Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR MONITORING REACTIONS IN REAL TIME
FIELD OF INVENTION
[0001 ] The present invention relates to signaling
aptamers for enzyme activity monitoring and inhibitor
screening.
BACKGROUND OF THE INVENTION
[0002] Aptamers are nucleic acids with ligand-binding
capabilities that are isolated from random-sequence nucleic
acid pools. Aptamers may comprise RNA, DNA or nucleotide
derivatives. Several reports have described fluorescence-
based signaling aptamers and signaling ribozymes and
deoxyribozymes for the detection of small molecules and
proteins in solution. There remained however a need for
dynamic reporters that can be used to monitor a chemical
reaction in real time as well as reporters that can be used to
screen for inhibitors of a chemical reaction.
[0003] Enzymes are abundant in nature. They catalyze
a large array of chemical transformations and act on a broad
scope of substrates. In addition, many artificial enzymes
including catalytic antibodies, ribozymes and DNAzymes have
been generated. This broadens the scope of the use of
enzymatic reactions in assays and creates new opportunities
for novel applications. The important biological roles of
enzymes and their increasing utility in a variety of applications
place a significant need for convenient analytical methods to
both characterize specific enzyme properties and to detect
enzymatic activities. Conventional methods for analyzing
enzymatic reactions often involve the use of sophisticated
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instrumentation such as high performance liquid
chromatography (HPLC). Although those methods are
accurate, the analytical processes are often time-consuming
and difficult to adapt to unconventional applications such as
high throughput robotic screening. Therefore, methods that are
capable of monitoring enzyme activities conveniently,
particularly in real time, are highly desirable.
SUMMARY OF THE INVENTION
[0004] The present invention exploits the use of signaling
aptamers as real-time probes to report enzyme activities.
Several reports have described the use of signaling aptamers
for the detection of small molecules and proteins in solution.
An aptamer binds to a target and may have different affinities
for related targets. The present invention discloses, for the
first time, nucleic acid aptamers that can be used to monitor
enzymatic reactions in real time.
[0005] In one aspect, the present invention provides a
method for monitoring a chemical reaction. The method
comprises providing a signaling aptamer having differential
affinities for a substrate or substrate target and a product
target. Intermediate targets may also be detected. The term
"substrate" is used herein to refer to any substance that is
acted on in a chemical reaction. The term "product target"
refers to a product of a chemical reaction. The signaling
aptamer is modified with a reporter molecule that provides a
signal when the aptamer binds to a target. .
[0006] In one aspect, the method of monitoring a
chemical reaction comprises interacting a substance with a
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signaling aptamer capable of binding to the substance and
monitoring for a change in the amplitude of the signal, wherein
a change in amplitude is indicative of transformation of the
substance and disruption of aptamer binding to the substance.
[0007] In another aspect, the method comprises
incubating substance A in the presence of a signaling aptamer
that has a first affinity for substance A and a second, different
affinity for product B, determining the amplitude of the signal
based on the affinity of the aptamer for substance A, providing
conditions favourable for the conversion of substance A to
product B, and monitoring for a change in amplitude of the
signal.
[0008] In a preferred embodiment, the signaling aptamer
comprises a fluorophore and a quencher in close proximity.
Upon binding to a targefi the quencher is separated from the
fluorophore and a fluorescent signal is generated. The
signaling aptamer generally expresses different affinities for
difFerent targets (i.e. substrate, intermediate, product). Thus,
the signal generated will be either increased or decreased as
the reaction proceeds.
[0009] In a particularly preferred embodiment, the
signaling aptamer is a signaling aptamer complex (SAC). The
SAC comprises a target binding oligonucleotide duplexed with
a fluorophore modified oligonucleotide and a quencher
modified oligonucleotide.
[0010] In another aspect, the method comprises
incubating a signaling aptamer in the presence of a substrate,
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determining a baseline fluorescence, adding a test sample and
monitoring for a change in fluorescence. A 'change in
fluorescence is generated as the substrate is converted to
product.
[0011] In one embodiment, the aptamer recognizes the
product target with a greater affinity and thus an increase in
fluorescence is seen as the reaction progresses.
[0012] In another embodiment, the aptamer has a
greater affinity for the substrate and a decrease in
fluorescence is seen.
[0013] In another preferred embodiment, the chemical
reaction is an enzymatic reaction. In a further preferred
embodiment, the method is useful to detect the activity of a
phosphatase, deaminase, adenyl cyclase or
phosphodiesfierase.
[0014] In yet another preferred embodiment, the
chemical reaction is a phosphorylation reaction.
[0015] In another aspect, a method for the detection of
an enzyme is provided. The method comprises incubating a
substrate with a signaling aptamer having an affinity for the
substrate, measuring the baseline fluorescence signal, adding
an enzyme test sample and monitoring for a change in
fluorescent signal, wherein a change in fluorescent signal is
indicative of enzyme activity.
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[0016] In a preferred embodiment an assay for the
detection of phosphatase is provided. A signaling ATP
aptamer is incubated in the presence of AMP, the baseline
fluorescence is recorded, a test sample is added and the
change in fluorescence is measured. An increase in
fluorescence is indicative of the presence of a phosphatase.
