U.S. patent application number 10/387314 was filed with the patent office on 2004-02-05 for methods and compositions for aptamers against anthrax.
Invention is credited to Kiel, Johnathan L., Vivekananda, Jeevalatha.
Application Number | 20040023266 10/387314 |
Document ID | / |
Family ID | 27538166 |
Filed Date | 2004-02-05 |
United States Patent
Application |
20040023266 |
Kind Code |
A1 |
Vivekananda, Jeevalatha ; et
al. |
February 5, 2004 |
Methods and compositions for aptamers against anthrax
Abstract
The present invention concerns methods of preparing nucleic acid
ligands against anthrax spores, compositions comprising anthrax
specific nucleic acid ligands and methods of use of such ligands
for detection and/or neutralization of anthrax spores.
Inventors: |
Vivekananda, Jeevalatha;
(San Antonio, TX) ; Kiel, Johnathan L.;
(Universal, TX) |
Correspondence
Address: |
Blakely Sokoloff Taylor & Zafman
Seventh Floor
12400 Wilshire Boulevard
Los Angeles
CA
90025-1030
US
|
Family ID: |
27538166 |
Appl. No.: |
10/387314 |
Filed: |
March 11, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10387314 |
Mar 11, 2003 |
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09978753 |
Oct 15, 2001 |
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6569630 |
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09978753 |
Oct 15, 2001 |
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09909492 |
Jul 19, 2001 |
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09909492 |
Jul 19, 2001 |
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09608706 |
Jun 30, 2000 |
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6303316 |
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60142301 |
Jul 2, 1999 |
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60199620 |
Apr 25, 2000 |
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60291371 |
May 15, 2001 |
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Current U.S.
Class: |
506/5 ;
435/252.31; 435/6.12; 506/9 |
Current CPC
Class: |
C12Q 2565/607 20130101;
C12Q 1/6837 20130101; C12Q 1/6825 20130101; C12Q 1/6837
20130101 |
Class at
Publication: |
435/6 ;
435/252.31 |
International
Class: |
C12Q 001/68; C12N
001/20 |
Goverment Interests
[0002] The Federal Government has rights to use the present
invention pursuant to contract F41624-00-D-7000 awarded by the
Department of the Air Force.
Claims
What is claimed is:
1. Amethod for preparing one or more nucleic acid ligands against
Bacillus anthracis (anthrax) spores comprising: a) obtaining
anthrax spores; b) obtaining a pool of nucleic acid ligands; c)
contacting said spores with said pool under conditions allowing
binding of one or more nucleic acid ligands to the anthrax spores;
d) separating ligands bound to the spores from ligands that do not
bind to the spores; and e) collecting one or more anthrax binding
nucleic acid ligands.
2. The method of claim 1, further comprising repeating (c) through
(e) until one or more anthrax binding nucleic acid ligands of a
desired degree of specificity or binding affinity against anthrax
spores is obtained.
3. The method of claim 2, wherein the nucleic acid ligand binds to
anthrax spores with high affinity.
4. The method of claim 2, wherein the nucleic acid ligand is highly
specific for anthrax spores.
5. The method of claim 4, wherein the nucleic acid ligand binds
only to anthrax spores.
6. The method of claim 1, wherein the pool of nucleic acid ligands
are attached to magnetic beads.
7. The method of claim 1, wherein the nucleic acid ligands are
operably linked to an organic semiconductor.
8. The method of claim 7, wherein the organic semiconductor is
diazoluminomelanin (DALM).
9. The method of claim 7, wherein the organic semiconductor is a
polyphenylene.
10. The method of claim 1, wherein said separating comprises
nitrocellulose filtration.
11. The method of claim 10, wherein nucleic acid ligands that bind
to nitrocellulose filters in the absence of anthrax spores are
removed from the pool of nucleic acid ligands before contacting the
anthrax spores with the pool.
12. The method of claim 1 or claim 2, wherein the nucleic acid
ligands comprise 40-mers of random sequence, the random 40-mers
attached at their 5' and 3' ends to primer binding sequences.
13. The method of claim 12, further comprising amplifying the
nucleic acid ligands using a 5' primer and a 3' primer.
14. The method of claim 12, wherein biotin residues are attached to
the 5' ends of the 5' primer binding sequences.
15. The method of claim 13, wherein biotin residues are attached to
the 5' ends of the 5' primers.
16. The method of claim 15, wherein ssDNA to be used for SELEX
screening is prepared by binding of biotin-labeled nucleic acid
ligands to streptavidin conjugated beads.
17. The method of claim 15, wherein anthrax binding nucleic acid
ligands are collected by binding to streptavidin conjugated beads.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. patent
application Ser. No. 09/978,753, filed Oct. 15, 2001, which was a
continuation-in-part of U.S. patent application Ser. No.
09/909,492, filed Jul. 19, 2001, which was a continuation-in-part
of U.S. patent application Ser. No. 09/608,706, filed Jun. 30, 2000
(now issued U.S. Pat. No. 6,303,316), which claimed the benefit
under 35 U.S.C. .sctn.119(e) of provisional Patent Application
Serial No. 60/142,301, filed Jul. 2, 1999 and 60/199,620, filed
Apr. 25, 2000. U.S. patent application Ser. No. 09/978,753 claimed
the benefit under 35 U.S.C. .sctn.119(e) of provisional Patent
Application Serial No. 60/291,371, filed May 30, 2001.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to the field of detection of
biological agents using novel compositions, methods and apparatus
comprising one or more nucleic acid ligands operably coupled to an
organic semiconductor. More particularly, the present invention
relates to the production and use of nucleic acid ligands against
anthrax spores.
[0005] 2. Description of Related Art
[0006] There is a great need for the development of methods,
compositions and apparatus capable of detecting and identifying
known or unknown chemical and biological agents (herein referred to
as analytes), which include but are not limited to nucleic acids,
proteins, illicit drugs, explosives, toxins, pharmaceuticals,
carcinogens, poisons, allergens, contaminants, pathogens and
infectious agents.
[0007] As one skilled in the art will readily appreciate, any
method, technique or device capable of such detection and
identification would have numerous medical, industrial forensic and
military applications. For instance, such methods, techniques and
devices could be employed in the diagnosis and treatment of
disease, to develop new compounds for pharmaceutical, medical or
industrial purposes, or to identify chemical and biological warfare
agents.
[0008] Current methods, techniques and devices that have been
applied to identification of chemical and biological analytes
typically involve capturing the analyte through the use of a
non-specific solid surface or through capture deoxyribonucleic
acids (DNA) or antibodies. A number of known binding agents must
then be applied, particularly in the case of biological analytes,
until a binding agent with a high degree of affinity for the
analyte is identified. A labeled antiligand (e.g., labeled DNA or
labeled antibodies) must be applied, where the antiligand causes,
for example, the color or fluorescence of the analyte to change if
the binding agent exhibits affinity for the analyte (i.e., the
binding agent binds with the analyte). The analyte may be
identified by studying which of the various binding agents
exhibited the greatest degree of affinity for the analyte.
[0009] There are a number of problems associated with current
methods of chemical and biological agent identification. It takes a
great deal of time and effort to repetitiously apply each of the
known labeled antiligands, until an antiligand exhibiting a high
degree of affinity is found. Accordingly, these techniques are not
conducive to easy automation. Current methods are also not
sufficiently robust to work in the heat, dust, humidity or other
environmental conditions that might be encountered, for example, on
a battlefield or in a food processing plant. Portability and ease
of use are also problems seen with current methods for chemical and
biological agent identification.
[0010] Within the field of biological warfare, there is a great
need for a rapid, sensitive method to detect and identify
pathogenic spores of Bacillus anthrax (hereafter "anthrax").
Anthrax is a highly pathogenic biological agent that is relatively
simple to produce and distribute in the field. Present methods for
detection of anthrax are not sufficiently rapid, sensitive, and
robust to allow early detection of exposure to anthrax under field
conditions, such as might be encountered on a battlefield. No good
method presently exists for neutralization of anthrax under field
conditions.
SUMMARY OF THE INVENTION
[0011] The present invention fulfills an unresolved need in the
art, by providing methods, compositions and apparatus for the
production of nucleic acid ligands capable of binding to,
identifying and/or neutralizing anthrax. The methods and
compositions disclosed herein provide substantial improvements over
earlier methods for anthrax detection (e.g., Reif et al., 1994;
Gatto-Menking et al., 1995; Bruno and Yu, 1996), by utilizing
anthrax-binding nucleic acid ligands.
[0012] The compositions of the present invention comprise a
recognition complex or a recognition complex system that are
capable of detecting, identifying, characterizing or purifying a
chemical or biological agent (hereafter, "analyte"), preparing or
purifying high affinity nucleic acid ligands for selected known
analytes, using high affinity nucleic acid ligands to measure the
concentration of analyte in a sample or to neutralize an analyte,
or to perform high through-put screening of libraries of compounds
or native plant extracts for compounds that are structural analogs
of known inhibitors, activators or binding agents of bioactive
molecules. The recognition complex and recognition complex system
and the corresponding techniques should be capable of full
automation.
[0013] Each recognition complex is comprised of a nucleic acid
ligand operably coupled to an organic semiconductor. In certain
embodiments, the organic semiconductor is DALM
(diazoluminomelanin), although the use of other organic
semiconductors, such as polyphenylenes, is contemplated within the
scope of the invention. In various embodiments, the organic
semiconductor may be attached to the nucleic acid ligand by either
covalent or non-covalent interaction.
[0014] In preferred embodiments, the nucleic acid ligand is DNA,
although it is contemplated within the scope of the invention that
other nucleic acids comprised of RNA or synthetic nucleotide
analogs could be utilized as well. In certain embodiments, the
nucleic acid ligand sequences are random, or may be generated from
libraries of random DNA sequences. In other embodiments, the
nucleic acid ligand sequences may not be random, but may rather be
designed to react with specific target analytes. In a preferred
embodiment, the nucleic acid ligand sequences are aptamers (Lorsch
and Szostak, 1996; Jayasena, 1999; U.S. Pat. Nos. 5,270,163;
5,567,588; 5,650,275; 5,670,637; 5,683,867; 5,696,249; 5,789,157;
5,843,653; 5,864,026; 5,989,823 and PCT application WO 99/31275,
each incorporated herein by reference).
[0015] In certain embodiments, the analyte to be identified may be
added in the form of a complex mixture that may include, for
example, aqueous or organic solvent, proteins, lipids, nucleic
acids, detergents, particulates, intact cells, bacteria, viruses
and spores, as well as other components. In other embodiments, the
analyte may be partially or fully purified before exposure to the
array. In particularly preferred embodiments, the analyte is
anthrax spore.
[0016] In certain embodiments, a recognition complex system,
comprising two or more recognition complexes, may be used in
methods for identifying an analyte. After the analyte is contacted
with the recognition complexes, certain recognition complexes will
bind the analyte, while others will not. Binding of analyte to a
recognition complex may be detected by changes in the
electrochemical properties of the nucleic acid ligand/organic
semiconductor couplet upon binding to the analyte. Nonlimiting
examples of electrochemical signals include photochemical,
fluorescent or luminescent signals, changes in color or changes in
electrical conductivity. The degree to which the electrochemical
properties change is a function of the degree to which the nucleic
acid ligand binds the analyte. Accordingly, the electrochemical
changes that occur across all of the recognition complexes, when
taken as a whole, can be used as a unique signature to identify the
analyte.
[0017] To facilitate detection of such electrochemical changes, the
recognition complex system may be associated with a detection unit
operably coupled to the recognition complexes. Non-limiting
examples of detection units include a charge coupled device (CCD),
a CCD camera, a photomultiplier tube, a spectrophotometer or a
fluorometer. The recognition complex system may also be associated
with system memory for storing electrochemical signals, as well as
a data processing unit that may comprise a neural network or lookup
tables. For embodiments where the binding of analyte is detected by
changes in electrical conductivity of the recognition complex, the
complexes may be positioned between a pair of electrodes attached
to a conductivity meter.
[0018] In addition to analyte identification, recognition complexes
may be used to screen for the presence or measure the amount of
analytes that are biological molecules, such as hormones,
cytokines, vitamins, metabolites or other compounds, in samples of
human tissue, fluids or extracts. Nucleic acid ligands with high
affinity for biological molecules of interest may be prepared as
described below. Upon exposure of recognition complexes
incorporating the high affinity ligands to a sample, the presence
of the biological molecule is indicated by its binding to the
ligand. Since binding of analyte to ligand results in an
electrochemical signal, the concentration of biological molecule in
the sample can be readily determined by quantifying the signal.
Where the biological molecule of interest is part of a
macromolecular complex, flow cytometry may also be used to detect
and quantify the amount of biological molecule in a sample.
[0019] In certain embodiments, the recognition complex system may
be used to enrich or purify analytes that bind to one or more
selected nucleic acid ligands. In a preferred embodiment, selected
nucleic acid ligands are attached to a surface and exposed to a
population of analytes. After binding of analyte to nucleic acid
ligand, the unbound analytes are removed and the enriched or
purified bound analyte is eluted from the ligand. Enrichment and
purification may occur using either an interative process, with
multiple cycles of binding, separation and elution, or by a
single-step process. Separation of bound from unbound analyte may
occur by any method known in the art. In a non-limiting example,
the ligands may be attached to a column chromatography resin or
other solid support and exposed to a mixture of analytes. Unbound
analyte may be removed by simple washing of the column or other
support. Bound analyte may be eluted by exposure to solutions
containing appropriate salt concentration, pH, detergent content,
chaotrophic agent or other substance that interferes with the
binding interaction. Depending on the affinity of analyte for
ligand and the stringency of the initial binding interaction, it
may be possible to obtain a relatively purified analyte with a
single binding step.
[0020] In certain embodiments, the recognition complexes may be
attached to a surface, such as a Langmuir-Blodgett film,
functionalized glass, germanium, silicon, PTFE, polystyrene,
gallium arsenide, gold, silver, membrane, nylon, glass bead,
magnetic bead or PVP. In preferred embodiments, the recognition
complex system of the present invention employs organic
semiconductor chip technology wherein nucleic acid ligands are
distributed across the surface of the chip so as to form an array
of recognition complexes. In other embodiments, the recognition
complexes of the present invention may be attached to a surface for
use in a flow cell apparatus.
[0021] In additional embodiments, the nucleic acid ligands are
attached to magnetic beads instead of to a chip. An array of
nucleic acid ligands may be assembled, each attached to a magnetic
bead. In certain embodiments, each nucleic acid ligand attached to
a single magnetic bead has the same nucleic acid sequence, while in
other embodiments a single magnetic bead may be attached to nucleic
acid ligands of different sequences. In a preferred embodiment, the
magnetic bead is attached to an organic semiconductor, such as
DALM, and the nucleic acid ligand is attached to the organic
semiconductor, forming an array of recognition complexes. Although
any method may be employed within the scope of the present
invention to attach the organic semiconductor to the magnetic bead
and the nucleic acid ligand to the organic semiconductor, in a
preferred embodiment the organic semiconductor is covalently
attached to the magnetic bead and the nucleic acid ligand is
non-covalently attached to the organic semiconductor. In a more
preferred embodiment, the attachment of nucleic acid ligand to
organic semiconductor is an electrostatic interaction, preferably
mediated by magnesium ion.
[0022] In certain embodiments, an array of recognition complexes
attached to magnetic beads is exposed to an analyte and binding of
analyte to nucleic acid ligand may be detected, for example, by
photochemical changes in the nucleic acid ligand/DALM couplet upon
binding to the analyte. The skilled artisan will realize that
magnetic beads would be particularly useful for separating
recognition complexes that bind to the analyte from recognition
complexes that do not bind the analyte. In one embodiment, a
magnetic flow cell, such as is described in U.S. Pat. No. 5,972,721
(incorporated herein by reference), could be used in conjunction
with the recognition complex system to identify and separate
analyte-binding recognition complexes from recognition complexes
that do not bind the analyte.
[0023] In certain preferred embodiments, flow cytometry is used to
separate recognition complexes that bind to an analyte from those
that do not bind. In such embodiments, the recognition complex may
be attached to a glass or other bead, or the analyte may comprise a
population of cells, spores or other large particles for analytical
or preparative procedures. Nucleic acid ligands that bind to the
target analyte, or analytes that bind to a specific nucleic acid
ligand, may be sorted, for example, by screening particles for
DALM-associated fluorescence in a flow cytometer.
[0024] In certain embodiments, the recognition complex system may
be subject to an iterative process to increase the specificity and
affinity of the nucleic acid ligands for an analyte of interest. In
such embodiments, nucleic acid ligand sequences that bind a
selected analyte are identified, separated, amplified (e.g., using
a polymerase chain reaction) and attached to organic semiconductor
to form a new recognition complex system. The nucleic acid ligand
sequences that do not bind to the analyte are discarded. The new
recognition complex system is exposed to the analyte and binding of
analyte to nucleic acid ligands produces an enhanced
electrochemical signature, as the nucleic acid ligand sequences
present will more specifically compliment the analyte. This
procedure may be repeated, with each iteration producing a more
unique or enhanced signature.
