Note: Descriptions are shown in the official language in which they were submitted.
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CAGED COMPOUND CLEAVING PROCESS
FIELD OF THE INVENTION
This invention relates to chemical complexes known as "caged compounds",.
and their use in initiating chemical and biochemical reactions. More
specifically, it
relates to procedures for cleaving caged compounds to release active chemical
or
biochemical components therefrom, and utilizing the released active entity in
chemical
or biochemical reactions such as biochemical assays.
BACKGROUND OF THE INVENTION AND PRIOR ART
Several chemical groups have the convenient property that they can be removed
or destroyed photochemically. Such photolabile chemical groups have been
widely
described and have been used in various applications. The photogeneration of
an
essential active chemical species in the course of a chemical reaction offers
a milder
method of cleavage than normally employed. As such, compounds containing
photolabile chemical groups have been widely employed in organic and
bioorganic
reactions.
The ability to prepare photolabile compounds that modulate or block the
activity
of a critical reagent, for example adenosine triphosphate (ATP), and thus
prevent its
biochemical function allows localized delivery of reagent. This has been
termed
"chemical caging". Essentially, photolysis instantaneously releases the
reactant in sitzt
allowing the reaction to proceed.
Caged compounds are synthetic entities whose biological or biochemical
activity is controlled by photocatalytic reaction. Caged compounds are most
commonly
designed by covalently coupling of a desired molecule (the "active moiety")
with a
suitable photoremovable protecting or "caging" group such that the activity of
the
active moiety is masked or caged. In one kind of chemical caging, on
photoirradiation
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(photolysis) of appropriate wavelength, the photolabile bond is broken
releasing an
active moiety that could participate in a chemical or biochemical reaction
such that it
initiates the chemical or biochemical reaction in the immediate surrounding
medium.
In another kind of chemical caging, the photolabile bond is broken upon ,
photoirradiation (photolysis) with the appropriate wavelength, releasing an
active
moiety that results in removal of one of the essential components of a
chemical
reaction. In other words, photocatalytic reactions employing caged compounds
could
either add or remove one essential component from a chemical reaction.
The term "caged " is utilized as an indication that a biologically or
biochemically active species is trapped and masked inside a larger chemical
"framework", and can be "released" upon illumination, thus uncaging the active
content. The term "caged " has become popular because it is brief and
pictorial, rather
than being strictly accurate (see Adams et.al., Annu. Rev. Physiol. 55: 755-
784, 1993.
One important application that utilizes photolabile chemical groups to cage
and
mask chemical moieties is the probing of biological systems. For example,
photolysis
of photolabile chemical groups of caged compolmds and the consequent release
of
active chemical moieties involved in enzyme systems is one of the best
techniques to
examine the fast kinetics or spatial heterogeneity of biochemical responses in
such
systems. Illumination can be easily controlled in timing, location and
amplitude. One
can exert temporal and spatial control over the introduction of
physiologically active
compounds into complex systems, by introducing an inert photolabile compound
such
as a caged compound into the biological system and then activating it very
rapidly with
light. This provides a means of causing abrupt and localized changes in
concentration
of active species, in controlled amplitudes. This is particularly valuable
when rapid
mechanical mixing is impractical, for example on the surface of or inside a
more or less
intact cell, tissue, or protein crystal.
Caged compounds have been utilized to determine the structures of short-lived
enzymatic intermediates, by using time-resolved Laue crystallography. In
studying
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rapid enzymatic processes, numerous investigators have used caged compounds to
mask an essential functional group, so that a chemical reaction may be
initiated by a
light pulse.
Many, but not all, of the photolabile protecting groups used in caging
compounds comprise aromatic rings. To be useful in a biochemical system, a
protecting group must satisfy several requirements. Caging of a compound with
a
photolabile protecting group should render the caged compound inert to the
biochemical system used. The photolabile protecting group should release the
active
chemical moiety in high yield and at sufficient speed by photolysis at
wavelengths not
detrimental to the biochemical preparation. Further, photoproducts other than
the active
moiety should not interact with or interfere with the system. In nearly all
useful caged
biological molecules reported to date, simple covalent bond formation
involving the
active moiety masks some feature that is important for biological recognition.
The
photochemical cleavage of that covalent bond releases the active species
(active
moiety) or photoproducts having altered affinity, usually much reduced or much
increased, to the active moiety.
