U.S. patent application number 12/931305 was filed with the patent office on 2012-10-25 for methods of sequencing 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 | 20120270221 12/931305 |
Document ID | / |
Family ID | 46719228 |
Filed Date | 2012-10-25 |
United States Patent
Application |
20120270221 |
Kind Code |
A1 |
Bruno; John G. ; et
al. |
October 25, 2012 |
Methods of sequencing fluorophore-quencher FRET-aptamers
Abstract
The present invention describes methods for the production and
selecting 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. Alternatively, single DNA or RNA intrachain
FRET-aptamers can be sequenced and the locations of F and Q within
the structure can be determined by nanopore sequencing and the
locations of F and Q within the structure can be verified by
nucleic acid "combing" coupled to high-powered fluorescence
microscopy.
Inventors: |
Bruno; John G.; (San
Antonio, TX) ; Chanpong; Joseph; (San Antonio,
TX) |
Assignee: |
PRONUCLEOTEIN BIOTECHNOLOGIES,
LLC
|
Family ID: |
46719228 |
Appl. No.: |
12/931305 |
Filed: |
January 28, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11433009 |
May 12, 2006 |
7906280 |
|
|
12931305 |
|
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Current U.S.
Class: |
435/6.12 ;
436/501; 977/781; 977/902 |
Current CPC
Class: |
C12N 2320/10 20130101;
G01N 33/5308 20130101; G01N 33/533 20130101; G01N 33/542 20130101;
C12N 2310/16 20130101; C12N 15/111 20130101 |
Class at
Publication: |
435/6.12 ;
436/501; 977/781; 977/902 |
International
Class: |
G01N 21/64 20060101
G01N021/64 |
Claims
1. A method of determining the locations of fluorophores ("F") or
quenchers ("Q") bound to a single-chain fluorescence resonance
energy transfer ("FRET")-aptamer that selectively binds to a target
molecule, comprising: selecting a FRET-aptamer 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; and determining the locations of fluorophores
("F") or quenchers ("Q") of said FRET-aptamer via single
FRET-aptamer sequencing.
2. The method of claim 1, wherein said single FRET-aptamer
sequencing comprises nanopore sequencing.
3. The method of claim 2, wherein said nanopore sequencing further
comprises: drawing an individual single-stranded DNA or RNA
molecule through a pore wherein said pore is greater than or equal
to 1 nm in diameter; measuring changes in optical properties as
each DNA or RNA base or nucleotide, fluorophore ("F"), or quencher
("Q") moves through said pore; and identifying said DNA bases, Fs,
and Q's based upon said measured optical property changes.
4. The method of claim 3, wherein said pore is in an inorganic
silicon nitride or biological phospholipid bilayer membrane.
5. The method of claim 3, wherein said drawing step is accomplished
by pulling said DNA or RNA molecule through said pore.
6. The method of claim 5, wherein said pulling of said DNA or RNA
molecule through said pore is accomplished via applying
electrophoresis or negative pressure to said DNA or RNA
molecule.
7. The method of claim 3, wherein said drawing step is accomplished
by pushing said DNA or RNA molecule through said pore.
8. The method of claim 7, wherein said pushing of said DNA molecule
through said pore is accomplished via applying electrophoresis or
positive pressure to said DNA molecule.
9. The method of claim 2, wherein said nanopore sequencing further
comprises: drawing an individual single-stranded DNA molecule
through a pore wherein said pore is greater than or equal to 1 nm
in diameter; measuring changes in electrical properties as each
base or nucleotide moves through said pore; and identifying said
DNA or RNA bases, Fs, and Q's based upon said measured electrical
property changes.
10. The method of claim 9, wherein said pore is in an inorganic
silicon nitride or biological phospholipid bilayer membrane.
11. The method of claim 9, wherein said drawing step is
accomplished by pulling said DNA molecule through said pore.
12. The method of claim 11, wherein said pulling of said DNA or RNA
molecule through said pore is accomplished via applying
electrophoresis or negative pressure to said DNA or RNA
molecule.
13. The method of claim 9, wherein said drawing step is
accomplished by pushing said DNA molecule through said pore.
14. The method of claim 13, wherein said pushing of said DNA or RNA
molecule through said pore is accomplished via applying
electrophoresis or positive pressure to said DNA or RNA
molecule.
15. The method of claim 1, wherein said single FRET-aptamer
sequencing comprises DNA or RNA combing.