[0025] In a further embodiment, this iterative process may be used
to identify and amplify one or more nucleic acid ligand sequences
that exhibit the highest degree of affinity for a specific analyte.
Production of a nucleic acid ligand that binds to the analyte with
high affinity (dissociation constant of 1.0 .mu.M or lower) would
have utility in a variety of applications. For certain embodiments,
production of a nucleic acid ligand with a dissociation constant of
100 nM or lower, more preferably 10 nM or lower, most preferably 1
nM or lower is preferred. This process also provides a method for
purifying a nucleic acid ligand that binds to a target analyte.
Purification may be less than 100%, the only requirement being that
the nucleic acid ligand of interest is present in significantly
greater proportion in the final mixture compared to the starting
material. A "purified" nucleic acid ligand may comprise 10% or
more, preferably 20% or more, more preferably 40% or more, more
preferably 60% or more, more preferably 80% or more, more
preferably 95% or more of the total nucleic acid content of the
"purified" fraction.
[0026] It is contemplated within the scope of the present invention
that separation of bound from unbound nucleic acid ligands may
occur using virtually any method that can separate bound from
unbound ligands. Non-limiting examples include use of nucleic acid
chips, use of magnetic beads and magnetic filters, use of glass or
other beads and flow cytometry, and flow cytometry using cells as
the target analyte. In any case, further iterations of the binding
and separation steps will result in progressive enrichment
(purification) of ligands that bind to the analyte. If desired, the
stringency of the binding interaction may be increased, for example
by increasing the temperature or by raising or lowering the salt
concentration or the pH of the solution.
[0027] In another embodiment, nucleic acid ligands that bind to the
analyte with high affinity can be reproduced (synthesized or
amplified) for use as a neutralizing agent to inactivate or destroy
the analyte. A high affinity nucleic acid ligand may be attached to
a variety of agents that could be used to neutralize the analyte,
such as toxic proteins, enzymes capable of activating protoxins, or
other molecules or reactive moieties including radioisotopes and
other organic or inorganic compounds. In certain embodiments, the
high affinity nucleic acid ligand can be attached to an organic
semiconductor, such as DALM. The DALM/nucleic acid ligand couplet,
after binding to the analyte, may be activated by a variety of
techniques, including exposure to sunlight, heat, or irradiation of
various types, including laser, microwave, radiofrequency,
ultraviolet and infrared. Activation of the DALM/nucleic acid
ligand couplet results in absorption of energy, which may be
transmitted to the analyte, inactivating or destroying it. See U.S.
Pat. No. 6,303,316, incorporated herein by reference.
[0028] In certain embodiments, the high affinity nucleic acid
ligand could be incorporated into an apparatus capable of being
carried into the field. For example, the high affinity nucleic acid
ligand could be incorporated into a patch or card to be worn by an
individual. Exposure of the individual to the specific analyte for
which the nucleic acid ligand exhibits high affinity could be
indicated by a color change of the patch, or by a change in the
electrical or photochemical properties of a nucleic acid
ligand/organic semiconductor couplet. Alternatively, the high
affinity nucleic acid ligand could be incorporated into an
apparatus to be carried by a vehicle that could be used to cover a
wide area to detect and identify unknown chemical or biological
agents.
[0029] The skilled artisan will realize that the scope of the
present invention is not limited to applications in chemical or
biological warfare, but rather includes a broad variety of
potential applications in industry and medicine, where early
detection and identification of exposure to chemical or biological
agents is desired. Non-limiting examples of such applications
include to detect explosives or illegal drugs in an airport
detection system, to detect air-borne pathogens in an air
conditioner monitoring system, to detect water-borne pathogens,
carcinogens, teratogens or toxins in a water quality monitoring
system, to detect pathogens in a hospital operating room monitoring
system, to screen for pathogens in samples of human tissues or
fluids, to detect allergens, pathogens or contaminants in a food
production monitoring system, to detect genetically modified
organisms, or to perform high through-put screening for
pharmaceutical compounds.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0031] FIG. 1 illustrates a recognition complex system in
accordance an exemplary embodiment of the present invention.
[0032] FIG. 2 illustrates another exemplary embodiment of a
recognition complex system, using recognition complexes attached to
magnetic beads. The flow chart illustrates the operational
relationships between the components of a preferred embodiment of a
recognition complex system.
[0033] FIG. 3 illustrates a process for separation of recognition
complexes, comprising magnetic beads, that bind analyte from those
that do not, as well as an iterative process for producing nucleic
acid ligands that bind to an analyte with high affinity.
[0034] FIG. 4 illustrates a PCR amplified product from an
anti-anthrax aptamer after ten cycles of selection.
[0035] FIG. 5A-FIG. 5B show the destruction of an anthrax spore
using DALM and a high power microwave pulse.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0036] Definitions
[0037] As used herein, "a" or "an" may mean one or more than one of
an item.
[0038] "Nucleic acid" means either DNA, RNA, single-stranded,
double-stranded or triple stranded and any chemical modifications
thereof. Virtually any modification of the nucleic acid is
contemplated by this invention. Non-limiting examples of nucleic
acid modifications are discussed in further detail below. "Nucleic
acid" encompasses, but is not limited to, oligonucleotides and
polynucleotides. "Oligonucleotide" refers to at least one molecule
of between about 3 and about 100 nucleotides in length.
"Polynucleotide" refers to at least one molecule of greater than
about 100 nucleotides in length. These terms generally refer to at
least one single-stranded molecule, but in certain embodiments also
encompass at least one additional strand that is partially,
substantially or fully complementary in sequence. Thus, a nucleic
acid may encompass at least one double-stranded molecule or at
least one triple-stranded molecule that comprises one or more
complementary strand(s) or "complement(s)." As used herein, a
single stranded nucleic acid may be denoted by the prefix "ss", a
double stranded nucleic acid by the prefix "ds", and a triple
stranded nucleic acid by the prefix "ts."
[0039] Within the practice of the present invention, a "nucleic
acid" may be of almost any length, from 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35,40, 45, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 175, 200, 225,
250, 275, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000,
2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10000,
15000, 20000 or even more bases in length. The term "nucleic acid"
will generally refer to at least one molecule or strand of DNA, RNA
or a derivative or mimic thereof, comprising at least one
nucleobase. A "nucleobase" refers to a heterocyclic base, for
example, a purine or pyrimidine base naturally found in DNA (e.g.
adenine "A," guanine "G," thymine "T" and cytosine "C") or RNA
(e.g. A, G, uracil "U" and C), as well as their derivatives and
mimics. A "derivative" refers to a chemically modified or altered
form of a naturally occurring molecule, while "mimic" and "analog"
refer to a molecule that may or may not structurally resemble a
naturally occurring molecule, but that functions similarly to the
naturally occurring molecule.
[0040] As used herein, a "moiety" generally refers to a smaller
chemical or molecular component of a larger chemical or molecular
structure.
[0041] A "nucleoside" is an individual chemical unit comprising a
nucleobase covalently attached to a nucleobase linker moiety. An
example of a "nucleobase linker moiety" is a sugar comprising
5-carbon atoms (a "5-carbon sugar"), including but not limited to
deoxyribose, ribose or arabinose, and derivatives or mimics of
5-carbon sugars. Examples of derivatives or mimics of 5-carbon
sugars include 2'-fluoro-2'-deoxyribose or carbocyclic sugars where
a carbon is substituted for the oxygen atom in the sugar ring.
[0042] A "nucleotide" refers to a nucleoside further comprising a
"backbone moiety" used for the covalent attachment of one or more
nucleotides to another molecule or to each other to form a nucleic
acid. The "backbone moiety" in naturally occurring nucleotides
typically comprises a phosphorus moiety covalently attached to a
5-carbon sugar. The attachment of the backbone moiety typically
occurs at either the 3'- or 5'-position of the 5-carbon sugar.
However, other types of attachments are known in the art,
particularly when the nucleotide comprises derivatives or mimics of
a naturally occurring 5-carbon sugar or phosphorus moiety.
[0043] "Nucleic acid ligand" means a non-naturally occurring
nucleic acid having a desirable action on a target. A desirable
action includes, but is not limited to, binding of the target,
catalytically changing the target, reacting with the target in a
way that modifies or alters the target or the functional activity
of the target, covalently attaching to the target, facilitating the
reaction between the target and another molecule, and neutralizing
the target. In a preferred embodiment, the action is specific
binding affinity for a target molecule, such target molecule being
a three dimensional chemical structure. The meaning of "nucleic
acid ligand" specifically excludes nucleic acids that bind to
another nucleic acid through a mechanism which predominantly
depends on Watson/Crick base pairing. Nucleic acid ligands include,
but are not limited to, nucleic acids that are identified by the
SELEX process discussed below.
[0044] "SELEX" (Systematic Evolution of Ligands by Exponential
enrichment) involves the combination of selection of nucleic acid
ligands which interact with a target in a desirable manner, for
example binding to the target, with amplification of those selected
nucleic acid ligands. Iterative cycling of the
selection/amplification steps allows selection of one or a small
number of nucleic acid ligands that interact most strongly with the
target from a pool that contains a very large number of nucleic
acid ligands. Cycling of the selection/amplification procedure is
continued until a selected goal is achieved. In certain embodiments
of the present invention, the goal may be to produce one or more
nucleic acid ligands that, for example, can be used to bind to and
detect, identify, quantify, neutralize or destroy an analyte.
Non-limiting examples of analytes include a toxin, poison,
allergen, virus, bacterium, spore or other biological or chemical
agent.
[0045] "Aptamer" means a nucleic acid that binds to another
molecule ("target," as defined below). This binding interaction
does not encompass standard nucleic acid/nucleic acid hydrogen bond
formation exemplified by Watson-Crick basepair formation (e.g., A
binds to U or T and G binds to C), but encompasses all other types
of non-covalent (or in some cases covalent) binding. Non-limiting
examples of non-covalent binding include hydrogen bond formation,
electrostatic interaction, Van der Waals interaction and
hydrophobic interaction. An aptamer may bind to another molecule by
any or all of these types of interaction, or in some cases by
covalent interaction. Covalent binding of an aptamer to another
molecule may occur where the aptamer or target molecule contains a
chemically reactive or photoreactive moiety. The term "aptamer"
refers to a nucleic acid that is capable of forming a complex with
an intended target substance. "Target-specific" means that the
aptamer binds to a target analyte with a much higher degree of
affinity than it binds to contaminating materials.
[0046] "Analyte," "target" and "target analyte" mean any compound
or aggregate of interest. Non-limiting examples of analytes include
a protein, peptide, carbohydrate, polysaccharide, glycoprotein,
lipid, hormone, receptor, antigen, allergen, antibody, substrate,
metabolite, cofactor, inhibitor, drug, pharmaceutical, nutrient,
toxin, cholera toxin, Shiga-like toxin, poison, explosive,
pesticide, chemical warfare agent, biohazardous agent, prion,
radioisotope, vitamin, heterocyclic aromatic compound, carcinogen,
mutagen, narcotic, amphetamine, barbiturate, hallucinogen, waste
product, contaminant or other molecule. Molecules of any size can
serve as targets. "Analytes" are not limited to single molecules,
but may also comprise complex aggregates of molecules, such as a
virus, bacterium, spore, mold, yeast, algae, amoebae,
dinoflagellate, unicellular organism, pathogen, cell or infectious
agent. In certain embodiments, cells exhibiting a particular
characteristic or disease state, such as a cancer cell, may be
target analytes. Virtually any chemical or biological effector
would be a suitable target. In particularly preferred embodiments,
the analyte is anthrax.
[0047] Non-limiting examples of infectious agents within the
meaning of "analyte" include the following.
1 Actinobacillus spp. Actinomyces spp. Adenovirus (types 1, 2, 3,
4, 5 et 7) Adenovirus (types 40 and 41) Aerococcus spp. Aeromonas
hydrophila Ancylostoma duodenale Angiostrongylus cantonensis
Ascaris lumbricoides Ascaris spp. Aspergillus spp. Bacillus
anthracis Bacillus cereus Bacteroides spp. Balantidium coli
Bartonella bacilliformis Blastomyces dermatitidis Bluetongue virus
Bordetella bronchiseptica Bordetella pertussis Borrelia burgdorferi
Branhamella catarrhalis Brucella spp. B. abortus B. canis, B.
melitensis B. suis Brugia spp. Burkholderia mallei Burkholderia
pseudomallei Campylobacter fetus subsp. fetus Campylobacter jejuni
C. coli C. fetus subsp. jejuni Candida albicans Capnocytophaga spp.
Chlamydia psittaci Chlamydia trachomatis Citrobacter spp.
Clonorchis sinensis Clostridium botulinum Clostridium difficile
Clostridium perfringens Clostridium tetani Clostridium spp.
Coccidioides immitis Colorado tick fever virus Corynebacterium
diphtheriae Coxiella burnetii Coxsackievirus Creutzfeldt-Jakob
agent, Kuru agent Crimean-Congo hemorrhagic fever virus
Cryptococcus neoformans Cryptosporidium parvum Cytomegalovirus
Dengue virus (1, 2, 3, 4) Diphtheroids Eastern (Western) equine
encephalitis virus Ebola virus Echinococcus granulosus Echinococcus
multilocularis Echovirus Edwardsiella tarda Entamoeba histolytica
Enterobacter spp. Enterovirus 70 Epidermophyton floccosum,
Microsporum spp. Trichophyton spp. Epstein-Barr virus Escherichia
coli, enterohemorrhagic Escherichia coli, enteroinvasive
Escherichia coli, enteropathogenic Escherichia coli,
enterotoxigenic Fasciola hepatica Francisella tularensis
Fusobacterium spp. Gemella haemolysans Giardia lamblia Giardia spp.
Haemophilus ducreyi Haemophilus influenzae (group b) Hantavirus
Hepatitis A virus Hepatitis B virus Hepatitis C virus Hepatitis D
virus Hepatitis E virus Herpes simplex virus Herpesvirus simiae
Histoplasma capsulatum Human coronavirus Human immunodeficiency
virus Human papillomavirus Human rotavirus Human T-lymphotrophic
virus Influenza virus Junin virus/Machupo virus Klebsiella spp.
Kyasanur Forest disease virus Lactobacillus spp. Legionella
pneumophila Leishmania spp. Leptospira interrogans Listeria
monocytogenes Lymphocytic choriomeningitis virus Marburg virus
Measles virus Micrococcus spp. Moraxella spp. Mycobacterium spp.
Mycobacterium tuberculosis, M. bovis Mycoplasma hominis, M. orale,
M. salivarium, M. fermentans Mycoplasma pneumoniae Naegleria
fowleri Necator americanus Neisseria gonorrhoeae Neisseria
meningitidis Neisseria spp. Nocardia spp. Norwalk virus Omsk
hemorrhagic fever virus Onchocerca volvulus Opisthorchis spp.
Parvovirus B19 Pasteurella spp. Peptococcus spp. Peptostreptococcus
spp. Plesiomonas shigelloides Powassan encephalitis virus Proteus
spp. Pseudomonas spp. Rabies virus Respiratory syncytial virus
Rhinovirus Rickettsia akari Rickettsia prowazekii, R. canada
Rickettsia rickettsii Ross river virus/O'Nyong-Nyong virus Rubella
virus Salmonella choleraesuis Salmonella paratyphi Salmonella typhi
Salmonella spp. Schistosoma spp. Scrapie agent Serratia spp.
Shigella spp. Sindbis virus Sporothrix schenckii St. Louis
encephalitis virus Murray Valley encephalitis virus Staphylococcus
aureus Streptobacillus moniliformis Streptococcus agalactiae
Streptococcus faecalis Streptococcus pneumoniae Streptococcus
pyogenes Streptococcus salivarius Taenia saginata Taenia solium
Toxocara canis, T. cati Toxoplasma gondii Treponema pallidum
Trichinella spp. Trichomonas vaginalis Trichuris trichiura
Trypanosoma brucei Ureaplasma urealyticum Vaccinia virus
Varicella-zoster virus Venezuelan equine encephalitis Vesicular
stomatitis virus Vibrio cholerae, serovar 01 Vibrio
parahaemolyticus Wuchereria bancrofti Yellow fever virus Yersinia
enterocolitica Yersinia pseudotuberculosis Yersinia pestis
[0048] "Binding" refers to an interaction or binding between a
target and a nucleic acid ligand or aptamer, resulting in a
sufficiently stable complex so as to permit separation of nucleic
acid ligand:target complexes from uncomplexed nucleic acid ligands
under given binding or reaction conditions. Binding is mediated
through hydrogen bonding, electrostatic interaction, hydrophobic
interaction, Van der Walls forces or other molecular forces. In
certain embodiments, binding may be covalent, for example where the
nucleic acid ligand or analyte contains a photoreactive or
chemically reactive moiety to promote covalent attachment of ligand
and analyte. Covalent binding may be desirable, for example, where
an analyte or ligand is labeled to facilitate purification of the
analyte:ligand pair.
