U.S. patent application number 12/931309 was filed with the patent office on 2012-08-30 for methods of running assays using intrachain fluorophore-quencher fret-aptamers.
This patent application is currently assigned to PRONUCLEOTEIN BIOTECHNOLOGIES, LLC. Invention is credited to John G. Bruno, Joseph Chanpong.
Application Number | 20120219961 12/931309 |
Document ID | / |
Family ID | 46719228 |
Filed Date | 2012-08-30 |
United States Patent
Application |
20120219961 |
Kind Code |
A1 |
Bruno; John G. ; et
al. |
August 30, 2012 |
Methods of running assays using intrachain fluorophore-quencher
FRET-aptamers
Abstract
The present invention describes methods for the production and
use of single chain (single-stranded) fluorescence resonance energy
transfer ("FRET") DNA or RNA aptamers containing fluorophores (F)
and quenchers (Q) at various loci within their structures, such
that when its specific matching analyte is bound and the
FRET-aptamers are excited by specific wavelengths of light, the
fluorescence intensity of the system is modulated (increased or
decreased) in proportion to the amount of analyte added. F and Q
are covalently linked to nucleotide triphosphates (NTPs), which are
incorporated by various nucleic acid polymerases such as Taq
polymerase during the polymerase chain reaction (PCR) and then
selected by affinity chromatographic, size-exclusion or molecular
sieving, and fluorescence techniques. Further separation of related
FRET-aptamers can be achieved by ion-pair reverse phase high
performance liquid chromatography (HPLC) or other types of
chromatography. Finally, FRET-aptamer structures and the specific
locations of F and Q within FRET-aptamer structures are determined
by digestion with exonucleases and mass spectral nucleotide
sequencing analysis.
Inventors: |
Bruno; John G.; (San
Antonio, TX) ; Chanpong; Joseph; (San Antonio,
TX) |
Assignee: |
PRONUCLEOTEIN BIOTECHNOLOGIES,
LLC
|
Family ID: |
46719228 |
Appl. No.: |
12/931309 |
Filed: |
January 28, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11433009 |
May 12, 2006 |
7906280 |
|
|
12931309 |
|
|
|
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Current U.S.
Class: |
435/7.1 ;
436/501 |
Current CPC
Class: |
C12N 2320/10 20130101;
C12N 2310/16 20130101; C12N 15/111 20130101; G01N 33/5308 20130101;
G01N 33/542 20130101; G01N 33/533 20130101 |
Class at
Publication: |
435/7.1 ;
436/501 |
International
Class: |
G01N 21/64 20060101
G01N021/64 |
Claims
1. A method of assaying a target molecule in a solution using
single-stranded fluorescence resonance energy transfer
("FRET")-aptamers, comprising: selecting FRET-aptamers that bind
with said target molecule; adding said selected FRET-aptamers to
said solution, wherein said FRET-aptamers have at least one
fluorophore ("F") and at least one quencher ("Q") incorporated into
said FRET-aptamers in the interior of said FRET-aptamers; wherein
said F and said Q are spectrally matched such that an absorption
spectrum of said Q overlaps significantly with an emission spectrum
of said F; wherein prior to binding said target molecule said
location of said F is within a Forster distance of said Q such that
said Q interacts with said F such that minimal fluorescence is
detectable; wherein said FRET-aptamers emit increased detectable
fluorescence after binding said target molecule due to an increase
in said Forster distance between said Q and said F, and said
detectable fluorescence changes proportionately in response to the
amount of said target molecule in said solution; measuring said
fluorescence light level; and determining the presence or absence
of said target molecules in said solution.
2. The method of claim 1, further comprising calculating the amount
of said target molecules in said solution
3. The method of claim 2, wherein said assay is used for detecting
and quantifying a target molecule, wherein said target molecule is
less than 1,000 Daltons.