[0017] In a preferred embodiment, alkaline phosphatase
activity is monitored using a signaling aptamer that displays a
sequential, differential fluorescent signal amplitude as ATP is
converted to ADP, ADP is converted to AMP and AMP is
converted to adenosine by alkaline phosphates (ALP). A
method of screening for ALP inhibitors comprises comparing
the sequential change in fluorescent intensity in the presence
of a potential inhibitor as compared to the control reaction.
[001 ~] In a further aspect of the invention, a method of
screening for an enzyme inhibitor is provided. The method
comprises obfiaining a signaling aptamer that exhibits a
differenfiial affinity and therefore a different fluorescent signal
in the presence of the enzyme substrate versus the product of
the enzyme reaction and establishing a threshold change in
the amplitude of the fluorescence signal that is indicative of a
conversion from the substrate to the product. The method
further comprises introducing a potential inhibitor into the
reaction, determining a change in the amplitude of the
fluorescent signal and comparing the amplitude change'to the
change in amplitude in the absence of the inhibitor. The
efficacy or strength of the inhibition can be determined by
comparing the fluorescent signal generated in the presence of
the inhibitor to a standard curve.
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[0019] It is clearly apparent that in all the methods of the
present invention, the order in which the components are
added can be varied.
[0020] In another aspect of the invention, enzyme
inhibitors identified by the method are provided. The use of the
identified inhibitors in diagnostics and therapeutics is also
encompassed.
[0021 ] The present invention also provides for the
identification and use of enzyme inhibitors.
[0022] New uses for known compounds, such as
aptamers and small molecules, are provided using the
methods of the present invention.
[0023] A method and assay system for the detection and
quantitation of an enzyme are also provided. The method
comprises establishing a standard curve of fluorescent values
generated by a signaling aptamer in response to
predetermined amounts of an enzyme, reacting a test sample
with the signaling aptamer, determining the fluorescent signal
generated and comparing that signal to the standard curve to
determine the amount of enzyme activity in the test sample.
[0024] In another aspect of the invention, an assay
system is provided. The assay system comprises a signaling
aptamer which has a target binding domain flanked by a
fluorophore and a quencher. The aptamer binding domain has
differential afFinities for a substrate and a product. Control
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reagents are also provided. The assay system may be
provided in the form of a kit.
[0025] In a preferred embodiment, a reaction monitoring
kit is provided. The kit comprises a substrate and a signaling
aptamer capable of binding to the substrate.
[0026] In another embodiment, a kit for detecting enzyme
activity is provided. This kit comprises a signaling aptamer, an
aptamer target and a control enzyme. The kit can also be
used to screen for enzyme inhibitors.
SRIEF DESCRIPTION OF THE DRAWINGS
[0027] These and other features of the invention will
become more apparent from the following description in which
reference is made to the appended drawings wherein:
[0023] Figure 1A illustrates the structure of an exemplary
signaling aptamer complex;
[0029] Figure 1 S demonstrates the ability of the aptamer
complex of Figure 1A to distinguish between adenosine, AMP,
ADP, and ATP;
[0030] Figure 1 C illustrates the fluorescent signaling
profile of the signaling aptamer in the presence of various
amounts of adenosine, AMP and AMP treated with calf ALP;
[0031 ] Figure 2 demonstrates the fluorescence intensity
differential of the aptamer, 0F, vs target (AMP or adenosine)
concentration.
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[0032] Figure 3 demonstrates the enzymatic conversion
of AMP to adenosine.
[0033] Figure 4A illustrates the signaling profile of the
aptamer in the presence of variable amounts of calf intestine
alkaline phosphatase;
[0034] Figure 4B demonstrates the signaling rate
constant vs. concentration of alkaline phosphatase;
[0035] Figure 5A illustrates the effect of various small
molecules on alkaline phosphatase activity;
[0036] Figure 5B demonstrates the results of inhibition
assays performed in a 96-well assay format;
[0037] Figure 6 illustrates the effect of an adenosine
deaminase inhibitor on enzyme activity as measured by
aptamer binding affinity.
DETAILED DESCRIPTION
[0038] The present invention relates to a method of
monitoring chemical reactions using nucleic acid aptamers
which have different affinities for various substrates and
products. Aptamers are oligonucleotides that have been
selected for specific binding to a variety of -molecular targets
including proteins and small molecules.
[0039] Aptamers that , have differential affinities for
substrates and products are used to monitor enzymatic
reactions in real time. The aptamers can be selected using a
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variety of approaches. For example, an aptamer can be
selected for specific binding to a particular target using known
in vitro selection techniques such as those described in the
following references: Tuerk, C. and Gold, L. (1990) Science
249, 505-510; Ellington, A. D. and Szostak, J. W. (1990)
Nature 346, 818-822; Famulok, M. (1999) Curr. Opin. Struct.
Biol. 9, 324-329. Each of these documents is hereby
incorporated by reference.
[0040] An aptamer with .specificity for a particular target
can also be selected using the aptamer selection technique
described in PCT/CA2004/000482 that is incorporated by
reference herein.
