U.S. patent application number 10/286518 was filed with the patent office on 2003-06-19 for screen for compounds with affinity for nucleic acids.
Invention is credited to Arenas, Jaime E., Lillie, James W., Pakula, Andrew.
Application Number | 20030113779 10/286518 |
Document ID | / |
Family ID | 21705721 |
Filed Date | 2003-06-19 |
United States Patent
Application |
20030113779 |
Kind Code |
A1 |
Arenas, Jaime E. ; et
al. |
June 19, 2003 |
Screen for compounds with affinity for nucleic acids
Abstract
The present invention provides methods for high-throughput
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
measuring 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
conformation.
Inventors: |
Arenas, Jaime E.;
(Lexington, MA) ; Lillie, James W.; (Wellesley,
MA) ; Pakula, Andrew; (Lexington, MA) |
Correspondence
Address: |
DAVID R PRESTON & ASSOCIATES
12625 HIGH BLUFF DRIVE
SUITE 205
SAN DIEGO
CA
92130
US
|
Family ID: |
21705721 |
Appl. No.: |
10/286518 |
Filed: |
November 1, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10286518 |
Nov 1, 2002 |
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09962432 |
Sep 24, 2001 |
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6503721 |
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09962432 |
Sep 24, 2001 |
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08709342 |
Sep 6, 1996 |
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6337183 |
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60003406 |
Sep 8, 1995 |
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Current U.S.
Class: |
435/6.11 |
Current CPC
Class: |
C12Q 1/6818 20130101;
C12Q 1/6818 20130101; C12Q 1/6811 20130101; C12N 15/1048 20130101;
C12Q 2565/1015 20130101; C12Q 2525/301 20130101; C12Q 2525/301
20130101; C12Q 2565/1015 20130101; C12Q 1/701 20130101; C40B 40/06
20130101; C40B 50/06 20130101; C12Q 1/6811 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Claims
What is claimed is:
1. A high-throughput 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 in the
presence of each of said test ligands to produce a test
combination; (c) incubating the target RNA sequence in the absence
of a test ligand to produce a control combination; (d) measuring
the conformation of the target RNA sequence in each combination;
(e) selecting as a ligand any test ligand that causes a measurable
change in the target RNA conformation in the test combination
relative to the target RNA conformation in the control combination;
and (f) repeating steps (b)-(c) with a plurality of said test
ligands to identify a ligand that binds to the target RNA
sequence.
2. The method of claim 1, wherein the measurable chance in the
target RNA conformation comprises a change in the target RNA
conformation from less folded to more folded, from more folded to
less folded, or from a first folded
3. The method of claim 1, further comprising subjecting the test
and control combinations to conditions that denature a detectable
fraction of the target RNA sequence in the control combination.
4. The method of claim 3, wherein the subjecting comprises at least
one of altering the temperature, altering the salt concentration,
adding denaturing compounds, and combinations thereof.
5. The method of claim 1, wherein the target RNA is from about 5 to
about 500 nucleotides in length.
6. The method of claim 1, wherein the target RNA comprises a label
selected from the group consisting of a radionuclide, a fluorescent
compound, an affinity label, and combinations thereof.
7. The method of claim 1, wherein the measuring step comprises the
steps of: (i) contacting the test and control combinations with an
oligonucleotide under conditions in which the oligonucleotide
preferentially hybridizes to a predetermined conformation of the
target RNA sequence; and (ii) measuring the fraction of the target
RNA sequence present in hybrids with the oligonucleotide, wherein
the fraction measured in (ii) indicates the fraction of the target
RNA in said predetermined conformation.
8. The method of claim 7, wherein the oligonucleotide comprises a
label selected from the group consisting of a radionuclide, a
fluorescent compound, an affinity label, and combinations
thereof.
9. The method of claim 1, wherein the target RNA sequence further
comprises a first fluorescence probe and a second fluorescence
probe, wherein (i) the fluorescence emission wavelength maximum of
the first probe overlaps the fluorescence absorption wavelength
maximum of the second probe, and (ii) the first probe and the
second probe are positioned in the target RNA so that fluorescence
energy transfer between the first and second probes occurs only
when the target RNA is in a predetermined conformation.
Description
[0001] This is a continuation of application Ser. No. 08/709,342,
filed Sep. 6, 1996, which claims priority pursuant to 35 U.S.C.
.sctn.119 from Provisional application Serial No. 60/003,406, filed
Sep. 8, 1995. Each of these prior applications is hereby
incorporated herein by reference, in its entirety.
FIELD OF INVENTION
[0002] This invention pertains to novel methods for high-throughput
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
[0003] 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.
[0004] 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.
[0005] 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.
[0006] 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 binding of small
molecules (reviewed in Wyatt and Tinoco, 1993).
[0007] 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).
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).
[0008] Existing assays for ligands of nucleic acids, such as, for
example, methods that use equilibrium dialysis, differential
scanning calorimetry, viscometric analyses, or UV melting, have not
been used in high-throughput applications. 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.
[0009] 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,
[0010] 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.
[0011] 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
[0012] The present invention encompasses high-throughput 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 in the presence of
each of the test ligands to produce a test combination; incubating
the target RNA sequence in the absence of a test ligand to produce
a control combination; measuring the conformation of the target RNA
in each combination; selecting as a ligand any test ligand that
causes a measurable change in the target RNA conformation in the
test combination relative to the control combination; and repeating
the method with a plurality of said test ligands to identify a
ligand that binds to the target RNA sequence. Ligands identified by
the methods of the present invention may cause the target RNA to
change from a less folded to more folded conformation, from a more
folded to less folded conformation, or from a first folded
conformation to a second, alternative, folded conformation.
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.
[0013] In practicing the present invention, the effect of test
ligands on the folding state of the target RNA is determined using
well-known methods, including without limitation hybridization with
complementary oligonucleotides, treatment with
conformation-specific nucleases, binding to matrices specific for
single-stranded or double-stranded nucleic acids, and fluorescence
energy transfer between fluorescence probes. In one embodiment, the
target RNA is radiolabelled and incubated with a biotinylated
oligonucleotide that preferentially hybridizes to a particular
conformation of the target RNA; following capture of biotinylated
molecules using immobilized streptavidin, the extent of
hybridization can be readily quantified by measurement of
immobilized radiolabel. In another embodiment, the target RNA
contains two different fluorescence probes in which the
fluorescence emission wavelength maximum of the first probe
overlaps the fluorescence absorption maximum of the second probe.
The probes are positioned within the target RNA so that the
efficiency of fluorescence energy transfer between the probes is
dependent upon the target RNA conformation.
[0014] A "measurable change" in target RNA 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
[0015] FIG. 1 is a graphic illustration of the reaction between a
biotinylated complementary oligonucleotide and a labelled target
RNA.
[0016] FIG. 2 is a graphic illustration of the use of
streptavidin-coated plates in conjunction with a biotinylated
oligonucleotide complementary to the target RNA.
[0017] FIG. 3 is a graphic illustration of different conformations
of a model target RNA.
[0018] FIG. 4 is a graphic illustration of the use of
intramolecular fluorescence energy transfer to monitor RNA
structure.
[0019] FIG. 5 is a graphic illustration of the use of
intermolecular fluorescence energy transfer to monitor
hybridization.
[0020] FIG. 6 is a graphic illustration of the effect of nuclease
digestion on alternative target RNA conformations.
[0021] FIG. 7 is a graphic illustration of the use of RNAase H
digestion, in conjunction with biotinylated and labelled target RNA
to measure RNA:DNA hybrids.
[0022] FIG. 8 is a graphic illustration of the use of S1 nuclease,
in conjunction with a biotinylated and labelled oligonucleotide, to
measure RNA:DNA hybrids.
[0023] FIG. 9 is a graphic illustration of the structure of AB-RNA
(top) and of the reaction of AB-RNA with either ATP or with a
complementary oligonucleotide bottom).
