,
nucleotides (including non-naturally occurring ones) and
combinations thereof. Typically, small organic molecules have a
molecular weight of more than 50 yet less than about 2,500 daltons,
and most preferably less than about 600 daltons. Preferably, test
ligands are not oligonucleotides. Complex mixtures of substances
such as natural product extracts, which may include more than one
test ligand, can also be tested, and the component (or multiple
component) that may bind the target RNA can be identifed and
purified from the mixture in a subsequent step.
[0025] Test ligands may be derived from large libraries of
synthetic or natural compounds. For example, synthetic compound
libraries are commercially available from Maybridge Chemical Co.
(Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon
Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.).
A rare chemical library is available from Aldrich (Milwaukee,
Wis.). Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant and animal extracts are available from Pan
Labs (Bothell, Wash.) or MycoSearch (NC), or are readily
producible. Additionally, natural and synthetically produced
libraries and compounds are readily modified through conventional
chemical, physical, and biochemical means. For example, the
compounds may be modified to enhance efficacy, stability,
pharmaceutical compatibility, and the like. For example, once a
peptide ligand has been identified using the present invention, it
may be modified in a variety of ways to enhance its stability, such
as using an unnatural amino acid, such as a D-amino acid,
particularly D-alanine, or by functionalizing the amino or carboxyl
terminus, e.g., for the amino group, acylation or alkylation, and
for the carboxyl group, esterification or amidification, or through
constraint of the peptide chain in a cyclic form, or through other
strategies well known to those skilled in the art.
[0026] As used herein, the term "target RNA" refers to a RNA
sequence for which identification of a ligand or binding partner is
desired. Target RNAs include without limitation sequences known or
believed to be involved in the etiology of a given disease,
condition or pathophysiological state, or in the regulation of
physiological function. Target RNAs may be derived from any living
organism, such as a vertebrate, particularly a mammal and even more
particularly a human, or from a plant, virus, bacterium, fungus,
protozoan, parasite or bacteriophage. Target RNA may comprise wild
type sequences, or, alternatively, mutant or variant sequences,
including those with altered stability, activity, or other variant
properties, or hybrid sequences to which heterologous sequences
have been added. Furthermore, target RNA as used herein includes
RNA that has been chemically modified, such as, for example, by
conjugation of biotin, peptides, fluorescent molecules, modified
nucleotides, and the like.
[0027] Target RNA sequences for use in the present invention are
typically between about 5 and about 500 nt, preferably between
about 30 and about 100 nt, and most preferably about 50 nt. Target
RNAs may be isolated from native sources, or, more preferably, are
synthesized in vitro using conventional polymerase-directed
cell-free systems such as those employing T7 RNA polymerase or SP6
RNA polymerase (Studier et al., Meth.Enzymol. 185:60 (1990).
[0028] As used herein, "test combination" refers to the combination
of a test ligand, one or more probes, and a target RNA. "Control
combination" refers to the combination of a target RNA with one or
more probes in the absence of a test ligand.
[0029] As used herein, the "folded state" of a target RNA refers to
a native (lowest Gibbs free energy state) conformation of the
sequence in the absence of denaturing conditions. The folded state
of an RNA encompasses both particular patterns of intramolecular
base-pairing, as well as higher-order structures stabilized by
covalent or non-covalent bonding. Without wishing to be bound by
theory, it is believed that certain target RNAs may achieve one of
several alternative folded states stabilized by the lowest Gibbs
free energy state, depending upon experimental conditions
(including buffer, temperature, presence of ligands, and the
like).
[0030] As used herein, the "unfolded state" of a target RNA refers
to a situation in which the RNA has been rendered partially or
completely single-stranded relative to its folded state(s) or
otherwise lacks elements of its structure that are present in its
folded state. The term "unfolded state" as used herein encompasses
partial or total denaturation and loss of structure.
[0031] As used herein, a "measurable change" in RNA stability or
conformation refers to a quantity that is empirically determined
and that will vary depending upon the method used to monitor RNA
stability or conformation. The present invention encompasses any
difference between the test and control combinations in any
measurable physical parameter, where the difference is greater than
expected due to random statistical variation.