The most frequently described caging compounds in the current literature are
those based on the photoisomerism of 2-nitrobenzyl derivatives. The
nitrobenzyl group
is incorporated into the active molecule by linkage through a heteroatom,
usually O, S
or N. A wide range of other photolabile protecting groups is also commercially
available.
In all of these photolabile protecting groups, the photoactivation process
requires a light source. Any suitable conventional light source may be
utilized to deliver
a pulse of light energy to uncage photolabile compounds and release the active
entity.
Most commonly, lasers emitting energy in the ultraviolet (UV) or the infrared
region
axe used. A brief, high flux emission of a light pulse results in the
photoremoval of the
photolabile protecting group. Also, UV flashlamps, which emit in the UV region
of the
spectrum or which their light output filtered to deliver UV radiation have
been widely
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applied in the photochemistry of caged compounds.
The wavelength and energy of the optical source has been tailored to generate
an appropriate pulse that breaks a particular photolabile bond of a caged
compound. A
non-exhaustive listing of photolabile chemical groups and the optimal
wavelength for
their removal may be found in the recently published (1998) Methods i~
Enzymology
Volume 291 "Caged Compounds. "
Also, the inclusion of the various photolabile chemical groups in a wide
variety
of chemical and biochemical entities of differing characteristics to produce
caging
compounds that mediate different functions in chemical or biochemical
reactions is also
to be found in the recent reference cited above.
Chemical caging with photolabile protecting groups has been employed in
connection with amino acids, nucleic acids, enzyme substrates, catalysts of
biochemical
reactions and binding molecules whose affinities change upon irradiation.
Among the
common examples of active moieties employed in a caged compound form for use
in
biochemical systems are calcium ions, adenosine triphosphate (ATP), guanosine
triphosphate (GTP), fluorescein and biotin.
A specific example of the use of caged compounds in biochemical processes is
in bioaffinity binding assays such as immunoassays, nucleic acid binding
assays and
receptor binding assays. In such binding assays, the specific binding of
affinity partners
results in a modulation of a characteristic that may be easily measured. The
modulated
characteristics may be an enzymatic activity or a change in affinity that
results in certain
enzymatic activity. The modulated activity could also be measured tluough a
change in
certain characteristics prior to the binding reaction. Such a change could be
the
generation of light, modulation of colour absorbance or the generation of
colour.
The affinity of binding of biotin to avidin or streptavidin is one of the
strongest
non-covalent bindings in chemistry. Both biotin and streptavidin have been
successfully
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caged and utilized in photolytic reactions where their binding needs to be
controlled in
spatial or temporal terms. Also, both molecules have been successfully
conjugated to
various compounds in order to utilize the high affinity of these binding
partners.
In United States patent 5,981,207 Burbaum et. al. disclose the utilization of
caged enzyme substrates as probes in genetically-modified cells during iiz
vivo cell-
based reporter gene binding assays where the reporter enzyme activity and, by
inference, the activator or suppressor activity of a compound under testing in
a cell-
based assay for drug discovery could be monitored. Also, in international
patent
application PCT/CA00/00718 Gawad discloses a process of monitoring i~ vitro
binding
assays by utilizing caged compounds. In both inventions, a chemiluminescent
reaction
is triggered by the photolytic release from a caged compound of an active
moiety that
participates in the binding assay. The resultant chemiluminescent signal is
collected and
monitored. In such photolabile reactions, the binding lcinetics are closely
controlled
A difficulty with such a process is the need for both a light source that
triggers
release of the active moiety needed for initiating the binding reaction signal
from the
caged compound and a light detection system to measure the emitted light
output of the
chemiluminescent reaction. Such light signals could interfere with each other,
causing
confusion between the triggering light signal (to cause uncaging of the caged
compound) and the emitted light resulting from the chemiluminescent reaction.
Moreover, the electronic detection system necessary to measure the light
emissions, in
the presence of a system providing light input, is complicated, cumbersome and
expensive, if enough light emission is to be collected for meaningful
measurements.
, Furthermore, the triggering light results in a decrease in the sensitivity
of the binding
assay due to a need for a light filtration process to separate the two
different light
signals.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a novel method of
conducting
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a process involving cleavage of a caged chemical compound to release an active
moiety
able to participate in a chemical reaction.
It is a further and more specific object of the present invention to provide a
novel method for conducting luminescent biochemical affinity assays.