[0024] FIG. 10 depicts an autoradiogram of a native polyacrylamide
gel in showing the effect of ATP and UTP on the hybridization of
radiolabelled AB-RNA to a complementary DNA oligonucleotide.
[0025] FIG. 11 is a graphic illustration of the effect of ATP and
UTP on the binding of AB-RNA:biotinylated DNA hybrids to
streptavidine-coated paramagnetic beads.
[0026] FIG. 12 is a graphic illustration of the effect of adding
increasing amounts of streptavidin (SA), streptavidin-alkaline
phosphatase (SA:AP), or streptavidin-beta galactosidase (SA:BG) on
the binding to nitrocellulose filters of hybrids between
radiolabelled RRE RNA and a biotinylated complementary DNA
oligonucleotide.
[0027] FIG. 13A is a graphic illustration of the effect of adding
increasing concentrations of oligonucleotide 91B on the binding to
nitrocellulose filters of hybrids between radiolabelled AB RNA and
biotinylated complementary 91B oligonucleotide.
[0028] FIG. 13B is a graphic illustration of the time course of the
formation of hybrids between radiolabelled AB RNA and biotinylated
complementary 91B oligonucleotide.
[0029] FIG. 13C is a graphic illustration of the lack of effect of
adding increasing concentrations of tRNA on the formation of
hybrids between radiolabelled AB RNA and biotinylated complementary
91B oligonucleotide.
[0030] FIG. 13D is a graphic illustration of the effect of adding
increasing concentrations of sodium chloride on the formation of
hybrids between radiolabelled AB RNA and biotinylated complementary
91B oligonucleotide.
[0031] FIG. 14A is a graphic illustration of the time course of
hybrid formation between radiolabelled HIV RRE RNA and biotinylated
oligonucleotides, using 100 nM target RNA and 250 nM
oligonucleotide.
[0032] FIG. 14B is a graphic illustration of the time course of
hybrid formation between radiolabelled HIV RRE RNA and biotinylated
oligonucleotides, using 25 nM target RNA and 50 nM
oligonucleotide.
[0033] FIG. 14C is a graphic illustration of the structure of HIV
RRE RNA target (full length shown at left) showing in bold-face
uppercase the sequences that are complementary to oligonucleotides
91B, 92A, 99A, 92B, and 92C.
[0034] FIG. 15 is a graphic illustration of the effect of
increasing concentrations of ATP or UTP on the formation of hybrids
between radiolabelled AB RNA and biotinylated complementary 67B
oligonucleotide. The data are shown as the percentage of inhibition
of hybrid formation when compared to reactions in the absence of
ligand.
[0035] FIG. 16 is a graphic illustration of the effect of
increasing concentrations of the indicated diphenylfuran
derivatives on the formation of hybrids between RRE RNA and a
biotinylated complementary oligonucleotide. The data are shown as
in FIG. 15.
[0036] FIG. 17 is a graphic illustration of the effect of
increasing concentrations of ATP on the formation of hybrids
between RRE RNA and a biotinylated complementary oligonucleotide
(closed squares); and the effect of increasing concentrations of
the diphenylfuran derivative ST36723 on the formation of hybrids
between radiolabelled AB RNA and biotinylated complementary 67B
oligonucleotide (open squares).
[0037] FIG. 18A is a graphic illustration of the effect of
increasing concentrations of DMSO on the formation of hybrids
between radiolabelled AB RNA and biotinylated complementary 67B
oligonucleotide.
[0038] FIG. 18B is a graphic illustration of the effect of
increasing concentrations of DMSO on the extent of inhibition by 5
mM ATP on the formation of hybrids between radiolabelled AB RNA and
biotinylated complementary 67B oligonucleotide.
[0039] FIG. 18C is a graphic illustration of the effect of
increasing concentrations of dithiothreitol on the formation of
hybrids between radiolabelled AB RNA and biotinylated complementary
67B oligonucleotide.
[0040] FIG. 18D is a graphic illustration of the effect of
increasing concentrations of ethanol and methanol on the formation
of hybrids between radiolabelled AB RNA and biotinylated
complementary 67B oligonucleotide.
[0041] FIG. 19 is a graphic illustration of the reproducibility of
the SCAN assay performed as in FIG. 18 above (5% DMSO).
[0042] FIG. 20 (top panel) is an illustration an ATP-binding target
RNA showing in bold-face uppercase the sequences complementary to
the biotinylated oligonucleotide used. The bottom panel is a
graphic illustration of the time course of hybrid formation (A) and
dissociation (D) under equilibrium conditions.
[0043] FIG. 21 is a graphic illustration of the effect of
temperature on the absorbance of AB RNA in the absence and presence
of ATP.
[0044] FIG. 22 is a graphic illustration of a simple stem-loop RNA
structure.
[0045] FIG. 23 is a graphic illustration of a complex RNA structure
containing fluorescein and rhodamine labels, which can fold into
mutually exclusive folding patterns.
[0046] FIG. 24 is a graphic illustration of a simple stem-loop RNA
structure containing fluorescein and rhodamine labels.
[0047] FIG. 25 is a graphic illustration of the hybridization
reaction between a complex RNA structure labelled with both
fluorescein and rhodamine and a complementary DNA
oligonucleotide.
[0048] FIG. 26 is a graphic illustration of the hybridization
reaction between a complex labelled RNA structure and a
biotinylated oligonucleotide.
[0049] FIG. 27 is a graphic illustration of the hybridization
reaction between a complex biotinylated RNA structure and
individual oligonucleotides complementary to different regions of
the RNA.
[0050] FIG. 28A is a graphic illustration of the time course of
change in fluorescence of fluorescein-labelled AB RNA during
incubation with an unlabelled complementary oligonucleotide
(67B).
[0051] FIG. 28B is a graphic illustration of the effect of ATP,
GTP, and UTP on the fluorescence increase shown in FIG. 28A.
DETAILED DESCRIPTION OF THE INVENTION
[0052] 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.
[0053] Definitions
[0054] 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 anywhere 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.
[0055] 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. Small organic molecules have a molecular
weight of more than 50 yet less than about 2,500 daltons, and most
preferably less than about 400 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 that binds the target
RNA can be purified from the mixture in a subsequent step.
[0056] 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.
[0057] 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 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, and the
like.
[0058] 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.
[0059] As used herein, "test combination" refers to the combination
of a test ligand and a target RNA. "Control combination" refers to
the target RNA in the absence of a test ligand.
[0060] As used herein, the "folded state" of a target RNA refers to
a native or alternative 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
particular higher-order structures. Without wishing to be bound by
theory, it is believed that certain target RNAs may achieve one of
several alternative folded states depending upon experimental
conditions (including buffer, temperature, presence of ligands, and
the like).
[0061] 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.
[0062] As used herein, a "measurable change" in RNA conformation
refers to a quantity that is empirically determined and that will
vary depending upon the method used to monitor RNA 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.
[0063] The present invention encompasses high-throughput screening
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 condition. 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.
[0064] 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. Experimental conditions are chosen so that the
target RNA is subjected to unfolding or rearrangement. If the test
ligand binds to the target RNA under these conditions, the relative
amount of folded:unfolded target RNA, the relative amounts of 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 in the
presence and absence of a test ligand. This is followed by analysis
of the absolute or relative amounts of folded vs. unfolded target
RNA, the relative amounts of specific folded conformations, or of
the rate of folding or unfolding of the target RNA.
[0065] 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.
[0066] 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. Experimental conditions are
determined empirically for each target RNA. When testing multiple
test ligands, incubation conditions are chosen so that most
ligand:target RNA interactions would be expected to proceed to
completion. 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, or, alternatively, can be
bound to a solid phase matrix.
[0067] The time necessary for binding of target RNA to ligand will
vary depending on the test ligand, target RNA and other conditions
used. In some cases, 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 is
maintained for a longer time e.g. up to 12-16 hours, before binding
is detected. 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.
[0068] Other experimental conditions that are optimized for each
RNA target include pH, reaction temperature, salt concentration and
composition, divalent cation concentration 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.