[0049] "Organic semiconductor" means a conjugated (alternating
double and single bonded) organic compound in which regions of
electrons and the absence of electrons (holes or positive charges)
can move with varying degrees of difficulty through the aligned
conjugated system (varying from insulator to conductor). An organic
semiconductor may be thought of as the organic equivalent of a
metal, in terms of electrical properties. Organic semiconductors
are distinguished from metals in their spectroscopic properties.
Organic semiconductors of use in the practice of the instant
invention may be fluorescent, luminescent, chemiluminescent,
sonochemiluminescent, thermochemiluminescent or
electrochemiluminescent or may be otherwise characterized by their
absorption, reflection or emission of electromagnetic radiation,
including infrared, ultraviolet or visible light. In certain
embodiments, the organic semiconductor is DALM, although
alternative forms of organic semiconductor are contemplated within
the scope of the invention.
[0050] "Recognition complex" refers to a nucleic acid ligand that
is operably coupled to an organic semiconductor. "Operably coupled"
means that the nucleic acid ligand and the organic semiconductor
are in close physical proximity to each other, such that binding of
an analyte to the nucleic acid ligand results in a change in the
properties of the organic semiconductor that is detectable as a
signal. In preferred embodiments, the signal is an electrochemical
signal, such as a photochemical signal, a fluorescent signal, a
luminescent signal, a change of color or a change in electrical
conductivity. In one preferred embodiment, the signal is a change
in the fluorescence emission profile of the organic
semiconductor/nucleic acid ligand couplet. Operable coupling may be
accomplished by a variety of interactions, including but not
limited non-covalent or covalent binding of the organic
semiconductor to the nucleic acid ligand. In another embodiment,
the nucleic acid ligand may be at least partially embedded in the
organic semiconductor. Virtually any type of interaction between
the organic semiconductor and the nucleic acid ligand is
contemplated within the scope of the present invention, so long as
the binding of an analyte to the nucleic acid ligand results in a
change in the properties of the organic semiconductor. In one
preferred embodiment, the nucleic acid ligand is electrostatically
linked to the organic semiconductor by a magnesium ion bridge. In
an alternate embodiment, the nucleic acid ligand is covalently
linked to the semiconductor by chemical cross-linking. A number of
suitable chemical cross-linking reagents are well known in the art,
such as EDC (1-ethyl-3-(2-dimethylaminopropyl)carbodiimide).
[0051] A "recognition complex system" comprises an array of
recognition complexes. In preferred embodiments, the array of
recognition complexes is operably coupled to a detection unit, such
that changes in the electrochemical properties of the organic
semiconductor that result from binding of analyte to nucleic acid
ligand may be detected by the detection unit. It is contemplated
within the scope of the present invention that detection may be an
active process or a passive process. For example, in embodiments
where the array of recognition complexes is incorporated into a
card or badge, the binding of analyte may be detected by a change
in color of the card or badge. In other embodiments, detection
occurs by an active process, such as scanning the fluorescence
emission profile of an array of recognition complexes.
[0052] "Electrochemical" is used in a broad sense to mean any
process involving a transfer of electrons, including
reduction-oxidation chemistry of any sort. "Electrochemical"
specifically includes photo-induced oxidation and reduction.
[0053] "Photochemical" means any light related or light induced
chemistry. A "photochemical signal" specifically includes, but is
not limited to, a fluorescent signal, a luminescent signal, a
change of color, a change in electrical conductivity,
photo-oxidation and photo-reduction.
[0054] "Magnetic bead," "magnetic particle" and "magnetically
responsive particle" are used herein to mean any particle
dispersible or suspendable in aqueous media, without significant
gravitational settling and separable from suspension by application
of a magnetic field. The particles comprise a magnetic metal oxide
core, often surrounded by an adsorptively or covalently bound
sheath or coat bearing functional groups to which various
molecules, such as DALM or DNA, may be covalently coupled or
adsorbed.
[0055] In certain embodiments, non-magnetic beads, such as
functionalized or non-functionalized glass, or functionalized or
non-functionalized polystyrene, may be used as surfaces for the
attachment of recognition complexes and the separation of
recognition complexes bound to analyte from complexes that do not
bind analyte.
[0056] Recognition Complex System
[0057] An embodiment of the instant invention relates to
compositions and apparatus capable of undergoing a process that
selectively amplifies nucleic acid ligands that bind to a target
analyte. This recognition complex system comprises an array of
recognition complexes, each recognition complex comprising a
nucleic acid ligand. In various embodiments, the nucleic acid
ligand may be attached to an organic semiconductor, such as DALM.
In certain embodiments, the recognition complexes are arranged in a
two-dimensional array, that may be attached to a glass or other
flat surface. In other embodiments, the recognition complexes
comprise nucleic acid ligands attached to magnetic bead or to
non-magnetic beads, such as glass, polystyrene, or polyacrylamide
beads, in a three-dimensional array. In a preferred embodiment, the
beads are suspended in a liquid medium.
[0058] The array of recognition complexes is exposed to analyte.
Binding of analyte to individual recognition complexes is detected
by, for example, changes in the electrical or photochemical
properties of the recognition complex upon binding to the analyte.
Where the recognition complexes comprise an organic semiconductor,
such as DALM, the changes in electrical or photochemical properties
may be detected by a variety of techniques, described in detail
below.
[0059] In certain embodiments, an iterative process may be used to
increase the specificity of the array of recognition complexes for
the analyte. In each round of iteration, the array is exposed to
the analyte. Recognition complexes that bind to the analyte are
separated from recognition complexes that do not bind to the
analyte. Methods for separating bound from unbound recognition
complexes are also described in detail below. The nucleic acid
ligands from recognition complexes that bind to the analyte are
amplified, for example by PCR, and used to make a new array of
recognition complexes. The new array will contain a higher
proportion of recognition complexes that bind to the analyte,
producing a stronger and more specific electrical or photochemical
signal. As discussed below, certain aspects of this process
resemble SELEX technology (Tuerk and Gold, 1990; Klug and Famulok,
1994; Tuerk, 1993, 1997, U.S. Pat. Nos. 5,270,163; 5,475,096;
5,567,588; 5,580,737; 5,595,877; 5,641,629; 5,650,275; 5,683,867;
5,696,249; 5,707,796; 5,763,177; 5,817,785; 5,874,218; 5,958,691;
6,001,577; 6,030,776; each incorporated herein by reference). With
each round of iteration, a set of nucleic acid ligands will be
produced that bind to the analyte with greater affinity. This
iterative process may also be used to produce nucleic acid ligands
that bind to the analyte with high affinity. Such high affinity
nucleic acid ligands will be useful in numerous applications,
described below. One such application involves production of a
neutralizing agent that can inactivate or destroy the target
analyte.
[0060] Embodiments Involving a Chip Type of Array
[0061] FIG. 1 illustrates a recognition complex system in
accordance with an exemplary embodiment of the present invention.
This embodiment of the recognition complex system includes a sample
collection unit 105, an analyte isolation unit 110, an organic
semiconductor chip based array of recognition complexes 115, a
detection unit 120 and a data storage and processing unit 125. In
general, the sample collection unit 105 is employed to actively
collect or passively receive samples containing the unknown analyte
to be identified. The analyte isolation unit 110 is employed to
filter the sample and isolate the unknown analyte from other
substances or compounds that might be present in the sample. The
sample collection unit 105 and the analyte isolation unit 110 may
be implemented in accordance with any number of known techniques
and/or components known in the art.
[0062] The array of recognition complexes 115 comprises one or more
individual recognition complexes 130. It will be understood that
the array of recognition complexes 115 is shown as comprising 15
recognition complexes for illustrative purposes only. In actuality,
the array 115 may contain significantly more than 15 recognition
complexes. Within the scope of the invention, the array may
comprise approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95, 100, 110, 120, 125, 130, 140, 150, 160, 170,
175, 180, 185, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400,
425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,
1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000,
8000, 9000, 10000, 15000, 20000, 30000, 40000, 50000, 75000, 10000,
20000, 30000, 40000, 50000, 100000, 200000, 500000, 10.sup.6,
10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12,
10.sup.14, 10.sup.16, 10.sup.18, 10.sup.20, 10.sup.22, up to
10.sup.24 recognition complexes or any number in between. In
certain embodiments, the nucleic acid ligand component of each
recognition complex differs in sequence from the nucleic acid
ligand component of the other recognition complexes in the array.
In other embodiments, some or all of the nucleic acid ligands may
be similar or identical in sequence.
[0063] Each of the recognition complexes 130 associated with the
array 115 comprises a nucleic acid ligand/organic semiconductor
couplet. In a preferred embodiment, the couplet is sandwiched
between a pair of electrodes, one of which is preferably
transparent. The recognition complexes may be sandwiched between
two electrodes with (for alternating current or forward and reverse
DC bias) or without (for DC only) intervening insulating layers.
This embodiment provides a recognition complex system formed from a
miniaturized array of light-emitting diodes. One of the electrodes
is transparent to allow for the passage of light. The other
electrode is made of a conductive substance such as copper,
aluminum, or gold.
[0064] In certain embodiments, the organic semiconductor used in
diazoluminomelanin (DALM). DALM is a polymer that exhibits slow
fluorescent, chemiluminescent, sonochemiluminescent,
thermochemiluminescent and electrochemiluminescent properties
(Bruno and Yu, 1996). However, other organic semiconductors may
serve as acceptable substitutes, in particular, polyphenylenes. A
non-limiting example of a polyphenylene that might be used within
the scope of the instant invention is poly(para-phenylenevinylene)
(Kugler et al., 1999).
[0065] As shown in FIG. 1, the recognition complex system comprises
an array 115 of recognition complexes, such as recognition complex
130. Each of these recognition complexes comprises a nucleic acid
ligand/organic semiconductor couplet. Separating each of the
recognition complexes is binding material. The nucleic acid ligand
sequences present at each of the recognition complexes may be
random sequences. In an exemplary embodiment, the nucleic acid
ligand sequences may be distributed across the array as a function
of charge and size, or alternatively as a function of charge and pI
(isoelectric point).
[0066] After collecting and isolating the unknown analyte, the
analyte is applied to each recognition complex associated with the
array 115. In those embodiments where the nucleic acid ligand
sequences are not identical, some of the nucleic acid ligands will
exhibit a high affinity for the analyte, some nucleic acid ligands
will exhibit less affinity for the analyte and some nucleic acid
ligands will exhibit no affinity for the analyte. The
electrochemical properties of the nucleic acid ligand/organic
semiconductor couplet will change depending on the degree to which
the nucleic acid ligands bind to the analyte. The electrotochemical
properties associated with some recognition complexes will change
significantly, while the electrochemical properties associated with
other recognition complexes may change very little, if at all, upon
exposure to a given analyte.
[0067] In accordance with one exemplary embodiment, one of the
electrodes associated with each recognition complex is transparent.
The transparency of this electrode permits excitation energy, such
as light, to be transmitted through each recognition complex. In a
preferred embodiment, ultra-violet light is employed. The passage
of ultra-violet or other frequency irradiation through each of the
recognition complexes 130 may permit detection unit 120 to more
easily detect and quantify any electrochemical changes that take
place at each recognition complex 130 as a result of binding to the
analyte. The electrochemical changes may involve changes in the
color of the nucleic acid ligand/organic semiconductor couplet
and/or changes in the color intensity. In preferred embodiments,
the detection unit 120 comprises a charge coupled device (CCD),
such as a CCD camera, digital camera, photomultiplier tube or any
other functionally equivalent detector.
[0068] The electrochemical signature of the analyte may consist of
a two-dimensional distribution of fluorescence resulting from
long-wavelength ultraviolet light excitation. Response of the array
115 at a specific spatial location 130 may be similar for two or
more different analytes, but by combining the fluorescence response
of many independent measurement locations, specificity can be high.
A typical consumer-type CCD-based color video camera has
768.times.494 discrete detectors. A miniaturized cell utilizing
such a camera with an array could have about 380,000 parallel
channels (single detectors). Practical considerations would group
detectors for lower but less spatially noisy resolution with fewer
channels. Hundreds to thousands of channels could easily be
achieved. Optimization of the number of channels would minimize
channels and thus computational load, while maximizing specificity
and classification accuracy.
[0069] Analysis of the photochemical signature, by data processing
unit 125, may involve a comparison of multiple channels of
fluorescence spectral signatures. Comparison of signatures by data
processing unit 125 may be implemented using artificial neural
networks (such as the Qnet v2000 neural net software package from
Vesta Services, Inc., 1001 Green Bay Rd., Winnetka, Ill. 60093).
This would provide a fast comparison of unknown analytes to a
database of previously recorded signatures of known analytes.
[0070] Any binding between the analyte and the nucleic acid ligand
associated with a given recognition complex may alter the
electrical properties of the corresponding nucleic acid
ligand/organic semiconductor couplet. In another exemplary
embodiment, a voltage is applied across each recognition complex of
the array 115 after the analyte has been introduced. The amount of
current that is able to flow across each recognition complex is a
function of the conductivity of the nucleic acid ligand/organic
semiconductor couplet. Changes in conductivity of each couplet upon
binding of analyte may be stored and analyzed to identify the
analyte.
[0071] In certain embodiments, voltage may be applied across each
of the recognition complexes in addition to exciting each
recognition complex with ultraviolet or other frequency
irradiation. In such embodiments, changes in both the electrical
properties and the photochemical properties of each recognition
complex may be detected and analyzed. These combined data may more
readily establish a unique signature for identifying the analyte.
In these embodiments, the detection unit 120 would have to include
the ability to detect both changes in current and photochemical
changes at each of the recognition complexes. Application of a
current flowing through the recognition complexes may result in the
enhancement of any photochemical changes that take place as a
result of analyte/nucleic acid ligand binding, thereby making it
easier for the detection unit 120 to detect and quantify those
photochemical changes.
[0072] In accordance with one aspect of the present invention,
unknown chemical and biological analytes may be detected and
identified in a single, automated binding step, as the reaction
between the analyte and the nucleic acid ligand sequences
distributed across the array 115 produces a relatively unique
change in the electrochemical properties of the array as a whole.
However, where two or more analytes share similar chemical
structures, they might cause the array 115 to produce a relatively
similar electrochemical response.
[0073] Thus, in accordance with another aspect of the present
invention, a more unique electrochemical response from the array
115 can be achieved to more clearly distinguish between
structurally similar analytes. To accomplish this, the nucleic acid
ligands associated with those recognition complexes that bind to
the analyte, as indicated by changes in electrochemical properties,
may extracted from the array.
[0074] In certain embodiments, individual recognition complexes 130
may be detached from the array 115 by hydrolysis, cleavage, heating
or other methods of dissociation applied to the array at the
location of each such recognition complex. The nucleic acid ligand
sequences exhibiting affinity for analyte may be separated from the
analyte by washing the nucleic acid ligand bound to analyte with
deionized water, salt solutions, detergents, chaotrophic agents,
solvents or other solutions that serve to separate the analyte from
ligand. The nucleic acid ligand sequences that exhibit no affinity
for the analyte can be discarded. The extracted nucleic acid ligand
sequences may be amplified and applied to a clean chip to produce a
new array 115. Since the new array 115 comprises only those nucleic
acid ligand sequences that were identified as binding to the
analyte, it should exhibit a greater degree of specificity and a
higher binding affinity for the analyte.
[0075] As the process of amplification inherently produces some
variation in the amplified nucleic acid ligand sequences, due to
the normal error rate of DNA or RNA polymerase, the amplified
nucleic acid ligands may exhibit some sequences that were not
present on the initial array, although they will generally be
identical or almost identical in sequence to the original nucleic
acid ligands. These sequence variants may also exhibit variability
in their binding affinity for the analyte, with some sequence
variants exhibiting an increased affinity for analyte. The
iterative process may be used to select for nucleic acid ligand
sequences that bind to analyte with higher affinity with each round
of iteration. The skilled artisan will realize that use of
polymerases with a greater inherent error rate, or manipulation of
amplification conditions to increase the error rate, may be
desirable in certain embodiments of the present invention.
[0076] Once a new array chip 115 is produced, analyte may be
introduced to each of the array recognition complexes 130, and the
electrochemical changes across the array may be detected and
analyzed, producing an even more unique signature that can be used
for analyte identification and to distinguish the analyte from
chemically or structurally similar species.
[0077] The production of chips for attachment of nucleic acid
ligands is well known in the art. The chip may comprise a
Langmuir-Bodgett film, functionalized glass, germanium, silicon,
PTFE, polystyrene, gallium arsenide, gold, silver, membrane, nylon,
PVP, or any other material known in the art that is capable of
haying functional groups such as amino, carboxyl, Diels-Alder
reactants, thiol or hydroxyl incorporated on its surface. In
certain embodiments, these groups may be covalently attached to
cross-linking agents so that binding interactions between analyte
and recognition complex occur without steric hindrance from the
chip surface. Typical cross-linking groups include ethylene glycol
oligomer, diamines and amino acids. Any suitable technique useful
for immobilizing a recognition complex on a chip is contemplated by
this invention, including sialinization. In certain embodiments,
DALM is attached to the chip surface and nucleic acid ligands are
then attached, covalently or non-covalently, to the DALM.