16. The method of claim 15, wherein said DNA or RNA combing further
comprises: straightening an individual single-stranded DNA or RNA
molecule; staining said individual single-stranded DNA or RNA
molecule with a nucleic acid-specific fluorescent dye; imaging said
straightened individual single-stranded DNA or RNA molecule with a
fluorescence microscope; detecting Fs along the length of said
individual single-stranded DNA or RNA molecule using their emission
color; measuring a distance from either end of said individual
single-stranded DNA or RNA molecule to determine a position of said
F in said individual single-stranded DNA or RNA molecule.
17. A method of sequencing single-chain fluorescence resonance
energy transfer ("FRET")-aptamers that contain fluorophores or
quenchers in the same single-stranded oligonucleotides, comprising:
incorporating fluorophores and quenchers into an aptamer population
using polymerase chain reaction ("PCR") wherein at least one of
said fluorophores and at least one of said quenchers are
incorporated into some of said oligonucleotides at random locations
in said oligonucleotides; exposing said aptamer population to a
population of target molecules; separating those FRET-aptamers that
have bound to a target molecule; selecting a preferred FRET-aptamer
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; and determining the
nucleotide sequence of said preferred FRET-aptamer via single
FRET-aptamer sequencing.
18. The method of claim 17, wherein said single FRET-aptamer
sequencing further comprises: drawing an individual single-stranded
DNA or RNA molecule through a pore wherein said pore is greater
than or equal to 1 nm in diameter; measuring changes in optical
properties as each DNA or RNA base, fluorophore ("F"), or quencher
("Q") moves through said pore; and identifying said DNA or RNA
bases, Fs, and Q's based upon said measured optical property
changes.
19. The method of claim 17, wherein said single FRET-aptamer
sequencing further comprises: drawing an individual single-stranded
DNA or RNA molecule through a pore wherein said pore is greater
than or equal to 1 nm in diameter; measuring changes in optical
properties as each DNA or RNA base, fluorophore ("F"), or quencher
("Q") moves through said pore; and identifying said DNA or RNA
bases, Fs, and Q's based upon said measured electrical property
changes.
20. The method of claim 17, wherein said nanopore sequencing
further comprises: straightening an individual single-stranded DNA
or RNA molecule; staining said individual single-stranded DNA or
RNA molecule with a nucleic acid-specific fluorescent dye; imaging
said straightened individual single-stranded DNA or RNA molecule
with a fluorescence microscope; detecting Fs along the length of
said individual single-stranded DNA or RNA molecule using their
emission color; measuring a distance from either end of said
individual single-stranded DNA or RNA molecule to determine a
position of said F in said individual single-stranded DNA or RNA
molecule.
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, or DNA
combing and nanopore sequencing, 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] An alternative method of sequencing the FRET-aptamer to
determine the absolute positions of F and Q within the nucleic acid
(DNA or RNA) chain which has the advantage of sequencing individual
nucleic acids is called "nanopore sequencing." This method can be
applied the FRET-aptamer to determine exactly where F and Q are
because as F and Q pass through the nanopore, they will register
unique electrical patterns that can be used to distinguish them
from the nucleotides (adenine, cytosine, guanine, uracil or
thymine). Nanopore sequencing is currently performed in two basic
ways: 1) through a silicon nitride plane or membrane with nanopores
created by electrons shot through the silicon nitride by an
electron gun or 2) the use of natural cell membranes (e.g.,
phospholipid bilayers or micelles) containing natural pore-like
proteins such as ion-channels or gates or bacterial alpha-hemolysin
through which single-stranded DNA and RNA can pass. Electrodes are
set up on either side of the membrane to measure electrical
activity according to Ohm's Law as the DNA passes through the
membrane and this method could be applied to sequencing of single
DNA or RNA molecules with additional information on which bases or
nucleotides had F and Q covalently conjugated to them. The nucleic
acid can be pushed or pulled through a nanopore by the application
of positive or negative fluid pressure or electrophoresis, etc.
While the electrical properties of nucleotides, F and Q are
currently used to identify these components as they pass through
the membrane, this does not preclude the use of other physical
means such as optics (fluorescence intensity, fluorescence lifetime
analysis, absorbance, or circular dichroism, etc.) to detect and
identify specific nucleotides, as well as F's and Q's attached to
nucleotides, as they pass through the membrane holes.
