U.S. patent application number 16/413989 was filed with the patent office on 2019-08-29 for method for generating aptamers with improved off-rates.
The applicant listed for this patent is SomaLogic, Inc.. Invention is credited to Chris Bock, Bruce Eaton, Larry Gold, Daniel J. Schneider, Sheri K. Wilcox, Dominic Zichi.
Application Number | 20190264208 16/413989 |
Document ID | / |
Family ID | 46331949 |
Filed Date | 2019-08-29 |
View All Diagrams
United States Patent
Application |
20190264208 |
Kind Code |
A1 |
Zichi; Dominic ; et
al. |
August 29, 2019 |
Method for Generating Aptamers with Improved Off-Rates
Abstract
The present disclosure describes improved SELEX methods for
producing aptamers that are capable of binding to target molecules
and improved photoSELEX methods for producing photoreactive
aptamers that are capable of both binding and covalently
crosslinking to target molecules. Specifically, the present
disclosure describes methods for producing aptamers and
photoaptamers having slower dissociation rate constants than are
obtained using prior SELEX and photoSELEX methods. The disclosure
further describes aptamers and photoaptamers having slower
dissociation rate constants than those obtained using prior
methods. In addition, the disclosure describes aptamer constructs
that include a variety of functionalities, including a cleavable
element, a detection element, and a capture or immobilization
element.
Inventors: |
Zichi; Dominic; (Boulder,
CO) ; Wilcox; Sheri K.; (Longmont, CO) ; Bock;
Chris; (Cleveland Heights, OH) ; Schneider; Daniel
J.; (Arvada, CO) ; Eaton; Bruce; (Longmont,
CO) ; Gold; Larry; (Boulder, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SomaLogic, Inc. |
Boulder |
CO |
US |
|
|
Family ID: |
46331949 |
Appl. No.: |
16/413989 |
Filed: |
May 16, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15878161 |
Jan 23, 2018 |
10316321 |
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16413989 |
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15410414 |
Jan 19, 2017 |
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15878161 |
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14610758 |
Jan 30, 2015 |
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15410414 |
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13113261 |
May 23, 2011 |
8975388 |
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14610758 |
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12175434 |
Jul 17, 2008 |
7947447 |
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13113261 |
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11623535 |
Jan 16, 2007 |
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12175434 |
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11623580 |
Jan 16, 2007 |
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11623535 |
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61051594 |
May 8, 2008 |
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61031420 |
Feb 26, 2008 |
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60950293 |
Jul 17, 2007 |
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60950281 |
Jul 17, 2007 |
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60950283 |
Jul 17, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/6429 20130101;
C12N 15/115 20130101; C12Q 1/6832 20130101; C12Q 1/6834 20130101;
C12N 2310/16 20130101; C12N 2310/322 20130101; C12Q 1/6811
20130101; G01N 33/5308 20130101; C12Q 1/6816 20130101; C12N 2330/31
20130101; C12Q 1/6832 20130101; C12N 2310/321 20130101; C12N
2310/334 20130101; C12N 2310/335 20130101; C12N 2310/314 20130101;
C12N 2310/315 20130101; C12Q 1/6816 20130101; C12Q 2525/161
20130101; C12Q 2525/205 20130101; C12Q 2525/161 20130101; C12Q
2537/101 20130101; C12N 15/1048 20130101; C12Q 2525/205 20130101;
C12N 2310/336 20130101; C12N 2310/333 20130101; C12Q 1/6834
20130101; C12Q 1/6811 20130101; G01N 33/58 20130101; C12Q 2525/205
20130101; C12Q 2565/514 20130101; C12Q 2523/313 20130101; C12Q
2525/205 20130101 |
International
Class: |
C12N 15/115 20060101
C12N015/115; C12Q 1/6816 20060101 C12Q001/6816; C12Q 1/6832
20060101 C12Q001/6832; C12Q 1/6834 20060101 C12Q001/6834; G01N
33/53 20060101 G01N033/53; G01N 33/58 20060101 G01N033/58; C12N
9/74 20060101 C12N009/74; C12Q 1/6811 20060101 C12Q001/6811; C12N
15/10 20060101 C12N015/10 |
Claims
1. A mixture comprising 500 protein targets, each protein target
being bound non-covalently by an aptamer, wherein the 500 protein
targets are the proteins of FIG. 7.
2. The mixture of claim 1, wherein each aptamer is capable of
binding to one of the 500 proteins of FIG. 7.
3. The mixture of claim 1, wherein the aptamer comprises a C-5
modified pyrimidine.
4. The mixture of claim 3, wherein the C-5 modified pyrimidine is
selected from FIG. 14.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/878,161, filed Jan. 23, 2018, which is a continuation of
U.S. application Ser. No. 15/410,414, filed Jan. 19, 2017, now
abandoned, which is a continuation of U.S. application Ser. No.
14/610,758, filed Jan. 30, 2015, now abandoned, which is a
continuation of U.S. application Ser. No. 13/113,261, filed May 23,
2011, which issued as U.S. Pat. No. 8,975,388 on Mar. 10, 2015,
which is a divisional of U.S. application Ser. No. 12/175,434,
filed Jul. 17, 2008, which issued as U.S. Pat. No. 7,947,447 on May
24, 2011, which claims the benefit of U.S. Provisional Application
Ser. No. 60/950,281, filed Jul. 17, 2007, U.S. Provisional
Application Ser. No. 60/950,293, filed Jul. 17, 2007, U.S.
Provisional Application Ser. No. 60/950,283, filed Jul. 17, 2007,
U.S. Provisional Application Ser. No. 61/031,420, filed Feb. 26,
2008 and U.S. Provisional Application Ser. No. 61/051,594, filed
May 8, 2008. U.S. application Ser. No. 12/175,434 is also a
continuation in part of U.S. application Ser. No. 11/623,580 and
U.S. application Ser. No. 11/623,535, each of which was filed on
Jan. 16, 2007, and each of which are now abandoned. Each of these
applications are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The present disclosure relates generally to methods for the
generation of aptamers and photoaptamers having improved properties
and the improved aptamers and photoaptamers generated thereby. In
particular, the present disclosure describes slow off-rate aptamers
that are highly specific to a target of interest. The disclosure
describes the composition of these slow off-rate aptamers as well
methods for their selection. Further the disclosure describes
aptamer constructs with improved functionalities for detection
methods. Further, the disclosure describes applications enabled by
these improved aptamers.
SEQUENCE LISTING
[0003] Incorporated by reference herein in its entirety is the
Sequence Listing entitled "sm1_19D_sequence_ST25.txt", created Jul.
28, 2018, size of 6 kilobytes.
BACKGROUND
[0004] The following description provides a summary of information
relevant to the present disclosure and is not a concession that any
of the information provided or publications referenced herein is
prior art to the claimed invention.
[0005] The SELEX process is a method for the in vitro selection of
nucleic acid molecules that are able to bind with high specificity
to target molecules and is described in U.S. Pat. No. 5,475,096
entitled "Nucleic Acid Ligands" and U.S. Pat. No. 5,270,163 (see
also WO 91/19813) entitled "Nucleic Acid Ligands" each of which is
specifically incorporated by reference herein. These patents,
collectively referred to herein as the SELEX Patents, describe
methods for making an aptamer to any desired target molecule.
[0006] The basic SELEX process has been modified to achieve a
number of specific objectives. For example, U.S. Pat. No.
5,707,796, entitled "Method for Selecting Nucleic Acids on the
Basis of Structure" describes the use of the SELEX process in
conjunction with gel electrophoresis to select nucleic acid
molecules with specific structural characteristics, such as bent
DNA. U.S. Pat. No. 5,580,737, entitled "High-Affinity Nucleic Acid
Ligands That Discriminate Between Theophylline and Caffeine"
describes a method for identifying highly specific aptamers able to
discriminate between closely related molecules, termed
Counter-SELEX. U.S. Pat. No. 5,567,588, entitled "Systematic
Evolution of Ligands by Exponential Enrichment: Solution SELEX"
describes a SELEX-based method which achieves highly efficient
partitioning between oligonucleotides having high and low affinity
for a target molecule. U.S. Pat. No. 5,496,938, entitled "Nucleic
Acid Ligands to HIV-RT and HIV-1 Rev" describes methods for
obtaining improved aptamers after SELEX has been performed. U.S.