[0041] The aptamers which are useful in the present
invention are signaling aptamers that have been engineered to
incorporate reporter molecules, such as chromophores,
fluorophores, isotopes and metals. Standard techniques can
be used to label the aptamers.
[0042] The signaling aptamer may be a molecular
beacon as described in Tyagi and Kramer, 1996, incorporated
herein by reference. A molecular beacon is an oligonucleotide
modified with a fluorophore at one end and a quencher at the
other end. In the absence of the target, the molecular beacon
adopts a closed state stem-loop configuration in which the
fluorophore and quencher are in close proximity and the
fluorescence is quenched. In the presence of target, the
beacon adopts an open state where the fluorophore and
quencher are separated and a fluorescent signal is generated.
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[0043] Alternatively, the signaling aptamer is preferably a
signaling aptamer complex as described in PCT application
CA03/00086, incorporated herein by reference. In one
example, a quencher modified oligonucleotide which has a
sequence complementary to part of the target-binding
sequence hybridizes to the aptamer sequence and another
oligonucleotide which is fluorophore-modified binds to a non-
target-binding sequence of the aptamer. In the absence of
target the quencher-modified oligonucleotide and the
fluorophore-modified oligonucleotide are in close proximity and
the fluorescent signal is quenched. In the presence of target,
they become separated as the quencher modified
oligonucleotide is displaced and a fluorescent signal is
generated. It is clearly apparent that the signaling aptamer
complex could have the fluorophore oligonucleotide hybridized
to the target binding sequence and vice versa. An example of
this type of signaling aptamer complex is illustrated in Figure 1
and discussed further in Example 1.
[0044] The present invention is based on the discovery
that some aptamers have differential affinities for specific
substrate targets and product targets. During the course of a
chemical reaction, a substrate target is converted to a product
target. The term product is used broadly herein to refer to any
transformed substrate resulting from a reaction. Since the
aptamer has a different affinity for the substrate target than for
the product target, the conversion from a substrate to a
product by can be measured by a change in the intensity of
the fluorescent signal as binding of the aptamer to the
substrate is disrupted.
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[0045] A signaling aptamer can be used to report a
simple chemical reaction, A-~B, in real time, if the signaling
aptamer exhibits different levels of fluorescence upon binding
to A and B. For example, if the signaling aptamer has a
greater affinity for B, there will be a higher fluorescence
readout for B than for A. The transformation of A to B can be
conveniently monitored by following the fluorescence intensity
increase upon binding of the signaling aptamer. On the other
hand, if the aptamer has a greater affinity for A, there will be a
decrease in the fluorescent signal as A is transformed to B. If
an enzyme mediates the chemical reaction, a fluorescent
aptamer reporter according to the present invention permits
real-time monitoring of the enzyme's activity. The signaling
aptamer complex can also be used in assays to screen for
inhibitors of the enzyme activity.
[0046] According to the invention, a chemical reaction in
which a substrate is converted to a product is monitored by
first obtaining a signaling aptamer that has a different affinity
for the substrate and the product. It should be understood that
a product of one reaction can be a substrate for a subsequent
reaction. For example, there can be a series of reactions in
which substance A is converted to substance B, then
substance B is converted to substance C, followed by
conversion of substance C to substance D. The signaling
aptamer distinguishes between the various substances based
on differences in affinity and thus the transition from one
substance to another is followed by a change in the amplitude
of the signal. Typically, a baseline signal for the signaling
aptamer is determined. When the substrate target is added,
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there is an increase in fluorescence as the signaling aptamer
binds to the target and the quencher and fluorophore are
separated. If a signaling aptamer complex is used, the
quencher modified oligonucleotide is displaced from the
duplex it had formed with the aptamer binding sequence. A
fluorescent signal is generated and when an enzyme is
introduced, the substrate is converted to a product. Since the
aptamer has a different affinity for the product, there will be a
change in fluorescence as the constant rate of aptamer
complex bound will be either increased or decreased. In this
manner, the conversion of substrate to product can be
monitored in real time. Signaling aptamer complexes are
particularly useful for monitoring chemical reactions due to
their ability to induce several fold increase in fluorescence
upon target binding. Thus, they can provide a sensitive
readout. The method of the invention can be used to follow
many different types of chemical reactions. For example,
addition, transfer or removal of a functional group can be
detected. Some examples of enzymatic reactions that can be
monitored include those mediated by a phosphatase, a
deaminase, an adenyl cyclase and a phosphodiesterase. It is
clearly apparent that any chemical reaction where a signaling
aptamer has a different affinity for the substrate and the
product can be monitored using the methods of the present
invention.
[0047] The invention also provides a method of detecting
enzyme activity. The presence, in a test sample, of an enzyme
capable of converting a substrate to a product can be
determined by obtaining a baseline signal for the signaling
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aptamer or signaling aptamer complex, adding the substrate
and determining the amplitude of the fluorescent signal. The
test sample is then added and the fluorescent signal is
monitored. A change in fluorescent signal amplitude is
indicative of the presence of an enzyme converting the
substrate to a product. The change may be an increase or a
decrease in amplitude depending on the relative affinity for the
substrate and the product.