4. The method of claim 3, wherein said target molecule is selected
from the group consisting of: pesticides, natural and synthetic
amino acids, histidine, histamine, homocysteine, DOPA, melatonin,
nitrotyrosine, short chain proteolysis products, cadaverine,
putrescine, polyamines, spermine, spermidine, nitrogen bases of DNA
or RNA, nucleosides, nucleotides, nucleotide cyclical isoforms,
cAMP, cGMP, cellular metabolites, urea, uric acid, pharmaceuticals,
therapeutic drugs, narcotics, hallucinogens, gamma-hydroxybutyrate,
cellular mediators, cytokines, chemokines, immune modulators,
neural modulators, inflammatory modulators, prostaglandins,
prostaglandin metabolites, explosives, trinitrotoluene, peptides,
macromolecules, proteins, bacterial surface proteins,
glycoproteins, lipids, glycolipids, nucleic acids, polysaccharides,
lipopolysaccharides, whole cells, and subcellular organelles or
cellular fractions.
5. The method of claim 2, wherein said assay is used for detecting
and quantifying a target molecule, wherein said target molecule is
equal to or greater than 1,000 Daltons and water- soluble.
6. A method of assaying a target molecule in a solution using
single-stranded fluorescence resonance energy transfer
("FRET")-aptamers, comprising: selecting FRET-aptamers that bind
with said target molecule; adding said selected FRET-aptamers to
said solution, wherein said FRET-aptamers have at least one
fluorophore ("F") and at least one quencher ("Q") incorporated into
said FRET-aptamers in the interior of said FRET-aptamers; wherein
said F and said Q are spectrally matched such that an absorption
spectrum of said Q overlaps significantly with an emission spectrum
of said F; wherein prior to binding said target molecule said
location of said F is beyond a Forster distance of said Q such that
said Q does not interact with said F such that said F emits
detectable fluorescence wherein said FRET-aptamers emit decreased
detectable fluorescence after binding said target molecule due to a
decrease in the Forster distance between said F and said Q, and
said detectable fluorescence changes proportionately in response to
the amount of said target molecule in said solution; measuring said
fluorescence light level; and determining the presence or absence
of said target molecules in said solution.
7. The method of claim 6, wherein said assay is used for detecting
a target molecule, wherein said target molecule is less than 1,000
Daltons.
8. The method of claim 7, wherein said target molecule is selected
from the group consisting of: pesticides, natural and synthetic
amino acids, histidine, histamine, homocysteine, DOPA, melatonin,
nitrotyrosine, short chain proteolysis products, cadaverine,
putrescine, polyamines, spermine, spermidine, nitrogen bases of DNA
or RNA, nucleosides, nucleotides, nucleotide cyclical isoforms,
cAMP, cGMP, cellular metabolites, urea, uric acid, pharmaceuticals,
therapeutic drugs, narcotics, hallucinogens, gamma-hydroxybutyrate,
cellular mediators, cytokines, chemokines, immune modulators,
neural modulators, inflammatory modulators, prostaglandins,
prostaglandin metabolites, explosives, trinitrotoluene, peptides,
macromolecules, proteins, bacterial surface proteins,
glycoproteins, lipids, glycolipids, nucleic acids, polysaccharides,
lipopolysaccharides, whole cells, and subcellular organelles or
cellular fractions.
9. The method of claim 6, wherein said assay is used for detecting
a target molecule, wherein said target molecule is equal to or
greater than 1,000 Daltons and water-soluble.
Description
[0001] This application is based upon and claims priority from U.S.
Utility application Ser. No. 11/433,009, which is incorporated
herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] Applicants' invention relates to the field of aptamer- and
nucleic acid-based diagnostics. More particularly, it relates to
methods for the production and use of single chain
(single-stranded) fluorescence resonance energy transfer ("FRET")
DNA or RNA aptamers containing fluorophores ("F") and quenchers
("Q") at various loci within their structures.