It has now been found that many if not all caged compounds can be cleaved to
release the caged chemical moiety, in active condition, by being subjected to
a pulse of
high energy electric current. In accordance with the invention, instead of
triggering
release of the desired active moiety by photolysis, a totally different input
is used,
namely a high energy electric pulse, so that in a system where the active
moiety is
released from a caged compound and then this active moiety participates in a
reaction
for signal generation, particularly involving light generation, the input to
release the
active moiety cannot be confused with the output from the reaction. This leads
to the
adoption of simpler detection systems, and to more accurate measurements of
light
output.
Furthermore, in light-generating reactions where the method of triggering the
release of the active moiety from the caged compound is other than a light
pulse, an
increase in the sensitivity of quantifying the reaction outcome can be
achieved using
less complicated machinery.
Thus according to a first aspect of the present invention, there is provided a
process of releasing an active moiety from a caged compound in which said
moiety is
held in inactive form, which comprises subjecting the caged compound to a
pulse of
high energy electric current.
According to a second and more specific aspect, there is provided a process of
conducting a biochemical binding assay for an analyte of interest, which
comprises
preparing, in a liquid medium, a mixture comprising a complex of said analyte
with a
specific binding partner for said analyte, and other components of a signal
generating
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system wherein one of the components is caged, releasing the active moiety
from the
caged compound in an active form by subjecting the caged compound to a high
energy
electrical pulse, and measuring the signal generated by the signal generating
system.
"Analyte" is a commonly used term of art, denoting a target compound whose
presence and/or quantity is to be determined, in a test medium. The analyte is
usually a
necessary reactant in a reaction scheme. In assays for such an analyte,
binding reactions
are commonly used, based on bioaffinity or enzymatically catalyzed reactions.
A
specific binding partner known to have specific binding affinity for the
analyte under
test, for example an appropriately chosen antibody, natural hormone binding
protein,
lectin, enzyme, receptor, DNA, RNA or peptide nucleic acid (PNA), or
artificial
antibody or nucleic probe, is used, to form a complex with the analyte, and
including a
label to quantify the complex. The process of the invention, i.e. the
subjection of a
caged compound to high energy electric current to release the active
ingredient from the
caged compound, can be applied to provide to the reaction medium any of the
active
components required either to form the complex of the analyte and binding
partner, or
to activate the signal generating system.
According to a third and yet more specific aspect, there is provided a process
of
conducting a binding assay for an analyte of interest where the signaling
mechanism of
the binding assay once activated results in the emission of light. According
to this
aspect, there is provided a process which includes the steps of preparing, in
an
electrolyte medium, a mixture of a fluid containing or suspected of containing
the said
analyte, one or more specific binding partners for said analyte and other
essential
components of a light-generating signaling mechanism where one of said
components
is caged, releasing the active moiety from the caged compound, in active form,
by
subjecting the medium to a high energy electrical pulse which results in
micaging of the
active moiety from the caged compound and thereby initiating the light
generating
reaction, and measuring the emitted light signal of the signaling mechanism.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
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Caged compounds, which will undergo release of active moiety in response to
subjection to high energy electric pulse for use in accordance with the
invention,
include substantially all of those previously reported in the literature
having an organic
protecting group and an active moiety that is photochemically releasable in
active form.
Preferred protective groups for caged compounds for use according to the
invention
include 2-nitrobenzyl; carboxy-2-nitrobenzyl; 2,2'-dinitrobenzhydryl; 1-(2-
nitrophenyl)ethyl; 4,5-dimethoxy-2-nitrobenzyl; 1-(4,5-dimethoxy-2-
nitrophenyl)ethyl;
5-carboxymethoxy-2-nitrobenzyl; ((5-carboxymethoxy-2-nitrobenzyl)oxy)carbonyl;
(1-
diazobenzyl)pyrene bromide; N-hydroxy-2-thiopyridone bromide; N-
hydroxysuccinimidyl bromide; p-azidobenzoate, N-hydroxysuccinimidyl ester of p-
azidobenzoylglycine bromide; N-hydroxysuccinimidyl bromide; (1-(2-nitro-4,5-
dimethoxy)phenyl-diazoethane; 1-(2-nitro)phenyl-diazoethane; 1-(2-nitro-
3,4,5,6-
tetramethyl-diazoethane; desoxybenzoinyl; hydroxyphenacyl; 6-
nitroveratryloxycarbonyl; 6-nitropiperonyloxy-carbonyl; alpha-dimethyl-
dimethoxybenzyloxycarbonyl; 1-(4,5-dimethoxy-2-nitrophenyl)-1,2-diaminoethane-
N,N,N,N-tetraacetic acid (DNMP); and 1-pyrenylmethyl. Some routine
experimentation
may be required, on the part of the skilled operator, to determine the best
combinations
of these protective groups with the preferred substrates for use in the
present invention
(ATP, GTP, Ca ions, etc), and to determine the optimum electric current pulse
characteristics for their cleavage, as further discussed below.