[0069] Binding of a test ligand to the target RNA is assessed by
comparing 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 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 folded,
and less unfolded, target RNA (and, thus, a higher ratio 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 folded, and more unfolded, target RNA than is
present in the absence of a test ligand. Another possibility is
that binding of the test ligand changes the pattern or 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.
[0070] In either case, determination of the absolute amounts of
folded and unfolded target RNA, the folded:unfolded ratio, or the
rates of folding or unfolding, may be carried out using any method,
including without limitation hybridization with complementary
oligonucleotides, treatment with conformation-specific nucleases,
binding to matrices specific for single-stranded or double-stranded
nucleic acids, and fluorescence energy transfer between adjacent
fluorescence probes. Other physico-chemical techniques may also be
used, either alone or in conjunction with the above methods; these
include without limitation measurements of circular dichroism,
ultraviolet and fluorescence spectroscopy, and calorimetry.
However, it will be recognized by those skilled in the art that
each target RNA may have unique properties that make a particular
detection method most suitable in a particular application.
[0071] 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 be manageable in practical terms. Second, the
number of positive compounds should reflect ligands with an
appreciable affinity 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.
[0072] Methods for Detection of RNA Folding
[0073] The present invention may be practiced using any of a large
number of detection methods well-known in the art. For example, an
oligonucleotide (whether DNA or RNA) 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. In some embodiments, hybridization
of an oligonucleotide to a target RNA is allowed to proceed in the
absence and presence of test ligands (i.e., in control and test
combinations, respectively), after which the extent of
hybridization is measured using any of the methods well-known in
the art. Typically, an increase or decrease in hybridization that
is greater than that expected due to random statistical variation
in the test vs. control combination indicates that the test ligand
binds the target RNA. Other useful methods to measure the extent of
folding of the target RNA include without limitation intramolecular
fluorescence energy transfer, digestion with conformation-specific
nucleases, binding to materials specific for either single-stranded
or double-stranded nucleic acids (such as, nitrocellulose or
hydroxylapatite), measurement of biophysical properties indicative
of RNA folded structure (such as UV, Raman, or CD spectrum,
intrinsic fluorescence, sedimentation rate, or viscosity),
measurement of the stability of a folded RNA structure to-heat
and/or formamide denaturation (using methods such as, spectroscopy
or nuclease susceptibility), and measurement of protein binding to
adjacent reporter RNA. Examples of these methods are disclosed in
the following articles: Kan et al., Eur. J. Biochem. 168:635, 1987
(NMR); Edy et al., Eur. J. Biochem. 61:563, 1976, Yeh et al., J.
Biol. Chem. 263:18213, 1988, Clever et al., J. Virol. 69: 2101,
1995, and Vigne et al., J. Mol. Evol. 10:77, 1977 (RNAases);
Millar, Biochim. Biophy. Acta 174:32, 1969 (thermal melting,
fluorescence polarization); and Zimmerman Biochem. Z. 344:386, 1966
and Dupont et al., C. R. Acad. Sci. Hebd. Seances Acad. Sci. D.
266:2234, 1968 (viscosity).
[0074] 1. Gel Shift
[0075] A gel shift protocol, though nominally a low-throughput
method, is an important tool for the initial selection of the assay
configuration for oligonucleotide-based methods to be used with
each particular target RNA. Typically, the target RNA is
transcribed in vitro, using as template a DNA that contains the
sequence for the T7-RNA polymerase promoter followed by a region
encoding the target RNA, and [.sup.32P]-UTP to produce
radiolabelled RNA. Conditions for the T7 RNA polymerase
transcription reaction using oligonucleotide templates have been
described by Milligan et al., Nuc. Acids Res., 15: 8783, 1987. The
[.sup.32P]-labelled target RNA is optionally heat denatured at
90.degree. C. for 2 min and chilled on ice for 2 min, after which
aliquots are incubated in the absence and presence of test ligands
and increasing concentrations of a complementary oligonucleotide.
The reaction mixtures are then resolved in polyacrylamide native
gels containing or lacking the test ligands.
[0076] If a test ligand binds the target RNA, it will inhibit the
formation of hybrids between the target and the complementary
oligonucleotide. An example of this method is described in Example
1 below.
[0077] 2. High Throughput Assays Using Streptavidin-Biotin
[0078] In these embodiments, a biotin moiety is introduced into the
complementary oligonucleotide or into the target RNA. This allows
the use of capture methods that are based on the strong interaction
between biotin and avidin, streptavidin (SA) or other derivatives
of biotin-binding proteins (Wilchek et al., Meth. Enzymol.,
184:5-45, 1990). FIG. 1 illustrates the use of a biotinylated
oligonucleotide and a labeled RNA target. Only those labelled RNA
target molecules that are hybridized to the biotinylated
oligonucleotide can become associated with the biotin binding
protein.
[0079] The biotin moiety can be located at any position in the
oligonucleotide, or there can be multiple biotin moieties per
oligonucleotide molecule. The SA (or its derivatives or analogues)
can be covalently attached to a solid support, or it can be added
free in solution. The target RNA can be radiolabelled, for example,
or labelled with a fluorophore such as fluorescein or rhodamine or
any other label that can be readily measured.
[0080] SA coated plates: FIG. 2 illustrates the use of 96-well
plates coated with SA, which are commercially available (Pierce,
Rockford, Ill.). The reactions are first set up in a regular
uncoated 96-well plate by mixing the labeled RNA target with the
biotinylated oligonucleotide and the ligands to be tested. After an
incubation period, the reaction mixture is transferred into a SA
coated plate to allow binding of the biotin moiety to SA. Target
RNA:oligonucleotide hybrids and free-biotinylated oligonucleotide
will bind to the wall of the plate through the SA-biotin
interaction. Unbound material, including target RNA that is not
associated with the oligonucleotide, is washed away with an excess
of buffer. The target RNA remaining in the plate after the wash is
quantified by an appropriate method, depending upon the nature of
the label in the target RNA.
[0081] SA coated beads: In this embodiment, the SA is covalently
attached to small beads of an inert material, such as, for example,
sepharose (Pharmacia, Uppsala, Sweden), agarose (Sigma Chemical
Co., St. Louis, Mo.), or Affigel (BioRad, Hercules, Calif.). A
fixed amount of beads is added to each well containing the reaction
mixture to allow binding of the hybrids. The beads are then washed
to remove unbound material before quantitation.
[0082] SA coated paramagnetic beads: The washing step required when
using SA-beads can be facilitated by using paramagnetic-SA coated
beads (PMP-SA). These beads are commercially available (Promega,
Madison, Wis.) and can be concentrated and held by a magnet.
Positioning the magnet under the plate during washing steps
concentrates and retains the beads at the bottom of the well during
the washing procedure thus preventing loss of beads and allowing
faster operation. An example of the use of this method is described
in Example 2 below.
[0083] SA coated SPA beads and scintillant containing plates:
Scintillation proximity assay (SPA) is a technology available from
Amersham Corp. (Arlington Heights, Ill.) that can be used for
measurement of hybridization. SA coated SPA beads contain a solid
phase scintillant that can be excited by low energy isotopes in
close proximity. In this embodiment, the target RNA is labelled
with .sup.3H (whose weak radiation energy is virtually undetectable
at distances of more than one micron unless the signal is amplified
by a scintillant). When using SPA beads, only those radiolabelled
molecules that bind to the SA-SPA-beads will be close enough to
cause the scintillant to emit a detectable signal, while those
molecules in solution will not contribute to the signal. Therefore,
by using a biotinylated oligonucleotide and a .sup.3H labelled RNA
target, it is possible to determine the amount of hybrids formed in
the reaction by adding SA-SPA beads to the reaction and then
counting in a LSC-counter after a brief binding incubation period.
Plates coated with SA (which contain a scintillant attached to the
surface of the wells) are also commercially available
(Scintiplates.RTM., Packard, Meriden, Conn.) can be used for this
assay in place of SA-SPA beads.