Pat. No. 5,705,337, entitled "Systematic Evolution of Ligands by
Exponential Enrichment: Chemi-SELEX" describes methods for
covalently linking an aptamer to its target.
[0007] The SELEX process encompasses the identification of
high-affinity aptamers containing modified nucleotides conferring
improved characteristics on the ligand, such as improved in vivo
stability or improved delivery characteristics. Examples of such
modifications include chemical substitutions at the ribose and/or
phosphate and/or base positions. SELEX process-identified aptamers
containing modified nucleotides are described in U.S. Pat. No.
5,660,985, entitled "High Affinity Nucleic Acid Ligands Containing
Modified Nucleotides" that describes oligonucleotides containing
nucleotide derivatives chemically modified at the 5'- and
2'-positions of pyrimidines. U.S. Pat. No. 5,580,737, see supra,
describes highly specific aptamers containing one or more
nucleotides modified with 2'-amino (2'-NH.sub.2), 2'-fluoro (2'-F),
and/or 2'-O-methyl (2'-OMe).
[0008] Further modifications of the SELEX process are described in
U.S. Pat. Nos. 5,763,177, 6,001,577, and 6,291,184, each of which
is entitled "Systematic Evolution of Nucleic Acid Ligands by
Exponential Enrichment: Photoselection of Nucleic Acid Ligands and
Solution SELEX"; see also, e.g., U.S. Pat. No. 6,458,539, entitled
"Photoselection of Nucleic Acid Ligands". These patents,
collectively referred to herein as "the PhotoSELEX Patents"
describe various SELEX methods for selecting aptamers containing
photoreactive functional groups capable of binding and/or
photocrosslinking to and/or photoinactivating a target molecule.
The resulting photoreactive aptamers are referred to as
photocrosslinking aptamers or photoaptamers.
[0009] Although these SELEX and photoSELEX processes are useful,
there is always a need for processes that lead to improved
properties of aptamers generated from in vitro selection
techniques. For example, a need exists for aptamers to target
molecules with better binding affinities than those achieved with
naturally occurring DNA or RNA nucleotides, as well as methods for
producing such aptamers. For many applications, such as for
example, in vitro assays, diagnostics, therapeutic, or imaging
applications, it is of interest to produce aptamers with slow
dissociation rates from the aptamer/target affinity complex.
Several techniques have been proposed for producing such reagents
(see, e.g., WO 99/27133 and US 2005/0003362). However, these
selection processes do not discriminate between the selection of
reagents that have fast association kinetics with the target (i.e.,
fast on-rates) and the selection of reagents that have slow
dissociation kinetics with the target (i.e., slow off-rates). Thus,
there is a need for novel processes and techniques that favor the
selection of slow off-rate aptamers while inhibiting the selection
of aptamers that simply have a fast association rate with the
target.
[0010] Finally, there is a need for aptamer constructs that include
different built-in functionalities. These functionalities may
include tags for immobilization, labels for detection, means to
promote or control separation, etc.
SUMMARY
[0011] The present disclosure describes novel aptamers, and methods
to produce and use such aptamers. In particular, the disclosure
describes slow off-rate (slow rate of dissociation) aptamers, slow
off-rate aptamers containing C-5 modified pyrimidines, and
processes for the selection of slow off-rate aptamers by dilution,
by the addition of a competitor, or by a combination of both
approaches. In addition, slow off-rate aptamers to various targets
such as proteins and peptides are described. Slow off-rate aptamers
with unique structural features and melting temperatures are also
described. The disclosure also describes slow off-rate aptamers
with photoreactive functional groups, aptamers that are refractory
to the presence of poly-anionic materials, and a selection process
for these aptamers, as well as aptamers constructed with a variety
of other functionalities to improve their utility in various
applications.
[0012] The present disclosure describes improved SELEX methods for
generating aptamers that are capable of binding to target
molecules. More specifically, the present disclosure describes
methods for producing aptamers and/or photoaptamers having slower
rates of dissociation from their respective target molecules than
aptamers and photoaptamers obtained with previous SELEX methods.
Generally, after contacting the candidate mixture with the target
molecule and allowing the formation of nucleic acid-target
complexes to occur, a slow off-rate enrichment process is
introduced wherein nucleic acid-target complexes with fast
dissociation rates will dissociate and not reform, while complexes
with slow dissociation rates will remain intact. Methods for
introducing a slow off-rate enrichment process include, but are not
limited to, adding competitor molecules to the mixture of nucleic
acids and target molecules, diluting the mixture of nucleic acids
and target molecules, or a combination of both of these. The
disclosure further describes aptamers and photoaptamers obtained
using these methods.
[0013] In one embodiment, the method comprises preparing a
candidate mixture of nucleic acids; contacting the candidate
mixture with a target molecule wherein nucleic acids with the
highest relative affinities to the target molecule preferentially
bind the target molecule, forming nucleic acid-target molecule
complexes; introducing a slow off-rate enrichment process to induce
the dissociation of nucleic acid-target molecule complexes with
relatively fast dissociation rates; partitioning the remaining
bound nucleic acid-target molecule complexes from free nucleic
acids in the candidate mixture; and identifying the nucleic acids
that were bound to the target molecule. The process may further
include the iterative step of amplifying the nucleic acids that
bind to the target molecule to yield a mixture of nucleic acids
enriched with nucleic acids that bind to the target molecule yet
produce nucleic acid-target molecule complexes having slow
dissociation rates.
[0014] In another embodiment, the candidate mixture of nucleic
acids includes nucleic acids containing modified nucleotide bases
that may aid in the formation of modified nucleic acid-target
complexes having slow dissociation rates. Improved methods for
performing SELEX with modified nucleotides, including nucleotides
which contain photoactive or other functional groups, or
nucleotides which contain placeholders for photoactive groups are
disclosed in U.S. application Ser. No. 12/175,388, filed Jul. 17,
2008, which is incorporated by reference herein in its entirety,
and entitled "Improved SELEX and PHOTOSELEX" which is being filed
concurrently with the instant application. Placeholder nucleotides
may also be used for the mid-SELEX or post-SELEX introduction of
modified nucleotides that are not photoreactive.
[0015] The various methods and steps described herein can be used
to generate an aptamer capable of either (1) binding to a target
molecule or (2) binding to a target molecule and subsequently
forming a covalent linkage with the target molecule upon
irradiation with light in the UV or visible spectrum.
[0016] In another aspect, the various methods and steps described
herein can be used to generate an aptamer capable of modifying the
bioactivity of a target molecule through binding and/or
crosslinking to the target molecule. In one embodiment, an aptamer
to a unique target molecule associated with or relevant to a
specific disease process is identified. This aptamer can be used as
a diagnostic reagent, either in vitro or in vivo. In another
embodiment, an aptamer to a target molecule associated with a
disease state may be administered to an individual and used to
treat the disease in vivo. The aptamers and photoaptamers
identified herein can be used in any diagnostic, imaging, high
throughput screening or target validation techniques or procedures
or assays for which aptamers, oligonucleotides, antibodies and
ligands, without limitation can be used. For example, aptamers and
photoaptamers identified herein can be used according to the
methods described in detail in the concurrently filed U.S.
application Ser. No. 12/175,446, entitled "Multiplexed Analyses of
Test Samples", which is incorporated by reference herein in its
entirety.
[0017] Previous aptamers that do not have the slow off-rate
properties of the aptamers of the present invention have been used
for a variety of purposes. In almost all such uses, slow off-rate
aptamers will have improved performance relative to aptamers not
selected to have slow off-rate properties.
[0018] The aptamer Macugen.RTM., (See, e.g., U.S. Pat. Nos.
6,168,778; 6,051,698; 6,426,335; and 6,962,784; each of which is
incorporated herein by reference in its entirety) has been approved
for the treatment of macular degeneration, and functions due to its
specific affinity for VEGF. Other aptamers have been studied and/or
are in development for use as therapeutic agents. Aptamers not
selected to have slow off-rate properties have also been used in
many diagnostic and imaging applications (See, e.g., U.S. Pat. Nos.
5,843,653; 5,789,163; 5,853,984; 5,874,218; 6,261,783; 5,989,823;
6,177,555; 6,531,286; each of which is incorporated herein by
reference in its entirety), high-thorough put screening (See, e.g.,
U.S. Pat. Nos. 6,329,145; 6,670,132; 7,258,980; each of which is
incorporated herein by reference in its entirety) and in PCR kits
(See, e.g., U.S. Pat. Nos. 6,183,967; 6,020,130; 5,763,173;
5,874,557; 5,693,502; each of which is incorporated herein by
reference in its entirety.) The slow off-rate aptamers of this
disclosure may be used in any diagnostic, therapeutic, imaging or
any other use for which antibodies, aptamers and ligand binding
pairs have been used.