[0032] As used herein, a "probe" is any molecule that is
intrinsically fluorescent or to which a fluorescent moiety is
attached, in which one or more fluorescence properties are
sensitive to the stability and/or conformation of the target RNA
and/or to the binding state of the probe. Suitable probe compounds
include without limitation nucleic acids, particularly
oligonucleotides; small RNA-binding molecules exemplified by
2-deoxystreptamine antibiotics, which bind the Rev-responsive
element in HIV RNA, or other compounds that specifically recognize
the major or minor groove of RNA; and proteins, and peptides
derived therefrom, that recognize particular RNA sequences or
conformations.
[0033] Oligonucleotide probes may comprise any purine- and
pyrimidine-containing polymers, including without limitation DNA,
RNA, protein-nucleic acid, or combinations thereof. Furthermore,
oligonucleotide probes may be modified so as to modulate their
affinity for the target RNA; this may be achieved, for example, by
changing the length of the olignucleotide; by introducing
mismatches between otherwise complementary sequences; by employing
nucleotide analogues that engage in stronger or weaker base-pairing
than the original nucleotide (Kutyavin et al., Biochemistry
35:11170, 1996; Woo et al., Nuc.Acids Res. 24:2470, 1996); and by
other chemical modifications known in the art. Similarly, small
molecule probes or peptide- or protein-derived probes may be
chemically modified using methods well known in the art in order to
change their affinity for the target RNA. Fluorescent moieties
which may be attached to the probe molecules include without
limitation: fluorescein, rhodamine, BODIPY.TM. compounds, and
derivatives thereof (Molecular Probes, Eugene Oreg.). Methods for
conjugating these moieties to nucleic acids, peptides, and small
organic molecules are disclosed in Haugland, Handbook of
Fluorescent Probes, 5th Ed. (Molecular Probes, Eugene Oreg.,
1992).
[0034] The present invention encompasses screening methods,
particularly high-throughput methods, for identifying a ligand that
binds a target RNA. If the target RNA to which the test ligand
binds is associated with or causative of a disease or condition,
the ligand may be useful for diagnosing, preventing or treating the
disease or conditioner for regulating the expression of a gene. A
ligand identified by the present method can also be one that is
used in a purification or separation method, such as a method that
results in purification or separation of the target RNA from a
mixture. The present invention also relates to ligands identified
by the present method and their therapeutic uses (for diagnostic,
preventive or treatment purposes) and uses in purification and
separation methods.
[0035] According to the present invention, a ligand for a target
RNA is identified by its ability to influence the extent or pattern
of intramolecular folding or the rate of folding or unfolding of
the target RNA and, more specifically, the energetic stability
(Gibbs free energy state) of the target RNA. If the test ligand
binds to the target RNA, the relative amounts of stabilized target
RNA, folded or unfolded target RNA, or one or another of multiple
alternative folded states of the target RNA, or the rate of folding
or unfolding of the target RNA in the presence of the test ligand
will be different, i.e., higher or lower, than that observed in the
absence of the test ligand. Thus, the present method encompasses
incubating the target RNA with probe in the presence and absence of
a test ligand. This is followed by analysis of the differential
stability and/or folding of the target RNA, preferably using
fluorescence polarization as an indicator.
[0036] An important feature of the present invention is that it may
detect any compound that binds to any region of the target RNA, not
only to discrete regions that are intimately involved in a
biological activity or function. It is contemplated that multiple
ligands may be used simultaneously on the same target RNA, and may
bind cooperatively or independently.
[0037] In practicing the present invention, the test ligand is
combined with a target RNA, and the mixture is maintained under
appropriate conditions and for a sufficient time to allow binding
of the test ligand to the target RNA. Equilibrium or
non-equilibrium conditions may be used. In general, the test ligand
is present in molar excess relative to the target RNA. As discussed
in more detail below, the target RNA can be in a soluble form,
conjugated to a larger molecule in solution, or bound to a solid
phase matrix. Preferably, the test solution contains monovalent and
divalent ions and has a pH of about 7.5, and the incubation is
conducted at temperatures between about 25.degree. C. and
40.degree. C. ("physiological" conditions).