[0078] The array-based chip design 115 may be distinguished from
conventional biochips (e.g., U.S. Pat. Nos. 5,861,242 and
5,578,832) by a number of characteristics, including the use of an
organic semiconductor, such as DALM. Additionally, conventional
biochips typically are constructed by attaching or synthesizing
nucleic acid ligands having affinities for known analytes on
specific identified locations on the chip. The presence of a target
analyte in a sample is detected by binding to the specific chip
locus containing a nucleic acid ligand with known affinity for that
analyte. In contrast, in certain embodiments of the present
invention the affinities of the nucleic acid ligand/organic
semiconductor couplets for various analytes are unknown at the time
they are initially attached to the chip. Target analytes are
identified by their pattern of binding to the entire chip, not by
their binding to a specific locus on the chip. This system provides
greater efficiency and flexibility, in that it is not necessary to
prepare nucleic acid ligands of known specificity before
construction of the chip. Further, previously unknown analytes may
be characterized by their pattern of interaction with the chip,
without having to clone and sequence their RNA or DNA or prepare
high-affinity aptamers in advance of chip production.
[0079] This is not meant to exclude the possibility of selecting
for the presence of one or more nucleic acid ligands with higher
affinity for the target. Such higher affinity nucleic acid ligands
may be used to generate a new array 115 with increased affinity or
specificity for the target. That capability further distinguishes
the present invention from conventional biochips, which do not
utilize iterative amplification of selected nucleic acid ligands to
generate new chips with higher specificity or affinity for a target
analyte.
[0080] Embodiments Involving Magnetic Beads
[0081] In an alternative embodiment, the nucleic acid ligand
sequences may be attached to magnetic beads instead of to a glass
or other flat surface. In this case, each recognition complex would
comprise a magnetic bead attached to one or more nucleic acid
ligands. In a preferred embodiment, each nucleic acid ligand
molecule attached to the same magnetic bead will have the same
sequence. In other embodiments, the nucleic acid ligand molecules
attached to a single bead may have different sequences. In certain
preferred embodiments, the nucleic acid ligands will also be
attached to an organic semiconductor, such as DALM. Attachment of
nucleic acid ligands to DALM would facilitate the detection and
quantitation of analyte binding to the nucleic acid ligands, as
described above.
[0082] The skilled artisan will realize that use of magnetic bead
technology would facilitate certain applications of the invention,
such as the iterative process for producing nucleic acid ligands of
higher specificity and greater binding affinity for the analyte.
With magnetic bead technology, the individual recognition complexes
are more easily manipulated and separated according to their
characteristics. For example, recognition complexes that bind to
the analyte may be separated from recognition complexes that do not
bind to the analyte by using a magnetic flow cell or filter block,
as disclosed in U.S. Pat. No. 5,972,721, incorporated herein by
reference in its entirety.
[0083] A diagram for use of magnetic beads in a recognition complex
system is shown in FIG. 2. Nucleic acid ligands of random or
non-random sequence may be synthesized or amplified and attached to
magnetic beads. The individual recognition complexes, each
corresponding to a magnetic bead attached to one or more nucleic
acid ligands, together comprise an array, similar to that described
above for FIG. 1. The array is added to the magnetic bead mixer
(FIG. 2) and analyte is added and allowed to bind to the nucleic
acid ligands. The mixture is then transferred to a
photo-electrochemical cell with a magnetic electrode, where the
mixture may be exposed to ultraviolet or other irradiation. A CCD,
photomultiplier tube, digital camera or other detection device may
be used to obtain absorption or emission spectra. As described
above, binding of analyte will result in characteristic changes in
the photochemical properties of individual recognition complexes.
These changes in photochemical properties will be detected and
analyzed to produce an analyte signature, as described above.
Although the suspension of recognition complexes in the bead mixer
is random, the use of a magnetic electrode in the
photo-electrochemical cell will provide a spatial distribution of
recognition complexes, analogous to the two-dimensional array 115
described above. Beads will deposit and separate on the surface of
the magnetic electrode according to their accumulated mass (from
binding analyte). This spatial distribution, along with the
detected photochemical changes, may be analyzed to produce a unique
signature that can be used to identify the analyte.
[0084] After detection, the recognition complexes may be
transferred to a magnetic filter (FIG. 2), where the recognition
complexes that bind to the analyte may be separated from those that
do not bind analyte. The recognition complexes that do not bind
analyte are transferred to the recycle bin (FIG. 2), where the
nucleic acid ligands may be detached from the magnetic beads. The
magnetic beads may be disposed of or recycled for attachment to new
nucleic acid ligands. Those recognition complexes that bind to the
analyte may be transferred to a PCR cycler (FIG. 2), where the
nucleic acid ligand sequences may be amplified. The new nucleic
acid ligand sequences are attached to magnetic beads and
transferred to the magnetic bead mixer (FIG. 2) for another
iteration of the process. This iterative process may be used to
produce nucleic acid ligands that bind with high affinity to the
analyte, or may be used to produce an array with greater
specificity for the target analyte. Certain components that may be
incorporated into a recognition complex system as shown in FIG. 2
include pumps and valves to facilitate fluid transfer between
different components of the recognition complex system. It is
anticipated that virtually any pump or valve capable of producing a
controlled fluid transfer between one component and another
component of the recognition complex system illustrated in FIG. 2
could be used.
[0085] Processes for the coupling of molecules to magnetic beads or
a magnetite substrate are well known in the art, i.e. U.S. Pat.
Nos. 4,695,393, 3,970,518, 4,230,685, and 4,677,055 herein
expressly incorporated by reference. Alternatively, an organic
semiconductor such as DALM may be attached directly to the magnetic
bead. Nucleic acid ligands, such as DNA, may be attached to DALM by
electrostatic interaction with magnesium ion (FIG. 3). This would
facilitate detachment of DNA from the DALM/magnetic bead, since DNA
would be released by addition of a chelating agent such as EDTA
(ethylene diamine tetraacetic acid). Alternatively, the nucleic
acid ligand may be covalently attached, for example by chemical
cross-linking to DALM through the use of any appropriate
cross-linking agent known in the art, such as EDC.
[0086] As shown in FIG. 3, the analyte may bind to one or more
recognition complexes. Those recognition complexes bound to the
analyte may be separated from unbound recognition complexes by mass
segregation, using a magnetic filter (see FIG. 2). The nucleic acid
ligands (indicated in FIG. 3 as DNA) with affinity for analyte may
be amplified by PCR or other methods described below. The amplified
nucleic acid ligands may be attached to DALM and/or magnetic beads
for another iteration of analyte binding and detection, or may be
collected and used for other purposes, such as analyte
neutralization or preparation of high-affinity diagnostic devices
for detecting analyte in the field (FIG. 3).
[0087] It is envisioned that particles employed in the instant
invention may come in a variety of sizes. While large magnetic
particles (mean diameter in solution greater than 10 .mu.m) can
respond to weak magnetic fields and magnetic field gradients, they
tend to settle rapidly, limiting their usefulness for reactions
requiring homogeneous conditions. Large particles also have a more
limited surface area per weight than smaller particles, so that
less material can be coupled to them. In preferred embodiments, the
magnetic beads are less than 10 .mu.m in diameter.
[0088] Various silane couplings applicable to magnetic beads are
discussed in U.S. Pat. No. 3,652,761, incorporated herein by
reference. Procedures for silanization known in the art generally
differ from each other in the media chosen for the polymerization
of silane and its deposition on reactive surfaces. Organic solvents
such as toluene (Weetall, (1976)), methanol, (U.S. Pat. No.
3,933,997) and chloroform (U.S. Pat. No. 3,652,761) have been used.
Silane deposition from aqueous alcohol and aqueous solutions with
acid have also been used.
[0089] Ferromagnetic materials in general become permanently
magnetized in response to magnetic fields. Materials termed
"superparamagnetic" experience a force in a magnetic field
gradient, but do not become permanently magnetized. Crystals of
magnetic iron oxides may be either ferromagnetic or
superparamagnetic, depending on the size of the crystals.
Superparamagnetic oxides of iron generally result when the crystal
is less than about 300 angstroms (.ANG.) in diameter; larger
crystals generally have a ferromagnetic character.
[0090] Dispersible magnetic iron oxide particles reportedly having
300 .ANG. diameters and surface amine groups were prepared by base
precipitation of ferrous chloride and ferric chloride
(Fe.sup.2+/Fe.sup.3+=1) in the presence of polyethylene imine,
according to U.S. Pat. No. 4,267,234. These particles were exposed
to a magnetic field three times during preparation and were
described as redispersible. The magnetic particles were mixed with
a glutaraldehyde suspension polymerization system to form magnetic
polyglutaraldehyde microspheres with reported diameters of 0.1
.mu.m. Polyglutaraldehyde microspheres have conjugated aldehyde
groups on the surface which can form bonds to amino containing
molecules such as proteins.
[0091] While a variety of particle sizes are envisioned to be
applicable in the disclosed method, in a preferred embodiment,
particles are between about 0.1 and about 1.5 .mu.m diameter.
Particles with mean diameters in this range can be produced with a
surface area as high as about 100 to 150 m.sup.2/gm, which provides
a high capacity for bioaffinity adsorbent coupling. Magnetic
particles of this size range overcome the rapid settling problems
of larger particles, but obviate the need for large magnets to
generate the magnetic fields and magnetic field gradients required
to separate smaller particles. Magnets used to effect separations
of the magnetic particles of this invention need only generate
magnetic fields between about 100 and about 1000 Oersteds. Such
fields can be obtained with permanent magnets which are preferably
smaller than the container which holds the dispersion of magnetic
particles and thus, may be suitable for benchtop use. Although
ferromagnetic particles may be useful in certain applications of
the invention, particles with superparamagnetic behavior are
usually preferred since superparamagnetic particles do not exhibit
the magnetic aggregation associated with ferromagnetic particles
and permit redispersion and reuse.
[0092] The method for preparing the magnetic particles may comprise
precipitating metal salts in base to form fine magnetic metal oxide
crystals, redispersing and washing the crystals in water and in an
electrolyte. Magnetic separations may be used to collect the
crystals between washes if the crystals are superparamagnetic. The
crystals may then be coated with a material capable of adsorptively
or covalently bonding to the metal oxide and bearing functional
groups for coupling with nucleic acid ligands or DALM.
[0093] Embodiments Involving Non-Magnetic Beads, Cells or Particles
and Flow Cytometry
[0094] In another embodiment, the recognition complexes or analyte
of interest may be non-covalently or covalently attached to
non-magnetic beads, such as glass, polyacrylamide, polystyrene or
latex. Receptor complexes may be attached to such beads by the same
techniques discussed above for magnetic beads. After exposure of
analyte to receptor complexes, those complexes bound to analyte may
be separated from unbound complexes by flow cytometry. Non-limiting
examples of flow cytometry methods are disclosed in Betz et al.
(1984), Wilson et al. (1988), Scillian et al. (1989), Frengen et
al. (1994), Griffith et al. (1996), Stuart et al. (1998) and U.S.
Pat. Nos. 5,853,984 and 5,948,627, each incorporated herein by
reference in its entirety. U.S. Pat. Nos. 4,727,020, 4,704,891 and
4,599,307, incorporated herein by reference, describe the
arrangement of the components comprising a flow cytometer and the
general principles of its use.
[0095] In the flow cytometer, beads, cells or other particles are
passed substantially one at a time through a detector, where each
particle is exposed to an energy source. The energy source
generally provides excitatory light of a single wavelength. The
detector comprises a light collection unit, such as photomultiplier
tubes or a charge coupled device, which may be attached to a data
analyzer such as a computer. The beads, cells or particles can be
characterized by their response to excitatory light, for example by
detecting and/or quantifying the amount of fluorescent light
emitted in response to the excitatory light. Changes in size due to
binding of analyte to ligand can also be incorporated into sorting
strategies. Beads or cells exhibiting a particular characteristic
can be sorted using an attached cell sorter, such as the FACS
Vantage.TM. cell sorter sold by Becton Dickinson Immunocytometry
Systems (San Jose, Calif.).
[0096] This system is well suited to use with an organic
semiconductor, such as DALM, that has well defined fluorescent and
luminescent properties. Using a flow cytometer, it is possible to
separate beads, cells or particles that are associated with
recognition complexes bound to analytes, from unbound complexes, by
detecting the presence of and characterizing the electrochemical
properties of the organic semiconductor. Because those properties
change upon binding of recognition complex to analyte, it is
possible to separate bead-attached recognition complexes that bind
to analyte from complexes that do not bind analyte. This process is
even simpler when the analyte is incorporated into a cell or cell
fragment, or attached to a bead. In this case, only analytes bound
to recognition complexes should show a fluorescent or other
spectroscopic signature associated with the organic semiconductor.
In an alternative embodiment, the analyte or ligand may be labeled
with a different fluorescent or other spectroscopic tag moiety.
Many examples of fluorescent or other tag moieties are known in the
art.
[0097] Flow cytometry may be used to purify or partially purify
analytes that bind to a particular nucleic acid ligand, or to
purify or partially purify ligands that bind to a particular
analyte. Other manipulations may include sorting for differences in
fluorescence and/or size that represent differences in binding
affinity or avidity of analyte for ligand or the number of ligands
bound to each analyte or of analyte bound to each ligand.
[0098] Nucleic Acids
[0099] Nucleic acid ligands within the scope of the present
invention may be made by any technique known to one of ordinary
skill in the art. Non-limiting examples of nucleic acid ligands
include synthetic oligonucleotides made by in vitro chemical
synthesis using phosphotriester, phosphite or phosphoramidite
chemistry and solid phase techniques (EP 266,032, incorporated
herein by reference) or via deoxynucleoside H-phosphonate
intermediates (Froehler et al., 1986, and U.S. Pat. No. 5,705,629,
each incorporated herein by reference). Examples of enzymatically
produced nucleic acid ligands include those produced by
amplification reactions such as PCR.TM. (e.g., U.S. Pat. Nos.
4,683,202 and 4,683,195, each incorporated herein by reference), or
the synthesis of oligonucleotides described in U.S. Pat. No.
5,645,897, incorporated herein by reference. Examples of a
biologically produced nucleic acid ligand include recombinant
nucleic acid production in living cells, such as recombinant DNA
vector production in bacteria (e.g., Sambrook et al. 1989).
[0100] Nucleobase, nucleoside and nucleotide mimics or derivatives
are well known in the art, and have been described in exemplary
references such as, for example, Scheit, Nucleotide Analogs (John
Wiley, New York, 1980). Purine and pyrimidine nucleobases encompass
naturally occurring purines and pyrimidines and derivatives and
mimics thereof. These include, but are not limited to, purines and
pyrimidines substituted with one or more alkyl, carboxyalkyl,
amino, hydroxyl, halogen (i.e. fluoro, chloro, bromo, or iodo),
thiol, or alkylthiol groups. The alkyl substituents may comprise
from about 1, 2, 3, 4, or 5, to about 6 carbon atoms.
[0101] Examples of purines and pyrimidines include deazapurines,
2,6-diaminopurine, 5-fluorouracil, xanthine, hypoxanthine,
8-bromoguanine, 8-chloroguanine, bromothymine, 8-aminoguanine,
8-hydroxyguanine, 8-methylguanine, 8-thioguanine, azaguanines,
2-aminopurine, 5-ethylcytosine, 5-methylcytosine, 5-bromouracil,
5-ethyluracil, 5-iodouracil, 5-chlorouracil, 5-propyluracil,
thiouracil, 2-methyladenine, methylthioadenine,
N,N-dimethyladenine, azaadenines, 8-bromoadenine, 8-hydroxyadenine,
6-hydroxyaminopurine, 6-thiopurine, 4-(6-aminohexyl/cytosine), and
the like. A list of exemplary purine and pyrimidine derivatives and
mimics is provided in Table 1.
2TABLE 1 Purine and Pyrimidine Derivatives or Mimics Abbr. Modified
base description ac4c 4-acetylcytidine chm5u 5-
(carboxyhydroxylmethyl)uridine Cm 2'-O-methylcytidine cmnm5s2u
5-carboxymethylaminometby- l-2- thioridine cmnm5u 5-
carboxymethylaminomethyluridine D Dihydrouridine Fm
2'-O-methylpseudouridine gal q beta,D-galactosylqueosine Gm
2'-O-methylguanosine I Inosine i6a N6-isopentenyladenosine m1a
1-methyladenosine m1f 1-methyipseudouridine m1g 1-methylguanosine
m1I 1-methylinosine m22g 2,2-dimethylguanosine m2a
2-methyladenosine m2g 2-methylguanosine m3c 3-methylcytidine m5c
5-methylcytidine m6a N6-methyladenosine m7g 7-methylguanosine mam5u
5-methylaminomethyluridine mam5s2u 5-methoxyaminomethyl-2-
thiouridine man q Beta,D-mannosylqueosine mcm5s2u
5-methoxycarbonylmethyl-2- thiouridine mcm5u
5-methoxycarbonylmethyluridine mo5u 5-methoxyuridine ms2i6a
2-methylthio-N6- isopentenyladenosine ms2t6a
N-((9-beta-D-ribofuranosyl-2- methylthiopurine-6-
yl)carbamoyl)threonine mt6a N-((9-beta-D-ribofuranosylpurine-
6-yl)N-methyl-carbamoyl)threonine mv Uridine-5-oxyaceticacid
methylester o5u Uridine-5-oxyacetic acid (v) osyw Wybutoxosine p
Pseudouridine q Queosine s2c 2-thiocytidine s2t
5-methyl-2-thiouridine s2u 2-thiouridine s4u 4-thiouridine t
5-methyluridine t6a N-((9-beta-D-ribofuranosylpurine-
6-yl)carbamoyl)threonine tm 2'-O-methyl-5-methyluridine um
2'-O-methyluridine yw Wybutosine x 3-(3-amino-3-
carboxypropyl)uridine, (acp3)u
[0102] An example of a nucleic acid ligand comprising nucleoside or
nucleotide derivatives and mimics is a "polyether nucleic acid",
described in U.S. Pat. No. 5,908,845, incorporated herein by
reference, wherein one or more nucleobases are linked to chiral
carbon atoms in a polyether backbone. Another example of a nucleic
acid ligand is a "peptide nucleic acid", also known as a "PNA",
"peptide-based nucleic acid mimics" or "PENAMs", described in U.S.