[0019] In another aspect, the disclosure provides aptamers and
photoaptamers identified by the improved methods disclosed herein,
diagnostic kits that include such aptamers and photoaptamers, and
therapeutic and diagnostic uses of such aptamers and photoaptamers.
The novel, slow off-rate aptamers and photoaptamers identified
using the described methods can be used in a variety of assays
including, assays that use planar arrays, beads, and other types of
solid supports. The assays may be used in a variety of contexts
including in life science research applications, clinical
diagnostic applications, (e.g., a diagnostic test for a disease, or
a "wellness" test for preventative healthcare); ALONA and UPS
assays, and in vivo imaging applications. For some applications,
multiplexed assays employing the described aptamers and
photoaptamers may be used.
[0020] In some embodiments, the slow off-rate aptamers (or
photoaptamers) described herein can be used as intravenous or oral
contrast agents for CAT scans and other imaging applications. CAT
scans are used in the diagnosis of muscle and bone disorders,
locating blood clots, detecting internal bleeding, monitoring
diseases such as cancer, etc. The slow off-rate aptamers may be
labeled with a CAT scan detectable component, such as, for example,
iodine, barium, or gastrograffin. In addition to carrying the
detectable component, the aptamer may be designed to direct that
component to a specific tissue or desired target. The aptamer may
serve to concentrate or localize the detectable component and thus
improve the signal to noise ratio by increasing available signal.
Because the off-rate of the aptamer can be sufficiently slow, the
duration of the scan can be increased, and the signal to noise
ratio of the scan may be improved. The specificity of the aptamer
for the target may also improve the signal to noise ratio in these
imaging applications.
[0021] In one embodiment, the slow off-rate aptamer is labeled with
a diamagnetic or paramagnetic material. In this embodiment, the
labeled aptamer may be used to improve the performance of magnetic
resonance imaging (MRI). MRI is particularly well suited to the
imaging of small, selective areas and tissues with high water
content or to monitoring blood flow. The specificity of the slow
off-rate aptamers may improve the localization of the MRI reagent
to a desired tissue section. Similarly, slow off-rate aptamers may
be modified with materials such as fluorine, carbon11, oxygen15, or
nitrogen13, for use in PET scans. In another embodiment, the
aptamers may be labeled with IR active materials that may be used
for infrared imaging. It is also contemplated that slow off-rate
aptamers may be labeled for use with other imaging modalities.
[0022] In one embodiment, the slow off-rate aptamers may be used as
very sensitive and specific reagents for incorporation into a
variety of in vitro diagnostic methods or kits. In some
embodiments, the slow off-rate aptamers are used as substitutes for
antibodies in a number of infectious, or other type of, disease
detection methods where the aptamer to the target of interest
includes either or both a detectable material and an immobilization
or capture component. In these embodiments, after the aptamer from
the kit is mixed with a clinical specimen, a variety of assay
formats may be utilized. In one embodiment, the aptamer also
includes a detectable label, such as a fluorophore. In other
embodiments, the assay format may include fluorescence quenching,
hybridization methods, flow cytometry, mass spectroscopy,
inhibition or competition methods, enzyme linked oligonucleotide
assays, SPR, evanescent wave methods, etc. In some embodiments, the
aptamer is provided in the kit in solution. In other embodiments,
the aptamer in the kit is immobilized onto a solid support used in
conjunction with the assay for testing the specimen. In various
embodiments, the solid support is designed for the detection of one
or more targets of interest. In other embodiments, the kit may
further include reagents to extract the target of interest,
reagents for amplifying the aptamer, reagents for performing
washing, detection reagents, etc.
[0023] In another embodiment, the slow off-rate aptamers may be
used in therapeutic imaging studies. During the development of new
therapeutic compounds, it is often difficult to assess certain
characteristics of the compound, such as, for example,
biodistribution, the washout rate, bioavailability, in vivo
drug/target interactions, etc. In many cases, if a suitable
detectable material was used to modify the therapeutic compound,
imaging studies could be used to assess all of these
characteristics. Though direct modification of a therapeutic
compound frequently inhibits its ability to interact with its
target and thus reduces efficacy, an aptamer's small size and
customizable specificity, render it potentially well-suited to
react with a therapeutic compound (for example, an antibody or
other protein-based therapeutic) while minimizing any undesirable
effects on the compound's therapeutic efficacy. To assess such
characteristics as biodistribution and the washout rate, the
aptamer/therapeutic complex may survive for an extended period of
time. These types of studies may be simplified in cases where the
therapeutic compound is a slow off-rate aptamer. In various
embodiments, aptamers used in therapeutic, imaging, and diagnostic
applications may include various modifications, such as, for
example, 2' fluoro and other modifications, to increase the
stability of the aptamer upon exposure to various components that
may be present in a test sample or in vivo, such as, for example,
nucleases and other sample or bodily fluid components.
BRIEF DESCRIPTION OF THE FIGURES
[0024] FIG. 1A illustrates an exemplary SELEX method and FIG. 1B
illustrates an exemplary SELEX method which includes the step of
incorporating a slow off-rate enrichment process or process.
[0025] FIG. 2 illustrates representative aptamer template, primer,
and complementary oligonucleotide sequences used in the disclosure.
The oligonucleotides were prepared by standard solid-phase
synthesis techniques. B=dT-biotin.
[0026] FIGS. 3A and 3B illustrate histograms of dissociation rate
constants for affinity aptamers selected without (FIG. 3A) and with
(FIG. 3B) a slow off-rate enrichment process as described in
Example 2.
[0027] FIGS. 4A and B show oligonucleotides that were used to
prepare the candidate mixtures or perform various steps in the
selection process described in Example 2. The oligonucleotides were
prepared by standard solid-phase synthesis techniques. BrdU
(5-bromo-dUTP), Anthraquinone (AQ), and psoralen (Psor)
chromophores were purchased as phosphoramidites and added to the 5'
terminus of the forward primer during synthesis.
4-azido-2-nitro-aniline (ANA) was prepared as a para-nitro-phenyl
carbonate derivative and coupled to a 5' hexylamine phosphoramidite
after synthesis. Two candidate mixture sequences were used in this
example, designated 1 and 2. B=dT-biotin. (FIG. 4A) Template 1 was
only used with candidate mixtures containing 5'-BrdU, AQ, and ANA,
and (FIG. 4B) Template 2 was only used with candidate mixtures
containing 5'-Psor for Example 2.
[0028] FIG. 5 illustrates chemical structures of the chromophores
coupled to the 5' terminus of the forward primer as illustrated in
FIGS. 4A and 4B.
[0029] FIG. 6 illustrates a PAGE analysis of crosslink activity of
TIMP-3 5'ANA/BndU enriched library using 5'-Fixed PhotoSELEX
described in Example 3. The gel illustrates the separation of free
aptamer (A.sub.f), intramolecular crosslinked aptamer (A.sub.f*),
and crosslinked protein:aptamer complexes (P:A).
[0030] FIG. 7 is a chart of over 500 targets for which aptamers
have been identified. Many of these aptamers have been designed to
have slow dissociation rates from their respective targets.
[0031] FIGS. 8A to 8D illustrate aptamer constructs that contain a
variety of different and optional functionalities including
immobilization tags, labels, photocrosslinking moieties, spacers,
and releasable moieties.
[0032] FIGS. 9A to 9F illustrate examples of aptamer constructs
including a cleavable or releasable element, a tag (for example
biotin), a spacer, and a label (for example Cy3).
[0033] FIG. 10 illustrates the aptamer and primer constructs
described in the disclosure. Cy3 represents a Cyanine 3 dye, PC a
photocleavable linker, ANA a photoreactive crosslinking group,
(AB).sub.2 a pair of biotin residues separated by dA residues, and
(T).sub.8 a poly dT linker. Primer constructs are complementary to
the complete 3' fixed region of the aptamer constructs.
[0034] FIGS. 11A to 11C illustrate dose response curves for slow
off-rate aptamers versus traditional aptamers for three different
targets.
[0035] FIGS. 12A and 12B illustrate performance curves for a slow
off-rate aptamer where the target was a peptide.