[0038] The time necessary for binding of target RNA to ligand will
vary depending on the test ligand, target RNA and other conditions
used. Preferably, binding will occur instantaneously (e.g.,
essentially simultaneous with combination of test ligand and target
RNA), while in others, the test ligand-target RNA combination can
be maintained for a longer time e.g. up to 12-16 hours, before
binding is measured. When many test ligands are employed, an
incubation time is chosen that is sufficient for most RNA:ligand
interactions, typically about one hour. The appropriate time will
be readily determined by one skilled in the art.
[0039] Other experimental conditions that are optimized for each
RNA target include pH, reaction temperature, salt concentration,
valency, and composition, amount of RNA, reducing agent
concentration and composition, and the inclusion of non-specific
protein and/or nucleic acid in the assay. An important
consideration when screening chemical or natural product libraries
is the response of the assay to organic solvents (e.g., dimethyl
sulfoxide, methanol or ethanol) commonly used to resuspend such
materials. Accordingly, each RNA is tested in the presence of
varying concentrations of each of these organic solvents. Finally,
the assay may be particularly sensitive to certain types of
compounds, in particular intercalating agents, that commonly appear
in chemical and especially natural product libraries. These
compounds can often have potent, but non-specific, inhibitory
activity. Some of the buffer components and their concentrations
will be specifically chosen in anticipation of this problem. For
example, bovine serum albumin will react with radicals and minimize
surface adsorption. The addition of non-specific DNA or RNA may
also be necessary to minimize the effect of nucleic acid-reactive
molecules (such as, for example, intercalating agents) that would
otherwise score as "hits" in the assay.
[0040] Binding of a test ligand to the target RNA is assessed by
comparing the stability (Gibbs free energy) and/or the absolute
amount of folded or unfolded target RNA in the absence and presence
of test ligand, or, alternatively, by determining the ratio of
folded:unfolded target RNA or change in the folded state of the
target RNA, or the rate of target RNA folding or unfolding, or
differential stability, in the absence and presence of test ligand.
If a test ligand binds the target RNA (i.e., if the test ligand is
a ligand for the target RNA), there may be significantly more
stabilized or more folded target RNA (and, thus, a higher ratio of
more stable to less stable or of folded to unfolded target RNA)
than is present in the absence of a test ligand. Alternatively,
binding of the test ligand may result in significantly less
stabilized or less folded target BNA than is present in the absence
of a test ligand. Another possibility is that binding of the test
ligand changes the pattern or stabilization properties of
alternative RNA folded structures. Similarly, binding of the test
ligand may cause the rate of target RNA folding or unfolding to
change significantly or may change the rate of acquisition of an
alternative structure.
[0041] In any case, determination of the absolute amounts or ratios
of stabilized and non-stabilized or folded and unfolded target RNA
may be carried out using probes which comprise one or more
fluorescent moieties (see below). Any stability-sensitive and/or
conformation-sensitive probe to which an appropriate fluorescence
moiety can be attached may be used in practicing the invention. For
example, an oligonucleotide can be designed so that it will
hybridize to a particular RNA target only when the RNA is in an
unfolded conformation or to single-stranded regions in an otherwise
folded conformation. A protein, or a peptide or structural domain
derived therefrom, can be isolated that binds to a particular RNA
target only when the RNA is in a particular folded conformation.
For example, antibodies (or antigen combining sites derived
therefrom) may be used that recognize specific RNA sequences
(and/or conformations of such sequences) (Deutscher et al.,
Proc.Natl.Acad. Sci. USA 85:3299, 1988); bind to double-stranded or
single-stranded RNA in a sequence-independent manner (Schonborn et
al., Nuc.Acids Res. 19:2993, 1991); or bind DNA:RNA hybrids
specifically (Stumph et al., Biochem. 17:5791, 1978).
[0042] For the purposes of high-throughput screening, the
experimental conditions are adjusted to achieve a threshold
proportion of test ligands identified as "positive" compounds or
ligands from among the total compounds screened. This threshold is
set according to two criteria. First, the number of positive
compounds should he manageable in practical terms. Second, the
number of positive compounds should reflect ligands with an
appreciable affinity and specificity towards the target RNA. A
preferred threshold is achieved when 0.1% to 1% of the total test
ligands are shown to be ligands of a given target RNA within a
specified binding affinity range.