[0084] Adsorption to nitrocellulose filters: This embodiment takes
advantage of the fact that most proteins tightly adsorb to
nitrocellulose filters. When using a biotinylated oligonucleotide
and a labelled target RNA, free SA or a SA derivative such as
SA:alkaline phosphatase conjugate (SA:AP), SA:.beta.-galactosidase
(SA:BG), or other SA-conjugate or fusion protein, is added to the
reaction to allow binding to the biotin moiety in the
oligonucleotide. Subsequently, the reaction is filtered through
96-well nitrocellulose filter plates (Millipore, Bedford, Mass.,
HATF or NC). Labeled target RNA hybridized to the biotinylated
oligonucleotide is retained in the filter through the adsorption of
SA or its derivative to the filter, while unhybridized RNA passes
through. SA has been found to bind poorly to nitrocellulose
filters; however, the present inventors have discovered that the
use of SA conjugates or fusion proteins increases the adsorption of
the protein to nitrocellulose. Therefore, the use of SA fusion
proteins or conjugates is preferred. An example of the use of this
method is described in Example 3 below.
[0085] 3. High-Throughput Assays Using Covalently Attached
Proteins
[0086] Polypeptides and proteins can be covalently attached to the
5'-end of nucleic acids that have been treated with a carbodiimide
to form an activated 5'-phosphorimidazolide derivative that will
readily react with amines including those in polypeptides and
proteins (Chu et al., Nuc. Acids Res., 11:6513, 1983). Using this
approach, any peptide or protein of choice can be covalently
attached to the 5'-end of the RNA target or the complementary
oligonucleotide used in the present invention. Non-limiting
examples of embodiments that use this technique are described
below.
[0087] Adsorption to nitrocellulose filters: With a peptide or
protein covalently attached to the oligonucleotide and a labeled
RNA target, or, conversely, with a peptide or protein covalently
attached to the RNA target and a labeled oligonucleotide,
hybridization can be quantified in essentially the same way as
described above for the SA based capture of biotin containing
hybrids in nitrocellulose filters. In this case, however, binding
to the filter is via the peptide or protein adduct in the RNA
target, or in the oligonucleotide.
[0088] Affinity binding to solid supports: All the techniques
described above for the use of SA-biotin can be duplicated by using
any of a number of other affinity pairs in place of SA and biotin.
The adduct "Y" attached to the oligonucleotide (or target RNA) is
capable of high affinity binding with a specific molecule "X" which
is attached to a solid support. Activated resins (beads) and
96-well plastic plates for attachment of macromolecules or their
derivatives are commercially available (Dynatech, Chantilly, Va.).
X and Y can be a number of combinations including antigen-antibody,
protein-protein, protein-substrate, and protein-nucleic acid pairs.
Some of these pairs are shown in the following table:
1 X Y Antigen/epitope Specific antibody Protein A Immunoglobulin
Glutathione Glutathione-S-transferase Maltose Maltose binding
protein RNA or DNA motif Specific motif binding protein
[0089] In most cases either component of the pair can be attached
to the RNA target (or oligonucleotide) while the other is attached
to the solid support. However, if a specific RNA or DNA binding
protein is used, it is preferable to attach the protein to the
solid support while the specific sequence motif that the protein
binds can be incorporated during synthesis at any convenient
position in the sequence of the RNA target or oligonucleotide.
Finally, attachment of the protein to solid support can be omitted
if adsorption to nitrocellulose filters is used instead (as
described above).
[0090] 4. High-Throughput Assays Using Fluorescence Energy Transfer
(FET)
[0091] Fluorophores such as fluorescein, rhodamine and coumarin
have distinctive excitation and emission spectra. Fluorescence
energy transfer occurs between pairs of fluorophores in which the
emission spectrum of one (donor) overlaps the excitation spectrum
of the other (acceptor). For appropriately chosen pairs of
fluorescent molecules, emission by the donor probe is reduced by
the presence of an acceptor probe in close proximity because of
direct energy transfer from the donor to acceptor. Thus, upon
excitation at a wavelength absorbed by the donor probe, a reduction
in donor emission and increase in acceptor emission relative to the
probes alone is observed if the probes are close in space. In
other-words, the donor's emission fluorescence is quenched by the
acceptor, which in turn emits a higher wavelength fluorescence.
FET, however, is effective only when donor and acceptor are in
close proximity. The efficiency of energy transfer is inversely
proportional to the sixth power of the distance between the donor
and acceptor probes, thus the extent of these effects can be used
to calculate the distance separating the probes. FET has been used
to probe the structure of transfer RNA molecules (Beardsley et al.,
Proc. Natl. Acad. Sci. USA 65:39, 1970), as well as for detection
of hybridization, for restriction enzyme assays, for DNA-unwinding
assays, and for other applications).
[0092] A. Intramolecular FET
[0093] FET is used in the present invention to monitor a change in
target RNA conformation, when the distance between the donor and
acceptor probes differs significantly between the different
conformations. In one embodiment an RNA molecule is used in which
an internal hybridization probe sequence has been engineered as in,
for example, FIG. 3. The solid region represents target RNA
sequences and the hatched region represents an internal probe
sequence that is complementary to a large portion of the target
sequence. In conformation 1, the probe and target sequences
hybridize, bringing the acceptor (A) and donor (D) fluorescent
probes into close proximity. In the presence of a ligand that binds
to a structured conformation of the target sequences, conformation
2 is stabilized; as a consequence, the probes are further apart. A
predominance of conformation 2 is reflected in a relative increase
in donor fluorescence and/or a decrease in acceptor
fluorescence.
[0094] B. Use of Oligonucleotide Hybridization and FET
[0095] Acceptor and donor in the same strand: In this embodiment,
the target RNA is designed so that the 5'- and 3'-ends of the RNA
stay in close-proximity when folded, allowing FET. FIG. 4 shows the
model in which the formation of hybrids with another DNA or RNA
oligonucleotide will result in a decrease in the FET efficiency due
to the larger distance between donor and acceptor.
[0096] Acceptor and donor in separate strands: This approach is
useful when the design of the RNA target does not allow the
incorporation of both donor and acceptor fluorophores in the same
strand. In this case, the donor and acceptor are in separate
strands and come in close proximity in the target:oligonucleotide
hybrid (FIG. 5). In this embodiment, the formation of the hybrid
results in an increase of FET.
[0097] 5. High-Throughput Assays Using Conformation-Specific
Nucleases
[0098] In practicing the present invention the ligand-induced
stabilization of a folded conformation of a target RNA by binding
decreases the fraction of the target RNA present in an unfolded
conformation. Conversely, in the absence of ligand, a greater
fraction of the RNA is found in the unfolded state than in the
presence of such a compound. Folded conformations of RNA are
characterized by double-stranded regions in which base pairing
between RNA strands occurs. A variety of nucleolytic enzymes, such
as S1 and mung bean nucleases, preferentially digest phosphodiester
bonds in single-stranded RNA relative to double stranded RNA. Such
enzymes can be used to probe the conformation of RNA target
molecules in the current invention.
[0099] In a typical assay, target RNA and test compound(s) are
preincubated to allow binding to occur. Next, an appropriate
nuclease is added, and the mixture is incubated under appropriate
conditions of temperature, nuclease concentration, ionic strength
and denaturant concentration to ensure that (in the absence of
ligand) about 75% of the RNA is digested according to the
specificity of the nuclease used, within a short incubation period
(typically 30 minutes). The extent of digestion is then measured
using any method well-known in the art for distinguishing between
free ribonucleoside monophosphates and oligonucleotides, including,
without limitation, acid precipitation and detection of labelled
RNA, FET of RNA containing donor and acceptor fluorescence probes,
and electrophoretic separation and detection of RNA by
autoradiography, fluorescence, UV absorbance, hybridization with
labeled nucleic acid probe or dye binding.