[0036] FIG. 13 illustrates a plot of the measured melting
temperature of a number of slow off-rate aptamers relative to the
predicted melting temperature.
[0037] FIG. 14 describes the base modifications of nucleotides
included in this disclosure. The R groups that may be used are
described in addition to the linkers (X) that may be used between
the nucleotide attachment point and the R group. The positions of
attachment for the various "R" groups are also indicated on the
respective R group.
[0038] FIG. 15 illustrates a plot used in the determination of the
binding constant for a slow off-rate aptamer containing C-5
modified pyrimidines to thrombin.
DETAILED DESCRIPTION
[0039] The practice of the invention disclosed herein employs,
unless otherwise indicated, conventional methods of chemistry,
microbiology, molecular biology, and recombinant DNA techniques
within the level of skill in the art. Such techniques are explained
fully in the literature. See, e.g., Sambrook, et al. Molecular
Cloning: A Laboratory Manual (Current Edition); DNA Cloning: A
Practical Approach, vol. I & II (D. Glover, ed.);
Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic
Acid Hybridization (B. Hames & S. Higgins, eds., Current
Edition); Transcription and Translation (B. Hames & S. Higgins,
eds., Current Edition).
[0040] All publications, published patent documents, and patent
applications cited in this specification are indicative of the
level of skill in the art(s) to which the invention pertains. All
publications, published patent documents, and patent applications
cited herein are hereby incorporated by reference to the same
extent as though each individual publication, published patent
document, or patent application was specifically and individually
indicated as being incorporated by reference.
[0041] As used in this specification, including the appended
claims, the singular forms "a," "an," and "the" include plural
references, unless the content clearly dictates otherwise, and are
used interchangeably with "at least one" and "one or more." Thus,
reference to "an aptamer" includes mixtures of aptamers, reference
to "a probe" includes mixtures of probes, and the like.
[0042] As used herein, the term "about" represents an insignificant
modification or variation of the numerical values such that the
basic function of the item to which the numerical value relates is
unchanged.
[0043] As used herein, the terms "comprises," "comprising,"
"includes," "including," "contains," "containing," and any
variations thereof, are intended to cover a non-exclusive
inclusion, such that a process, method, product-by-process, or
composition of matter that comprises, includes, or contains an
element or list of elements does not include only those elements
but may include other elements not expressly listed or inherent to
such process, method, product-by-process, or composition of
matter.
[0044] As used herein, "nucleic acid ligand" "aptamer" and "clone"
are used interchangeably to refer to a non-naturally occurring
nucleic acid that has or may have a desirable action on a target
molecule. 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 (as in a suicide inhibitor), and facilitating the reaction
between the target and another molecule. In one embodiment, the
action is specific binding affinity for a target molecule, such
target molecule being a three dimensional chemical structure, other
than a polynucleotide, that binds to the aptamer through a
mechanism which is predominantly independent of Watson/Crick base
pairing or triple helix binding, wherein the aptamer is not a
nucleic acid having the known physiological function of being bound
by the target molecule. Aptamers include nucleic acids that are
identified from a candidate mixture of nucleic acids, the aptamer
being a ligand of a given target, by the method comprising: (a)
contacting the candidate mixture with the target, wherein nucleic
acids having an increased affinity to the target relative to other
nucleic acids in the candidate mixture may be partitioned from the
remainder of the candidate mixture; (b) partitioning the increased
affinity and/or slow off-rate nucleic acids from the remainder of
the candidate mixture; and (c) amplifying the increased affinity,
slow off-rate nucleic acids to yield a ligand-enriched mixture of
nucleic acids, whereby aptamers to the target molecule are
identified. It is recognized that affinity interactions are a
matter of degree; however, in this context, the "specific binding
affinity" of an aptamer for its target means that the aptamer binds
to its target generally with a much higher degree of affinity than
it may binds to other, non-target, components in a mixture or
sample. An "aptamer" or "nucleic acid ligand" is a set of copies of
one type or species of nucleic acid molecule that has a particular
nucleotide sequence. An aptamer can include any suitable number of
nucleotides. "Aptamers" refer to more than one such set of
molecules. Different aptamers may have either the same number or a
different number of nucleotides. Aptamers may be DNA or RNA and
maybe single stranded, double stranded, or contain double stranded
regions.
[0045] As used herein, "slow off-rate" or "slow rate of
dissociation" or "slow dissociation rate" refers to the time it
takes for an aptamers/target complex to begin to dissociate. This
can be expressed as a half life, t.sub.1/2, or the point at which
50% of the aptamer/target complex has dissociated. The off-rate or
dissociation rate of a slow off-rate aptamer, expressed as
t.sub.1/2 values, can be about .gtoreq.30 min., .gtoreq.about 60
min., .gtoreq.about 90 min., .gtoreq.about 120 min. .gtoreq.about
150 min. .gtoreq.about 180 min. .gtoreq.about 210 min., and
.gtoreq.about 240 min.
[0046] In one embodiment, a method for producing a synthetic
library of nucleic acids comprises: 1) synthesizing the nucleic
acids; 2) deprotecting the nucleic acids; 3) purifying the nucleic
acids; and 4) analyzing the nucleic acids. In the synthesis step, a
monomer mixture is prepared where the ratio of the various
nucleotides in the mix is optimized to yield equal ratios of each
nucleotide in the final product. One or more of the monomers in the
mixture may comprise a modified nucleotide. Amidite protection
groups are used in this procedure and in one embodiment, the
monomer concentration is 0.1M. During synthesis, the five prime
protecting group is retained in the product nucleic acid. Synthesis
is conducted on a solid support (controlled pore glass, CPG) and at
least about 80 cycles are completed to synthesize the final
product.
[0047] After the synthesis process, the nucleic acid product is
deprotected. A 1.0 M aqueous lysine buffer, pH 9.0 is employed to
cleave apurinic sites while the product is retained on the support
(controlled pore glass, CPG). These cleaved truncated sequences are
washed away with deionized (dI) water two times. 500 uL of dl water
are added after the two washes in preparation for the deprotection
step. This step involves the treatment with 1.0 mL of
t-butylamine:methanol:water, 1:1:2, for 5 hours at 70.degree. C.,
is followed by freezing, filtration, and evaporation to dryness.
The nucleic acid product is purified based on the hydrophobicity of
the protecting group on a PRP-3 HPLC column (Hamilton). Appropriate
column fractions are collected and pooled, desalted, and evaporated
to dryness to remove the volatile elution buffers. The final
product is washed with water by a centrifugation process and then
re-suspended. Finally the resuspended material is treated to
deprotect the final product. Final product is characterized by base
composition, primer extension, and sequencing gel.
[0048] A candidate mixture of nucleic acids, or a library of
nucleic acids, may also be produced by an enzymatic method using a
solid phase. In one embodiment, this method comprises the same
basic steps described above. In this case the goal is the synthesis
of an antisense library and these libraries are produced with a 5'
biotin modification. All remaining synthetic processes are as
described above. Once the synthetic library is prepared, the
nucleic acids maybe used in a primer extension mix containing one
or more modified nucleotides to produce the final candidate mixture
in a classic primer extension method.
[0049] Aptamers may be synthesized by the same chemistry that is
used for the synthesis of a library. However, instead of a mixture
of nucleotides, one nucleotide is introduced at each step in the
synthesis to control the final sequence generated by routine
methods. Modified nucleotides may be introduced into the synthesis
process at the desired positions in the sequence. Other
functionalities may be introduced as desired using known chemical
modifications of nucleotides.
[0050] As used herein, "candidate mixture" is a mixture of nucleic
acids of differing sequence from which to select a desired ligand.
The source of a candidate mixture can be from naturally-occurring
nucleic acids or fragments thereof, chemically synthesized nucleic
acids, enzymatically synthesized nucleic acids or nucleic acids
made by a combination of the foregoing techniques. Modified
nucleotides, such as nucleotides with photoreactive groups or other
modifications, can be incorporated into the candidate mixture. In
addition, a SELEX process can be used to produce a candidate
mixture, that is, a first SELEX process experiment can be used to
produce a ligand-enriched mixture of nucleic acids that is used as
the candidate mixture in a second SELEX process experiment. A
candidate mixture can also comprise nucleic acids with one or more
common structural motifs. As used herein, a candidate mixture is
also sometimes referred to as a "pool" or a "library." For example,
an "RNA pool" refers to a candidate mixture comprised of RNA.