[0004] 2. Background Information
[0005] FRET-aptamers are a new class of compounds, consisting in
part of single-stranded oligonucleotides, desirable for their use
in rapid (within minutes), one-step, homogeneous assays involving
no wash steps (simple bind and detect quantitative assays). Several
individuals and groups have published and patented FRET-aptamer
methods for various target analytes that consist of placing the F
and Q moieties either on the 5' and 3' ends respectively to act
like a "molecular (aptamer) beacon" or placing only F in the heart
of the aptamer structure to be "quenched" by another proximal F or
the DNA or RNA itself. These preceding FRET-aptamer methods are all
highly engineered and based on some prior knowledge of particular
aptamer sequences and secondary structures, thereby enabling clues
as to where F might be placed in order to optimize FRET
results.
[0006] Until now, no individual or group has described a method for
natural selection of single chain (intrachain) FRET-aptamers that
contain both fluorophore-labeled deoxynucleotides ("F-dNTPs") and
highly efficient spectrally matched quencher ("Q-dNTP") moieties in
the heart of an aptamer binding loop or pocket by polymerase chain
reaction ("PCR"). The advantage of this F and Q "doping" method is
two-fold: 1) the method allows nature to take its course and select
the most sensitive FRET-aptamer target interactions in solution,
and 2) the positions of F and Q within the aptamer structure can be
determined via exonuclease digestion of the FRET-aptamer followed
by mass spectral analysis of the resulting fragments, thereby
eliminating the need to "engineer" the F and Q moieties into a
prospective aptamer binding pocket or loop. Sequence and mass
spectral data can be used to further optimize the FRET-aptamer
assay performance after natural selection as well.
[0007] Others have described nucleic acid-based "molecular beacons"
that snap open upon binding to an analyte or upon hybridizing to a
complementary sequence, but beacons are always end-labeled with F
and Q at the 3' and 5' ends. FRET-aptamers may be labeled anywhere
in their structure that places the F and Q within the Forster
distance of approximately 60-85 Angstroms to achieve quenching
prior to or after target analyte binding to the aptamer "binding
pocket" (typically a "loop" in the secondary structure).
[0008] "Signaling aptamers" do not include a Q in their structures,
but rather appear to rely upon the "self-quenching" of two adjacent
fluorophores or the mild quenching ability of the nucleic acid
itself. Both of these methods of quenching are relatively poor,
because eventually F-emitted photons escape into the environment
and are detectable, thereby contributing to background light and
limiting the sensitivity of the FRET assay. True quenchers such as
dabcyl ("D"), the "Black Hole Quenchers" ("BHQs"), and the QSY
family of dyes (QSY-5, QSY-7, or QSY-9) are broad spectrum
absorbing molecules that appear dark or even black in color,
because they absorb many wavelengths of light and do not re-emit
photons. The inclusion of a Q in the intrachain FRET-aptamer
structure or the competitive aptamer FRET format, reduces
background fluorescence intensity significantly, thereby increasing
signal-to-noise ratios and improving assay sensitivity.
[0009] In addition to the novelty of the quencher introduction into
the assay formats and advantages conferred in terms of sensitivity
by cutting background fluorescence, the method of selecting single
intrachain FRET-aptamers based on differential molecular weight and
fluorescence intensity of the target analyte-aptamer bound subset
fractions is a novel FRET-aptamer development method. The F and Q
molecules used can include any number of appropriate fluorophores
and quenchers as long as they are spectrally matched so the
emission spectrum of F overlaps significantly (almost completely)
with the absorption spectrum of Q.
SUMMARY OF THE INVENTION
[0010] The present invention describes a single chain
(single-stranded intrachain) FRET assay approach in which F and Q
are incorporated into an aptamer population via their nucleotide
triphosphate derivatives (for example, ALEXA FLUOR.TM.-NTPs,
CASCADE BLUE.RTM.-NTPs, CHROMATIDE.RTM.-NTPs, fluorescein-NTPs,
rhodamine-NTPs, RHODAMINE GREEN.TM.-NTPs,
tetramethylrhodamine-dNTPs, OREGON GREEN.RTM.-NTPs, and TEXAS
RED.RTM.-NTPs may be used to provide the fluorophores, while
dabcyl-NTPs, Black Hole Quencher or BHQ.TM.-NTPs, and QSY.TM.
dye-NTPs may be used for the quenchers) by PCR after several rounds
of selection and amplification without the F- and Q-modified bases.