[0100] Changes in conformation between two or more alternative
folded RNA conformations can also be detected using nuclease
digestion. In this embodiment, each of the conformations typically
contains some regions of double stranded RNA. If the alternate
conformations involve differing amounts of double-stranded regions,
they can be distinguished by measuring the amount of
nuclease-resistant material. If the overall double-stranded content
of these structures is comparable, it is necessary to distinguish
between the nuclease-resistant fragments yielded by nuclease
digestion of different target RNA conformations. FIG. 6 illustrates
the effect of single-strand specific nuclease digestion of two
alternate target RNA conformations that yields stable products
differing in size and sequence content. For example, although
regions B and B' are found among the nuclease resistant fragments
of both conformations 1 and 2, region A is not found after
digestion of conformation 2. Specific RNA fragments may be detected
and quantified by any method well-known in the art, including,
without limitation, labelling of target RNA, hybridization with
target-specific probes, amplification using target-specific primers
and reverse-transcriptase-cou- pled PCR, and size determination of
digestion products (if digestion products of a specific RNA
conformation have characteristic sizes that distinguish them from
the digestion products of other conformations).
[0101] Nucleases that are specific for different nucleic acid
structures may also be used to quantify hybridization of
complementary oligonucleotides.
[0102] RNAse H: RNAse H is a commercially available nuclease that
specifically degrades the RNA strand of RNA:DNA hybrids. A 5'-end
or 3'-end biotinylated RNA target is also labeled at the other end
with a radionuclide or a fluorophore such as fluorescein, rhodamine
or coumarin. RNAase H digestion of the RNA:DNA hybrids formed
during the reaction results in physical separation of the biotin
moiety (on one end) from the fluorophore or radionuclide (on the
other end) (FIG. 7). RNA target strands not involved in hybrid
formation will not be digested by RNAse H and can be quantified
after streptavidin binding as described above. In this embodiment,
the signal obtained will increase if the test ligand binds the
target RNA.
[0103] Nuclease S1: Single stranded nucleic acids can be
specifically digested with the commercially available nuclease S1
(Promega, Madison, Wis.). This enzyme can be used in the present
invention if the DNA oligonucleotide carries the biotin moiety as
well as the label at an internal position (FIG. 8). Labelled
strands forming hybrids resist digestion by S1 nuclease and are
quantified by SA-mediated capture as described above. The label can
also be in the section of RNA that participates in hybrid
formation. Alternatively, the same approach can be carried out with
single strand specific RNases such as RNAse T1 or RNAase ONE.TM.
(Promega), in which case the label must be located in the RNA
target.
[0104] 6. Conformation Specific Binding
[0105] A variety of materials bind with greater affinity to one or
another type of RNA structure. A prime example of this phenomenon
is hydroxyapatite, which has greater affinity for double-stranded
than for single-stranded nucleic acids. Nitrocellulose, by
contrast, has higher affinity for single-stranded than for
double-stranded RNA. These and other similar materials can be used
to distinguish between different conformations of RNA, particularly
where ligand binding stabilizes one conformation that differs
significantly from other conformations in its single-stranded
content. These methods are generally useful when ligand binding
stabilizes folded RNA conformations relative to the unfolded
state.
[0106] Antibodies that recognize RNA may also be used in a
high-throughput mode to identify ligands according to the present
invention. Useful antibodies may recognize specific RNA sequences
(and/or conformations of such sequences) (Deutscher et al., Proc.
Natl. Acad. Sci. USA 85:3299, 1988), may bind to double-stranded or
single-stranded RNA in a sequence-independent manner (Schonborn et
al., Nuc. Acids Res. 19:2993, 1991), or may bind DNA:RNA hybrids
specifically (Stumph et al., Biochem. 17:5791, 1978). In these
embodiments, binding of antibodies to the target RNA is measured in
the presence and absence of test ligands.
[0107] 7. Biophysical Measurements
[0108] A variety of biophysical measurements can be used to examine
the folded and unfolded conformation(s) of RNA molecules and detect
the relative amounts of such conformations, including, without
limitation, UV absorbance, CD spectrum, intrinsic fluorescence,
fluorescence of extrinsic covalent or noncovalent probes,
sedimentation rate, and viscosity. Each of these properties may
change with changing RNA conformation. In these embodiments,
measurements are-performed on mixtures of target RNA and
appropriate buffer, salt and denaturants in the presence and
absence of test ligand(s). A change in a measurable property,
particularly one that suggests conversion of unfolded to folded
forms of the RNA, is indicative of ligand binding.
[0109] 8. Changes in Conformational Stability
[0110] Any of the structural measurements described above can be
used to examine the stabilization of a conformation by ligand
binding. The stability of such a conformation is defined as the
free energy difference between that conformation and alternative
(typically unfolded) conformations. Conformational stability can be
measured under constant conditions, with and without test
ligand(s), or over a range of conditions. For example, the effect
of increasing temperature on-structure, as measured by one of the
methods above, can be measured,in the presence and absence
(control) of test ligand(s). An increase in the temperature at
which structure is lost is indicative of ligand binding.
[0111] 9. Disruption of Protein Binding to Adjacent RNA
[0112] A variety of proteins are known that bind to specific RNA
sequences in a manner that is dependent on the three-dimensional
structure of the RNA. In these embodiments, protein binding is used
as a probe of RNA structure and its alteration upon ligand binding.
A target RNA sequence and an RNA sequence to which a protein binds
are incorporated within the same RNA molecule. The interaction of a
binding protein with its binding sequence is measured in the
presence and absence of test ligands. Ligand-induced changes in the
RNA conformation that alter the conformation of the protein binding
site are detected by measurement of protein binding.
[0113] Applications
[0114] 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. 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
incubating radiolabelled ligand with unlabelled target, and then
separating RNA-bound and unbound ligand. Furthermore, the ligand
can be tested for its ability to influence, either positively or
negatively, a known biological activity of the target RNA.
[0115] Non-Limiting examples of RNA targets to which the present
invention can be applied are shown in the following table:
[0116] Therapeutic
2 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 Rheumatoid Alternative splicing of CD23 Arthritis
Cancer Metastatic behavior is conferred by alternatively-spliced
CD44; mRNAs encode proto-oncogenes CNS RNA editing alters glutamate
receptor-B, changing calcium ion permeability Neurofibromatosis RNA
editing introduces stop codon at 5' end of NFl type I GAP-related
domain to inactivate NFl epigenetically Cardiovascular RNA editing
influences amount of ApoB-100, strongly associated with
atherosclerosis
[0117] The following examples are intended to further illustrate
the invention without limiting it thereof.
EXAMPLE 1
Detection of RNA Folding Using a Gel Shift Assay
[0118] A. Rationale
[0119] The RNA molecule shown in FIG. 9 (top), designated AB-RNA,
was shown to bind ATP with high affinity (Sassanfor et al., Nature,
364:550, 1993). In the experiments described below, this RNA was
used to illustrate different embodiments of the present
invention.
[0120] As depicted in FIG. 9 (bottom), in practicing the present
invention, incubation of this RNA (AB-RNA) with ATP and a
competitor oligonucleotide allows the formation of an ATP:AB-RNA
complex as well as an oligo:AB-RNA hybrid. In the absence of ATP,
formation of the hybrid is favored. By contrast, in the presence of
ATP, the formation of the hybrid is less favored due to the
formation of a stable ATP:RNA complex. Thus, measurement of the
amount of oligonucleotide:RNA hybrids in the reaction mixture
indicates the presence or absence of an RNA-binding ligand.
[0121] B. Methods
[0122] AB-RNA was transcribed using as template the DNA
oligonucleotide RBS-87-8A, (5'-GGAAC CTTGC CGGCA CCGAA GTGCC GCAGT
TTCTT CCAA GGTTC CTATA GTGAG TCGTA TTA-3'), which contains the
sequence for the T7-RNA polymerase promoter followed by a region
encoding AB-RNA. [.sup.32P]-labelled AB-RNA was obtained by
including [.sup.32P]-UTP in the transcription reaction. Conditions
for the T7 RNA polymerase transcription reaction using
oligonucleotide templates have been described by Milligan et al.,
Nuc. Acids Res., 15:8783, 1987.