[0048] Enzymatic activity can be quantified by comparing
the amplitude of a change in fluorescence .with a standard
curve. The standard curve is obtained by incubation of a
known amount of substrate with predetermined amounts of the
specific enzyme to be detected. It is apparent that the
signaling aptamer complexes can also be used as sensitive
reporters to distinguish between closely related compounds.
[0049] The present invention also provides a method of
screening 'for enzyme inhibitors. An enzyme assay can be set
up as described above. The optimal substrate and enzyme
concentrations to get a peak change in fluorescence amplitude
are determined. A test compound is added and if the
compound inhibits the enzyme, the characteristic change in
fluorescence is not seen. The relative efficacy and required
dose of the inhibitor can be determined by evaluating the
degree of inhibition. The method can be used to screen for.
inhibitors of various enzymes.
(0050] The invention can be demonstrated using an ATP
aptamer signaling complex. The structure-switching ATP
reporter shown in Figure 1 A is able to generate different
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fluorescence signals when adenosine and its 5'-
phosphorylated species are used as targets. For example, at
750 ~,M of adenosine, AMP, ADP (adenosine 5'-diphosphate),
and ATP (adenosine 5'-triphosphate), the signaling aptamer
produced a fluorescence intensity increase of 8.1, 5.3, 8.3,
and 6.9-fold, respectively, over the background reading as
shown in Figure 1 B. Each target was added after the signaling
aptamer mixture was incubated for 10 min, indicated by the
first dashed line. These observations demonstrate that this
signaling aptamer is well suited as a reporter for nucleotide-
dephosphorylating enzymes such as alkaline phosphatase
(abbreviated as ALP), which is known to remove the 5'-
phosphate groups from ATP, ADP, AMP and ultimately
convert each of them into adenosine.
[0051 ] This efficiency of the reporter system was
demonstrated by adding ALP to each target-aptamer mixture
minutes after the target addition as shown by the second
dashed line in Figure 1 B. In the adenosine solution, the
addition of ALP did not result in any intensity change as would
be expected since no chemical reaction was expected to
occur. The addition of ALP did generate a rapid fluorescence
intensity increase in the AMP solution, as the AMP was
converted to adenosine for which the aptamer had a higher
affinity. ALP also promoted an intensity change in the ADP
solution. The intensity initially decreased, then slowly
recovered to the original level. Since ADP contains two
phosphate groups (a and ~3 phosphates), two
dephosphorylation reactions should occur. The initial intensity
drop was consistent with the removal of the [i-phosphate
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(generation of AMP from ADP, coupled with fluorescence
intensity decrease); the eventual intensity recovery is due to
the accumulation of AMP and the subsequent removal of the
a-phosphate (generation of adenosine from AMP, coupled
with fluorescence enhancement). When added to the ATP
solution, ALP induced a slow fluorescence increase. This is
consistent with the signaling behaviours associated with three
dephosphorylation reactions: the removal of the y-phosphate
from ATP (generation of ADP, accompanied by fluorescence
increase), the removal of the [i-phosphate (generation of AMP,
accompanied by fluorescence decrease), and the removal of
the a-phosphate (generation of adenosine, accompanied by
fluorescence increase). It appeared that the fluorescence gain
(resulting from the first and the third reactions) always
surpassed the fluorescence loss (associated with the second
reaction) during the entire course of incubation. These
observations indicate that the ATP reporter can be utilized as
a unique real-time probe to report the reaction catalyzed by
ALP.
[0052] The AMP-aptamer system was selected to
demonstrate that a signaling aptamer can be used to quantify
enzyme activities according to the method of the present
invention. Initially, titration curves for adenosine (circles), AMP
(squares) and AMP treated with ALP (triangles) (Figure 1 C)
were established. As expected, AMP treated with ALP gave
the same fluorescence vs. concentration curve as adenosine.
[0053] To establish the AMP concentration that
generates the largest signal for AMP-to-adenosine transition,
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the fluorescence intensity differential (0F, defined as Fadenosine
FAMP) was determined as a function of the target
concentration. A bell-shaped curve was observed with ~F
peaking at a target concentrations around 500-1000 ~,M as
shown in Figure 2.
[0054] The conversion of adenosine 5'-monophosphate
(AMP) into adenosine by alkaline phosphatase (ALP) was
used as a model reaction to demonstrate enzyme activity.
This specific reaction is shown in greater detail in Figure 3. A
structure-switching signaling DNA aptamer was used as the
fluorescent reporter. The signaling aptamer, exhibiting a higher
affinity for adenosine than for AMP, generates a unipue two-
leg signaling profile. The first leg of the signal profile is
generated upon addition of AMP (indicative of the formation of
the substrate-aptamer complex) and the second leg is
generated upon addition of ALP (reporting the enzymatic
conversion of the substrate to the product). In other words,
ALP activity can be detected in real time by monitoring
changes in fluorescence. In the absence of ALP activity, there
will be no change in fluorescence. If a compound inhibits ALP
activity, fihen there will be no change in fluorescence.
[0055] The effect of enzyme concentration on the second
leg signaling responses was determined in the presence of
0.75 mM AMP and variable amounts of calf intestine ALP. In
total, seven different amounts of ALP were tested. The ALP
samples were added to a 500~,L aptamer-AMP complex at the
25th minute as shown in Figure 4A. The ALP amounts ranged
from 10 to 10-5 units with progressive 10-fold dilution steps.