The process of the invention utilizes a high energy electrical pulse to
release an
active moiety from the caged compound in active form through either breaking a
chemical bond or through a change of the chemical affinity of the caging
molecule
before and after applying the electrical pulse. Preferably the electrical
pulse is a direct
current DC pulse, since use of an alternating AC current pulse entails
detailed tuning of
the frequency of the current to effect most efficient cleavage~of the caged
compound.
No such problems are encountered with DC current, provided that the energy is
sufficiently high to cleave the photolabile caging group, but not high enough
to destroy
one or more components of the chemical reaction. A minimum amount of direct
electrical current is used to cleave the photolabile chemical moiety. Several
parameters
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determine the level of energy delivered to the caging compound; the electrical
current,
the voltage, and the physical parameters of the system in which the reaction
is
conducted such as the shape of the electrodes to deliver the electrical
current, the nature
of the electrolyte, the material of the electrodes and the shape of the
reaction vessel. All
these factors determine the density of electrical current delivered to' the
photolabile
chemical group. The amount of energy required to be furnished to effect the
desired
bond cleavage is related to the bond energy of the selected bond, but the
relationship is
not straightforward because of factors such as the nature of the electrolyte
and the
amount of the applied electrical energy which the electrolyte will absorb and
hence will
not reach the photolabile bonds.
The total energy supplied according to preferred embodiments of the invention
is from about 0.01 m.joules to about 15 joules. The energy supplied is
dependent upon
the time for which the current is delivered, as well as the strength of the
current. For
example, if the DC current supplied is of high voltage (300 volts and above),
the
duration of the pulse required to cleave the caged compound can be as short as
one
microsecond. When a lower voltage is used, e.g. 70 volts, a pulse duration of
one
microsecond will only release a portion of the active moiety from the caged
compound,
and repeated pulses of such duration are required to'release all of the caged
compound.
There are occasions when the release of only a portion of the caged active
moiety is
desirable in order to control various aspects of the chemical reaction. Longer
pulses do
not appear to cause significant problems. A low voltage (4.8V, for example)
for a
longer pulse (3.3 seconds) has been satisfactorily used in practice. Use of
such low
voltages and longer times minimizes the loss of energy, which might otherwise
heat up
the liquid medium. Some routine experimentation with the chosen system, to
determine
the optimum electrical input, may be desirable, but such experimentation is
well within
the skill of the art.
In practice, a preferred method of conducting the process of the present
invention is to utilize a reaction cell containing two spaced-apart current-
delivering
electrodes between which the current can be passed through an electrolyte. The
cell is
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filled to an appropriate extent with an electrolyte medium containing all the
needed
reaction components including the caged compound. Upon delivery of the DC
current,
the photolabile chemical group of the caged compound releases the active
moiety
needed to initiate the desired chemical reaction. In light-generating chemical
reactions
in which one of the reaction components is a caged compound, a light receiving
detection system is provided, to receive and quantify light emissions from the
reaction
solution.
Appropriate electrical circuitry to provide pulsed DC electric current, of
predetermined voltage and duration, and hence energy level, is connected to
the
electrodes, and activated to cause cleavage of the caged compound. Incident
light is not
used to cause cleavage of the caged compound, and so no special measures such
as
light-proof shutters or light filtering or beam-splitting devices, are needed
to collect
light emitted in the light-producing chemical reaction.
The process of the invention shows utility not only in causing luminescent
emissions from a chemical reaction in binding assays as described above, but
also in
other areas where a detectable change due to uncaging of a caged compound and
release
of an active moiety which is essential for the progression of a chemical
reaction is
observed.
Further, the process of the invention provides controlled spatial or temporal
delivery of one component of a reaction system, which obviates designing
complicated
mechanical delivery mechanisms of such a component. The variety of chemical
reactions, which might benefit from the process of the invention include many
binding
assays. For example, determination of bacterial contaminants in food, where
antibodies
to the bacteria can be bound to a solid substrate and bind selectively to a
chosen
enzyme which subsequently reacts with the released active component from the
caged
compound and a detectable change in the enzyme activity is measured.
The invention is further described, for illustrative purposes only, in the
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following specific experimental examples.
Specific Embodiments of the Method of the Invention.