[0123] [.sup.32P]-labelled AB-RNA (approx. 30,000 cpm) in a 360
.mu.l mixture containing 37.5 mM Tris pH 7.5, 150 mM NaCl and 7.5
mM MgCl.sub.2 was heat denatured at 90.degree. C. for 2 min and
then chilled on ice for 2 min. The mixture was split into three 120
.mu.l aliquots which were supplemented with 20 .mu.l of water, 300
.mu.M ATP, or 300 .mu.M UTP. Nine 12 .mu.l aliquots of each of
these mixtures were then supplemented with varying amounts of the
complementary RBS-87-8A oligonucleotide and water so that the final
volume of each was 20 .mu.l. The resulting reaction mixtures
contained final oligonucleotide concentrations ranging from 0 to
1.97 .mu.M and either one of 30 .mu.M ATP, 30 .mu.M UTP, or no
ligand. The reaction mixtures were then supplemented with 3 .mu.l
of loading dye and loaded onto a 12% polyacrylamide native gel. The
gel and running buffers contained 1.times.TBE buffer in addition to
37.5 mM Tris pH 7.5, 150 mM NaCl,7.5 mM MgCl.sub.2 and the
corresponding ligand concentration.
[0124] C. Results
[0125] Resolution of hybridized and unhybridized RNA by gel
electrophoresis indicated that the conversion of 50% of the AB-RNA
into the RNA:DNA hybrid requires approximately 24 nM RBS-87-8A
oligonucleotide in the absence of ligand, and conversion of 100%
requires at least 72.9 nM oligonucleotide (FIG. 10, top panel). In
contrast, in presence of 30 .mu.M ATP, 656 nM to 1970 nM RBS-87-8A
oligonucleotide concentrations were required to induce the
formation of a similar amount of hybrids (FIG. 10, middle panel).
This effect is the result of competition between ATP and the
oligonucleotide for binding to the AB-RNA. Furthermore, this
experiment shows that UTP is unable to compete with the
oligonucleotide, demonstrating that the observed effect is
ligand-specific (FIG. 10, bottom panel).
EXAMPLE 2
High-Throughput Detection of RNA Ligands Using Streptavidin-Coated
Paramagnetic Beads
[0126] A. Methods
[0127] The [.sup.32P]-labelled ATP-binding RNA described in Example
1 above (AB-RNA) was heat denatured at 90.degree. C. for 2 min in
binding buffer (50 mM Tris pH 7.5, 200 mM NaCl, and 10 mM
MgCl.sub.2) and chilled on ice for 2 min. 37.5 .mu.l aliquots of
the heat denatured AB-RNA were then mixed with 12.5 .mu.l of a
solution containing 12.5 pmol of the 5'-biotinylated
oligonucleotide RBS-96-60 (having a nucleotide sequence identical
to RBS-87-8A described in Example 1 above) and varying amounts of
ATP or UTP. After a brief incubation at room temperature, the
mixture was transferred into 96-well plates containing 0.5 mg
paramagnetic beads coated with streptavidin (PMP-SA) (Promega,
Madison, Wis.). Plates were incubated at room temperature for 15
min to allow binding. A strong magnet was placed under the plate,
after which the PMP-SA beads were washed with 50 .mu.l of binding
buffer. [.sup.32P]-labelled AB-RNA bound to the beads was then
quantitated by Cerenkov counting in a Microbetta LSC-counter
(Wallac).
[0128] B. Results
[0129] FIG. 11 shows the amounts of [.sup.32P]-AB-RNA bound to the
PMP-SA beads in the presence of increasing concentrations of either
ATP or UTP, representing the hybrids formed between the
biotinylated oligonucleotide (RBS-96-60) and the target RNA
(AB-RNA). The results show that the amount of hybrids formed can be
reduced by 50% by adding as little as 25 .mu.M ATP. As expected, no
effect was seen when UTP was used as the ligand.
EXAMPLE 3
High-Throughput Detection of RNA Ligands Using Nitrocellulose
Filter Binding
[0130] A. Rationale:
[0131] This embodiment of the present invention takes advantage of
the fact that most proteins adsorb tightly to nitrocellulose
filters. When a biotinylated oligonucleotide and a labelled target
RNA are used (as described in Example 2 above), free streptavidin
(SA) or a SA derivative is added to the reaction to allow binding
to the biotin moiety in the oligonucleotide, after which the
reaction is filtered through 96-well nitrocellulose filter plates.
In this way, labelled target RNA hybridized to the biotinylated
oligonucleotide is retained in the filter, while non-hybridized RNA
is lost.
[0132] B. Method:
[0133] The target RNA was an RNA molecule containing the binding
site for the HIV Rev protein (Rev Responsive Element, RRE; 5'-GAAUA
CUAUG GGCGC AGCGU CAAUG ACGCU GACGG UACAG GCCAG ACAAU UAUUG UCUGG
UAUAG U-3') which had been labelled with .sup.32P as described in
Example 1 above. The biotinylated oligonucleotide, designated
RBS-79-91b-B, is complementary to positions 5 thru 28 of the RRE
target RNA and has the sequence 5'-biotin-CGTCA TTGAC GCTGC GCCCA
TAGTG C-3'. 0.1 pmol of [.sup.32P]-RRE RNA were incubated with 5
pmol of RBS-79-91b-B in 40 .mu.l of buffer containing 50 mM Tris pH
7.5 and 50 mM NaCl. After 15 min at room temperature, varying
amounts of streptavidin alone (SA), streptavidin conjugated to
.beta.-galactosidase (SA:BG), or streptavidin conjugated to
alkaline phosphatase (SA:AP) were added to the mixture and allowed
to bind for 20 min before filtration through Millipore HATF
nitrocellulose filter plates. The filtrate was further washed with
400 .mu.l of the same buffer and the amount of RRE retained in the
filter was determined by liquid scintillation counting.
[0134] C. Results
[0135] FIG. 12 shows that SA-conjugated proteins increase the
efficiency of retention of labelled RNA:oligonucleotide hybrids. If
an RRE-specific ligand is included in the reaction, the relative
amount of labelled RRE RNA bound to the filters should
decrease.
EXAMPLE 4
Characterization of Nitrocellulose Filter Binding Assay
[0136] The following experiments were performed to determine the
effect of various experimental parameters on the high-throughput
assay described in Example 3 above.
[0137] It was first determined that 2 .mu.g of SAAP, with a free
biotin binding activity of 20 pMol/.mu.g of protein, is sufficient
to ensure quantitative retention of hybrids on NC-filters.
[0138] It was then determined that, typically, a two-fold molar
excess of oligonucleotide over the RNA target is sufficient to
achieve substantial hybrid formation. A titration of the
biotinylated oligonucleotide 67B in a 30 min SCAN reaction
containing 100 nM radiolabeled AB-RNA, 50 mM Tris pH 7.5, 200 mM
NaCl, and 10 mM MgCl.sub.2 is shown in FIG. 13A. Addition of the
oligonucleotide at concentrations above 500 nM resulted in a
gradual loss of retention, suggesting that the capacity of the SAAP
is saturated. The ability to use minimal amounts of
oligonucleotides decreases the amount of SAAP required for
quantitative NC-filter retention and therefore reduces the cost of
the assay.
[0139] FIG. 13B shows a time course for reactions which contain 250
nM oligonucleotide. The results indicate that this amount of
oligonucleotide (2.5-fold molar excess over RNA target) is
sufficient to mediate the quantitatively retention of hybrids on
the filter, even when the SCAN reaction goes to completion.
[0140] FIG. 13C shows 10-minute SCAN reactions in which increasing
concentrations of yeast tRNA have been added as a non-specific
competitor. The results show that even a 100-fold molar excess of
yeast tRNA did not affect the retention of hybrids on
nitrocellulose filters. The addition of a large excess of a
non-specific RNA competitor to SCAN assays is expected to reduce
the incidence of unwanted positive screening results caused by
ligands that interact non-specifically with nucleic acids.