This process is generally referred to as "doping" with F-NTPs and
Q-NTPs.
[0011] Thereafter, the single chain or intrachain FRET-aptamers in
the population that still bind the intended target (after the
doping process) are purified by size-exclusion chromatography
columns, spin columns, gel electrophoresis or other means. Once
bound and separated based on weight or other physical properties,
the brightest fluorescing FRET-aptamer-target complexes are
selected because they are clearly the optimal FRET candidates. The
FRET-aptamers are separated from the targets by heating or chemical
means (urea, formamide, etc.) and purified again by size-exclusion
chromatography or other means.
[0012] These intrachain FRET-aptamers cannot be cloned for
sequencing due to the need for determining the locations of F and Q
in their structures. Cloning would lead to replication of the
FRET-aptamer insert in the plasmid and either dilution of the
desired FRET-aptamer or alteration of its F and Q locations within
the aptamer. Therefore, the candidate FRET-aptamers are separated
based on physical properties such as charge or weak interactions by
various types of high performance liquid chromatography ("HPLC"),
digested at each end with specific exonucleases (snake venom
phosphodiesterase on the 3' end and calf spleen phosphodiesterase
on the 5' end). The resulting oligonucleotide fragments, each one
base shorter than the predecessor, are subjected to mass spectral
analysis which can reveal the nucleotide sequences as well as the
positions of F and Q within the FRET-aptamers. Once the
FRET-aptamer sequence is known with the positions of F and Q, it
can be further manipulated during solid-phase DNA or RNA synthesis
in an attempt to make the FRET assay more sensitive and
specific.
[0013] There are a number of uses of the single-chain FRET-aptamers
developed by the present invention, including quantifiable
fluorescence assays for small molecules including pesticides,
natural and synthetic amino acids and their derivatives (e.g.,
histidine, histamine, homocysteine, DOPA, melatonin, nitrotyrosine,
etc.), short chain proteolysis products such as cadaverine,
putrescine, the polyamines spermine and spermidine, nitrogen bases
of DNA or RNA, nucleosides, nucleotides, and their cyclical
isoforms (e.g., cAMP and cGMP), cellular metabolites (e.g., urea,
uric acid), pharmaceuticals (therapeutic drugs), drugs of abuse
(e.g., narcotics, hallucinogens, gamma-hydroxybutyrate, etc.),
cellular mediators (e.g., cytokines, chemokines, immune modulators,
neural modulators, inflammatory modulators such as prostaglandins,
etc.), or their metabolites, explosives (e.g., trinitrotoluene) and
their breakdown products or byproducts, peptides and their
derivatives. Other uses of the single-chain FRET-aptamers include
use in quantifiable fluorescence assays for macromolecules
including proteins such as biotoxins including botulinum toxins,
Shiga toxins (See FIG. 2), staphylococcal enterotoxins, other
bacterial toxins, prions such as bovine spongiform encephalopathies
("BSEs") and transmissible spongiform encephalopathies ("TSEs"),
glycoproteins, lipids, glycolipids, triglycerides, nucleic acids,
polysaccharides, lipopolysaccharides, etc. The single-chain
FRET-aptamers may also be used in quantifiable fluorescence assays
for subcellular and whole cell targets including subcellular
organelles such as ribosomes, Golgi apparatus, vesicles,
microfilaments, microtubules, etc., viruses, virions, rickettsiae,
bacteria, protozoa, plankton, parasites such as Cryptosporidium
species, Giardia species, mammalian cells such as various classes
of leukocytes, neurons, stem cells, cancer cells, etc.