[0051] In various embodiments, each nucleic acid in a candidate
mixture may have fixed sequences on either side of a randomized
region, to facilitate the amplification process. The nucleic acids
in the candidate mixture of nucleic acids can each further comprise
fixed regions or "tail" sequences at their 5' and 3' termini to
prevent the formation of high molecular weight parasites during the
amplification process.
[0052] As used herein, "nucleic acid," "oligonucleotide," and
"polynucleotide" are used interchangeably to refer to a polymer of
nucleotides of any length, and such nucleotides may include
deoxyribonucleotides, ribonucleotides, and/or analogs or chemically
modified deoxyribonucleotides or ribonucleotides. The terms
"polynucleotide," "oligonucleotide," and "nucleic acid" include
double- or single-stranded molecules as well as triple-helical
molecules.
[0053] If present, chemical modifications of a nucleotide can
include, singly or in any combination, 2'-position sugar
modifications, 5-position pyrimidine modifications (e.g.,
5-(N-benzylcarboxyamide)-2'-deoxyuridine,
5-(N-isobutylcarboxyamide)-2'-deoxyuridine,
5-(N-tryptaminocarboxyamide)-2'-deoxyuridine,
5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2'-deoxyuridine
chloride, 5-(N-naphthylmethylcarboxamide)-2'-deoxyuridine, or
5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2'-deoxyuridine),
modifications at exocyclic amines, substitution of 4-thiouridine,
substitution of 5-bromo- or 5-iodo-uracil, backbone modifications,
methylations, unusual base-pairing combinations such as the
isobases, isocytidine and isoguanidine, and the like.
[0054] In one embodiment, the term "C-5 modified pyrimidine" refers
to a pyrimidine with a modification at the C-5 position including,
but not limited to those moieties illustrated in FIG. 14. Examples
of a C-5 modified pyrimidine include those described in U.S. Pat.
Nos. 5,719,273 and 5,945,527. Examples of a C-5 modification
include substitution of deoxyuridine at the C-5 position with a
substituent selected from: benzylcarboxyamide (alternatively
benzylaminocarbonyl) (Bn), naphthylmethylcarboxyamide
(alternatively naphthylmethylaminocarbonyl) (Nap),
tryptaminocarboxyamide (alternatively tryptaminocarbonyl) (Trp),
and isobutylcarboxyamide (alternatively isobutylaminocarbonyl)
(iBu) as illustrated immediately below.
##STR00001##
As delineated above, representative C-5 modified pyrimidines
include: 5-(N-benzylcarboxyamide)-2'-deoxyuridine (BndU),
5-(N-isobutylcarboxyamide)-2'-deoxyuridine (iBudU),
5-(N-tryptaminocarboxyamide)-2'-deoxyuridine (TrpdU) and
5-(N-naphthylmethylcarboxyamide)-2'-deoxyuridine (NapdU).
[0055] Modifications can also include 3' and 5' modifications, such
as capping or pegylation. Other modifications can include
substitution of one or more of the naturally occurring nucleotides
with an analog, internucleotide modifications such as, for example,
those with uncharged linkages (e.g., methyl phosphonates,
phosphotriesters, phosphoamidates, carbamates, etc.) and those with
charged linkages (e.g., phosphorothioates, phosphorodithioates,
etc.), those with intercalators (e.g., acridine, psoralen, etc.),
those containing chelators (e.g., metals, radioactive metals,
boron, oxidative metals, etc.), those containing alkylators, and
those with modified linkages (e.g., alpha anomeric nucleic acids,
etc.). Further, any of the hydroxyl groups ordinarily present in a
sugar may be replaced by a phosphonate group or a phosphate group;
protected by standard protecting groups; or activated to prepare
additional linkages to additional nucleotides or to a solid
support. The 5' and 3' terminal OH groups can be phosphorylated or
substituted with amines, organic capping group moieties of from
about 1 to about 20 carbon atoms, or organic capping group moieties
of from about 1 to about 20 polyethylene glycol (PEG) polymers or
other hydrophilic or hydrophobic biological or synthetic polymers.
If present, a modification to the nucleotide structure may be
imparted before or after assembly of a polymer. A sequence of
nucleotides may be interrupted by non-nucleotide components. A
polynucleotide may be further modified after polymerization, such
as by conjugation with a labeling component.
[0056] Polynucleotides can also contain analogous forms of ribose
or deoxyribose sugars that are generally known in the art,
including 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose,
carbocyclic sugar analogs, .alpha.-anomeric sugars, epimeric sugars
such as arabinose, xyloses or lyxoses, pyranose sugars, furanose
sugars, sedoheptuloses, acyclic analogs and abasic nucleoside
analogs such as methyl riboside. As noted above, one or more
phosphodiester linkages may be replaced by alternative linking
groups. These alternative linking groups include embodiments
wherein phosphate is replaced by P(O)S ("thioate"), P(S)S
("dithioate"), (O)NR.sub.2 ("amidate"), P(O)R, P(O)OR', CO or
CH.sub.2 ("formacetal"), in which each R or R' is independently H
or substituted or unsubstituted alkyl (1-20 C) optionally
containing an ether (--O--) linkage, aryl, alkenyl, cycloalky,
cycloalkenyl or araldyl. Not all linkages in a polynucleotide need
be identical. Substitution of analogous forms of sugars, purines,
and pyrimidines can be advantageous in designing a final product,
as can alternative backbone structures like a polyamide backbone,
for example.
[0057] In one embodiment, the variable region of the aptamer
includes nucleotides that include modified bases. Certain modified
aptamers may be used in any of the described methods, devices, and
kits. These modified nucleotides have been shown to produce novel
aptamers that have very slow off-rates from their respective
targets while maintaining high affinity to the target. In one
embodiment, the C-5 position of the pyrimidine bases may be
modified. Aptamers containing nucleotides with modified bases have
a number of properties that are different than the properties of
standard aptamers that include only naturally occurring nucleotides
(i.e., unmodified nucleotides). In one embodiment, the method for
modification of the nucleotides includes the use of an amide
linkage. However, other suitable methods for modification may be
used. It has been surprisingly observed that the structure of the
identified slow off-rate aptamers does not appear to be entirely in
accordance with the structure predicted by standard base pairing
models. This observation is supported by the fact that the measured
melting temperatures of the slow off-rate aptamers are not
consistent with the melting temperatures predicted by the models,
see FIG. 13. As shown, there appears to be no correlation between
the measured and predicted melting temperatures of the slow
off-rate aptamers. On average, the calculated melting temperature
(Tm) is 6.degree. C. lower than the measured Tm. The measured
melting temperatures indicate that slow off-rate aptamers including
these modified nucleotides are more stable than may be predicted
and potentially possess novel secondary structures. These modified
aptamers also have different circular dichorism spectra than
corresponding aptamers that include only unmodified nucleotides. In
the case of many targets, slow off-rate aptamers to the target are
more likely to be identified when modified nucleotides are used in
the production of the initial library or candidate mixture.
[0058] As used herein, "modified nucleic acid" refers to a nucleic
acid sequence containing one or more modified nucleotides. In some
embodiments it may be desirable that the modified nucleotides are
compatible with the SELEX process.
[0059] "Polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to polymers of amino acids of any
length. The polymer may be linear or branched, it may comprise
modified amino acids, and/or it may be interrupted by non-amino
acids. The terms also encompass an amino acid polymer that has been
modified naturally or by intervention; for example, disulfide bond
formation, glycosylation, lipidation, acetylation, phosphorylation,
or any other manipulation or modification, such as conjugation with
a labeling component. Also included within the definition are, for
example, polypeptides containing one or more analogs of an amino
acid (including, for example, unnatural amino acids, etc.), as well
as other modifications known in the art. Polypeptides can be single
chains or associated chains.