[0014] A second method, referred to as "DNA combing" (RNA can be
combed as well) could be used to linearize convoluted, folded or
globular nucleic acids and "comb" them out into single lines much
like matted hair is combed out into individual linear strands by a
comb or hair brush. One can actually image individual fluorescent
DNA or RNA molecules that have been combed out on glass, silicon,
of plastic microscope slides or other planar substrates using a
high-powered fluorescence or optical microscope and determine the
relative position of F from the 3' or 5' ends, thereby enabling
determination of which base or nucleotide to which F is attached.
It is envisioned that similar absorbance or circular dichroism or
optical polarization techniques can be devised to determine which
bases quenchers are attached to relative to the 3' or 5' ends for
verification of nanopore sequencing data of intrachain
FRET-aptamers.
[0015] 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.
[0016] 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
[0017] FIG. 1. is a schematic illustration of the single, chain
(intrachain) FRET-aptamer selection method.
[0018] 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.
[0019] FIG. 3. is a schematic illustration that illustrates a
comparison of possible nucleic acid FRET assay formats.
[0020] FIG. 4. illustrates sample aptamer sequences.
[0021] FIGS. 4A-4B. are schematic illustrations of the secondary
structures of selected aptamer sequences shown in FIG. 4.
[0022] FIG. 5. is a line graph correlating absorbance with BoNT A
concentration.
[0023] FIGS. 6A-6B. are line graphs mapping fluorescence intensity
against time.
[0024] FIG. 7. illustrates how and where nanopore DNA or RNA
sequencing and nucleic acid "combing" fit into the overall
intrachain FRET-aptamer process depicted in FIG. 1.
[0025] FIG. 8. illustrates how nanopore nucleic acid sequencing
works to sequence the nucleotides and determine the positions of F
and Q on a single intrachain-FRET aptamer by electrical
measurements.
[0026] FIGS. 9A-9D. illustrate variations on performance of the
combing process on top of an inverted fluorescence microscope
objective.
[0027] FIG. 10. illustrates how DNA or RNA combing could linearize
and determine the position of F relative to one end of the
aptamer.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0028] 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).
[0029] 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 an appropriately chosen
(based on known fractionation data) 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-dNTPs 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.
[0030] 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.
[0031] 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 260 nm and 280 nm 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.).
[0032] 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.
[0033] 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.
[0034] 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 260 nm and 280 nm similar to
that graphed in FIG. 1 (18). The fractions showing the highest
absorbance at the given wavelengths, such as 260 nm and 280 nm, are
then further analyzed for fluorescence and those fractions
exhibiting the greatest fluorescence are selected for separation
and sequencing.
[0035] 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.
[0036] The final FRET-aptamers are able to act as one-step "lights
on" or "lights off" binding and detection components in assays.
[0037] Intrachain FRET-aptamers that are to be used in assays with
long shelf-lives may be lyophilized (freeze dried) and
reconstituted as needed.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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).
[0042] 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.
[0043] 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.
[0044] FIG. 7. is an extension of FIG. 1 illustrating how and where
the alternative processes of nanopore sequencing and nucleic acid
combing verification fit into the overall intrachain FRET-aptamer
process for dealing with a single molecule of user chosen
"preferred" FRET-aptamers or a very small number (e.g., less than
ten) of preferred intrachain FRET-aptamers. FIG. 7 is a further
indication of how and where nanopore DNA or RNA sequencing and
nucleic acid "combing" fit into the overall intrachain FRET-aptamer
process depicted in FIG. 1 to aid in determining where F and Q are
in intrachain FRET-aptamers. In the case of a "lights on" FRET
assay, "preferred" means the FRET-aptamer and target complex
produces an increased emission of detectable fluorescence after the
FRET-aptamer is bound to the target molecule as compared to the
amount of detectable fluorescence emitted before the two are bound.
And, in the case of a "lights off" FRET assay, "preferred" means
the FRET-aptamer and target complex produces a decreased emission
of detectable fluorescence after the FRET-aptamer is bound to the
target molecule as compared to the amount of detectable
fluorescence emitted before the two are bound. However, it should
be noted that "preferred" refers to a FRET-aptamer with a
detectable change in detectable fluorescence (either increased or
decreased as per the type of assay) as chosen by the user.