[0043] Detection of RNA Ligands Using Fluorescence
Polarization/Anisotropy
[0044] Fluorescence polarization or anisotropy is a highly
sensitive method for detecting RNA ligands according to the present
invention. When fluorescent molecules are excited with plane
polarized light, they emit a majority of light in the same
polarized plane, provided that the molecule remains stationary
during the lifetime of the excited state (4 nanoseconds in the case
of fluorescein). However, if the molecule rotates or tumbles out of
the plane of the exciting polarized light during the excited state,
light is emitted in a different plane from that of the initial
excitation. The degree to which the fluorescence emission vector
moves from, e.g., a vertical to a horizontal plane is directly
related to the mobility of the fluorescently labeled molecule. That
is, if the fluorescently labeled molecules are large, they move
very little and the emitted light remains highly polarized with
respect to the excitation plane. By contrast, if the fluorescently
labeled molecules are small, they rotate or tumble faster, and the
resulting emitted light is depolarized relative to the excitation
plane (Lackowicz, Principles of Fluorescence Spectroscopy, Plenum
Press, NY, 1983; Methods in Fluorescence Polarization, Panvera
Corp, Madison Wis.).
[0045] Fluorescence polarization is defined as: 1 P = Int parallel
- Int vertical Int parallel + Int vertical
[0046] where Int.sub.parallel is the intensity of emission light
parallel to the excitation plane and Int.sub.vertical is the
intensity of emission light perpendicular to the excitation plane.
P is a dimensionless number (expressed as "polarization units" or
"millipolariztion units (mP)) and has a maximum value of 0.5 for
fluorescein.
[0047] Fluorescence anisotropy is related to polarization in the
following way: 2 A = Int parallel - Int vertical Int parallel + 2
Int vertical and A = 2 P 3 - P
[0048] Polarization/anisotropy is related to the speed at which a
fluorescently labeled molecule rotates, which, in turn, is related
to the size (molecular volume) of the fluorescent entity. Thus,
when a fluorescently labeled oligonucleotide probe, for example,
binds to a target RNA, the molecular volume of the fluorescent
entity increases, and, the fluorescence polarization value of the
sample will be higher. In this embodiment, hybridization of the
probe to the target RNA is allowed to proceed in the absence and
presence of test ligands (i.e., in control and test combinations,
respectively), and fluorescence polarization measurements are used
to quantify the level of hybridization in test and control
combinations. Ligands are identified as those compounds that cause
a depolarization of the test combination relative to the control
combination.
[0049] The present invention encompasses the use of more than one
fluorescent moiety attached to a single probe (see, e.g., FIG. 2),
as well as the use of two or more probes (each having a different
fluorophore) in a single reaction. In either case, the only
requirement is that the different flurorescence properties of the
different fluorophores should be such that their fluorescence
polarization can in principle be measured simultaneously. This may
be achieved, for example, by choosing fluorophores whose excitation
and emission wavelength maxima do not significantly overlap.
[0050] The present invention is also particularly advantageous in
allowing a direct determination of where in the target RNA the
ligand binds. By individually conjugating a fluorophore to
different regions of the probe molecule, it may be possible to
ascertain what part of the target RNA structure is affected by
ligand binding. Alternatively, multiple probes directed to
different sites on the target RNA may be used.
[0051] Detection of ligands by measuring fluorescence polarization
is particularly suitable for high-throughput applications. For
example, two instruments exist that can measure polarization of
samples present in 96-well microtiter dishes; these instruments are
marketed by Panvera Corp (Madison Wis.) and Jolley Instruments
(Chicago Ill.). Furthermore, the RNA target may be immobilized
(either on beads or on the walls of the microtiter wells), or may
be conjugated to a larger molecule in solution (such as, for
example, to streptavidin via a biotin moiety attached to the target
RNA), thereby enhancing differences in polarization of the
fluorescent probe subsequent to ligand binding.
[0052] Applications
[0053] Binding to a given target RNA is a prerequisite for
pharmaceuticals intended to modify directly the action of that RNA.