[0060] As used herein, "photoreactive nucleotide" means any
modified nucleotide that is capable of photocros slinking with a
target, such as a protein, upon irradiation with certain
wavelengths of light. For example, photoaptamers produced by the
photoSELEX process can include a photoreactive group selected from
the following: 5-bromouracil (BrU), 5-iodouracil (IU),
5-bromovinyluracil, 5-iodovinyluracil, 5-azidouracil, 4-thiouracil,
5-bromocytosine, 5-iodocytosine, 5-bromovinylcytosine,
5-iodovinylcytosine, 5-azidocytosine, 8-azidoadenine,
8-bromoadenine, 8-iodoadenine, 8-azidoguanine, 8-bromoguanine,
8-iodoguanine, 8-azidohypoxanthine, 8-bromohypoxanthine,
8-iodohypoxanthine, 8-azidoxanthine, 8-bromoxanthine,
8-iodoxanthine, 5-bromodeoxyuridine, 8-bromo-2'-deoxyadenine,
5-iodo-2'-deoxyuracil, 5-iodo-2'-deoxycytosine,
5-[(4-azidophenacyl)thio]cytosine, 5-[(4-azidophenacyl)thio]uracil,
7-deaza-7-iodoadenine, 7-deaza-7-iodoguanine,
7-deaza-7-bromoadenine, and 7-deaza-7-bromoguanine. A
"photoreactive pyrimidine" means any modified pyrimidine that is
capable of photocrosslinking with a target upon irradiation of
certain wavelengths. Exemplary photoreactive pyrimidines include
5-bromo-uracil (BrdU), 5-bromo-cytosine (BrdC), 5-iodo-uracil
(IdU), and 5-iodo-cytosine (IdC). In various embodiments, the
photoreactive functional group will absorb wavelengths of light
that are not absorbed by the target or the non-modified portions of
the oligonucleotide.
[0061] "SELEX" refers to a process that combines the selection of
nucleic acids that interact with a target in a desirable manner
(e.g., binding to a protein) with the amplification of those
selected nucleic acids. Optional iterative cycling of the
selection/amplification steps allows selection of one or a small
number of nucleic acids that interact most strongly with the target
from a pool that contains a very large number of nucleic acids.
Cycling of the selection/amplification procedure is continued until
a selected goal is achieved. The SELEX methodology is described in
the SELEX Patents. In some embodiments of the SELEX process,
aptamers that bind non-covalently to their targets are generated.
In other embodiments of the SELEX process, aptamers that bind
covalently to their targets are generated.
[0062] As used herein the term "amplification" or "amplifying"
means any process or combination of process steps that increases
the amount or number of copies of a molecule or class of
molecules.
[0063] "SELEX target" or "target molecule" or "target" refers
herein to any compound upon which a nucleic acid can act in a
desirable manner. A SELEX target molecule can be a protein,
peptide, nucleic acid, carbohydrate, lipid, polysaccharide,
glycoprotein, hormone, receptor, antigen, antibody, virus,
pathogen, toxic substance, substrate, metabolite, transition state
analog, cofactor, inhibitor, drug, dye, nutrient, growth factor,
cell, tissue, any portion or fragment of any of the foregoing,
etc., without limitation. In one embodiment, a SELEX target does
not include molecules that are known to bind nucleic acids, such
as, for example, known nucleic acid binding proteins (e.g.
transcription factors). Virtually any chemical or biological
effector may be a suitable SELEX target. Molecules of any size can
serve as SELEX targets. A target can also be modified in certain
ways to enhance the likelihood or strength of an interaction
between the target and the nucleic acid. A target can also include
any minor variation of a particular compound or molecule, such as,
in the case of a protein, for example, minor variations in amino
acid sequence, disulfide bond formation, glycosylation, lipidation,
acetylation, phosphorylation, or any other manipulation or
modification, such as conjugation with a labeling component, which
does not substantially alter the identity of the molecule. A
"target molecule" or "target" is a set of copies of one type or
species of molecule or multimolecular structure that is capable of
binding to an aptamer. "Target molecules" or "targets" refer to
more than one such set of molecules. Embodiments of the SELEX
process in which the target is a peptide are described in U.S. Pat.
No. 6,376,190, entitled "Modified SELEX Processes Without Purified
Protein," incorporated herein by reference in its entirety. FIG. 7
lists over 500 targets for which aptamers have been produced
including a variety of slow off-rate aptamers.
[0064] As used herein, "competitor molecule" and "competitor" are
used interchangeably to refer to any molecule that can form a
non-specific complex with a non-target molecule. In this context,
non-target molecules include free aptamers, where, for example, a
competitor can be used to inhibit the aptamer from binding
(re-binding), non-specifically, to another non-target molecule. A
"competitor molecule" or "competitor" is a set of copies of one
type or species of molecule. "Competitor molecules" or
"competitors" refer to more than one such set of molecules.
Competitor molecules include, but are not limited to
oligonucleotides, polyanions (e.g., heparin, herring sperm DNA,
salmon sperm DNA, tRNA, dextran sulfate, polydextran, abasic
phosphodiester polymers, dNTPs, and pyrophosphate). In various
embodiments, a combination of one or more competitor can be
used.
[0065] As used herein, "non-specific complex" refers to a
non-covalent association between two or more molecules other than
an aptamer and its target molecule. A non-specific complex
represents an interaction between classes of molecules.
Non-specific complexes include complexes formed between an aptamer
and a non-target molecule, a competitor and a non-target molecule,
a competitor and a target molecule, and a target molecule and a
non-target molecule.
[0066] As used herein, the term "slow off-rate enrichment process"
refers to a process of altering the relative concentrations of
certain components of a candidate mixture such that the relative
concentration of aptamer affinity complexes having slow
dissociation rates is increased relative to the concentration of
aptamer affinity complexes having faster, less desirable
dissociation rates. In one embodiment, the slow off-rate enrichment
process is a solution-based slow off-rate enrichment process. In
this embodiment, a solution-based slow off-rate enrichment process
takes place in solution, such that neither the target nor the
nucleic acids forming the aptamer affinity complexes in the mixture
are immobilized on a solid support during the slow off-rate
enrichment process. In various embodiments, the slow off-rate
enrichment process can include one or more steps, including the
addition of and incubation with a competitor molecule, dilution of
the mixture, or a combination of these (e.g., dilution of the
mixture in the presence of a competitor molecule). Because the
effect of an slow off-rate enrichment process generally depends
upon the differing dissociation rates of different aptamer affinity
complexes (i.e., aptamer affinity complexes formed between the
target molecule and different nucleic acids in the candidate
mixture), the duration of the slow off-rate enrichment process is
selected so as to retain a high proportion of aptamer affinity
complexes having slow dissociation rates while substantially
reducing the number of aptamer affinity complexes having fast
dissociation rates. The slow off-rate enrichment process may be
used in one or more cycles during the SELEX process. When dilution
and the addition of a competitor are used in combination, they may
be performed simultaneously or sequentially, in any order. The slow
off-rate enrichment process can be used when the total target
(protein) concentration in the mixture is low. In one embodiment,
when the slow off-rate enrichment process includes dilution, the
mixture can be diluted as much as is practical, keeping in mind
that the nucleic acids are recovered for subsequent rounds in the
SELEX process. In one embodiment, the slow off-rate enrichment
process includes the use of a competitor as well as dilution,
permitting the mixture to be diluted less than might be necessary
without the use of a competitor.
[0067] In one embodiment, the slow off-rate enrichment process
includes the addition of a competitor, and the competitor is a
polyanion (e.g., heparin or dextran sulfate (dextran)). Heparin or
dextran have been used in the identification of specific aptamers
in prior SELEX selections. In such methods, however, heparin or
dextran is present during the equilibration step in which the
target and aptamer bind to form complexes. In such methods, as the
concentration of heparin or dextran increases, the ratio of high
affinity target/aptamer complexes to low affinity target/aptamer
complexes increases. However, a high concentration of heparin or
dextran can reduce the number of high affinity target/aptamer
complexes at equilibrium due to competition for target binding
between the nucleic acid and the competitor. By contrast, the
presently described methods add the competitor after the
target/aptamer complexes have been allowed to form and therefore
does not affect the number of complexes formed. Addition of
competitor after equilibrium binding has occurred between target
and aptamer creates a non-equilibrium state that evolves in time to
a new equilibrium with fewer target/aptamer complexes. Trapping
target/aptamer complexes before the new equilibrium has been
reached enriches the sample for slow off-rate aptamers since fast
off-rate complexes will dissociate first.
[0068] In another embodiment, a polyanionic competitor (e.g.,
dextran sulfate or another polyanionic material) is used in the
slow off-rate enrichment process to facilitate the identification
of an aptamer that is refractory to the presence of the polyanion.
In this context, "polyanionic refractory aptamer" is an aptamer
that is capable of forming an aptamer/target complex that is less
likely to dissociate in the solution that also contains the
polyanionic refractory material than an aptamer/target complex that
includes a non-polyanionic refractory aptamer. In this manner,
polyanionic refractory aptamers can be used in the performance of
analytical methods to detect the presence or amount or
concentration of a target in a sample, where the detection method
includes the use of the polyanionic material (e.g. dextran sulfate)
to which the aptamer is refractory.