"Preferred" does not necessarily refer to the single FRET-aptamer
that produces most or least detectable fluorescence after binding
with a target molecule, nor the greatest change in detectable
fluorescence after binding with a target molecule. Thus, there may
be multiple "preferred" FRET-aptamers in a population, or specific
to a target.
[0045] One weakness or obstacle to industrial implementation of the
intrachain FRET-aptamer approach is that selecting the "preferred"
FRET-aptamer or FRET-aptamers from the remaining aptamer population
from the pool by chromatography as shown in FIG. 7, generally
leaves only a very small amount of FRET-aptamer DNA. In theory, the
number of "preferred" FRET-aptamer molecules could be as few as one
F- and Q-labeled DNA molecule which would be difficult or even
impossible to sequence by the combination of 3' and 5' exonuclease
degradation (base by base or nucleotide by nucleotide cleavage)
followed by mass spectral analyses.
[0046] Once selected, the FRET-aptamer may be separated from the
target molecule by heating or chemical means. After the
FRET-aptamer-target molecule dissociation, there may only be a few
or even a single selected FRET-aptamer remaining from the original
aptamer population. If the user determines the "preferred"
FRET-aptamer to be one that has been found in a very small amount,
sequencing individual DNA FRET-aptamers or small numbers of
FRET-aptamers is still possible via 1) nanopore DNA or RNA
sequencing, or 2) DNA or RNA "combing" combined with fluorescence
microscopy to determine or verify the relative positions of F and Q
within the FRET aptamer's structure after the aptamer is linearized
or "combed."
[0047] FIG. 8. illustrates a method for sequencing individual DNA
FRET-aptamers or small numbers of FRET-aptamers. It is a conceptual
illustration of how nanopore nucleic acid sequencing works to
sequence the nucleotides and determine the positions of F and Q on
a single intrachain-FRET aptamer by electrical measurements. This
method can be used when there is a large population of the selected
FRET-aptamer. However, it is also useful in the event that the
selected "preferred" FRET-aptamer has been found in a very small
amount.
[0048] It is still possible, via nanopore DNA sequencing or DNA
combing combined with fluorescence microscopy, to determine or
verify the relative positions of F and Q within the intrachain
FRET-aptamer's structure by tracking optical changes or electrical
patterns. For example, when Ohm's law is applied, a graph of
resistance versus current will elicit distinct electrical waveforms
or patterns that can be tracked and identified as indicating F's or
Q's attached to the various nucleic acid bases. The method of
nanopore DNA sequencing of individual linearized intrachain F- and
Q-labeled FRET-aptamer molecules allows the user to determine both
the sequence of the DNA strand as well as the locations of any F's
and Q's bound to the strand. In short, the "preferred" FRET-aptamer
is moved through a hole of greater than or equal to 1 nanometer in
diameter in a membrane to sequentially read nucleotide identities
and determine which nucleotides have F or Q covalently attached to
them. Identification of the bases, as well as the F and Q's is
accomplished by measuring changes in electrical conductivity,
voltage, resistance, optical absorbance, polarization,
birefringence, circular dichroism, fluorescence intensity, or
fluorescence lifetime as each base passes through the pore.
[0049] Nanopore sequencing involves drawing or pushing an
individual single-stranded DNA molecule through a pore (.gtoreq.1
nm in diameter) in an inorganic silicon nitride or biological
phospholipid bilayer membrane and monitoring changes in optical
(for example, fluorescence, absorbance, polarization, birefringence
or circular dichroism) or electrical properties as each base or
nucleotide moves through the pore. Nanopore sequencing, such as
described herein, may more broadly be referred to as "single
FRET-aptamer sequencing."
[0050] Single or multiple DNA molecules can be pulled or pushed
through the nanopores by electrophoresis, applied pressure or other
means, although if multiple DNA molecules are used then they are
processed a single molecule at a time. The changes in electrical
properties are essentially related to the Ohm's Law equation of
E=IR, or voltage=conductance.times.resistance. Therefore, any of
the components of Ohm's Law can be plotted as functions of the
other two components (as shown in FIG. 8). Each of the DNA bases,
adenine (A), cytosine (C), guanine (G) and thymine (T), or uridine
(U instead of T in the case of RNA) perturbs the electric field
differently as it passes through the pore's capacitor and can be
identified by its electrical plot signature, thereby determining
the sequence of the DNA strand. Likewise then, if a fluorophore (F)
or quencher (Q) in an intrachain FRET-aptamer is covalently
attached to any of the bases, it too will produce unique and
characteristic optical or electrical patterns that can then be used
to identify which base it is attached to and at which position in
the overall aptamer sequence. In this way, the DNA or RNA
FRET-aptamer's nucleotide sequence--and the locations of F and Q in
the FRET-aptamer structure--can be simultaneously determined.