[0014] The use of unlabeled aptamer sequence information and
secondary stem-loop structures may aid in the determination and
engineered optimization of F or Q placement within the aptamer
structure to maximize FRET assay sensitivity and specificity. As
described and claimed herein, "within" and "interior" are defined
as directing F or Q placement, or labeling, interior to, or
between, the ends of the aptamer; the ends of the aptamer being the
3' and 5' ends. Although, an anticipated step in the present method
of natural selection of F- and/or Q-labeled aptamers to form
solution phase interactions with their target analytes, additional
sequence and secondary structure information can be used to confirm
and enhance F and Q placement to optimize assay performance.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1. is a schematic illustration of the single chain
(intrachain) FRET-aptamer selection method.
[0016] FIG. 2. is a bar graph showing toxin concentration mapped
with fluorescence intensity and illustrating a "lights off" FRET
with shiga-like toxin 1 and round 5 aptamers.
[0017] FIG. 3. is a schematic illustration that illustrates a
comparison of possible nucleic acid FRET assay formats.
[0018] FIG. 4. illustrates sample aptamer sequences.
[0019] FIGS. 4A-4B. are schematic illustrations of the secondary
structures of selected aptamer sequences shown in FIG. 4.
[0020] FIG. 5. is a line graph correlating absorbance with BoNT A
concentration.
[0021] FIGS. 6A-6B. are line graphs mapping fluorescence intensity
against time.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0022] Referring to the figures, FIG. 1. illustrates a single chain
(intrachain) FRET-aptamer selection method. This method consists of
several steps. First the random DNA library of oligonucleotides
(randomized region of 20 or more bases flanked by known primer
regions) is "doped" with F-dNTPs Q-dNTPs by the PCR (10). The F and
Q doped library is then exposed to a protein or other target
molecule (12). Some members of the doped library will bind to the
target protein (14).
[0023] If the target molecule is a larger water-soluble molecule
such as a protein, glycoprotein, or other water soluble
macromolecule, then exposure of the nascent F-labeled and Q-labeled
DNA or RNA random library to the free target analyte is done in
solution. If the target is a soluble protein or other larger
water-soluble molecule, then the optimal FRET-aptamer-target
complexes are separated by size-exclusion chromatography. The
FRET-aptamer-target complex population of molecules is the heaviest
subset in solution and will emerge from a size-exclusion column
first, followed by unbound FRET-aptamers and unbound proteins or
other targets. Among the subset of analyte-bound aptamers there
will be heterogeneity in the numbers of F- and Q-NTPs that are
incorporated as well as nucleotide sequence differences, which will
again effect the mass, electrical charge, and weak interaction
capabilities (e.g., hydrophobicity and hydrophilicity) of each
analyte-aptamer complex. These differences in physical properties
of the aptamer-analyte complexes can then be used to separate out
or partition the bound from unbound analyte-aptamer complexes.
[0024] If the target is a small molecule (generally defined as a
molecule with molecular weight of .ltoreq.1,000 Daltons), then
exposure of the nascent F-labeled and Q-labeled DNA or RNA random
library to the target is done by immobilizing the target. The small
molecule can be immobilized on a column, membrane, plastic or glass
bead, magnetic bead, or other matrix. If no functional group is
available on the small molecule for immobilization, the target can
be immobilized by the Mannich reaction (formaldehyde-based
condensation reaction) on a device similar to a PHARMALINK.TM.
column. Elution of bound DNA from the small molecule affinity
column, membrane, beads or other matrix by use of 0.2-3.0M sodium
acetate at a pH ranging between 3 and 7, although the optimal pH is
approximately 5.2.
[0025] These complexes can be separated from the non-binding doped
DNA molecules by running the aptamer-protein aggregates (or
selected aptamers-protein aggregates) through a size-exclusion
column, by means of size-exclusion chromatography using
Sephadex.TM. or other gel materials in the column (16). Since they
vary in weight due to variations in aptamers sequences and degree
of labeling, they can be separated into fractions with different
fluorescence intensities. Purification methods such as capillary
electrophoresis or preparative gel electrophoresis are possible as
well. Small volume fractions (.ltoreq.1 mL) can be collected from
the column and analyzed for absorbance at 260nm and 280nm which are
characteristic wavelengths for DNA, RNA and proteins. The heaviest
materials come through a size-exclusion column first. Therefore,
the DNA-protein complexes or RNA-protein complexes will come out of
the column before either the DNA or protein alone when using an
appropriate grade of column matrix material (e.g., various grades
of Sephadex, Superdex, Sepharose, or Sephacryl, etc.).