Pat. Nos. 5,786,461, 5,891,625, 5,773,571, 5,766,855, 5,736,336,
5,719,262, 5,714,331, 5,539,082, and WO 92/20702, each of which is
incorporated herein by reference. A peptide nucleic acid generally
comprises at least one nucleobase and at least one nucleobase
linker moiety that is not a 5-carbon sugar and/or at least one
backbone moiety that is not a phosphate group. Examples of
nucleobase linker moieties described for PNAs include aza nitrogen
atoms, amido and/or ureido tethers (see for example, U.S. Pat. No.
5,539,082). Examples of backbone moieties described for PNAs
include an aminoethylglycine, polyamide, polyethyl, polythioamide,
polysulfinamide or polysulfonamide backbone moiety.
[0103] Peptide nucleic acids generally have enhanced sequence
specificity, binding properties, and resistance to enzymatic
degradation in comparison to molecules such as DNA and RNA (Egholm
et al., Nature 1993, 365, 566; PCT/EP/01219). In addition, U.S.
Pat. Nos. 5,766,855, 5,719,262, 5,714,331 and 5,736,336 describe
PNAs comprising nucleobases and alkylamine side chains with further
improvements in sequence specificity, solubility and binding
affinity. These properties promote double or triple helix formation
between a target and the PNA.
[0104] The skilled artisan will realize that the present invention
is not limited to the examples disclosed herein, but may include
nucleobases, nucleotides and nucleic acids produced by any other
means known in the art.
[0105] Amplification
[0106] In certain embodiments, the nucleic acid ligands of the
recognition complex system may be amplified to provide a source of
high affinity nucleic acid ligands for neutralizing analytes.
Amplification may also be of use in the iterative process for
generating arrays with greater specificity or binding affinity for
the analyte. Within the scope of the present invention,
amplification may be accomplished by any means known in the art.
Exemplary embodiments are described below.
[0107] Primers
[0108] The term primer, as defined herein, is meant to encompass
any nucleic acid that is capable of priming the synthesis of a
nascent nucleic acid in a template-dependent process. Typically,
primers are oligonucleotides from ten to twenty base pairs in
length, but longer sequences may be employed. Primers may be
provided in double-stranded or single-stranded form, although the
single-stranded form is preferred.
[0109] Amplification Methods
[0110] A number of template dependent processes are available to
amplify the marker sequences present in a given template sample.
One of the best known amplification methods is the polymerase chain
reaction (referred to as PCR) which is described in detail in U.S.
Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, and in Innis et al.,
1990, each of which is incorporated herein by reference.
[0111] Briefly, in PCR, two primer sequences are prepared which are
complementary to regions on opposite complementary strands of, for
example, a nucleic acid ligand. An excess of deoxynucleoside
triphosphates are added to a reaction mixture along with a DNA
polymerase, e.g., Taq polymerase. Examples of polymerases that may
be used for purposes of nucleic acid amplification are provided in
Table 2 below. If the marker sequence is present in a sample, the
primers will bind to the marker and the polymerase will cause the
primers to be extended along the marker sequence by adding on
nucleotides. By raising and lowering the temperature of the
reaction mixture, the extended primers will dissociate from the
nucleic acid ligand to form reaction products, excess primers will
bind to the nucleic acid ligand and to the reaction products and
the process is repeated.
[0112] A reverse transcriptase PCR amplification procedure may be
performed in order to quantify the amount of mRNA amplified.
Methods of reverse transcribing RNA into cDNA are well known and
described in Sambrook et al., 1989. Alternative methods for reverse
transcription utilize thermostable DNA polymerases. Polymerase
chain reaction methodologies are well known in the art.
[0113] Another method for amplification is the ligase chain
reaction ("LCR"), disclosed in European Application No. 320,308. In
LCR, two complementary probe pairs are prepared, and in the
presence of the nucleic acid ligand sequence, each pair will bind
to opposite complementary strands of the target such that they
abut. In the presence of a ligase, the two probe pairs will link to
form a single unit. By temperature cycling, as in PCR, bound
ligated units dissociate from the nucleic acid ligand and then
serve as templates for ligation of excess probe pairs. U.S. Pat.
No. 4,883,750 describes a method similar to LCR for binding probe
pairs to a nucleic acid ligand sequence.
[0114] Qbeta Replicase, described in PCT Application No.
PCT/US87/00880, may also be used as still another amplification
method in the present invention. In this method, a replicative
sequence of RNA that has a region complementary to that of a
nucleic acid ligand is added to a sample in the presence of an RNA
polymerase. The polymerase will copy the replicative sequence which
may then be detected.
[0115] An isothermal amplification method, in which restriction
endonucleases and ligases are used to achieve the amplification of
nucleic acid ligand molecules that contain nucleotide
5'-[alpha-thio]-triphosphates in one strand of a restriction site
may also be useful in the amplification of nucleic acid ligands in
the present invention. Walker et al., (1992).
[0116] Strand Displacement Amplification (SDA) is another method of
carrying out isothermal amplification of nucleic acid ligands that
involves multiple rounds of strand displacement and synthesis,
i.e., nick translation. A similar method, called Repair Chain
Reaction (RCR), involves annealing several probes throughout a
region targeted for amplification, followed by a repair reaction in
which only two of the four bases are present. The other two bases
may be added as biotinylated derivatives for easy detection. A
similar approach is used in SDA. Target specific sequences may also
be detected using a cyclic probe reaction (CPR). In CPR, a probe
having 3' and 5' sequences of non-specific DNA and a middle
sequence of specific RNA is hybridized to DNA that is present in a
sample. Upon hybridization, the reaction is treated with RNase H,
and the products of the probe identified as distinctive products
that are released after digestion. The original template is
annealed to another cycling probe and the reaction is repeated.
[0117] Still other amplification methods described in GB
Application No. 2 202 328, and in PCT Application No.
PCT/US89/01025, may be used in accordance with the present
invention. In the former application, "modified" primers are used
in a PCR like, template and enzyme dependent synthesis. The primers
may be modified by labeling with a capture moiety (e.g., biofin)
and/or a detector moiety (e.g., enzyme). In the latter application,
an excess of labeled probes are added to a sample. In the presence
of the target sequence, the probe binds and is cleaved
catalytically. After cleavage, the nucleic acid ligand sequence is
released intact to be bound by excess probe. Cleavage of the
labeled probe signals the presence of the nucleic acid ligand
sequence.
[0118] Other nucleic acid ligand amplification procedures include
transcription-based amplification systems (TAS), including nucleic
acid sequence based amplification (NASBA) and 3SR. Kwoh et
al.,(1989) and PCT Application WO 88/10315. In NASBA, the nucleic
acid ligands may be prepared for amplification by standard
phenol/chloroform extraction, heat denaturation of a clinical
sample, treatment with lysis buffer and minispin columns for
isolation of DNA and RNA or guanidinium chloride extraction of RNA.
These amplification techniques involve annealing a primer which has
nucleic acid ligand specific sequences. Following polymerization,
DNA/RNA hybrids are digested with RNase H while double stranded DNA
molecules are heat denatured again. In either case the single
stranded DNA is made fully double stranded by addition of second
nucleic acid ligand specific primer, followed by polymerization.
The double-stranded DNA molecules are then multiply transcribed by
a polymerase such as T7 or SP6. In an isothermal cyclic reaction,
the RNA's are reverse transcribed into double stranded DNA, and
transcribed once against with a polymerase such as T7 or SP6. The
resulting products, whether truncated or complete, indicate nucleic
acid ligand specific sequences.
[0119] Davey et al., European Application No. 329,822 disclose a
nucleic acid amplification process involving cyclically
synthesizing single-stranded RNA ("ssRNA"), ssDNA, and
double-stranded DNA (dsDNA), which may be used in accordance with
the present invention. The ssRNA is a first template for a first
primer oligonucleotide, which is elongated by reverse transcriptase
(RNA-dependent DNA polymerase). The RNA is then removed from the
resulting DNA:RNA duplex by the action of ribonuclease H (RNase H,
an RNase specific for RNA in duplex with either DNA or RNA). The
resultant ssDNA is a second template for a second primer, which
also includes the sequences of an RNA polymerase promoter
(exemplified by T7 RNA polymerase) 5' to its homology to the
template. This primer is then extended by DNA polymerase
(exemplified by the large "Klenow" fragment of E. coli DNA
polymerase I), resulting in a double-stranded DNA ("dsDNA")
molecule, having a sequence identical to that of the original RNA
between the primers and having additionally, at one end, a promoter
sequence. This promoter sequence may be used by the appropriate RNA
polymerase to make many RNA copies of the DNA. These copies may
then re-enter the cycle leading to very swift amplification. With
proper choice of enzymes, this amplification may be done
isothermally without addition of enzymes at each cycle. Because of
the cyclical nature of this process, the starting sequence may be
chosen to be in the form of either DNA or RNA.
[0120] Miller et al., PCT Application WO 89/06700 disclose a
nucleic acid sequence amplification scheme based on the
hybridization of a promoter/primer sequence to a target
single-stranded DNA ("ssDNA") followed by transcription of many RNA
copies of the sequence. This scheme is not cyclic, i.e., new
templates are not produced from the resultant RNA transcripts.
Other amplification methods include "race" and "one-sided PCR."
Frohman, (1990) and Ohara et al., (1989).
[0121] Methods based on ligation of two (or more) oligonucleotides
in the presence of nucleic acid having the sequence of the
resulting "di-oligonucleotide", thereby amplifying the
di-oligonucleotide, may also be used in the amplification step of
the present invention. Wu et al., Genomics 4:560 (1989).
[0122] Labels
[0123] For certain embodiments, it may be desirable to incorporate
a label into nucleic acid ligands, amplification products, probes
or primers. A number of different labels may be used, such as
fluorophores, chromophores, radioisotopes, enzymatic tags,
antibodies, chemiluminescent, electroluminescent, affinity labels,
etc. One of skill in the art will recognize that these and other
label moieties not mentioned herein can be used in the practice of
the present invention.
[0124] Examples of affinity labels include an antibody, an antibody
fragment, a receptor protein, a hormone, biotin, DNP, and any
polypeptide/protein molecule that binds to an affinity label.
[0125] Examples of enzymatic tags include urease, alkaline
phosphatase or peroxidase. Colorimetric indicator substrates can be
employed with such enzymes to provide a detection means visible to
the human eye or spectrophotometrically.
[0126] The following fluorophores are contemplated to be useful in
practicing the present invention. Alexa 350, Alexa 430, AMCA,
BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR,
BODIPY-TRX, Cascade Blue, Cy2, Cy3, Cy5,6-FAM, Fluorescein, HEX,
6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514,
Pacific Blue, REG, Rhodamine Green, Rhodamine Red, ROX, TAMRA, TET,
Tetramethylrhodamine, and Texas Red.
[0127] Imaging Agents and Radioisotopes
[0128] In certain embodiments, the claimed nucleic acid ligands of
the present invention may be attached to imaging agents of use for
imaging, treatment and diagnosis of various diseased organs or
tissues. Many appropriate imaging agents are known in the art, as
are methods for their attachment to nucleic acids. Certain
attachment methods involve the use of a metal chelate complex
employing, for example, an organic chelating agent such a DTPA
attached to the nucleic acid.
[0129] Non-limiting examples of paramagnetic ions of potential use
as imaging agents include chromium (III), manganese (II), iron
(III), iron (II), cobalt (II), nickel (II), copper (II), neodymium
(III), samarium (III), ytterbium (III), gadolinium (III), vanadium
(II), terbium (III), dysprosium (III), holmium (III) and erbium
(III), with gadolinium being particularly preferred. Ions useful in
other contexts, such as X-ray imaging, include but are not limited
to lanthanum (III), gold (III), lead (II), and especially bismuth
(III).
[0130] Radioisotopes of potential use as imaging or therapeutic
agents include astatine.sup.211, .sup.14carbon, .sup.51chromium,
.sup.36chlorine, .sup.57cobalt, .sup.58cobalt, copper.sup.67,
.sup.152Eu, gallium.sup.67, .sup.3hydrogen, iodine.sup.123,
iodine.sup.125, iodine.sup.131, indium.sup.111, .sup.59iron,
.sup.32phosphorus, rhenium.sup.186, rhenium.sup.188,
.sup.75selenium, .sup.35sulphur, technicium.sup.99m and
yttrium.sup.90. .sup.125I is often being preferred for use in
certain embodiments, and technicium.sup.99m and indium.sup.111 are
also often preferred due to their low energy and suitability for
long range detection.
[0131] Methods of Immobilization
[0132] In various embodiments, the nucleic acid ligands of the
present invention may be attached to a solid surface
("immobilized"). In a preferred embodiment, immobilization may
occur by attachment of an organic semiconductor, such as DALM, to a
solid surface, such as a magnetic, glass or plastic bead, a plastic
microtiter plate or a glass slide. Nucleic acid ligands may be
attached to the DALM by electrostatic interaction with magnesium
ion (FIG. 3). This system is advantageous in that the attachment of
nucleic acid ligand to DALM may be readily reversed by addition of
a magnesium chelator, such as EDTA.
[0133] Immobilization of nucleic acid ligands may alternatively be
achieved by a variety of methods involving either non-covalent or
covalent interactions between the immobilized nucleic acid ligand,
comprising an anchorable moiety, and an anchor. In an exemplary
embodiment, immobilization may be achieved by coating a solid
surface with streptavidin or avidin and the subsequent attachment
of a biotinylated polynucleotide (Holmstrom, 1993). Immobilization
may also occur by coating a polystyrene or glass solid surface with
poly-L-Lys or poly L-Lys, Phe, followed by covalent attachment of
either amino- or sulfhydryl-modified polynucleotides, using
bifunctional crosslinking reagents (Running, 1990; Newton,
1993).
[0134] Immobilization may take place by direct covalent attachment
of short, 5'-phosphorylated primers to chemically modified
polystyrene plates ("Covalink" plates, Nunc) Rasmussen, (1991). The
covalent bond between the modified oligonucleotide and the solid
phase surface is formed by condensation with a water-soluble
carbodiimide. This method facilitates a predominantly 5'-attachment
of the oligonucleotides via their 5'-phosphates.
[0135] Nikiforov et al. (U.S. Pat. No. 5,610,287 incorporated
herein by reference) describes a method of non-covalently
immobilizing nucleic acid ligand molecules in the presence of a
salt or cationic detergent on a hydrophilic polystyrene solid
support containing an --OH, --C.dbd.O or --COOH hydrophilic group
or on a glass solid support. The support is contacted with a
solution having a pH of about 6 to about 8 containing the nucleic
acid ligand and the cationic detergent or salt. The support
containing the immobilized nucleic acid ligand may be washed with
an aqueous solution containing a non-ionic detergent without
removing the attached molecules.
[0136] Another commercially available method for immobilization is
the "Reacti-Bind.TM. DNA Coating Solutions" (see
"Instructions--Reacti-Bind.T- M. DNA Coating Solution" January
1997). This product comprises a solution that is mixed with DNA and
applied to surfaces such as polystyrene or polypropylene. After
overnight incubation, the solution is removed, the surface washed
with buffer and dried, after which it is ready for hybridization.
It is envisioned that similar products, i.e. Costar "DNA-BIND.TM."
or Immobilon-AV Affinity Membrane (IAV, Millipore, Bedford, Mass.)
may be used in the practice of the instant invention.
[0137] Cross-Linkers
[0138] Bifunctional cross-linking reagents may be of use in various
embodiments of the claimed invention, such as attaching an organic
semiconductor to a nucleic acid ligand, attaching an organic
semiconductor to a substrate, attaching various functional groups
to a nucleic acid ligand, or attaching a nucleic acid ligand or an
analyte to a bead or particle. Homobifunctional reagents that carry
two identical functional groups are highly efficient in inducing
cross-linking. Heterobifunctional reagents contain two different
functional groups. By taking advantage of the differential
reactivities of the two different functional groups, cross-linking
can be controlled both selectively and sequentially. The
bifunctional cross-linking reagents can be divided according to the
specificity of their functional groups, e.g., amino, guanidino,
indole, or carboxyl specific groups. Of these, reagents directed to
free amino groups have become especially popular because of their
commercial availability, ease of synthesis and the mild reaction
conditions under which they can be applied.