The method of the invention is the trigger of a chemical reaction upon
delivery
of an electric pulse to the reaction medium. As a prerequisite to employ the
method of
the invention for the initiation the chemical reaction is the presence of
caged chemical
compound, which is either inactive when in the caged condition or carry a
trigger
compound rendering the trigger compound inaccessible to activate the reaction.
Upon
employing the method of the invention of activation of the reaction by an
electrical
current pulse, the chemical reaction is initiated by releasing an active
compound from
the caged inactive precursor. Caged compounds offer a chemical entrapment
method
for delivery of reagents, which is superior to other delivery methods such as
physical
entrapment of chemical reagents (e.g. in liposomes) or delivery by mechanical
means.
Certain chemical reactions could benefit from the method of the invention of
applying an electric current to release active compounds from caged compounds
needed
for the chemical reaction to proceed by either speeding up the reaction,
control of reaction
dynamics or by simplifying the machinery involved in timed-sensitive addition
of reagents.
A general scheme of a simple chemical reaction is a follow:
A+B ~ C
Whether C is the final reaction product and can be measured, or whether C is
linked
to another chemical reaction for generating a measurable signal that
quantifies this
reaction, the method of the invention can be employed. All the reaction
components are
essential for a measurable outcome and any or all of the components can be
caged with
photolabile bond and can be released by a high-energy direct current
electrical pulse in
accordance with the invention, supplied by appropriate electrical circuitry.
A requisite of the reaction to benefit from the method of the invention is
that
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mixing all of the reactants together, with one or more of the reagents being
caged,
facilitates no progression of the reaction. Release of the caged component as
an active
moiety that is needed for the chemical reaction to proceed by means of the
method of the
invention results in the production of a measurable product. Depending on the
specifics of
the electrical pulse supplied in the process of the invention, the rate of the
reaction can be
controlled.
Examples of various chemical reactions that result in a measurable outcome and
can benefit from the method of the invention of releasing caged compounds,
include light-
producing chemical reactions e.g. those involving enzymes and visible outcome
chemical
reactions. Specific examples of the various kinds of chemical reactions that
can benefit
from the method of the invention are as follows:
Example 1: Light producing chemiluminescent reactions.
Numerous chemiluminescent reactions are known and utilized in research and
clinical laboratories. Caging of one or more compound needed to initiate a
chemiluminescent reaction and the release of the needed compound by the method
of the
invention offers control over the speed and amplitude of light generation and
also over the
timing of release of an active chemical compound.
For example, in the chemiluminescent reactions, which use photoproteins,
initiation
of the reaction depends on the addition of Ca to the reaction mixture. A
photoprotein
cliemiluminescent reaction can be illustrated by the following equation:
Photoprotien + Luciferin + Ca + Oxygen ~ Light
Addition of Ca to the charged photoprotein (photoprotein already bound to
luciferin) results in an instantaneous chemical reaction and the generation of
light.
Substituting Ca by a caged Ca compound and triggering the release of the caged
Ca by
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electrical pulse according to the invention will initiate the chemiluminescent
reaction and
the generation of light.
Depending on the electrical pulse characteristics, the light generation from
the
chemiluminescent reaction can be a single flash of light, or several flashes,
or a steady light
emission depending on the amounts of released Ca as determined by the
characteristics of
the electrical pulse used in the method of the invention.
To confirm that the electrical current pulse altered the photolabile caging
compound and caused release of Ca to trigger photoprotein chemiluminescence,
one of the
reaction components was omitted from the reaction chamber. When the
photolabile Ca
caging compound was omitted from the reaction, no chemiluminescent light
generation
occurred. When the photoprotein is omitted, no chemiluminescent light emission
occurs.
However, adding the photoprotein after the electrical pulse generates
chemiluminescent
light.
Further, to confirm that this process is specific to the alteration of the
caging
compound and release of Ca to trigger the reaction, a non-photolabile Ca
chelating agent
(EDTA), which has a higher affinity for Ca than the photoprotein, was added to
the
reaction cell that contains all other reaction components, including the Ca-
caging
compound. Under such condition, no light generation occurred as the released
Ca is
chelated by the non-photolabile Ca chelating agent.
Further, to confirm the specificity of unloading of the photolabile trigger
compound
by this form of energy, another from of energy was utilized in an attempt to
trigger the
same chemiluminescent reaction in the presence of a Ca-caging compound.
Triggering an
electromagnetic field of an electromagnet through the same electric circuitry
resulted in no
generation of light.