[0141] FIG. 13D illustrates the effect of NaCl (in absence of
MgCl.sub.2) under the same conditions. As expected, the formation
of hybrids is stimulated by the addition of NaCl. Interestingly, in
presence of 10 mM MgCl.sub.2, no NaCl was required to obtain
efficient retention. Thus, hybridization rates show dependence on
ionic strength. Additional experiments indicated that the ionic
strength dependence varied according to the RNA-target and
oligonucleotide being used.
[0142] Similar results were obtained in SCAN reactions using other
RNA targets, including HIV RRE and TAR elements. By varying
parameters such as ionic strength, oligonucleotide concentration
and design, and target-RNA concentration, it was possible to
modulate the hybridization kinetics in order to achieve a suitable
extent of hybrid formation in a time appropriate for
high-throughput screening, i.e., at room temperature, 30-50%
retention in reactions containing 10-100 nM RNA target and 2-4-fold
molar excess of biotinylated oligonucleotide over the target RNA in
reaction times of 0.5 to 2 hours.
EXAMPLE 5
Parameters for the Design of Oligonucleotides for Use in Scan
Reactions
[0143] The experiments described below were performed to identify
optimal oligonucleotide structures for use in high-throughput SCAN
reactions. The RNA target was a radiolabeled fragment of the HIV
Rev response element (RRE) containing the high affinity binding
site for Rev protein. It was hypothesized that the region including
positions 9-12 of the RNA target (see FIG. 14C) may have a higher
probability to unfold and may therefore provide a preferred site of
entry (nucleation site) for oligonucleotide binding. Therefore,
various oligonucleotides with full or partial complementarity to
this region were tested in the reaction.
[0144] In order to avoid differences due to,accessibility of the
biotin moiety in the hybrids, all oligonucleotides used in this
series had identical 5'-biotinylated ends while their 3'-ends
varied in length. The full length of each oligonucleotide was
complementary to the corresponding region of the RNA target (shown
in uppercase in FIG. 14C). The full length of the RNA-target used
in all reactions is shown to the far left in FIG. 6C while the
regions of RNA complementary to oligonucleotides 92A, 99A, 92B and
92C are shown on partial structures.
[0145] Time course reactions containing 100 nM radiolabeled
RNA-target and 250 nM oligonucleotide (FIG. 14A), or 25 nM
radiolabeled RNA-target and 50 nM oligonucleotide (FIG. 14B)
evidenced dramatic differences in hybrid formation. Comparison of
the reactions containing oligonucleotides 91B, 99A and 92A
indicated that, contrary to expectation, a longer oligonucleotide
will not necessarily show faster kinetics. Instead, the results
indicate that the positioning of the 3' terminus of the
oligonucleotide, and the number of complementary bases within the
region 9-12, both play a dominant role in determining hybrid
formation rates. Most dramatic is the difference between
oligonucleotides 92A, 99A, and 92B, whose lengths differ by one
base at their 3' termini.
[0146] These observations are consistent with region 9-12 acting as
a nucleation site, and suggest that the kinetics of hybrid
formation are largely dependent on the number of possible base
pairs that could form between the oligonucleotide and the
nucleation site. The results further suggest that nucleation
initiating with the 3' terminus of the oligonucleotide is preferred
over an internal oligonucleotide site (compare 91B and 92A).
Similar results supporting this interpretation have also been
obtained in SCAN reactions using other RNA-targets including the
HIV TAR RNA.
[0147] Knowledge of the rate-limiting factors in SCAN reactions is
important in guiding the design of oligonucleotides. The results
described above suggest that a major rate-limiting factor is the
initial interaction of the oligonucleotides with nucleation sites
on the target RNA, including those within hairpin loops, bulges,
internal loops and other regions. This property of the SCAN assay
can be used to advantage, since loops and bulges are the preferred
recognition sites of most RNA-binding proteins and may also be
involved in tertiary RNA-RNA interactions such as pseudoknots. Such
RNA-protein and RNA-RNA interactions are critical for the
regulatory function of most RNA structures. Therefore, by careful
choice of oligonucleotides, the SCAN assay can be made highly
sensitive to ligands that interact with these regions. Furthermore,
in larger multi-domain RNA structures, different oligonucleotides
can be used to target different sub-domains of the RNA.
EXAMPLE 6
Identification of Specific RNA-Binding Ligands
[0148] SCAN reactions containing 100 nM radiolabeled AB-RNA and 250
nM biotinylated oligonucleotide 67B were incubated with increasing
amounts of ATP or UTP as test ligands.
[0149] In the absence of test ligand, 70% of AB-RNA was retained on
NC-filters. Inhibition of RNA retention on NC-filters (%
inhibition) was obtained as (1-(R/R.sub.0).times.100), where
R.sub.0 is retention without test ligand and R is retention with
test ligand. While 5 mM ATP inhibited retention by 50%, 500 mM UTP
was needed to achieve the same effect showing a 100-fold
specificity factor (FIG. 15).
[0150] Similarly, addition of RRE ligands ST36723, ST46172, and
ST51378 inhibited RRE retention on NC-filters by 50% at 2.5-15
.mu.M (FIG. 16). These ligands have been shown in separate
experiments to interact specifically with RRE and to inhibit the
Rev-RRE interaction at low micromolar concentrations.
[0151] The differential response of the AB RNA-containing SCAN
assay to ATP and other NTPs reflects the specificity of the assay.
In order to confirm this selectivity, known RNA ligands were tested
in SCAN reactions using heterologous RNA targets. As shown in FIG.
17, ATP had no effect on RRE based SCAN reactions (closed squares).
Conversely, the RRE ligand ST36723 did not affect the AB-RNA SCAN
system (open squares).
EXAMPLE 7
Optimization of High-Throughput Screening Method
[0152] The effect of different solvents and reagents on the high
throughput screening reactions described above was tested. DMSO is
typically used as a solvent for distribution of compounds into
assay plates. In practicing the present invention, DMSO is
typically present at a concentration of 5% (v/v). Methanol and
ethanol are also used as solvents for some compound collections
including natural products. Dithiothreitol may be added to provide
a reducing environment to prevent unwanted reactivity of some
compounds.
[0153] Reactions containing 50 mM Tris pH 7.5, 200 mM NaCl, 10 mM
MgCl.sub.2, 100 nM AB-RNA, 1 .mu.M yeast tRNA and 250 nM
oligonucleotide 67B were performed in the presence of varying
amounts of the reagents. Addition of increasing amounts of DMSO to
the reactions resulted in a reduction in hybrid formation (FIG.
18A, closed circles), however the reaction remained sensitive to
the addition of a specific ligand (FIG. 18A, open circles).
Furthermore, the extent of inhibition obtained at various DMSO
concentrations with 5 .mu.M ATP as shown in FIG. 18A was not
significantly affected by DMSO (FIG. 18B).
[0154] FIGS. 18C and 18D show that the SCAN reactions are not
affected by various concentrations of DTT, methanol, or ethanol.
Similar results were obtained using RRE as the RNA target.
[0155] These results show that the SCAN reactions have a good
tolerance to solvents and reagents usually used in high throughput
screening procedures.
[0156] The SCAN assay is fully compatible with a high throughput
format involving automatic equipment and room temperature
conditions. Delivery of reagents and filtration is performed with
96-well format automatic pipettors (Quadra-96 from Wallac Oy,
Turku, Finland) equipped with a multiscreen vacuum manifold
(Millipore, Bedford, Mass.), and scintillation counting is
performed directly on the 96-well nitrocellulose filter plates
(Millipore, Bedford, Mass.) in a 96-well format Micro-beta
scintillation counter (Wallac Oy, Turku, Finland). Reaction
conditions and other parameters have been optimized in order to
keep reaction times within 0.5 and 2 hours. This procedure design
allows a single scientist to screen up to 8,000 compounds per
week.