[0139] Exemplary methods for cross-linking molecules, such as DALM,
nucleic acid ligands or analytes, are described in U.S. Pat. No.
5,603,872 and U.S. Pat. No. 5,401,511. Various ligands can be
covalently bound to surfaces through the cross-linking of amine
residues. Amine residues may be introduced onto a surface through
the use of aminosilane, as discussed above. Coating with
aminosilane provides an active functional residue, a primary amine,
on the surface for cross-linking purposes. Ligands are bound
covalently to discrete sites on the surfaces. The surfaces may also
have sites for non-covalent association. To form covalent
conjugates of ligands and surfaces, cross-linking reagents have
been studied for effectiveness and biocompatibility. Cross-linking
reagents include glutaraldehyde (GAD), bifunctional oxirane (OXR),
ethylene glycol diglycidyl ether (EGDE), and a water soluble
carbodiimide, preferably 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC). Through the complex chemistry of cross-linking,
linkage of the amine residues of the silane-coated surface and free
DALM, nucleic acid ligand or analyte may be accomplished.
[0140] Separation and Quantitation Methods
[0141] It may be desirable to separate nucleic acid ligands of
different lengths for the purpose of quantitation, analysis or
purification.
[0142] Gel Electrophoresis
[0143] In one embodiment, amplification products are separated by
agarose, agarose-acrylamide or polyacrylamide gel electrophoresis
using standard methods (Sambrook et al., 1989). Separation by
electrophoresis is based upon the differential migration through a
gel according to the size and ionic charge of the molecules in an
electrical field. High resolution techniques normally use a gel
support for the fluid phase. Examples of gels used are starch,
acrylamide, agarose or mixtures of acrylamide and agarose.
Separated nucleic acids may be visualized by staining, for example
with ethidium bromide. The gel may be a single concentration or
gradient in which pore size decreases with migration distance. In
gel electrophoresis of polynucleotides, mobility depends primarily
on molecular size. In pulse field electrophoresis, two fields are
applied alternately at right angles to each other to minimize
diffusion mediated spread of large linear polymers.
[0144] Agarose gel electrophoresis facilitates the size-based
separation of DNA or RNA in a matrix composed of a highly purified
form of agar. Nucleic acids tend to become oriented in an end on
position in the presence of an electric field. Migration through
the gel matrices occurs at a rate inversely proportional to the
loglo of the number of base pairs (Sambrook et al., 1989).
[0145] Chromatographic Techniques
[0146] Alternatively, chromatographic techniques may be employed to
effect separation. There are many kinds of chromatography which may
be used in the present invention: adsorption, partition,
ion-exchange and molecular sieve, and many specialized techniques
for using them including column, paper, thin-layer and gas
chromatography (Freifelder, 1982). In yet another alternative, cDNA
products labeled with biotin or antigen can be captured with beads
bearing avidin or antibody, respectively.
[0147] Microfluidic Techniques
[0148] Microfluidic techniques include separation on a platform
such as microcapillaries, designed by ACLARA BioSciences Inc., or
the LabChip.TM. liquid integrated circuits made by Caliper
Technologies Inc. These microfluidic platforms require only
nanoliter volumes of sample, in contrast to the microliter volumes
required by other separation technologies. Miniaturizing some of
the processes involved in genetic analysis has been achieved using
microfluidic devices. For example, published PCT Application No. WO
94/05414 reports an integrated micro-PCR.TM. apparatus for
collection and amplification of nucleic acids from a specimen. U.S.
Pat. No. 5,856,174 describes an apparatus that combines the various
processing and analytical operations involved in nucleic acid
analysis and is incorporated herein by reference.
[0149] Capillary Electrophoresis
[0150] In some embodiments, it may be desirable to provide an
additional, or alternative means for analyzing nucleic acid
ligands, such as microcapillary arrays. Microcapillary array
electrophoresis generally involves the use of a thin capillary or
channel that may or may not be filled with a particular separation
medium. Electrophoresis of a sample through the capillary provides
a size based separation profile for the sample. The use of
microcapillary electrophoresis in size separation of nucleic acids
has been reported in, e.g., Woolley and Mathies, 1994.
Microcapillary array electrophoresis generally provides a rapid
method for size-based sequencing, PCR.TM. product analysis and
restriction fragment sizing. The high surface to volume ratio of
these capillaries allows for the application of higher electric
fields across the capillary without substantial thermal variation
across the capillary, consequently allowing for more rapid
separations. Furthermore, when combined with confocal imaging
methods, these methods provide sensitivity in the range of
attomoles, which is comparable to the sensitivity of radioactive
sequencing methods.
[0151] Microfabrication of microfluidic devices including
microcapillary electrophoretic devices has been discussed in detail
in, e.g., Jacobsen et al., 1994; Effenhauser et al., 1994; Harrison
et al., 1993; Effenhauser et al., 1993; Manz et al., 1992; and U.S.
Pat. No. 5,904,824, incorporated herein by reference. Typically,
these methods comprise photolithographic etching of micron scale
channels on silica, silicon or other crystalline substrates or
chips, and can be readily adapted for use in the present invention.
In some embodiments, the capillary arrays may be fabricated from
the same polymeric materials described for the fabrication of the
body of the device, using injection molding techniques.
[0152] Tsuda et al., 1990, describes rectangular capillaries, an
alternative to the cylindrical capillary glass tubes. Some
advantages of these systems are their efficient heat dissipation
due to the large height-to-width ratio and, hence, their high
surface-to-volume ratio and their high detection sensitivity for
optical on-column detection modes. These flat separation channels
have the ability to perform two-dimensional separations, with one
force being applied across the separation channel, and with the
sample zones detected by the use of a multi-channel array
detector.
[0153] In many capillary electrophoresis methods, the capillaries,
e.g., fused silica capillaries or channels etched, machined or
molded into planar substrates, are filled with an appropriate
separation/sieving matrix. Typically, a variety of sieving matrices
are known in the art may be used in the microcapillary arrays.
Examples of such matrices include, e.g., hydroxyethyl cellulose,
polyacrylamide, agarose and the like. Generally, the specific gel
matrix, running buffers and running conditions are selected to
maximize the separation characteristics of the particular
application, e.g., the size of the nucleic acid fragments, the
required resolution, and the presence of native or undenatured
nucleic acid molecules. For example, running buffers may include
denaturants, chaotropic agents such as urea or the like, to
denature nucleic acid ligands in the sample.
[0154] DALM
[0155] In certain embodiments, an organic semiconductor like DALM
is used to attach nucleic acid ligands to a surface and/or to
promote electrochemical detection of binding of analyte to nucleic
acid ligand. Production and use of diazoluminomelanin (DALM) has
previously been described in U.S. Pat. Nos. 5,856,108 and
5,003,050, incorporated herein by reference. DALM is prepared by
reacting 3AT (3-amino-L-tyrosine) with an alkali metal nitrite,
such as sodium nitrite, and thereafter reacting the resulting
diazotized product with luminol. At some point in the reaction, the
alaninyl portion of the 3AT rearranges to provide the hydroxyindole
portion of the final product. It is believed that such
rearrangement occurs following coupling of the luminol to the
diazotized 3AT.
[0156] The reaction between 3AT and the alkali metal nitrite is
carried out in aqueous medium. Since diazotization reactions are,
in general, exothermic, it may be desirable to carry out this
reaction under isothermal conditions or at a reduced temperature,
such as, for example, at ice bath temperatures. The reaction time
for the diazotization can range from about 1 to 20 minutes,
preferably about 5 to 10 minutes.
[0157] Because of the relative insolubility of luminol in aqueous
medium, the luminol is dissolved in an aprotic solvent, such as
dimethylsulfoxide (DMSO), then added, with stirring, to the aqueous
solution of diazotized 3AT. This reaction is carried out, at
reduced temperature, for about 20 to 200 minutes. The solvent is
then removed by evaporation at low pressure, with moderate heating,
e.g., about 30.degree. to 37.degree. C.
[0158] The reaction mixture is acidic, having a pH of about 3.5.
The coupling of the luminol and the diazotized 3AT can be
facilitated by adjusting the pH of the reaction mixture to about
5.0 to 6.0.
[0159] The product DALM may be precipitated from the reaction
mixture by combining the reaction mixture with an excess of a
material that is not a solvent for the DALM, e.g., acetone. After
centrifuging the precipitate and discarding the supernatant, the
solid material may be dried under vacuum.
[0160] In general, the quantities of the 3AT, alkali metal nitrite
and luminol reactants are equimolar. It is, however, within the
scope of the invention to vary the quantities of the reactants. The
molar ratio of 3AT:luminol may be varied over the range of about
0.6:1 to 3:1.
[0161] DALM is water soluble, having an apparent pKa for solubility
about pH 5.0. DALM does not require a catalyst for
chemiluminescence. The duration of the reaction is in excess of 52
hours. In contrast, luminol requires a catalyst. With micro
peroxidase as the catalyst, luminol has shown peak luminescence at
1 sec and half-lives of light emission of 0.5 and 4.5 sec at pH 8.6
and 12.6, respectively. The chemiluminescence yield of DALM is
better at pH 7.4 than at pH 9.5, although it still provides a
strong signal at strongly basic pHs. DALM also produces
chemiluminescence at pH 6.5 which is about the same intensity as
that produced at pH 9.5.
[0162] DALM can be used for chemiluminescent immunoassays for
biological and chemical agents; in radiofrequency and ionizing
radiation dosimeters; and for RNA/DNA hybridization assays for
viruses and genetic detection.
[0163] Aptamers
[0164] In certain preferred embodiments, the nucleic acid ligands
to be used in the practice of the invention are aptamers. Methods
of constructing and determining the binding characteristics of
aptamers are well known in the art. For example, such techniques
are described in Lorsch and Szostak (1996) and in U.S. Pat. Nos.
5,582,981, 5,595,877 and 5,637,459, each incorporated herein by
reference.
[0165] Aptamers may be prepared by any known method, including
synthetic, recombinant, and purification methods, and may be used
alone or in combination with other aptamers specific for the same
target. Further, the term "aptamer" specifically includes
"secondary aptamers" containing a consensus sequence derived from
comparing two or more known aptamers that bind to a given
target.
[0166] In general, a minimum of approximately 3 nucleotides,
preferably at least 5 nucleotides, are necessary to effect specific
binding. The only apparent limitations on the binding specificity
of the target/nucleic acid ligand complexes of the invention
concern sufficient sequence to be distinctive in the binding
nucleic acid ligand and sufficient binding capacity of the target
substance to obtain the necessary interaction. Oligonucleotides of
sequences shorter than 10 bases may be feasible if the appropriate
interaction can be obtained in the context of the environment in
which the target is placed. Although the nucleic acid ligands
described herein are single-stranded or double-stranded, it is
contemplated that aptamers may sometimes assume triple-stranded or
quadruple-stranded structures.
[0167] The specifically binding nucleic acid ligands need to
contain the sequence that confers binding specificity, but may be
extended with flanking regions and otherwise derivatized. In
preferred embodiments of the invention, the analyte-binding
sequencess of aptamer binding will be flanked by known, amplifiable
sequences, facilitating the amplification of the nucleic acid
ligands by PCR or other amplification techniques. In a further
embodiment, the flanking sequence may comprise a specific sequence
that preferentially recognizes or binds a moiety to enhance the
immobilization of the aptamer to a substrate.
[0168] The nucleic acid ligands found to bind to the targets may be
isolated, sequenced, and/or amplified or synthesized as
conventional DNA or RNA molecules. Alternatively, nucleic acid
ligands of interest may comprise modified oligomers. Any of the
hydroxyl groups ordinarily present in nucleic acid ligands may be
replaced by phosphonate groups, phosphate groups, protected by a
standard protecting group, or activated to prepare additional
linkages to other nucleotides, or may be conjugated to solid
supports. The 5' terminal OH is conventionally free but may be
phosphorylated. Hydroxyl group substituents at the 3' terminus may
also be phosphorylated. The hydroxyls may be derivatized by
standard protecting groups. One or more phosphodiester linkages may
be replaced by alternative linking groups. These alternative
linking groups include, exemplary embodiments wherein P(O)O is
replaced by P(O)S, P(O)NR.sub.2, P(O)R, P(O)OR', CO, or CNR.sub.2,
wherein R is H or alkyl (1-20C) and R' is alkyl (1-20C); in
addition, this group may be attached to adjacent nucleotides
through O or S. Not all linkages in an oligomer need to be
identical.
[0169] The nucleic acid ligands used as starting materials in the
process of the invention to determine specific binding sequences
may be single-stranded or double-stranded DNA or RNA. In a
preferred embodiment, the sequences are single-stranded DNA. The
use of DNA eliminates the need for conversion of RNA aptamers to
DNA by reverse transcriptase prior to PCR amplification.
Furthermore, DNA is less susceptible to nuclease degradation than
RNA. In preferred embodiments, the starting nucleic acid ligand
will contain a randomized sequence portion, generally including
from about 10 to 400 nucleotides, more preferably 20 to 100
nucleotides. The randomized sequence is flanked by primer sequences
that permit the amplification of nucleic acid ligands found to bind
to the analyte. The flanking sequences may also contain other
convenient features, such as restriction sites. These primer
hybridization regions generally contain 10 to 30, more preferably
15 to 25, and most preferably 18 to 20, bases of known
sequence.
[0170] Both the randomized portion and the primer hybridization
regions of the initial oligomer population are preferably
constructed using conventional solid phase techniques. Such
techniques are well known in the art, such methods being described,
for example, in Froehler, et al., (1986a, 1986b, 1988, 1987).
Nucleic acid ligands may also be synthesized using solution phase
methods such as triester synthesis, known in the art. For synthesis
of the randomized regions, mixtures of nucleotides at the positions
where randomization is desired are added during synthesis.
[0171] Any degree of randomization may be employed. Some positions
may be randomized by mixtures of only two or three bases rather
than the conventional four. Randomized positions may alternate with
those that have been specified. Indeed, it is helpful if some
portions of the candidate randomized sequence are in fact
known.
[0172] SELEX Technology
[0173] A preferred method of selecting for nucleic acid ligand
specificity involves the SELEX process. The SELEX process is
described in U.S. Pat. No. 5,475,096, and U.S. Pat. No. 5,270,163,
(see also WO91/19813), each incorporated by reference.
[0174] The SELEX method involves selection from a mixture of
candidate nucleic acid ligands and step-wise iterations of binding,
partitioning and amplification, using the same general selection
scheme, to achieve virtually any desired criterion of binding
affinity and selectivity. Starting from a mixture of nucleic acid
ligands, preferably comprising a segment of randomized sequence,
the method includes the following steps. Contacting the mixture
with the target under conditions favorable for binding.
Partitioning unbound nucleic acid ligands from those nucleic acid
ligands that have bound specifically to target analyte.
Dissociating the nucleic acid ligand-analyte complexes. Amplifying
the nucleic acid ligands dissociated from the nucleic acid
ligand-analyte complexes to yield mixture of nucleic acid ligands
that preferentially bind to the analyte. Reiterating the steps of
binding, partitioning, dissociating and amplifying through as many
cycles as desired to yield highly specific, nucleic acid ligands
that bind with high affinity to the target analyte.
[0175] In the SELEX process, a candidate mixture of nucleic acid
ligands of differing sequence is prepared. The candidate mixture
generally includes regions of fixed sequences (i.e., each of the
nucleic acid ligands contains the same sequences) and regions of
randomized sequences. The fixed sequence regions are selected to:
(a) assist in the amplification steps; (b) mimic a sequence known
to bind to the target; or (c) promote the formation of a given
structural arrangement of the nucleic acid ligands. The randomized
sequences may be totally randomized (i.e., the probability of
finding a given base at any position being one in four) or only
partially randomized (i.e., the probability of finding a given base
at any location can be any level between 0 and 100 percent).
[0176] The candidate mixture is contacted with the selected analyte
under conditions favorable for binding of analyte to nucleic acid
ligand. The interaction between the target and the nucleic acid
ligands can be considered as forming nucleic acid ligand-target
pairs with those nucleic acid ligands having the highest affinity
for the analyte.
[0177] The nucleic acid ligands with the highest affinity for the
analyte are partitioned from those nucleic acid ligands with lesser
affinity. Because only a small number of sequences (possibly only
one molecule of nucleic acid ligand) corresponding to the highest
affinity nucleic acid ligands exist in the mixture, it is generally
desirable to set the partitioning criteria so that a significant
amount of nucleic acid ligands in the mixture (approximately 5-50%)
are retained during partitioning. Those nucleic acid ligands
selected during partitioning as having higher affinity for the
target are amplified to create a new candidate mixture that is
enriched in higher affinity nucleic acid ligands.