[0069] Thus, in one embodiment, a method for producing a
polyanionic refractory aptamer is provided. In this embodiment,
after contacting a candidate mixture of nucleic acids with the
target,the target and the nucleic acids in the candidate mixture
are allowed to come to equilibrium. A polyanionic competitor is
introduced and allowed to incubate in the solution for a period of
time sufficient to insure that most of the fast off-rate aptamers
in the candidate mixture dissociate from the target molecule. Also,
aptamers in the candidate mixture that may dissociate in the
presence of the polyanionic competitor will be released from the
target molecule. The mixture is partitioned to isolate the high
affinity, slow off-rate aptamers that have remained in association
with the target molecule and to remove any uncomplexed materials
from the solution. The aptamer can then be released from the target
molecule and isolated. The isolated aptamer can also be amplified
and additional rounds of selection applied to increase the overall
performance of the selected aptamers. This process may also be used
with a minimal incubation time if the selection of slow off-rate
aptamers is not needed for a specific application.
[0070] Thus, in one embodiment a modified SELEX process is provided
for the identification or production of aptamers having slow (long)
off-rates wherein the target molecule and candidate mixture are
contacted and incubated together for a period of time sufficient
for equilibrium binding between the target molecule and nucleic
acids contained in the candidate mixture to occur. Following
equilibrium binding an excess of competitor molecule, e.g.,
polyanion competitor, is added to the mixture and the mixture is
incubated together with the excess of competitor molecule for a
predetermined period of time. A significant proportion of aptamers
having off rates that are less than this predetermined incubation
period will dissociate from the target during the predetermined
incubation period. Re-association of these "fast" off-rate aptamers
with the target is minimized because of the excess of competitor
molecule which can non-specifically bind to the target and occupy
aptamer binding sites on the target. A significant proportion of
aptamers having longer off rates will remain complexed to the
target during the predetermined incubation period. At the end of
the incubation period, partitioning nucleic acid-target complexes
from the remainder of the mixture allows for the separation of a
population of slow off-rate aptamers from those having fast off
rates. A dissociation step can be used to dissociate the slow
off-rate aptamers from their target and allows for isolation,
identification, sequencing, synthesis and amplification of slow
off-rate aptamers (either of individual aptamers or of a group of
slow off-rate aptamers) that have high affinity and specificity for
the target molecule. As with conventional SELEX the aptamer
sequences identified from one round of the modified SELEX process
can be used in the synthesis of a new candidate mixture such that
the steps of contacting, equilibrium binding, addition of
competitor molecule, incubation with competitor molecule and
partitioning of slow off-rate aptamers can be iterated/repeated as
many times as desired.
[0071] The combination of allowing equilibrium binding of the
candidate mixture with the target prior to addition of competitor,
followed by the addition of an excess of competitor and incubation
with the competitor for a predetermined period of time allows for
the selection of a population of aptamers having off rates that are
much greater than those previously achieved.
[0072] In order to achieve equilibrium binding, the candidate
mixture may be incubated with the target for at least about 5
minutes, or at least about 15 minutes, about 30 minutes, about 45
minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours,
about 5 hours or about 6 hours.
[0073] The predetermined incubation period of competitor molecule
with the mixture of the candidate mixture and target molecule may
be selected as desired, taking account of factors such as the
nature of the target and known off rates (if any) of known aptamers
for the target. Predetermined incubation periods may be chosen
from: at least about 5 minutes, at least about 10 minutes, at least
about 20 minutes, at least about 30 minutes, at least 45 about
minutes, at least about 1 hour, at least about 2 hours, at least
about 3 hours, at least about 4 hours, at least about 5 hours, at
least about 6 hours.
[0074] In other embodiments a dilution is used as an off rate
enhancement process and incubation of the diluted candidate
mixture, target molecule/aptamer complex may be undertaken for a
predetermined period of time, which may be chosen from: at least
about 5 minutes, at least about 10 minutes, at least about 20
minutes, at least about 30 minutes, at least about 45 minutes, at
least about 1 hour, at least about 2 hours, at least about 3 hours,
at least about 4 hours, at least about 5 hours, at least about 6
hours.
[0075] Embodiments of the present disclosure are concerned with the
identification, production, synthesis and use of slow off-rate
aptamers. These are aptamers which have a rate of dissociation
(t.sub.1/2) from a non-covalent aptamer-target complex that is
higher than that of aptamers normally obtained by conventional
SELEX. For a mixture containing non-covalent complexes of aptamer
and target, the t.sub.1/2 represents the time taken for half of the
aptamers to dissociate from the aptamer-target complexes. The
t.sub.1/2 of slow dissociation rate aptamers according to the
present disclosure is chosen from one of: greater than or equal to
about 30 minutes; between about 30 minutes and about 240 minutes;
between about 30 minutes to about 60 minutes; between about 60
minutes to about 90 minutes, between about 90 minutes to about 120
minutes; between about 120 minutes to about 150 minutes; between
about 150 minutes to about 180 minutes; between about 180 minutes
to about 210 minutes; between about 210 minutes to about 240
minutes.
[0076] A characterizing feature of an aptamer identified by a SELEX
procedure is its high affinity for its target. An aptamer will have
a dissociation constant (k.sub.d) for its target that is chosen
from one of: less than about 1 .mu.M, less than about 100 nM, less
than about 10 nM, less than about 1 nM, less than about 100 pM,
less than about 10 pM, less than about 1 pM.
[0077] "Tissue target" or "tissue" refers herein to a certain
subset of the SELEX targets described above. According to this
definition, tissues are macromolecules in a heterogeneous
environment. As used herein, tissue refers to a single cell type, a
collection of cell types, an aggregate of cells, or an aggregate of
macromolecules. This differs from simpler SELEX targets that are
typically isolated soluble molecules, such as proteins. In some
embodiments, tissues are insoluble macromolecules that are orders
of magnitude larger than simpler SELEX targets. Tissues are complex
targets made up of numerous macromolecules, each macromolecule
having numerous potential epitopes. The different macromolecules
which comprise the numerous epitopes can be proteins, lipids,
carbohydrates, etc., or combinations thereof. Tissues are generally
a physical array of macromolecules that can be either fluid or
rigid, both in terms of structure and composition. Extracellular
matrix is an example of a more rigid tissue, both structurally and
compositionally, while a membrane bilayer is more fluid in
structure and composition. Tissues are generally not soluble and
remain in solid phase, and thus partitioning can be accomplished
relatively easily. Tissue includes, but is not limited to, an
aggregate of cells usually of a particular kind together with their
intercellular substance that form one of the structural materials
commonly used to denote the general cellular fabric of a given
organ, e.g., kidney tissue, brain tissue. The four general classes
of tissues are epithelial tissue, connective tissue, nerve tissue
and muscle tissue.
[0078] Examples of tissues which fall within this definition
include, but are not limited to, heterogeneous aggregates of
macromolecules such as fibrin clots which are a cellular;
homogeneous or heterogeneous aggregates of cells; higher ordered
structures containing cells which have a specific function, such as
organs, tumors, lymph nodes, arteries, etc, and individual cells.
Tissues or cells can be in their natural environment, isolated, or
in tissue culture. The tissue can be intact or modified. The
modification can include numerous changes such as transformation,
transfection, activation, and substructure isolation, e.g., cell
membranes, cell nuclei, cell organelles, etc.
[0079] Sources of the tissue, cell or subcellular structures can be
obtained from prokaryotes as well as eukaryotes. This includes
human, animal, plant, bacterial, fungal, and viral structures.
[0080] As used herein, the term "labeling agent," "label," or
"detectable moiety", or "detectable element" or "detectable
component" refers to one or more reagents that can be used to
detect a target molecule/aptamer complex. A detectable moiety or
label is capable of being detected directly or indirectly. In
general, any reporter molecule that is detectable can be a label.