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From these tests, the signaling rate constant (reflecting the
speed of fluorescence intensity increase in the initial linear
signaling range following the ALP addition as a way to
measure the enzyme activity) was determined as a function of
effective ALP concentration. The results are shown in Figure
4B. A linear relationship was observed when the enzyme
concentration was varied over 6 orders of magnitude. These
data clearly demonstrate indicate that the reporter can be
utilized as a sensitive probe to quantify effective ALP
concentrations and that the methods of the present invention
are broadly useful to quantitate enzyme activities. While ALP
is used as an example it is clearly apparent that other
enzymes can be measured in the same manner.
[0056] As discussed above, the present invention also
provides a method and means for screening for inhibitors of a
chemical reaction. As a model system, the ATP structure
switching aptamer complex and the AMP - adenosine reaction
were used to demonstrate the utility of the method of the
invention and the utility of structure-switching signaling
aptamers as unique reporting probes for screening small
molecules as enzyme inhibitors.
[0057] Levamisole and its racemic mixture tetramisole
are known inhibitors of porcine ALP (as well as other
mammalian ALPs but not calf intestine ALP) with dC; in the low
mM range. This suggested that if an appropriate amount of
levamisole or tetramisole was added into the anti-ATP
signaling aptamer mixture containing AMP, the activity of
porcine ALP should be attenuated and this would lead to a
slower rate of fluorescence intensity increase in the signaling
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aptamer solution. To test this, 0.1 mM levamisole or
tetramisole was added to the signaling aptamer mixture
containing 0.75 mM AMP. The rate of transformation of AMP
to adenosine promoted by porcine ALP was considerably
reduced relative to the uninhibited reaction as shown in Figure
5A. As controls, nine other randomly chosen non-inhibiting
chemical compounds were also tested at the same
concentration. The chemical structures of the small molecules
used for screening are shown below.
H3C
~~,,.. O
~~N (HCI ~~N HCI~ ~ il N' /NH
1 2 °°
/ z Hcl
N
HOOC~CHg
O Y YIH
NH CH3
pjHa ~ HCI
NHZ CHI
CHI
N~ 7 N N HaCO HCI
O
HCI H,C-S-OH
O H3C0
8
6 ~H
HaC
F
,,,NH~CHy O=S=O
HyCO ' / HCI
O
H3C0 OCH3 V \oCH3
OH 8Hy0 11 NNZ
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The chemical names of the small molecules are as follows:
1. Levamisole: (-) 2,3,5,6-Tetrahydro-6-phenylimidazo[2,1-
b]thiazole hydrochloride;
2. Tetramisole: (~) 2,3,5,6-Tetrahydro-6-
phenylimidazo[2,1-b]thiazole hydrochloride;
3. H-7 Dihydrochloride:1-(5-Isoquinolinylsulfonyl)-2-
methylpiperazine dihydrochloride;
4. Antipain Hydrochloride: N-[Na-carbonyl-Arg-Val-Arg-al]-
Phe hydrochoride;
5. Bestatin Hydrochloride: [(2S, 3R)-3-amino-2-hydroxy-4-
phenylbutanoyl]-L-leucine hydrochloride;
6. Desipramine Hydrochloride: 10,11-Dihydro-5-(3-
(methylamino)propyl)-5H-dibenz(b,f)azepine hydrochloride;
7. 9-CP-Ade: 9-Cyclopentyladenine;
3. Colchicine: (S)-N-(5,6,7,9-Tetrahydro-1,2,3,10-
tetramethoxy-9-oxobenzo[a]heptalen-7-yl)acetamide;
9. Papaverine Hydrochloride: 6,7-Dimethoxy-1-(3,4-
dimethoxybenzyl)isoquinoline hydrochloride;
10. AEBSF: 4-(2-Aminoethyl)benzenesulfonyl fluoride
hydrochloride;
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11. Ouabain: Acocantherin. As expected, no significant rate
reduction was observed for these compounds.
[0058] Using the same eleven compounds, the inhibition
assay was performed in a 96-well plate in order to
demonstrate the feasibility of exploring structure-switching
signaling aptamers for high-throughput screening of small
molecules as enzyme inhibitors (Figure 5B). A mixture was
produced for each well that contained the anti-ATP signaling
aptamer, AMP (0.75 mM) and one of the eleven compounds
(0.1 mM). Two fluorescence readings were taken from each
well: the first was recorded when the porcine ALP was added
(Finit) and the second was recorded 60 minutes later (Ffina~). A
control well containing the signaling aptamer and AMP but
lacking any test compound was also examined. The inhibition
effect exhibited by each compound (or the residue activity of
the enzyme, % Activity; Figure 5B) was then calculated by the
following equation: (FfinaI~Finit)compound~(FfinaI~Finit)control~ The results
shown in Figure 5B clearly indicate that structure-switching
signaling aptamers and methods of the present invention are
suitable for high throughput screening.