Thus, if a test ligand is shown, through use of the present method,
to bind an RNA that reflects or affects the etiology of a
condition, it may indicate the potential ability of the test ligand
to alter RNA function and to be an effective pharmaceutical or lead
compound for the development of such a pharmaceutical.
Alternatively, the ligand may serve as the basis for the
construction of hybrid compounds containing an additional component
that has the potential to alter the RNA's function. In this case,
binding of the ligand to the target RNA serves to anchor or orient
the additional component so as to effectuate its pharmaceutical
effects.
[0054] The fact that the present method is based on
physico-chemical properties common to most RNAs gives it widespread
application. The present invention can be applied to large-scale
systematic high-throughput procedures that allow a cost-effective
screening of many thousands of test ligands. Once a ligand has been
identified by the methods of the present invention, it can be
further analyzed in more detail using known methods specific to the
particular target RNA used. For example, the ligand can be tested
for binding to the target RNA directly, such as, for example, by
using equilibrium dialysis or chromatographic frontal analysis.
Furthermore, the ligand can be tested for its ability to influence,
either positively or negatively, a known biological activity of the
target RNA, such as (but not limited to) protein translation or RNA
splicing.
[0055] Non-Limiting examples of RNA targets to which the present
invention can be applied are shown in the following table:
1 Therapeutic Area RNA Targets Antivirals HBV epsilon sequence; HCV
5' untranslated region; HIV packaging sequence, RRE, TAR;
picornavirus internal translation enhancer Antibacterials RNAse P,
tRNA, rRNA (16S and 23S), 4.5S RNA Antifungals Similar RNA targets
as for antibacterials, as well as snRNA Rheumatoid Alternative
splicing of CD23 Arthritis Cancer Metastatic behavior is conferred
by alternatively-spliced CD44; mRNAs encode proto-oncogenes;
telomere length CNS RNA editing alters glutamate receptor-B,
changing calcium ion permeability Neurofibromatosis RNA editing
introduces stop codon at 5' end of NF1 type I GAP-related domain to
inactivate NF1 epigenetically Cardiovascular RNA editing influences
amount of ApoB-100, strongly associated with atherosclerosis
[0056] The following example is intended to further illustrate the
invention without limiting it thereof.
EXAMPLE 1
[0057] Use of Fluorescence Polarization to Identify RNA-Binding
Ligands
[0058] The following experiment was performed to demonstrate that
measurements of fluorescence polarization can be used to
discriminate different types of probe binding to target RNA. The
target is a 48-nucleotide RNA that forms a stem-loop structure as
shown in FIG. 4 (bottom), containing a large distal loop and a
smaller proximal loop, also known as an assymetric bulge (see FIG.
4). The probes, designated Oligo A and Oligo B, are 27-nucleotide
DNA molecules which hybridize to nucleotides 8-34 of the target
RNA. Oligo A and Oligo B differ only in the region complementary to
the distal loop; Oligo B has mismatches (Xs) in place of canonical
Watson-Crick base pairing at two sites. The probes were
fluorescently labeled at their 5' ends using BODIPY-Texas Red
(Molecular Probes, Eugene Oreg.), using the manufacturer's
recommended conditions.
[0059] Reaction mixtures were set up containing: target RNA at
concentrations ranging from 0-64 nM; and Oligo A or Oligo B at a
concentration of 1 nM, in a solution of 50 mM Tris-HCl, pH 7.5, 150
mM KCl. The fluorescence polarization of each reaction was
monitored at 25.degree. C. for the indicated times (FIG. 5) using a
Jolley Instruments FPM-1 fluorescence polarimeter (Chicago Ill.).
The ordinate data are expressed as millipolarization units
(mP).
[0060] The results (FIG. 5) indicate that Oligo A, which fully
hybridizes to the target RNA, attains a higher polarization value
than Oligo B, which does not fully hybridize (and in which the
fluorescent moiety is presumably more free to rotate in solution).
The reaction containing Oligo B is analogous to one in which a
ligand binds to the target RNA at the distal loop, which would
prevent the binding of a probe such as Oligo A.
[0061] Many variations of the present invention will suggest
themselves to those skilled in the art in light of the
above-detailed description. Such obvious variations are within the
full intended scope of the appended claims.
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