[0157] FIG. 19 shows sample data obtained from 475 RRE SCAN
reactions containing 5% DMSO in the absence of ligand. The assay is
highly reproducible, with an average inhibition between of 0+/-8%
in the absence of ligands. With a standard screening compound
concentration of 20 .mu.M, a "hit" is expected to inhibit hybrid
formation by 50% and will be clearly distinguishable above the
background "noise" level.
EXAMPLE 8
Scan Assay Under Equilibrium Conditions
[0158] The following experiment was performed to demonstrate a
screening assay in which the olignucleotide and ligand reach an
equilbrium with the target RNA, i.e., the population of RNA:ligand
and RNA:oligonucleotide complexes depends upon their relative
dissociation constants. A screening assay having this configuration
has the advantage of removing time constraints for various steps in
the assay. In addition, thermodynamic parameters associated with
ligand binding can be more easily derived from the results of the
assay.
[0159] Using a series of oligonucleotides of various length and
target sites comprising AB RNA, a group of oligonucleotides has
been identified that fully equilibrate with ATP (an example is
shown in FIG. 20, top panel). The oligonucleotide used is
complementary to the sequence shown in bold-faced letters (FIG. 20,
top panel).
[0160] Target RNA:oligonucleotide complexes were pre-formed by
incubated a solution containing 100 nM AB RNA and 500 nM
oligonucleotide for 2 h at 25.degree. C. Dissociation was initiated
by diluting the reaction 5-fold in the presence or absence of 100
.mu.M ATP (resulting in final concentrations of AB RNA, 20 nM;
oligonucleotide, 100 nM; ATP, 100 .mu.M). Binding of hybrids to
nitrocellulose filters was performed as described above.
[0161] The results (presented as % oligo:RNA complex) indicate that
approximately 60% inhibition by ATP is observed, comparable to what
is found using a kinetic assay (i.e., under non-equilibrium
conditions).
EXAMPLE 9
Ligand Binding Induces Conformational Changes in RNA
[0162] The experiment described below was performed to monitor the
stabilization of an RNA structure upon ligand binding. In this
experiment, absorbance at 260 nM was monitored as the temperature
of the sample was increased. The increase in temperature causes the
RNA to unfold, thereby allowing individual bases to absorb more
ultraviolet light.
[0163] The results are graphically illustrated as the first
derivative of the absorbance with respect to change in temperature
(FIG. 21). The data indicate a new and obvious transition centered
at 40.degree. C. which occurs only in the presence of ATP. These
results indicate that the ATP ligand stabilizes the RNA
structure.
EXAMPLE 10
High-Throughput Detection RNA Ligands Using Fluorescence Energy
Transfer
[0164] Fluorescence energy transfer (FET) is based on the existence
of pairs of fluorescence donor and acceptor molecules in which
fluorescence emission by the donor probe is transferred directly to
an acceptor probe in close proximity to the donor probe. Thus, upon
excitation of the donor probe, a reduction in donor emission and
increase in acceptor emission relative to the probes alone is
observed if the probes are sufficiently close. The efficiency of
energy transfer is inversely proportional to the sixth power of the
distance between the donor and acceptor probes, thus the extent of
these effects can be used to calculate the distance separating the
probes. Intramolecular FET has been used to probe the structure of
transfer RNA molecules (Beardsley et al., Proc. Natl. Acad. Sci.
USA, 65:39, 1970). In the present invention, FET is used to monitor
a change in target RNA conformation. The only requirement is that
the distance between the donor and acceptor probes must differ
significantly between the different conformations.
[0165] A. Several embodiments of the present invention in which FET
is used are described below.
[0166] 1) FIG. 22 shows a relatively simple ABC stem-loop RNA
structure. A synthetic RNA target is prepared containing
complementary sequences B'A' and C'B' (FIG. 23). This synthetic
substrate is also double-labelled with Fluorescein (F) and
Rhodamine (R) at the 5' and 3'-ends, respectively. This RNA may
achieve two possible structures (FIG. 23, left and right). One of
these structures positions the fluorescent dyes in close proximity
favoring FET.
[0167] The RNA is designed so that the distance between F and R
(FIG. 23, right) is minimal. This ensures maximum FET difference
between the two structures and obviates the need for a
complementary DNA oligonucleotide to maintain an appropriate F-R
distance in the structure in FIG. 23, left.
[0168] 2) FIG. 24 shows a similar ABC stem-loop structure in its
folded (left) and unfolded (right) configurations. The lack of an
alternative structure requires that the stem-and-loop structure
must be intrinsically unstable, so that a difference in FET can be
detected when the stem-loop is stabilized by a binding of a
ligand.
[0169] 3) FIG. 25 shows a target RNA molecule containing multiple
discrete target structures (X, Y and Z). In this case, the use of a
relatively long DNA oligonucleotide will increase the FET
difference between the folded RNA and the hybrid.
[0170] Alternatively, a "capture measure" method may be used, as
illustrated in FIG. 26. In this embodiment, the target RNA is
labelled with a radioisotope or a fluorofor, and the DNA
oligonucleotide is biotinylated. The amount of hybrid is measured
by capture with streptavidin-coated beads in conjunction with SPA,
or any one of the SA-based methods described above.
[0171] To avoid potential problems caused by the formation of
partial hybrids, multiple discrete DNA strands directed to each of
the target sites could be used. In this embodiment, the RNA carries
the biotin tag, while each DNA strand is labeled with a different
label such as Fluorescein (F), texas red (R) and P.sup.32 (P) (FIG.
27). In this way, multiple targets within the same RNA molecule
could be tested simultaneously by measuring each one with a
different method.
[0172] B. Experimental results: While developing a SCAN assay based
on FET between 3'-fluorescein labeled AB-RNA (AB-RNA-F) and a
5'-rhodamine labeled oligonucleotide, it was unexpectedly observed
in a control experiment that a substantial increase in fluorescein
emission at 535 nm occurred upon addition of an oligonucleotide
carrying no fluorescent labels (FIG. 28A). Reactions containing 50
mM Tris pH 7.5, 200 mM NaCl, 10 mM MgCl.sub.2, and 60 nM AB-RNA-F
were monitored at 535 nm emission (485 nm excitation) in a
CytoFluor II fluorescence multi-well plate reader (PerSeptive
Biosystems, Framingham, Mass.) for increasing times after the
addition of oligonucleotide 67B.
[0173] The results suggested that the fluorescein emission was
quenched as a result of the association of the fluorescein moiety
with the structure of AB-RNA. In order to estimate the maximum
achievable change in fluorescence emission at 535 nm resulting from
the association of the fluorescein moiety and the RNA, the emission
at 535 nm was measured during the course of RNAse A digestion of
AB-RNA-F; it was found that it increased to a maximum of 2-fold
over the initial value.
[0174] Because of the high reproducibility and sensitivity of
fluorescence spectrophotometers, a 2-fold increase in fluorescence
represents more that sufficient change in signal to use it as a
sensitive assay. Therefore, the effect of ATP, GTP and UTP was
tested in SCAN reactions containing AB-RNA-F and the unlabeled
oligonucleotide 67B (FIG. 28B).
[0175] The results indicate that the addition of ATP specifically
inhibited the fluorescence increase observed in the absence of
added ligand. The IC50 value (5 mM) for ATP in this assay was
identical to the value obtained with the SAAP based SCAN assay. As
expected, UTP had some effect at much higher concentrations while
GTP had no effect at all. Accordingly, these data demonstrate the
suitability of this approach for measuring hybrid formation in SCAN
reactions.
Sequence CWU 1
1
3 1 64 DNA Artificial Sequence oligonucleotide 1 ggaaccttgc
ccggcaccga agtgccgcag tttcttccca aggttcctat agtgagtcgt 60 atta 64 2
66 DNA Artificial Sequence oligonucleotide 2 gaauacuaug ggcgcagcgu
caaugacgcu gacgguacag gccagacaau uauugucugg 60 uauagu 66 3 25 DNA
Artificial Sequence oligonucleotide 3 cgtcattgac gctgcgccca tagtg
25
* * * * *