[0059] The results shown in the model system
demonstrate that, based on the ability to distinguish between
adenosine, AMP, ADP and ATP, a signaling aptamer complex
can be used as a unique reporter to monitor the catalytic
activities of nucleotide-dephosphorylating enzymes such as
alkaline phosphatase.
[0060] While adenosine has been used as an exemplary
product in these experiments, it is clearly apparent that the
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conversion of other substrate targets to product targets as well
as intermediate targets can be detected using the method of
the present invention. It is also apparent that reporter
molecules other than those generating a fluorescent signal can
be used i.e. colorimetric, radioactive, etc...).
[0061] To further demonstrate the broad applicability of
the method, a different enzyme system was assessed.
Adenosine deaminase transforms adenosine into inosine. The
signaling ATP structure switching aptamer complex has
virtually no affinity for inosine. Thus conversion from
adenosine to inosine results in a decrease in fluorescent
signal. This model system was used to demonstrate that
inhibitors of adenosine deaminase can be detected using the
method of the present invention. EHNA is a known inhibitor of
adenosine deaminase. As shown in Figure 6, the rate of
deamination is reduced in the presence of 100 mM EHNA and
is virtually stopped in the presence of 10pM EHNA.
[006] ~epending on the enzyme and substrates
involved,conversion a substrate product may
of to a be
detectedas an increaseor a decrease fluorescence.
in It is
also possible to follow the enzymatic activity as a substrate or
substrate target is converted to an intermediate target and
then a product target.
[0063] The present invention can also be used to
measure the opposite reaction. For example, rather than
measuring phosphatase activity, a phosphorylation reaction
can be monitored.
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[0064] It is clearly apparent that the methods of the
present invention can be used to monitor a variety of chemical
reactions in real time. The methods are especially useful to
monitor stages in a signaling pathways.
[0065] The methods of the present invention are
generally applicable to a broad range of biological and
chemical systems for kinetic and mechanistic studies. The
components of the assay system can be provided as a kit for
the detection of enzyme activity. A kit for detecting
modification of a substrate includes a signaling aptamer and a
substrate. The kit may optionally include an enzyme control.
A kit for screening for inhibitors includes a substrate, a
signaling aptamer and an enzyme.
[0066] A large number of structure-switching aptamer
reporters can be created by the combination of in vitro
selection and aptamer engineering for use in the method of the
present invention to monitor a chemical reaction of interest. In
addition, several structure-switching signaling aptamers
carrying different fluorophores or quenchers can be
conveniently created. These aptamer reporters can be used to
set up various forms of multiplexed assays for real-time
monitoring of either multi-step enzymatic reactions or different
enzymatic activities in the same solution.
[0067] The two-leg fluorescence-based assay can be
equally effective even for the compounds with fluorescence-
quenching (Papaverine or Desipramine) or enhancing (9-CP-
Ade) properties.
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WO 2005/003377 PCT/CA2004/000966
[0068] The present invention demonstrates that
structure-switching signaling aptamers can be used as
reporters for chemical reactions in real time. Novel methods
that exploit signaling aptamers as fluorescent reagents for
reporting a chemical reaction in real time are provided. In
addition, the present invention provides methods to quantify
enzyme activities with a very large detection dynamic range
and very low detection limit. The invention provides for the use
of structure-switching signaling aptamers as enzyme-mediated
chemical reaction reporters that are compatible with high-
throughput screening technology for the identification of
enzymatic inhibitors. Moreover, the reporting system of the
present invention has a built-in checking mechanism (two-leg
signal profiling) for quality control that reduces the chance of
reporting false positives.
[0069] It is clearly apparent that, while an existing DNA
aptamer was used to report a series of different reactions for
exemplary purposes, the method could be applied to any other
enzymatic transformations for which a differential signaling
aptamer can be obtained. The broad applicability of the
methods is readily inferred due to the fact that in vitro selection
allows convenient selection of DNA aptamers with the ability to
distinguish structurally similar compounds, and the structure-
switching approach allows easy design of signaling aptamer
reporters with large signaling magnitudes. For example, the
above anti-ATP signaling aptamer can be used to perform
screening assays to search inhibitors for several important
enzymes such as adenosine deaminases (which transform
adenosine into inosine for which the aptamer has no affinity at
23
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WO 2005/003377 PCT/CA2004/000966
all) , adenyl cyclases (that transform ATP into cAMP for which
the ATP aptamer has very low affinity) or phosphodiesterases
(that transform cAMP into AMP). In addition, several structure-
switching signaling aptamers carrying different fluorophores or
quenchers can be generated through the combination of in
vitro selection and signaling aptamer engineering. These
aptamers could be used to establish various forms of
multiplexed assays for real-time monitoring of either multi-step
enzymatic reactions or different enzymatic activities occurring
in the same solution. It is clearly apparent that other signaling
aptamers that have differential affinities for substrates and
products can be used to monitor chemical reactions in real
time and to screen for inhibitors of the reaction.