[0178] By repeating the partitioning and amplifying steps, each
round of candidate mixture contains fewer and fewer weakly binding
sequences. The average degree of specificity and affinity of the
nucleic acid ligands to anthrax spores will generally increase with
each cycle. The SELEX process can ultimately yield a mixture
containing one or a small number of nucleic acid ligands having the
highest specificity and affinity for the target analyte. In
different embodiments, the desired degree of specificity and
binding affinity of the nucleic acid ligand(s) for anthrax spores
may vary from relatively low to quite high. It is expected that at
the lowest degree of specificity, the nucleic acid ligands will
have a higher affinity for anthrax spores than for any other
component of a sample. In preferred embodiments, the nucleic acid
ligands will bind primarily to anthrax spores, with only minor
binding exhibited for any other sample components. In the most
preferred embodiments, the nucleic acid ligands will bind only to
anthrax spores in a sample. Methods of determining specificity and
binding affinity are well known in the art and may be assessed,
within the scope of the present invention, by any such method.
[0179] Nucleic acid ligands produced for SELEX may be generated on
a commercially available DNA synthesizer. The random region is
produced by mixing equimolar amounts of each nitrogenous base
(A,C,G, and T) at each position to create a large number of
permutations (i.e., 4.sup.n, where "n" is the oligo chain length)
in a very short segment. Thus a randomized 40 mer (40 bases long)
would consist of 4.sup.30 or maximally 10.sup.24 different nucleic
acid ligands. This provides dramatically more possibilities to find
high affinity nucleic acid ligands when compared to the 10.sup.9 to
10.sup.11 variants of murine antibodies produced by a single mouse.
The random region is flanked by two short Polymerase Chain Reaction
(PCR) primer regions to enable amplification of the small subset of
nucleic acid ligands that bind tightly to the target analyte.
[0180] Another advantage of DNA-based binding is that simple
heating to .gtoreq.94.degree. C. can drive off the bound analyte.
Two potential technical hurdles associated with SELEX might be: 1)
there are potential electrostatic repulsions between the negatively
charged phosphate backbone of the nucleic acid ligands and
negatively charged target molecules, but this has not been a
significant problem in other recognized SELEX work, and 2) cloning,
which is necessary to obtain the DNA sequence of each high affinity
binding nucleic acid ligand. One final consideration is that many
RNA nucleic acids have performed well due to their propensity to
form secondary and tertiary structure "binding pockets", but RNAses
abound in nature making RNA nucleic acids less desirable for field
use. Fortunately, many single and double stranded DNA nucleic acid
ligands have also demonstrated specificity and high affinity
binding to their intended targets.
[0181] Nucleic Acid Chips and Aptamer Arrays
[0182] Nucleic acid chips and aptamer array technology provide a
means of rapidly screening analytes for their ability to hybridize
to a potentially large number of single stranded nucleic acid
ligand probes immobilized on a solid substrate. In preferred
embodiments, the nucleic acid ligands are DNA. Specifically
contemplated are chip-based DNA technologies such as those
described by Hacia et al., 1996 and Shoemaker et al., 1996. These
techniques involve quantitative methods for analyzing large numbers
of samples rapidly and accurately. The technology capitalizes on
the binding properties of single stranded DNA to screen samples.
(Pease et al., 1994; Fodor et al., 1993; Southern et al., 1994;
Travis, 1997; Lipshutz et al., 1995; Matson et al., 1995; each of
which is incorporated herein by reference.)
[0183] A nucleic acid ligand chip or array consists of a solid
substrate upon which an array of single stranded nucleic acid
ligand molecules have been attached. For screening, the chip or
array is contacted with a sample containing analyte that is allowed
to bind to the ligands. The degree of stringency of binding may be
manipulated as desired by varying, for example, salt concentration,
temperature, pH and detergent content of the medium. The chip or
array is then scanned to determine which nucleic acid ligands have
bound to the analyte. Prior to the present invention, DNA chips
were typically used to bind to target DNA or RNA molecules in a
sample.
[0184] A variety of DNA chip formats are described in the art, for
example U.S. Pat. Nos. 5,861,242 and 5,578,832, incorporated herein
by reference. The structure of a nucleic acid ligand chip or array
comprises: (1) an excitation source; (2) an array of probes; (3) a
sampling element; (4) a detector; and (5) a signal
amplification/treatment system. A chip may also include a support
for immobilizing the probe.
[0185] In particular embodiments, a nucleic acid ligand may be
tagged or labeled with a substance that emits a detectable signal,
for example, DALM. The tagged or labeled species may be
fluorescent, phosphorescent, luminescent, chemiluminescent or
electrochemiluminescent or it may emit Raman energy or it may
absorb energy. In certain embodiments, detection may occur by
enhanced chemiluminescent (ECL) detection (AP Biotech, Piscataway,
N.J.). When the nucleic acid ligand binds to a targeted analyte, a
signal is generated that is detected by the chip. The signal may
then be processed in several ways, depending on the nature of the
signal.
[0186] The nucleic acid ligand may be immobilized onto an
integrated microchip that also supports a phototransducer and
related detection circuitry. Alternatively, a nucleic acid ligand
may be immobilized onto a membrane or filter that is then attached
to the microchip or to the detector surface itself.
[0187] The nucleic acid ligands may be directly or indirectly
immobilized onto a transducer detection surface to ensure optimal
contact and maximum detection. The ability to directly synthesize
on or attach polynucleotide probes to solid substrates is well
known in the art. See U.S. Pat. Nos. 5,837,832 and 5,837,860,
incorporated by reference. A variety of methods have been utilized
to either permanently or removably attach the nucleic acid ligands
to the substrate. Exemplary methods are described above under the
section on immobilization. When immobilized onto a substrate, the
nucleic acid ligands are stabilized and may be used repeatedly.
[0188] Exemplary substrates include nitrocellulose, nylon membrane
or glass. Numerous other matrix materials may be used, including
reinforced nitrocellulose membrane, activated quartz, activated
glass, polyvinylidene difluoride (PVDF) membrane, polystyrene
substrates, polyacrylamide-based substrate, other polymers such as
poly(vinyl chloride), poly(methyl methacrylate), poly(dimethyl
siloxane) and photopolymers which contain photoreactive species
such as nitrenes, carbenes and ketyl radicals capable of forming
covalent links with target molecules (U.S. Pat. Nos. 5,405,766 and
5,986,076, each incorporated herein by reference).
[0189] Binding of nucleic acid ligand to a selected support may be
accomplished by any of several means. For example, DNA is commonly
bound to glass by first silanizing the glass surface, then
activating with carbodiimide or glutaraldehyde. Alternative
procedures may use reagents such as
3-glycidoxypropyltrimethoxysilane (GOP) or
aminopropyltrimethoxysilane (APTS) with DNA linked via amino
linkers incorporated either at the 3' or 5' end of the molecule
during DNA synthesis. DNA may be bound directly to membranes using
ultraviolet radiation. With nitrocellulose membranes, the DNA
probes are spotted onto the membranes. A UV light source
(Stratalinker, from Stratagene, La Jolla, Calif.) is used to
irradiate DNA spots and induce cross-linking. An alternative method
for cross-linking involves baking the spotted membranes at
80.degree. C. for two hours in vacuum. Further, it is specifically
contemplated that the nucleic acid ligand may be bound to an
immobilized indicator species. Therefore, in a preferred embodiment
of the invention, DALM is immobilized to a solid substrate and the
nucleic acid ligands attached to the immobilized DALM.
Alternatively, the DALMI nucleic acid ligand complex may be bound
via the DALM or the polynucleotide to the substrate.
[0190] Specific nucleic acid ligands may first be immobilized onto
a membrane and then attached to a membrane in contact with a
transducer detection surface. This method avoids binding the
nucleic acid ligand onto the transducer and may be desirable for
large-scale production. Membranes particularly suitable for this
application include nitrocellulose membrane (e.g., from BioRad,
Hercules, Calif.) or polyvinylidene difluoride (PVDF) (BioRad,
Hercules, Calif.) or nylon membrane (Zeta-Probe, BioRad) or
polystyrene base substrates (DNA.BIND.TM. Costar, Cambridge,
Mass.).
[0191] Biological Sensors and In Vivo Aptamer Production
[0192] The lac operon regulates the transcription of DNA into mRNA
for translation into .beta.-galactosidase, permease, and
transacetylase. These three enzymes are necessary for the bacteria
to metabolize lactose. The expression of .beta.-galactosidase in a
variety of cells including E. coli has become an invaluable tool
for marking transfection (the insertion of foreign genes) and
expression of genes. By using a medium that contains a substrate
(x-gal) for .beta.-galactosidase that turns blue upon the action of
the enzyme, one can detect the insertion of foreign genes into the
.beta.-galactosidase gene. In the absence of an insert into
.beta.-galactosidase, expression of the lac operon results in a
blue color on X-gal, while the presence of an insert results in a
white bacterial plaque.
[0193] The lac repressor gene within the lac operon encodes a
protein that prevents the enzymes in the lac operon from being
expressed. The repressor protein is inactivated by binding to an
inducer or de-repressor, resulting in expression of
.beta.-galactosidase and causing a blue color to form on x-gal. In
the absence of an inducer or de-repressor, only the repressor is
translated from the lac operon and no lactose (or color-producing
substrate) metabolism occurs. The repressor gene is always
translated first, before the enzymes in the operon. Therefore, if
the transcription of the repressor gene is altered too much, the
downstream genes will not be expressed (no blue color).
[0194] This method can be carried one step further. By inserting a
marker gene in place of the .beta.-galactosidase gene, induction or
derepression of the lac operon results in the expression of the new
protein in place of .beta.-galactosidase. Other markers used to
replace galactosidase include green fluorescent protein (GFP),
chloramphenicol acetyltransferase (CAT), luciferase, and nitrate
reductase (U.S. Pat. No. 5,902,728). The GFP makes the cells
fluoresce green, CAT converts radiolabeled chloramphenicol to a
more soluble product that appears in a different place on a
thin-layer chromatographic plate, luciferase produces
bioluminescence in transfected cells, and nitrate reductase can
produce colorimetric, fluorescent, or luminescent products in
cells.
[0195] A mutagen assay based on the lac operon has been
incorporated into cultured animal cells and whole transgenic
animals (Big Blue.TM. mice and rats). Mutations in the repressor
gene allow for unrestricted expression of .beta.-galactosidase and
production of blue colored substrate. Thus, mutagenic activity can
be assayed by measuring the level of blue plaques obtained in the
absence of induction. Further, by replacing the promoter of the lac
operon with another promoter that is responsive to different
regulatory factors, one can test for factors that bring about
expression of any gene of choice, using marker gene expression.
[0196] The problem of using the above methods is that a specific
promoter must be found for each agent (regulatory factor) that is
to be detected. To do this, microbes that already have the
appropriate metabolic machinery to detect the presence of a
specific agent must be found or genetically engineered. This has
been done at Oak Ridge National Laboratory for detection of
explosives using genetically engineered pseudomonads. The presence
of the specific agent (explosive) induces expression of a gene
encoding GFP. Thus, the pseudomonad produces GFP when spread out
over ground containing landmines (leaking explosives).
[0197] It would be much more convenient to genetically engineer the
lac operon of a microbe like E. coli to detect a variety of agents
(analytes). By using aptamers that can be selected to bind to
almost any desired target, this problem may be solved. DNA
sequences comprising nucleic acid ligands may be incorporated into
the repressor gene or its promoter in such a way that when the
target analyte binds to the nucleic acid ligand, expression of the
repressor protein is inhibited and .beta.-galactosidase or another
marker gene is expressed. Thus, blue colonies or other markers will
appear in the presence of the designed inducer (i.e. the target
analyte). Since aptamers with high affinity against virtually any
target analyte can be prepared and sequenced using the methods
described herein, it would be possible to design an appropriate
biosensor microorganism that is capable of detecting almost any
molecule in the environment.
[0198] It is envisioned within the scope of the invention that the
target analyte could bind to the nucleic acid ligand either within
the intact repressor gene or its promoter, or in the mRNA
transcript of the repressor gene, prior to its translation into
protein. High-affinity binding of analyte to mRNA would interfere
with ribosomal binding and mRNA translation. For this reason, in
preferred embodiments it may be desirable for the ligand insertion
site to be close to the ribosomal binding site of the repressor
gene sequence, allowing for steric hindrance of ribosomal
binding.
[0199] This process can be extended to a large library of aptamers,
each of which is inserted into the same site of the repressor gene
or its promoter. The process can thus be used to select an
appropriate nucleic acid ligand for a target analyte of choice by
selecting for a bacterial clone that is colored blue or otherwise
marked only in the presence of the target analyte. Amplification of
the selected clone and DNA sequencing would result in the
identification of aptamer sequences that can bind with high
affinity to the target analyte. The normal inducer will also work
because it acts on the repressor gene product (the repressor
protein itself) rather than the machinery to translate the gene
into protein (like the aptamer). This is an important positive
control to confirm the fidelity of the system. This method would
allow for screening of nucleic acid ligand libraries and selection
and amplification of nucleic acid ligands with high affinity for a
target analyte, as an alternative to the SELEX process. Purified
nucleic acid ligands of appropriate binding specificity may be
obtained either by chemical synthesis or by PCR or other
amplification processes using primers selected to flank the ligand
insertion site.
[0200] The process may also be adapted for use with a recognition
complex system. By cloning in E. coli (see U.S. Pat. No. 5,902,728,
incorporated herein by reference) or another appropriate host that
has been genetically engineered to produce the organic
semiconductor, such as DALM, then growth on an appropriate medium
will result in the production of aptamers that are already
operatively linked to the organic semiconductor.
[0201] Formulations and Routes for Administration to Patients
[0202] Where clinical applications are contemplated, it will be
necessary to prepare pharmaceutical compositions--such as
therapeutic nucleic acid ligands--in a form appropriate for the
intended application. Generally, this will entail preparing
compositions that are essentially free of pyrogens, as well as
other impurities that could be harmful to humans or animals.
[0203] Aqueous compositions of the present invention comprise an
effective amount of the ligand to cells, dissolved or dispersed in
a pharmaceutically acceptable carrier or aqueous medium. Such
compositions also are referred to as innocula. The phrase
"pharmaceutically or pharmacologically acceptable" refers to
molecular entities and compositions that do not produce adverse,
allergic, or other untoward reactions when administered to an
animal or a human. As used herein, "pharmaceutically acceptable
carrier" includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
nucleic acid ligands of the present invention, its use in
therapeutic compositions is contemplated. Supplementary active
ingredients also can be incorporated into the compositions.
[0204] The active compositions of the present invention may include
classic pharmaceutical preparations. Administration of these
compositions according to the present invention will be via any
common route so long as the target tissue is available via that
route. This includes oral, nasal, buccal, rectal, vaginal or
topical. Alternatively, administration may be by orthotopic,
intradermal, subcutaneous, intramuscular, intraperitoneal or
intravenous injection. Such compositions normally would be
administered as pharmaceutically acceptable compositions, described
supra.
[0205] The active compounds also may be administered parenterally
or intraperitoneally. Solutions of the active compounds as free
base or pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions also can be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0206] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), suitable mixtures thereof, and vegetable
oils. The proper fluidity can be maintained, for example, by the
use of a coating, such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. The prevention of the action of microorganisms can be
brought about by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal,
and the like. In many cases, it will be preferable to include
isotonic agents, for example, sugars or sodium chloride. Prolonged
absorption of the injectable compositions can be brought about by
the use in the compositions of agents delaying absorption, for
example, aluminum monostearate and gelatin.
[0207] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0208] The compositions of the present invention may be formulated
in a neutral or salt form. Pharmaceutically-acceptable salts
include the acid addition salts which are formed by reaction of
basic groups with inorganic acids such as, for example,
hydrochloric or phosphoric acids, or such organic acids as acetic,
oxalic, tartaric, mandelic, and the like. Salts formed with free
acidic groups can also be derived from inorganic bases such as, for
example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like.
[0209] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms such as injectable solutions, drug
release capsules and the like. For parenteral administration in an
aqueous solution, for example, the solution should be suitably
buffered if necessary and the liquid diluent first rendered
isotonic with sufficient saline or glucose. These particular
aqueous solutions are especially suitable for intravenous,
intramuscular, subcutaneous and intraperitoneal administration. In
this connection, sterile aqueous media which can be employed will
be known to those of skill in the art in light of the present
disclosure. For example, one dosage could be dissolved in 1 ml of
isotonic NaCl solution and either added to 1000 ml of
hypodermoclysis fluid or injected at the proposed site of infusion,
(see for example, "Remington's Pharmaceutical Sciences" 15th
Edition, pages 1035-1038 and 1570-1580). Some variation in dosage
will necessarily occur depending on the condition of the subject
being treated. The person responsible for administration will, in
any event, determine the appropriate dose for the individual
subject. Moreover, for human administration, preparations should
meet sterility, pyrogenicity, general safety and purity standards
as required by FDA Office of Biologics standards.
EXAMPLES
[0210] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventors to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
[0211] In Vitro Selection of High Affinity Nucleic Acid Ligands for
Anthrax Spores
[0212] Functional nucleic acid ligands can be selected from
random-sequence nucleic acid pools by a process known as SELEX.