Examples ofphotoproteins are aequorin, obelin, mnemiopsin, berovin, phosalin,
luciferase
of ostracods and cypiridina.
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Another example chemiluminescent reaction where the method of the invention is
useful is chemiluminescent reactions that employ the luciferases. Firefly
luciferase-
mediated chemical reaction, which generate light could be exemplified as
follows:
Luciferase + Luciferin + ATP + Mg + Oxygen ~ Light
During luciferase-mediated chemiluminescent light-generating reactions, one or
more essential components of the reaction can be in a caged form. With all the
reaction
components present, utilizing the method of the invention for uncaging of a
caged
molecule induces the generation of light from the luciferase chemiluminescent
reaction.
Upon triggering the reaction by the method of the invention of electrical
current pulse to
trigger the reaction, a controlled release of the caged component or
components occurs and
the released active moieties will trigger the reaction and result in light
generation.
Modulating the current electrical pulse to release one or more of the caged
components in
a controlled way would result in light emission that can be monitored.
Normally, a luciferase-mediated chemiluminescent reaction results in the
release
of a burst of light which is difficult to measure and monitor as i't lasts
only a second at
most. Several prior art patents have disclosed methods to alter the light
output by adding
one or more cofactors to the reaction. By utilizing the method of the
invention, controlling
the release of the caged compound controls the amount of released light and
also simplifies
the machinery needed to monitor the light emission. Several components of the
chemiluminescent system of the luciferase enzyme are already commercially
available in
a caged form, such as the luciferase enzyme itself, luciferin, and ATP. Also,
caged
chelating agents can be utilized to cage Mg. Most of these are available from
Molecular
Probes (Eugene).
Example 2: Non-chemiluminescent light emitting reactions.
In fluorescent binding reactions, the sensitivity of binding assays is limited
by the
fluorescence of the medium where the reaction is carried out as well as the
container. The
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high non-specific background signal limits the lower limit of detection of
fluorescent
assays. It has been suggested that bleaching the non-specific fluorescence of
the reaction
medium before stimulating the fluorescence of the specific signal would result
in a lower
background, thus lowering the lower limit of detection. Several caged
fluorescent
S compounds have been developed for this purpose. Irradiating the medium where
a caged
fluorescent compound is present would result in bleaching of fluorescence of
the medium
and at the same time maintain the caged fluorescent compound without
exhaustion.
Uncaging fluorescent compounds using the method of the invention will then
simplify the
machinery needed to gather the emitted signal, since otherwise the caged
compounds need
to be irradiated with UV light and most of the stimulation spectra of the
modern
fluorescent compounds are in the visible range. Therefore, utilizing the
method of the
invention would simplify the optical components of various detection systems.
Example 3: Colour Enzymatic Reactions.
The method of the invention of unloading or uncaging caged compounds to
release
the active moiety through he utilization of a high energy electrical pulse can
be employed
in binding assays with enzyme-mediated color changes. Numerous binding
reactions and
binding assays utilize enzymes to result into a measurable colour changes that
indicate the
quantity of the chemical entity under study. In most of these reactions, an
enzyme-
catalyzed process results in the conversion of a substrate from one color to
another. The
amount of color change then indicates the quantity of the chemical entity.
During such
reactions, adding one or more components to the system trigger initiation of
the reaction.
Commonly, this step is carried out mechanically. Replacing the mechanical step
with the
method of the invention would result in simplifying the measuring machinery.
The method of the invention can also be utilized in binding assays where a
caged
compound can be released by an electrical pulse and when the measured property
is not an
electrical signal. In almost any biological system where the measured output
is not an
electrical signal, the method of the invention can be utilized to trigger the
cleavage of the
photolabile bonds of various caging compounds. For example, the method of the
invention
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could be employed in cell mediated binding assays where a caged compound can
be
released by an electrical pulse. The list of applications in this area is very
broad.
Experimental Results
The method of the invention was demonstrated by carrying out several
chemiluminescent reaction experiments.
Experiment 1
In one experiment, in a total reaction volume of 10 ~L, all the components of
a
photoprotein chemiluminescence reaction were added in suitable electrical cell
(Aequorin,
native or recombinant, and recombinant Obelin were utilized in amounts varying
from 0.5
- 6 micrograms). The reaction cell also contained Ca-caging compound loaded
with Ca to
such an extent that the level of free Ca does not trigger light emission.