[0070] The above disclosure generally describes the
present invention. It is believed that one of ordinary skill in the
art can, using the preceding description, make and use the
compositions and practice the methods of the present
invention. A more complete understanding can be obtained by
reference to the following specific examples. These Examples
are described solely to illustrate preferred embodiments of the
present invention and are not intended to limit the scope of the
invention. Changes in form and substitution of equivalents are
contemplated as circumstances may suggest or render
expedient. Other generic configurations will be apparent to one
skilled in the art. All journal articles and other documents, such
as patents or patent applications, referred to herein are hereby
incorporated by reference.
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EXAMPLES
[0071 ] Although specific terms have been used in these
examples, such terms are intended in a descriptive sense and
not for purposes of limitation. Methods of molecular biology
and chemistry referred to but not explicitly described in the
disclosure and these examples are reported in the scientific
literature and are well known to those skilled in the art.
Example 1. DNA Oliaonucleotides and Chemical Reagents.
[0072] Both standard and modified DNA oligonucleotides
were prepared by automated DNA synthesis using cyanoethyl-
phosphoramidite chemistry (Keck Biotechnology Resource
Laboratory, Yale University; Central Facility, McMaster
University). 5'-Fluorescein and 3'-DABCYL (4-(4-
dimethylamino-phenyla~o)benzoic acid) moieties (in FDNA
and QDNA, respectively) were introduced using 5'-fluorescein
phosphoramidite and 3'-DABCYL-derivatized controlled pore
glass (CPG) (Glen Research, Sterling, Virginia) and were
purified by reverse phase HPLC. HPLC separation was
performed on a Beckman-Coulter HPLC System Gold with a
168 Diode Array detector. The HPLC column was an Agilent
Zorbax ODS C18 Column, with dimensions of 4.5 mm x 250
mm and a 5-~m bead diameter. A two-solvent system was
used for the purification of all DNA species, with solvent A
being 0.1 M triethylammonium acetate (TEAR, pH 6.5) and
solvent B being 100% acetonitrile. The best separation results
were achieved by a non-linear elution gradient (10% B for 10
min, 10%B to 40%B over 65 min) at a flow rate of 0.5 mL/min.
It was found that the main peak had very strong absorption at
CA 02530237 2005-12-21
WO 2005/003377 PCT/CA2004/000966
both 260 nm and 491 nm. The DNA within 2/3 of the peak-
width was collected and dried under vacuum. Unmodified DNA
oligonucleotides were purified by 10% preparative denaturing
(8 M urea) polyacrylamide gel electrophoresis (PAGE),
followed by elution and ethanol precipitation. Purified
oligonucleotides were dissolved in water and their
concentrations were determined spectroscopically.
[0073] Calf intestine alkaline phosphatase (calf intestine
ALP) was purchased from MBI-Fermentas and porcine ALP
from kidney was purchased from Sigma. Both were used
without further purification. Adenosine 5'-triphosphate (ATP),
adenosine 5'-diphosphate (ADP), adenosine 5'-
monophosphate (AMP), and adenosine were purchased from
Sigma and their solution concentrations were determined by
standard spectroscopic methods. All other chemical reagents
were also obtained from Sigma.
Example 2. General Procedures for Fluorescence
Measurements.
[0074] The following concentrations of oligonucleotides
were used for fluorescence measurements (DNA sequences
are shown in Figure 1A): 40 nM for FDNA, 80 nM for the
aptamer (MAP) and 120 nM for the quencher (QDNA). The
ratio of FDNA:MAP:QDNA was set to be 1:2:3 to ensure a low
background signal. Under this setting, the vast majority of
FDNA molecules would form a duplex structure with MAP and
the resulting FDNA-MAP duplexes would also be able to
engage a QDNA molecule for fluorescence quenching. The
assay buffer contained 300 mM NaCI, 5 mM MgCl2 and 20 mM
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WO 2005/003377 PCT/CA2004/000966
Tris~HCl (pH 8.3). The fluorescence intensities were recorded
on a Cary Eclipse Fluorescence Spectrophotometer (Varian)
with excitation at 490 nm and emission at 520 nm. The sample
volume in all cases was 500 ~,L except that 150 ~,L was used
in the 96-well microplate based assay. Measurements of
fluorescence intensities from specific samples are detailed
below.
Example 3 Generation of a two lea signal upon target addition
followed by enzyme addition.
[0075] 495 ~,L of FDNA-QDNA-MAP signaling mixture in
the assay buffer was incubated in the absence of any target
for 10 min at 22 °C, followed by the addition of adenosine
(filled green triangles), AMP (filled red circles), ADP (filled
purple triangles), and ATP (filled blue squares) to a final
concentration of 0.75 mM (achieved using 100~c stock); water
was added into the control sample (open circles). The
resultant aptamer-target mixtures were incubated at the same
temperature for 10 more minutes. At this point, 0.5 units of calf
intestine ALP were introduced (affording a final concentration
of 0.001 unit/p,L), and the resultant solution was further
incubated for 50 additional minutes. A fluorescence reading
was recorded every minute. The raw fluorescence data are
shown in Figure 1 B.