[0051] FIGS. 9A-9D. illustrate various methods of DNA combing. FIG.
9A shows a receding meniscus in which a drop containing the
FRET-aptamer is flowed across a polystyrene or other plastic or
coated-glass surface leaving in its wake combed DNA. The 3' and 5'
ends of DNA naturally tend to adhere strongly to the surface,
thereby tethering or anchoring at least one end of the molecule to
assist in combing. FIG. 9B illustrates chemical immobilization of
the 3' or 5' end to the planar substrate followed by fluid flow to
straighten out the intrachain FRET-aptamer. FIG. 9C illustrates
flowing liquid past one laser-trapped (optical "tweezer" potential
energy well) bead having the DNA of interest immobilized at one end
on its surface. FIG. 9D illustrates linearizing the DNA between two
optically trapped beads such that the ends of the single DNA
molecule are immobilized on one or the other bead and the DNA can
be stretched out.
[0052] FIG. 10. illustrates an alternate method to determine, or
verify, the location of a fluorophore in a single intrachain
FRET-aptamer molecule. It is a conceptual diagrams showing how DNA
or RNA combing could linearize and determine the position of F
relative to one end of the aptamer, thereby indicating which base
or nucleotide F is attached too. Initially, the FRET-aptamer is
folded in three dimensions. The folded intrachain aptamer is
straightened by "DNA combing" (or "RNA combing"). Intrachain
FRET-aptamers can be straightened, or linearized, by application of
heat or cationic solutes such as .gtoreq.30 mM formamide or urea,
or as illustrated in FIGS. 9A-9D. Once straightened, the DNA or RNA
is imaged under a high-powered (typically .gtoreq.1,000.times.
magnification) fluorescence microscope. The DNA or RNA itself can
be stained with a nucleic acid-specific fluorescent dye, such as
acridine orange ("AO") or ethidium bromide ("EtBr") to help locate
the polymer molecule in the microscopic field of view. The
PCR-incorporated fluorophore-dNTP (F; having a different emission
color than AO or EtBr) can be detected along the length of the
intrachain FRET-aptamer at one or more locations based on the
difference in its colored fluorescence emission. By then measuring
the distance from either end of the intrachain FRET-aptamer, one
can determine which base F is covalently attached to by
proportionality (if the DNA or RNA sequence of the aptamer is
already known). This entire technique may be described as nucleic
acid "combing" and can be used to verify the positions of F and
possibly Q within an intrachain FRET-aptamer. DNA combing or RNA
combing, such as described herein, may be more broadly referred to
as "single FRET-aptamer sequencing."
[0053] It is anticipated that a user could employ any method of
nucleic acid "combing" by straightening an intrachain FRET-aptamer
across a polystyrene, silanized glass or other planar surface to
linearize single intrachain FRET-aptamer molecules. An examination
with a fluorescence microscope follows the combing to determine the
relative position of covalently attached internal fluorophores (Fs)
from either end (3' or 5') of the aptamer.
EXAMPLE 1
Single (Intrachain) Chain FRET-Aptamer Assay for a Protein (E. Coli
Shiga-Like Toxin I).
[0054] 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
[0055] Use of Unlabeled Aptamer Nucleotide Sequences and Secondary
(Stem-Loop) Structures that can Confirm, Enhance, and Optimize
FRET-Aptamer Assays.
[0056] 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.
[0057] Aptamers were incorporated into plasmids. The plasmids were
purified and sequenced by capillary electrophoresis following
PCR.
[0058] 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).
[0059] FIGS. 7-10 illustrate the basic concepts of nanopore
sequencing and nucleic acid combing (collectively referred to as
"single FRET-aptamer sequencing") to deal with determining the
nucleotide sequences and exact positions of F and Q in rare or very
low abundance, but optimal, intrachain FRET-aptamers. Nanopore
sequencing and combing can be used to sequence and verify the
placement of F (and possibly Q) within even one individual
intrachain FRET-aptamer polymer molecule, where "polymer molecule"
refers to either a DNA or an RNA molecule.
[0060] 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
* * * * *