Specifically, the Ca-
caging compound was DNMP saturated to an extent of 50% - 77% with Ca. Two
spaced
metal electrodes were connected to a suitable circuitry to deliver a DC
electrical pulse. The
electrical pulse characteristics were changed and the light emission from
reaction was
monitored. Various metals were used in the different experiments, namely
silver,
aluminum and steel, and various different shapes of electrode, cylindrical, U-
shaped, etc
were used. A variety of different buffered electrolyte solutions (to decrease
changes in pH
due to the released compounds), were employed. These included MOPS buffer with
80mM
KCl (pH7.4 and 7.2), serum and plasma. All experiments were operated
successfully. The
following table summarizes the results.
Pulse Pulse Peals Number of pulses/
voltage. duration. current. Light flashes
(V) (S)
320 0.012 70 1
150 0.52 47 1
100 1.1 54 1
70 1.15 22.8 >5
63 1.1 19.5 15
60 0.52 29.6 11
50 2.4 17.6 >5
46 2.6 15.9 >8
24 2.4 40.9 >6
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12 2.2 30.1 3
3.3 5.7 3
5 In each of the previous experiments, altering the shape andlor material of
the
electrodes to deliver an electrical pulse of a certain voltage caused the
characteristics of
the light emission profile to change, but in all cases the experiment
proceeded
successfully. Replacing the electrolyte medium with pure water resulted in
unsuccessful
experiments.
Experiment 2
To confirm that the electrical pulse induces unloading of the photolabile Ca-
caging compound and release of Ca, which triggers light emission of
phortoproteins,
one of the reaction components was omitted from the reaction. When the
photolabile
Ca-caging compound was omitted, no chemiluminescent light generation occurred.
When the photoprotein (Aequorin or Obelin) was omitted from the reaction, no
chemiluminescent light emission also occurred. However, adding the
photoprotein after
the electrical pulse of the caged Ca initiated chemiluminescent light
generation. In these
experiments, the total electrical energy used is 5.94J.
Experiment 3
Further, to confirm that this process is specific to unloading of the
photolabile
Ca-caging compound and release of Ca to trigger the chemiluminescent reaction,
a non-
photolabile Ca-chelating agent (EDTA), which has a higher affinity to Ca than
photoproteins, was added to the reaction cell that contains all the other
reaction
components. Under such condition, no light generation occurred as the released
Ca is
chelated by the non-photolabile Ca chelating agent. In this experiments, the
electrical
energy used was 5.94J.
Experiment 4
To confirm the specificity of the destruction of the photolabile compound to
this
form of energy, a magnetic field was generated from pulsed electromagnet
through the
same electric circuitry used to generate chemiluminescence. In case of the
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electromagnetic field pulse, no light generation occurred.
Experiment 5
The generation of light from a chemiluminescent reaction that employ the
method
of the invention depends on the characteristics of the electrical pulse with
regard to its
duration as well as amplitude. In order to demonstrate this dependency, a
different
electrical circuitry was employed. An electrical circuit that relies on the
fast discharge of
electrical capacitor was employed to generate light of a chemiluminescent
reaction by the
method of the invention. Capacitors with voltage values between 100-330 Volts
and
capacitances of between 1-220 ~,F were utilized. The shape of the electrical
pulse of the
capacitor discharge determines the characteristics and frequency of the light
flash or
emission. Further, another kind of electrical circuitry was employed to
demonstrate the
method of the invention. A DC power supply source with an output voltage
between 3 and
150V and a switching circuit to control the duration of the pulse at which a
certain voltage
was applied was employed to trigger light generation of photoproteins Aequorin
and
Obelin chemiluminescence. Applying different electric pulses with
characteristics as listed
in the previous table resulted in triggering of light emission. Furthermore,
applying a direct
current electrical pulse below the required characteristic generated no light
from the same
reaction in the same electrical cell. Several pulses had to be applied before
light emission.
Experiment 6:
The method of the invention was also employed for triggering light emission
from
a chemiluminescence reaction that employs other caged reagents. Light emission
of the
chemiluminescence reaction of the luciferase was utilized with various caged
compounds
that are needed to trigger the reaction. A typical firefly luciferase
chemiluminescent
reaction needs all the following essential components; Luciferase enzyme,
Luciferin,
Magnesium and ATP, in the presence of oxygen to generate light according to
the
following reaction:
Luciferase + D-Luciferin + ATP + Mg++ ~ Oxidized Luciferin + pyrophosphate +
C02 + Light.