This technique allows repetitive cycles of selection and
amplification of single-stranded nucleic acids in vitro (Tuerk and
Gold, 1990; Ellington and Szostak, 1990). SELEX methodology was
used to develop high affinity single stranded DNA (ssDNA) ligands
that bind to live anthrax spores. The mixture of ssDNA and target
(anthrax spores) was allowed to interact. Nucleic acid ligands that
bound to the spores were separated from unbound ligands using a
nitrocellulose filter method. Aptamers that bound to anthrax spores
were separated from the spores and used for the next round of
selection.
[0213] Libraries and Primers: The starting material for SELEX
preparation of anti-anthrax spores comprised synthetic DNA
containing fixed sequences for primer annealing in a PCR
amplification reaction. The starting nucleic acid ligand library
was composed of 86-mers, containing 40-mer random DNA sequences
(N40) attached to 5' and 3' fixed primer annealing sequences, as
shown in Table 2 below.
3TABLE 2 5' Fixed sequences for primer 3' Fixed sequences for
annealing Random sequences primer annealing 5'-CCCCTGCAGGTGATTTT
NNNN---NNNN (40N) 5'-AGTATCGCTAATCA GCTCAAGT-3' GGCGGAT-3' (SEQ ID
NO:1) (SEQ ID NO:2)
[0214] In the Table above, N represents an equal mixture of all
four nucleotides (A, G, T and C). The 5' end of the 5' fixed
sequence was covalently attached to three biotin residues to
facilitate binding of the nucleic acid ligands to streptavidin. The
oligonucleotide library and corresponding PCR primers were
purchased from Genosys (The Woodland, Texas). Taq polymerase was
obtained from Display Systems Biotech (Vista, Calif.). A DNTP
mixture was purchased from Applied Biosystems (Foster City,
Calif.). Ultra pure urea, bis-acrylamide, fluor-coated TLC plates
and buffer saturated phenol were from Ambion (Austin, Tex.).
Glycogen and streptavidin-linked beads were purchased from Roche
Molecular Biochemicals (Indianapolis, Ind.). Spin columns and
10.times.TBE (Tris-borate-EDTA) buffer were from BioRad (Hercules,
Calif.). Nitrocellulose discs were from Millipore (Bedford, Mass.).
All other reagent grade chemicals were purchased from Sigma (St.
Louis, Miss.). Anthrax Spore Vaccine, a non-encapsulated live
culture, was supplied by the Colorado Serum Company (Denver,
Colo.).
[0215] Anthrax Spores: Anthrax spore vaccine was transferred from
the manufacturer's vial to sterile centrifugation tubes that had
been chilled on ice. The spores were pelleted by centrifuging at
9500.times.g for 10 min at 4.degree. C. and the pellet was washed
with ice cold sterile distilled water. Spores were resuspended in
ice cold, sterile distilled water and stored temporarily at
4.degree. C.
[0216] AK sporulation agar was used to make agar plates according
to the manufacturer's instructions. Sterile cotton-tipped swabs
were used to streak each agar plate with the anthrax spore
suspension. Plates were incubated at 37.degree. C. for 4 days and
then checked for complete sporulation under a light microscope.
Spores were harvested from the plates by using sterile cotton
tipped swabs wetted with distilled water. The swab was run across
the plate and placed into sterile ice-cold distilled water. The
entire layer of anthrax growth was removed and transferred to
distilled water. The spore suspension was then vacuum filtered
using a sterile Buchner funnel and Whatman filter paper into a
sterile flask in an ice bath. The spores are filtered through the
filter paper while vegetative debris is trapped on the filter
paper. The filtrate consisted almost entirely of spores. The spores
were heat treated at 65.degree. C. for 30 min and cooled
immediately in an ice bath. The suspension was centrifuged at
9500.times.g for 10 min, resuspended in ice cold sterile distilled
water and stored at 4.degree. C. until use. Stock spore suspension
concentration was determined from the average colony forming units
(CFUs) obtained from triple replicates at five different dilutions
of stock suspension.
[0217] The initial nucleic acid ligand library was amplified by
PCR. The 5' primer used was identical to SEQ ID NO:1, disclosed
above, with 3 biotin residues attached to the 5' end of the primer.
The 3' primer was complementary to the 3' fixed sequence disclosed
in Table 2 and is shown below as SEQ ID NO:3. PCR conditions were
checked in 200 .mu.L reaction mixture, using 5 pmol of template and
0.1 .mu.M of each primer, 20 .mu.L of 10.times.PCR reaction buffer,
2 .mu.L of 10 mM dNTP mix and 5 units of display TAQ polymerase,
with distilled water added to 200 .mu.L. Optimal PCR conditions
were determined to be denaturation at 94.degree. C. for 3 min,
annealing at 45.degree. C. for 30 sec, and extension at 72.degree.
C. for 1 min, with a final extension at 72.degree. C. for 3 min.
The reaction was performed using a Robocycler Model 96 thermal
cycler with a "Hot Top" assembly (Stratagene, La Jolla, Calif.).
The PCR product was checked every third cycle and the optimal
number of cycles determined. After obtaining optimal conditions,
the original library was amplified to prepare 25 ml of reaction mix
(125 reactions at 200 .mu.L each). The amplified DNA pool was
recovered by ethanol precipitation in the presence of glycogen and
the final DNA pellet was resuspended in sterile TE buffer
[Tris-HCl, EDTA, pH 8.0] and used for streptavidin binding.
[0218] 5'-ATCCGCCTGATTAGCGATACT-3' (SEQ ID NO:3)
[0219] Streptavidin Binding: Resuspended double stranded DNA was
mixed with streptavidin agarose beads and incubated at room
temperature to allow binding of biotin labeled DNA to streptavidin.
The mixture was transferred to spin columns and denatured by
addition of 0.2 M NaOH. The biotin labeled DNA strand remained in
the column along with the streptavidin beads, while the unlabeled
strand passed through the column and was collected. The eluate was
neutralized with 3 M sodium acetate, pH 5.0, ethanol precipitated
overnight and recovered by centrifugation at 4.degree. C. at 13,000
rpm. The ssDNA pellet was resuspended in TE buffer and used for gel
purification.
[0220] Gel Purification of ssDNA: The ssDNA was mixed with a
denaturing 2.times.sample buffer containing 90% formamide, 1 mM
EDTA and 0.1% bromophenol blue and heated at 90.degree. C. for 5
min. After cooling to room temperature, the contents were separated
by electrophoresis in a 6% acrylamide/bis (19:1) gel, with 7M urea
in 1.times.TBE buffer for 2 hours at 150 volts. The ssDNA was
visualized under UV light and the bands cut out and eluted
overnight in 0.3 M sodium chloride. Eluted DNA was ethanol
precipitated overnight and collected by centrifugation. The DNA
pellet was resuspended in TE buffer and used for in vitro
selection.
[0221] In vitro Selection by SELEX: To exclude filter-binding ssDNA
sequences from the pool, the DNA was initially passed over a 0.45
.mu.m HAWP filter (Millipore, Bedford, Mass.) and washed with TE
buffer. The filtrate containing non-binding DNA was used for in
vitro selection. In general, the final yield of ssDNA was in the
.mu.mole range. One hundred pmol of ssDNA was incubated with live
anthrax spores (0.5.times.10.sup.6 spores) in binding buffer (20 mM
Tris-HCI, pH 7.5, 45 mM sodium chloride, 3 mM magnesium chloride, 1
mM EDTA, 1 mM diothiothreitol in a final volume of 250 .mu.L)
according to Hesselberth et al. (2000). The binding reaction
mixture was incubated for one hour at room temperature, then vacuum
filtered through a HAWP filter at 5 psi and washed twice with 0.2
ml of binding buffer. DNA that bound to anthrax spores was retained
on the filter, while nucleic acid ligands that did not bind to
anthrax passed through the filter. The anthrax-binding ssDNA was
eluted 2.times. with 0.2 ml of 7 M urea, 100 mM MES
(4-morpholine-ethansulfonic acid, Roche Molecular Biochemicals), pH
5.5, 3 mM EDTA for 5 min at 100.degree. C. The eluted
anthrax-binding ssDNA was ethanol precipitated overnight and
collected by centrifugation. The pelleted DNA was resuspended and
used for the next round of SELEX selection.
[0222] Results: The methods described above resulted in the
production of ssDNA nucleic acid ligands that bind with high
affinity to live anthrax spores (Bacillus anthracis Sterne strain).
In vitro selection was performed using the SELEX procedure as
described above. (Robertson and Joyce, 1990; Tuerk and Gold, 1990;
Ellington and Szostak, 1990). Nucleic acid ligands containing 40 bp
random DNA sequences were screened for binding to live anthrax
spores. Anthrax-binding nucleic acid ligands were eluted, amplified
by PCR and subjected to further rounds of SELEX screening. A total
of seven rounds of SELEX screening were performed. Gel
electrophoresis analysis showed that the PCR amplification products
after each round were the same size (86-mer) as the original pool,
demonstrating that the primers were amplifying nucleic acid ligand
sequences, not anthrax genomic sequences. Controls performed in the
absence of anthrax spores, or in the presence of spores but the
absence of the ssDNA pool, showed no PCR amplification product,
demonstrating that the SELEX procedure resulted in the production
of anthrax-binding nucleic acid ligands.
[0223] FIG. 4 shows a representative gel electrophoresis analysis
of anthrax-binding nucleic acid ligands after five rounds of SELEX
selection (FIG. 4, lane 4). The amplification product (after 10 PCR
cycles) is present as essentially a single band. A zero
amplification control (FIG. 4, lane 3) shows that the band is not
observed in the absence of amplification. A positive PCR control
(FIG. 4, lane 7) shows that the anthrax-binding amplification
product is the same size as the PCR amplification products of the
initial random nucleic acid library. The positive control was run
on the gel after seven cycles of PCR amplification.
[0224] The sequences of anthrax-binding nucleic acid ligands
identified by the disclosed methods were as shown below.
4 SEQ ID NO:4 5'-GGATGAAATTATGAAGGAGTAATAGTGTGATGGAGTGGT- A-3' SEQ
ID NO:5 5'-ACCCGGTTAATTCGTAGTAGAGGAGG- GTCGTTTGGAGTCA-3' SEQ ID
NO:6 5'-AGAGGAATGTATAAGGATGTTCCGGGCGTGTGGGTAAGTC-3'
Example 2
[0225] Neutralization of Anthrax Spores Using DALM
[0226] In a preferred embodiment of the instant invention, nucleic
acid ligands with high affinity for anthrax spores are produced and
purified using the disclosed methods. Such nucleic acid ligands may
be used to neutralize biohazardous agents, such as viruses,
microbes, spores or potentially single molecules. More preferably,
high-affinity nucleic acid ligands against anthrax may be used to
neutralize anthrax spores in the field.
[0227] High affinity nucleic acid ligands may be produced as
disclosed in the preceding example. Such nucleic acid ligands may
be attached to a compound such as DALM. The nucleic acid ligand
provides specificity of binding to the target. The DALM-nucleic
acid ligand couplet is then used essentially as a photochemical
transducer.
[0228] DALM is capable of absorbing electromagnetic radiation
within a broad range of wavelengths and transmitting the absorbed
energy to molecules or targets to which it is attached. DALM
attached to a target via a bound nucleic acid ligand is irradiated
with a pulse of electromagnetic radiation. The radiation may be
transmitted in the form of visible light or infrared radiation, but
other forms of irradiation, such as microwave, laser or
radio-frequency are contemplated within the scope of the present
invention. Irradiation results in absorption of energy by DALM,
which is transmitted to the target. The resulting heating and
production of reactive chemical species produces an explosive
surface reaction that destroys the target.
[0229] DALM activated by hydrogen peroxide and bicarbonate and
pulsed with microwave radiation acts as a photochemical transducer,
releasing an intense pulse of visible light (not shown). High power
pulsed microwave radiation (HPM), applied to solutions containing
dissolved carbon dioxide (or bicarbonate), hydrogen peroxide and
DALM generates sound, pulsed luminescence and electrical discharge.
Microbes exposed to these conditions experience damage comparable
to short time, high temperature insults, even though measurable
localized temperatures were insufficient to cause the observed
effects.
[0230] Anthrax Spores: Steme strain veterinary vaccine anthrax
spores (Thraxol-2, Mobay Corp., Shawnee, Kans.) were streaked onto
blood agar plates and incubated at 37.degree. C. for 5 days to
promote extensive sporulation and autolysis of vegetative cells.
Colonies were gently washed and scraped from blood agar plates into
10 ml of filter-sterilized deionized water. The resultant
suspension consisted almost exclusively of spores. Vegetative cell
debris appeared to be largely removed by three washes in 10 ml of
filter-sterilized deionized water with resuspension and
centrifugation at 9,300.times.G for 10 min, as determined by
phase-contrast microscopy. Stock spore suspension concentration was
determined by the average of four hemocytometer counts to be
6.5.times.10.sup.6 spores/ml (standard
deviation=0.24.times.10.sup.6) using phase-contrast microscopy at
600.times.magnification.
[0231] DALM Mediated Neutralization of Anthrax Spores: Bacillus
anthracis spores were incubated with DALM and exposed to a high
power microwave (HPM) pulse. Bacillus anthracis (Sterne strain)
spore vaccine (Thraxol.TM., Mobay Corp., Animal Health Division,
Shawnee, Kans.) was centrifuged, the supernatant decanted and the
button washed with chilled deionized water. Dilute powdered milk
solution was made to a concentration of 25 mg of powdered milk
solids/ml of deionized water, filtered through a 0.2 micron filter.
The anthrax pellet was resuspended in 1 ml of sterile milk solution
to form an anthrax spore suspension.
[0232] For pulsed microwave exposure, 0.5 ml of anthrax spore
suspension was placed into 0.2 micron-filter centrifuge tubes
(Microfilterfuge.TM., Rainin Instrument Co., Inc., Woburn, Mass.).
The spores were centrifuged onto the filter at 16,000.times.g for
15 min. The tubes were refilled with 1.5 ml of a reaction mixture
consisting of 0.9 ml saturated sodium bicarbonate/luminol solution,
0.1 ml of 1:10 biosynthetic DALM, 0.6 ml of 1:10 diazoluminol, and
0.33 ml 3% hydrogen peroxide. All dilutions were made in saturated
sodium bicarbonate/luminol solution. The final dilution of DALM was
1:1000. A detailed description of the reaction mixture has been
published (Kiel et al., 1999a; Kiel et al., 1999b).
[0233] The filter, with the anthrax spores, was inserted into the
tube to a level just below the meniscus of the fluid. The solution
was exposed to 10 pulses per second of HPM (1.25 GHz, 6 .mu.sec
pulse, 2 MW peak incident power), starting at 3 minutes and 22
seconds after placing the reaction mixture in front of the
microwave waveguide. The exposure lasted for 13 min and 28 sec.
Total radiation exposure was for 48 msec. The temperature of the
sample, continuously monitored with a non-perturbing,
high-resistance temperature probe (Vitek.TM.), began at
25.3.degree. C. and reached an end point of 64.degree. C., below
the lethal temperature for anthrax spores.
[0234] Results: FIGS. 5A-5B shows the result of this procedure. The
control spore (FIG. 5A) was exposed to HPM in the absence of DALM.
It remained intact. The anthrax spore shown in FIG. 5B was exposed
to HPM in the presence of DALM. The spore lysed, with its contents
spread around the remnants of the spore (FIG. 5B). The effect of
HPM and DALM on anthrax spores shows that DALM coupled to nucleic
acid ligands may be used to neutralize biohazardous agents, such as
anthrax, against which high affinity nucleic acid ligands are
prepared by the methods disclosed herein.
[0235] All of the COMPOSITIONS, METHODS and APPARATUS disclosed and
claimed herein can be made and executed without undue
experimentation in light of the present disclosure. While the
compositions and methods of this invention have been described in
terms of preferred embodiments, it will be apparent to those of
skill in the art that variations may be applied to the
COMPOSITIONS, METHODS and APPARATUS and in the steps or in the
sequence of steps of the methods described herein without departing
from the concept, spirit and scope of the invention. More
specifically, it will be apparent that certain agents that are both
chemically and physiologically related may be substituted for the
agents described herein while the same or similar results would be
achieved. All such similar substitutes and modifications apparent
to those skilled in the art are deemed to be within the spirit,
scope and concept of the invention as defined by the appended
claims.
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Sequence CWU 1
1
6 1 25 DNA Artificial Artificial 1 cccctgcagg tgattttgct caagt 25 2
21 DNA Artificial Artificial 2 agtatcgcta atcaggcgga t 21 3 21 DNA
Artificial Artificial 3 atccgcctga ttagcgatac t 21 4 40 DNA
Artificial Artificial 4 ggatgaaatt atgaaggagt aatagtgtga tggagtggta
40 5 40 DNA Artificial Artificial 5 acccggttaa ttcgtagtag
aggagggtcg tttggagtca 40 6 40 DNA Artificial Artificial 6
agaggaatgt ataaggatgt tccgggcgtg tgggtaagtc 40
* * * * *