[0026] Means of separating FRET-aptamer-target complexes from
solution by alternate techniques (other than size-exclusion
chromatography) include, without limitation, various molecular
weight cut off ("MWCO") spin columns, dialysis, gel
electrophoresis, thin layer chromatography ("TLC"), and
differential centrifugation or ultracentrifugation using density
gradient materials.
[0027] A preferred FRET-aptamer is selected. A preferred
FRET-aptamer is one that exhibits a measurable change in
fluorescence between the fluorescence of said FRET-aptamer when it
is not bound to said target molecule compared to the fluorescence
of said FRET-aptamer when it is bound to said target molecule.
[0028] The optimal (most sensitive or highest signal to noise
ratio) FRET-aptamers among the bound class of FRET-aptamer-target
complexes are identified by assessment of fluorescence intensity
for various fractions of the FRET-aptamer-target class. The
separated DNA-protein complexes will exhibit the highest absorbance
at established wavelengths, such as 260nm and 280nm similar to that
graphed in FIG. 1 (18). The fractions showing the highest
absorbance at the given wavelengths, such as 260nm and 280nm, are
then further analyzed for fluorescence and those fractions
exhibiting the greatest fluorescence are selected for separation
and sequencing (20).
[0029] These-similar FRET-aptamers may be further separated using
techniques such as ion pair reverse-phase HPLC, ion-exchange
chromatography ("IEC", either low pressure or HPLC versions of
IEC), TLC, capillary electrophoresis, or similar techniques.
[0030] The final FRET-aptamers are able to act as one-step "lights
on" or "lights off' binding and detection components in assays.
[0031] Intrachain FRET-aptamers that are to be used in assays with
long shelf-lives may be lyophilized (freeze dried) and
reconstituted as needed.
[0032] FIG. 2. is a bar graph showing toxin concentration mapped
with fluorescence intensity and illustrating a "lights off' FRET
response with shiga-like toxin 1 and round 5 aptamers. If the
fluorescence intensity of the DNA aptamers is correlated to the
concentration of the surface protein and the fluorescence intensity
decreases as a function of increasing analyte concentration, then
it is referred to as a "lights off' assay. If the fluorescence
intensity increases as a function of increasing analyte
concentration, then it is referred to as a "lights on" assay.
Intrachain FRET-aptamer assay data are shown for detection of E.
coli shiga-like toxin I protein resulting in a "lights off' FRET
reaction as a function of toxin concentration. Fluorescence
readings were obtained within five minutes of toxin addition.
[0033] FIG. 3. illustrates a comparison of possible nucleic acid
FRET assay formats. Upper left is a molecular beacon (30) which may
or may not be an aptamer, but is typically a short oligonucleotide
used to hybridize to other DNA or RNA molecules and exhibit FRET
upon hybridizing. Molecular beacons are only labeled with F and Q
at the ends of the DNA molecule. Lower left is a signaling aptamer
(32), which does not contain a quencher molecule, but relies upon
fluorophore self-quenching or weak intrinsic quenching capacity of
the DNA or RNA to achieve limited FRET. Upper right is an
intrachain FRET-aptamer (34) containing F and Q molecules built
into the interior structure of the aptamer. Intrachain
FRET-aptamers are naturally selected and characterized by the
processes described herein. Lower right shows a competitive aptamer
FRET (36) motif in which the aptamer contains either F or Q and the
target molecule (38) is labeled with the complementary F or Q.