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The utility of method of the invention was demonstrated by canying out the
previous luciferase chemiluminescent reactions with one of the components of
the reaction
being caged. Upon delivering the needed electrical pulse, the caged compound
is uncaged
causing an instantaneous release of an active compound and the triggering of
light
generation.
A caged ATP was utilized to demonstrate the method of the invention of
controlling the trigger of light generation by a pulse of electric current.
Caged ATP is not
an active substrate of the luciferase chemiluminescence, however, functional
ATP, which
acts as a substrate for the luciferase reaction could be delivered by the
method of the
invention.
In a total reaction volume of 12 ~L, the following are mixed in a suitable
electric
reaction cell: Luciferase/D-Luciferin solution mix (6~.L), SmM Mg Citrate in
PBS (3 ~L),
caged ATP solution (3 ~L). The electrodes were connected to a power supply
circuitry and
an electric pulse was triggered to uncage ATP and initiate light generation.
Various
voltages and pulse durations were utilized. Under these experimental
conditions,
employing the method of the invention resulted in the generation of light from
the
luciferase chemiluminescence reaction.
Also, in order to demonstrate that the method of the invention can be employed
with various caging compound, as long as the electrical pulse shape is
altered, another
caged compound employed in luciferase chemiluminescence reaction was utilized.
In this
experiment, caged D-Luciferin was utilized to control the reaction kinetics.
Functional D-
Luciferin was delivered to the reaction from caged D-Luciferin upon exposure
of the
reaction components to an electric pulse.
In a total reaction volume of 25 ~,L, the following components were added as
solutions to a suitable electric cell: Luciferase solution (10 ~,L), SmM Mg
Citrate in PBS
(S~,L), 1mM ATP solution (S~.L) and Caged D-Luciferin solution (SQL). A
suitable
electrical circuitry was connected to the cell and an electric pulse was
delivered. Upon
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delivery of the electric pulse, the caged compound released the active
component needed
to trigger light generation of the luciferase chemiluminescence reaction. The
experimental
results of both the luciferase chemiluminescence reactions with the two caged
compounds;
caged ATP and caged Luciferin are summarized as follows:
Pulse pulse durationPeak current.Nmnber of pulses/
voltage (S) (mA) Light flashes
3 5 0 0.184 40.9 1
100 0.300 32.2 1
50 1.1 29.3 1
In these experiments of the luciferase chemiluminescence, an electrical
circuit that
relies on discharge of an electrical capacitor at various voltages was
employed. Capacitors
of a voltage value between 50-350 volts and capacitances of between 10-150 uF
were
utilized to alter the shape of the electric pulse. It was observed that a time
lag of about 0.5
second is needed before light emission start from the reaction. It was assumed
that light
generation start immediately, however the amounts of released active trigger
compound
from the caged compound need to accumulate before substantial enzyme-activated
reaction
and therefore light emission could be observed.
Reagents:
DM-EDTA (1-(4,5-Dimethoxy-2nitrophenyl)-1,2-diaminoethane-N,-N,-N,-N tetra
acetic acid), Molecular Probes, Eugene, OR , USA (catalog # D-6814).
- Recombinant Aequorin, Aqualite, Molecular Probes, Eugene, OR, USA (Catalog
# A-6785).
- Native Aequorin, Friday Harbor Photoproteins, Friday Harbor, WA, USA.
- Recombinant Obelin, curtsey of Dr. Eugene Vysotslci, Dept. of Biochemistry,
University of Georgia, Georgia, USA.
- A lyophilized mix of Luciferase/D-Luciferin is dissolved in Tricine
reconstitution
buffer [50 mM N-Tris(hydroxymethyl) methylglycine, adjusted with NaOH to pH
7.8, Lot 1418], both supplied by Kikkoman as assay kit (CheckLite HS Plus,
Catalog # 60342).
- Luciferase enzyme dissolved in Tricine buffer pH 7.8 [(50 mM N-Tris
(hydroxymethyl) methylglycine] adjusted with NaOH, supplied by Kikkoman
Catalog LUC T).
- 5 mM Mg Citrate solution in Phosphate buffered saline (PBS, pH 7.4).
- Caged ATP in methanol, Smg in 300 ~.L (Molecular Probes, Eugene, OR, USA
Catalog # A-1049).
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Caged D-Luciferin Smg dissolved in 300 ~L of Dimethylsulfoxide (DMSO)
(Molecular Probes, Eugene, OR, LTSA, Catalog # L-7085)
100mM ATP solution, pH 7.5 (Amersham Pharmacia, Catalog # 272056).