Example 4 Comparison of fluorescent siginals for adenosine,
AMP and AMP + ALP
[0076] Fluorescence readings were taken at 22 °C for
FDNA-QDNA-MAP solutions that contained either adenosine
(red circles) or AMP (green sequences) at 0, 0.1, 0.25, 0.5,
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WO 2005/003377 PCT/CA2004/000966
0.75, 1, or 3 mM. Each experiment was done in triplicate to
obtain an average reading. The results are shown in Figure
1 C. A fluorescence differential, OF, defined as Fadenos~ne FAMP
was calculated for each concentration. OF is plotted as a
function of the target concentration as shown in Figure 2.
Each AMP solution was also treated with 5 units of calf
intestine ALP (affording a final concentration of 0.01 unit/~.L)
for 15 minutes and the resultant solutions were measured
again for fluorescence intensity (blue triangles in Figure 1 C).
The new measurement afforded readings that matched those
of the solutions that contained adenosine at the same
concentrations (red circles in Figure 1 C). This indicates that all
AMP molecules were converted into adenosine. Each
adenosine solution was also treated with calf intestine ALP,
which did not induce any fluorescence intensity change as
expected (data not shown).
Example 5 Monitoring ALP activity in the transition from AMP
to adenosine.
(0077] Dephosphorylation of adenosine monophosphate
(AMP) by alkaline phosphatase (AP) was used as a model
reaction. A structure-swifiching signaling DNA aptamer,~ which
exhibits a moderate affinity for the substrate (AMP) and a
higher affinity for the product (adenosine), was used as the
reporter. A unique two-phase signaling profile can be obtained
with such a system. A first signal is produced upon the
addition of the substrate target, indicative of the formation of
substrate-aptamer complex and a second signal is generated
upon the addition of the enzyme, reporting the conversion of
AMP to adenosine. The reaction is shown in Figure 3.
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WO 2005/003377 PCT/CA2004/000966
[0078] The same procedures described for Example 4
were used to obtain the data shown in Figure 4 with three
variations: (1 ) only 0.75 mM AMP was used as the target, (2)
calf intestine ALP was used at seven different amounts from
10-~-10~ units (affording final concentrations of 2x10-8-2x10-2
unit/p.L), and (3) the incubation time after calf intestine ALP
addition was extended to 1200 minutes. Each experiment was
done in puadruplicate; however only one set of data is shown.
Example 6 Screening of small molecules as enzyme
inhibitors.
[0079] The ATP reporter and 0.75 mM AMP (500 pL)
were incubated for 10 min at room temperature before a test
compound was added at a final concentration of 0.1 mM. The
resulting mixture was incubated for another 10 min, followed
by the addition of 5 units of porcine ALP (resulting in a
concentration of 0.01 units/pL) and further incubation for 75
more minutes. The fluorescence intensity (F~~P) was monitored
continuously. For fihe same compound, the fluorescence
intensity (FNoALP) of a control reaction with the addition of
porcine ALP was also recorded in the same way. Figure 5A
plots Fp~p/FNoALP vS. reaction time for eleven compounds
tested .
Example 7. Inhibition assay in micro-well plate
[0080] The experiment procedure described in Example
6 was performed, in duplicate, in a 96-well plate (150 pL
solution was used for each well). The concentrations of all
components remained the same. The initial fluorescence
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WO 2005/003377 PCT/CA2004/000966
intensity (F;n;t) was taken when porcine ALP was added. The
fluorescence intensity (Ffinai) was again recorded 60 minutes
after the addition of the ALP. For each tested compound, an
Ff;~ai~F~n~t value was calculated. The inhibition effect (% ALP
aC'tlVlty) was calculated aS (Fq;naI~Finit)compound~(FfinaI~Finit)control. The
control reaction contained no tested compound. The results
are shown in Figure 5B.
Example 8. Adenosine deaminase activity
[0081 ] To demonstrate the versatility of the methods to
monitor enzymatic reactions generally, adenosine deaminase
activity was monitored. Three solutions of 500 pL containing
20nM fluorescent aptamer, 40nM antisense DNA labelled with
the quencher (called QDNA), 1 mM Adenosine, 300mM NaC1,
5mM MgCl2 and 25mM HEPES pH 8.0 were equilibrated at
22°C for the first 5 minutes. Fluorescence reading, made each
minute, reveal steady high fluorescence levels corresponding
to the complex state of the aptamer with adenosine. After 3
minutes, different concentrations of EHNA.HC1, an adenosine
deaminase inhibitor, in DMS~ were introduced: no inhibitor
(blue line), 100nM inhibitor (green line) and 10pM inhibitor (red
line). The solutions were further incubated for 5 more minutes.
No changes in fluorescence can be observed, suggesting that
the inhibitor or DMSO has no influence on the state of the
aptamer. Finally, the solutions were exposed to an enzyme,
called Adenosine Deaminase (ADA), which transforms
adenosine into inosine. The aptamer has no affinity for the
inosine, so the transformation should induce the disruption of
the aptamer - adenosine complex state and the formation of a
duplex state with the QDNA. The phenomenon can be
CA 02530237 2005-12-21
WO 2005/003377 PCT/CA2004/000966
monitored by following the decrease in fluorescence levels due
to fluorescence quenching (blue line). However, when the
inhibitor of ADA is present, the observed rate of deamination is
slower at 100nM EHNA.HC1 and almost no deamination is
observed at 10pM EHNA.HC1.
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