Introduction of unlabeled target molecules (40) then shifts the
equilibrium so that some labeled target molecules (38) are
liberated from the labeled aptamer (36) and modulate the
fluorescence level of the solution up or down thereby achieving
FRET. A target analyte (40) is either unlabeled or labeled with a
quencher (Q). F and Q can be switched or swapped from placement in
the aptamer (36) to placement in the target analyte (40) and vice
versa.
[0034] FIG. 4 illustrates sample aptamer sequences for botulinum
toxin A (BoNT A) in which all sequences are arranged 5' to 3' from
left to right. The actual degenerate (randomized) aptamer regions
are bolded. Clear consensus regions are bolded and italicized.
Flanking sequences match with the primers used in the PCR scheme or
the complementary strand primer sequences except in highlighted
cases. Most sequences end in a 3' A (added by Taq, underlined).
Aptamer sequences that bind and inhibit the action of botulinum A
(BoNT A) 150 kD holotoxin and the 50 kD enzymatic light chain or
subunit of BoNT A, and which may be useful in single chain
FRET-aptamer or competitive aptamer-FRET assays for detection and
quantification of BoNT A.
[0035] FIGS. 4A-4D. illustrate secondary structures of selected
aptamer sequences listed in FIG. 4. Various botulinum A (BoNT A)
DNA aptamer secondary (two dimensional) stem-loop structures that
bind the holotoxin (FIG. 4A, which exemplifies a sequence that
occurred in four different clones), and bind and inhibit (See FIGS.
6A and 6B) the small (50 kD) enzymatic subunit (FIGS. 4B-4D,
showing the secondary structures for three different sequences that
produced similar secondary structures).
[0036] FIG. 5. is a line graph correlating absorbance with BoNT A
concentration. It illustrates that aptamer-peroxidase colorimetric
plate binding assay results using polyclonal BoNT A aptamers and
BoNT A holotoxin. Two different trials or runs are shown.
Absorbance was quantified at 405 nm using standard ABTS substrate
and hydrogen peroxide activator reagents. The curves illustrate
binding and sensitive detection of BoNT A by the aptamers at a
level of at least 12.5 ng/mL.
[0037] FIGS. 6A-6B. are line graphs mapping the fluorescence
intensity of the DNA aptamers such as those shown in FIGS. 4A-4D
against time in minutes. DNA aptamers, such as those shown in FIGS.
4A-4D, bind and inhibit the enzymatic activity of BoNT A. Here the
inhibition of BoNT A's enzymatic activity is further proof of tight
aptamer binding to the toxin. FIG. 6A shows assay results using the
BoNT A holotoxin and FIG. 6B shows results using the isolated 50 kD
enzymatic subunit of BoNT A. The positive control line shows
greater fluorescence intensity over time for the uninhibited BoNT
SNAPTIDE.TM. assay and the "Test with Aptamer" line shows
consistent suppression of the fluorescence intensity of the
SNAPTIDE.TM. assay further proving aptamer binding and
aptamer-mediated inhibition of BoNT A enzymatic activity.
EXAMPLE 1
[0038] Single (Intrachain) Chain FRET-Aptamer Assay for a Protein
(E. coli Shiga-Like Toxin I).
[0039] Following five rounds of systematic evolution of ligands by
exponential enrichment ("SELEX") an aptamer family was subjected to
PCR in the presence of 3 .mu.M CHROMATIDE.TM.-dUTP and 40.mu.M
Dabcyl-dUTP using a standard PCR mix formulation and Taq enzyme at
1 Unit per 50 .mu.L reaction. This led to incorporation of the FRET
(F and Q) pair which demonstrated the lowest background
fluorescence of all F:Q ratios tested (nearly 1,200 fluorescence
units for the baseline reading without the toxin target).
Fluorescence readings in FIG. 2 were taken with a handheld
fluorometer. Error bars in FIG. 2 represent the standard deviation
of three trials and the bar heights represent the means of the 3
measurements. At the level of 40,000 picograms per milliliter
(pg/mL) or 40 nanograms (ng) of Shiga-like toxin I, a definitive
"lights off` FRET effect is noted. Since the mean fluorescence at
40 ng of added toxin is far greater than two standard deviations
below any of the other treatment groups, it must be considered
statistically significant.
EXAMPLE 2
[0040] Use of Unlabeled Aptamer Nucleotide Sequences and Secondary
(Stem-Loop) Structures that Can Confirm, Enhance, and Optimize
FRET-Aptamer Assays.
[0041] The present method enables the natural selection of
FRET-aptamers. However, the method can be confirmed and enhanced by
knowledge of the unlabeled aptamer sequences and structures that
were selected from several rounds of SELEX before the aptamer
population was "doped" with F-dNTPs and/or Q-dNTPs. FIG. 4 gives an
example of BoNT A aptamer sequences that are claimed as unlabeled
sequences, resulting in secondary stem-loop structures from energy
minimization software using 25.degree. C. as the nominal binding
temperature. The stem-loop structures shown in FIGS. 4A-4D may be
especially useful in determining if the F and Q locations are
indeed logical (i.e., fall in or near a binding loop structure). In
addition, if F and/or Q loci are found to be distal, information
such as the secondary structures in FIGS. 4A-4D could be
instrumental in slightly relocating the F and Q moieties to enhance
or optimize the FRET assay results in terms of assay sensitivity
and specificity.
[0042] Aptamers were incorporated into plasmids. The plasmids were
purified and sequenced by capillary electrophoresis following
PCR.
[0043] The BoNT A functionality of the aptamer sequences (ability
to bind and inhibit BoNT A) shown in FIGS. 4 and 4A-4D were
confirmed by colorimetric plate assay binding data (FIG. 5) and
SNAPTIDE.TM. FRET assay data showing inhibition of BoNT A enzymatic
activity by the "polyclonal" family of BoNT A aptamers (FIG.
6).
[0044] Although the invention has been described with reference to
`specific embodiments, this description is not meant to be
construed in a limited sense. Various modifications of the
disclosed embodiments, as well as alternative embodiments of the
inventions will become apparent to persons skilled in the art upon
the reference to the description of the invention. It is,
therefore, contemplated that the appended claims will cover such
modifications that fall within the scope of the invention.
Sequence CWU 1
1
11160DNAArtificial Sequencechemically synthesized 1catccgtcac
acctgctctg ctatcacatg cctgctgaag tggtgttggc tcccgtatca
60260DNAArtificial Sequencechemically synthesized 2catccgtcac
acctgctctg ctatcacatg cctgctgaag tggtgttggc tcccgtatca
60360DNAArtificial Sequencechemically synthesized 3catccgtcac
acctgctctg ctatcacatg cctgctgaag tggtgttggc tcccgtatca
60460DNAArtificial Sequencechemically synthesized 4catccgtcac
acctgcycyg ctatcacatg cctgctgaag tggtgttggc tcccgtatca
60560DNAArtificial Sequencechemically synthesized 5catccgtcac
acctgctctg gggatgtgtg gtgttggctc ccgtatcaag ggcgaattct
60661DNAArtificial Sequencechemically synthesized 6catccgtcac
acctgctctg atcagggaag acgccaacac gtggtgttgg ctcccgtatc 60a
61761DNAArtificial Sequencechemically synthesized 7catccgtcac
acctgctctg ggtggtgttg gctcccgtat caagggcgaa ttctgcagat 60a
61860DNAArtificial Sequencechemically synthesized 8catccgtcac
acctcctctg ctatcagatg cctggtgaag tggtgttggc tcccgtatca
60961DNAArtificial Sequencechemically synthesized 9catccgtcac
acctgctctg atcagggaag acgccaacac gtggtgttgg ctcccgtatc 60a
611060DNAArtificial Sequencechemically synthesized 10catccgtcac
acctgctctg gggatgtgtg gtgttggctc cggtatcaag ggcgaattct
601161DNAArtificial Sequencechemically synthesized 11catccgtcac
acctcctctg ggtggtgttg ggtccggtat caagggccaa ttctgcagat 60a 61
* * * * *