Labels include, for example, (i) reporter molecules that can be
detected directly by virtue of generating a signal, (ii) specific
binding pair members that may be detected indirectly by subsequent
binding to a cognate that contains a reporter molecule, (iii) mass
tags detectable by mass spectrometry, (iv) oligonucleotide primers
that can provide a template for amplification or ligation, and (v)
a specific polynucleotide sequence or recognition sequence that can
act as a ligand, such as, for example, a repressor protein, wherein
in the latter two instances the oligonucleotide primer or repressor
protein will have, or be capable of having, a reporter molecule,
and so forth. The reporter molecule can be a catalyst, such as an
enzyme, a polynucleotide coding for a catalyst, promoter, dye,
fluorescent molecule, quantum dot, chemiluminescent molecule,
coenzyme, enzyme substrate, radioactive group, a small organic
molecule, amplifiable polynucleotide sequence, a particle such as
latex or carbon particle, metal sol, crystallite, liposome, cell,
etc., which may or may not be further labeled with a dye, catalyst
or other detectable group, a mass tag that alters the weight of the
molecule to which it is conjugated for mass spectrometry purposes,
and the like. The label can be selected from electromagnetic or
electrochemical materials. In one embodiment, the detectable label
is a fluorescent dye. Other labels and labeling schemes will be
evident to one skilled in the art based on the disclosure
herein.
[0081] A detectable moiety (element or component) can include any
of the reporter molecules listed above and any other chemical or
component that may be used in any manner to generate a detectable
signal. The detectable moiety may be detected via a fluorescent
signal, a chemiluminescent signal, or any other detectable signal
that is dependent upon the identity of the moiety. In the case
where the detectable moiety is an enzyme (for example, alkaline
phosphatase), the signal may be generated in the presence of the
enzyme substrate and any additional factors necessary for enzyme
activity. In the case where the detectable moiety is an enzyme
substrate, the signal may be generated in the presence of the
enzyme and any additional factors necessary for enzyme activity.
Suitable reagent configurations for attaching the detectable moiety
to a target molecule include covalent attachment of the detectable
moiety to the target molecule, non-covalent association of the
detectable moiety with another labeling agent component that is
covalently attached to the target molecule, and covalent attachment
of the detectable moiety to a labeling agent component that is
non-covalently associated with the target molecule. Universal
protein stains (UPS) are described in detail in U.S. patent
application Ser. No. 10/504,696, filed Aug. 12, 2004, entitled
"Methods and Reagents for Detecting Target Binding by Nucleic Acid
Ligands".
[0082] "Solid support" refers herein to any substrate having a
surface to which molecules may be attached, directly or indirectly,
through either covalent or non-covalent bonds. The substrate
materials may be naturally occurring, synthetic, or a modification
of a naturally occurring material. Solid support materials may
include silicon, graphite, mirrored surfaces, laminates, ceramics,
plastics (including polymers such as, e.g., poly(vinyl chloride),
cyclo-olefin copolymers, polyacrylamide, polyacrylate,
polyethylene, polypropylene, poly(4-methylbutene), polystyrene,
polymethacrylate, poly(ethylene terephthalate),
polytetrafluoroethylene (PTFE or Teflon.RTM.), nylon, poly(vinyl
butyrate)), germanium, gallium arsenide, gold, silver, etc., either
used by themselves or in conjunction with other materials.
Additional rigid materials may be considered, such as glass, which
includes silica and further includes, for example, glass that is
available as Bioglass. Other materials that may be employed include
porous materials, such as, for example, controlled pore glass
beads. Any other materials known in the art that are capable of
having one or more functional groups, such as any of an amino,
carboxyl, thiol, or hydroxyl functional group, for example,
incorporated on its surface, are also contemplated.
[0083] The solid support may take any of a variety of
configurations ranging from simple to complex and can have any one
of a number of shapes, including a strip, plate, disk, rod,
particle, including bead, tube, well, and the like. The surface may
be relatively planar (e.g., a slide), spherical (e.g., a bead),
cylindrical (e.g., a column), or grooved. Exemplary solid supports
that may be used include microtitre wells, microscope slides,
membranes, paramagnetic beads, charged paper, Langmuir-Blodgett
films, silicon wafer chips, flow through chips, and microbeads.
[0084] As used herein, "partitioning" means any process whereby one
or more components of a mixture are separated from other components
of the mixture. For example, aptamers bound to target molecules can
be partitioned from other nucleic acids that are not bound to
target molecules and from non-target molecules. More broadly
stated, partitioning allows for the separation of all the nucleic
acids in a candidate mixture into at least two pools based on their
relative affinity and/or dissociation rate to the target molecule.
Partitioning can be accomplished by various methods known in the
art, including filtration, affinity chromatography, liquid-liquid
partitioning, HPLC, etc. For example, nucleic acid-protein pairs
can be bound to nitrocellulose filters while unbound nucleic acids
are not. Columns that specifically retain nucleic acid-target
complexes can also be used for partitioning. For example,
oligonucleotides able to associate with a target molecule bound on
a column allow the use of column chromatography for separating and
isolating the highest affinity aptamers. Beads upon which target
molecules are conjugated can also be used to partition aptamers in
a mixture. If the beads are paramagnetic, the partitioning can be
achieved through application of a magnetic field. Surface plasmon
resonance technology can be used to partition nucleic acids in a
mixture by immobilizing a target on a sensor chip and flowing the
mixture over the chip, wherein those nucleic acids having affinity
for the target can be bound to the target, and the remaining
nucleic acids can be washed away. Liquid-liquid partitioning can be
used as well as filtration gel retardation and density gradient
centrifugation. Affinity tags on the target molecules can also be
used to separate nucleic acid molecules bound to the tagged target
from aptamers that are free in solution. For example, biotinylated
target molecules, along with aptamers bound to them, can be
sequestered from the solution of unbound nucleic acid sequences
using streptavidin paramagnetic beads. Affinity tags can also be
incorporated into the aptamer during preparation.
[0085] As used herein, "photoSELEX" is an acronym for Photochemical
Systematic Evolution of Ligands by Exponential enrichment and
refers to embodiments of the SELEX process in which
photocrosslinking aptamers are generated. In one embodiment of the
photoSELEX process, a photoreactive nucleotide activated by
absorption of light is incorporated in place of a native base in
either RNA- or in ssDNA-randomized oligonucleotide libraries, the
nucleic acid target molecule mixture is irradiated causing some
nucleic acids incorporated in nucleic acid-target molecule
complexes to crosslink to the target molecule via the photoreactive
functional groups, and the selection step is a selection for
photocrosslinking activity. The photoSELEX process is described in
great detail in the PhotoSELEX Patents.
[0086] As used herein, "photoaptamer" and "photoreactive aptamer"
are used interchangeably to refer to an aptamer that contains one
or more photoreactive functional groups that can covalently bind to
or "crosslink" with a target molecule. For example, a naturally
occurring nucleic acid residue may be modified to include a
chemical functional group that confers photoreactivity upon the
nucleic acid residue upon exposure to a radiation source of an
appropriate wavelength. In some embodiments, a photoreactive
aptamer is identified initially. In other embodiments, an aptamer
is first identified and is subsequently modified to incorporate one
or more photoreactive functional groups, thereby generating a
photoaptamer. In these embodiments, one or more photoreactive
nucleic acid residues can be incorporated into an aptamer either by
substituting a photoreactive nucleic acid residue in the place of
one or more other nucleotides, such as one or more of the thymidine
and/or cytidine nucleotides in the aptamer, for example, or by
modifying one or more nucleic acid residues to include a
photoreactive functional group.
[0087] Exemplary photoreactive functional groups that may be
incorporated by a photoaptamer include 5-bromouracil, 5-iodouracil,
5-bromovinyluracil, 5-iodovinyluracil, 5-azidouracil, 4-thiouracil,
5-thiouracil, 4-thiocytosine, 5-bromocytosine, 5-iodocytosine,
5-bromovinylcytosine, 5-iodovinylcytosine, 5-azidocytosine,
8-azidoadenine, 8-bromoadenine, 8-iodoadenine, 8-aziodoguanine,
8-bromoguanine, 8-iodoguanine, 8-azidohypoxanthine,
8-bromohypoxanthine, 8-iodohypoxanthine, 8-azidoxanthine,
8-bromoxanthine, 8-iodoxanthine, 5-[(4-azidophenacyl)thio]cytosine,
5-[(4-azidophenacyl)thio]uracil, 7-deaza-7-iodoadenine,
7-deaza-7-iodoguanine, 7-deaza-7-bromoadenine, and
7-deaza-7-bromoguanine.
[0088] In addition to these exemplary nucleoside-based
photoreactive functional groups, other photoreactive functional
groups that can be added to a terminal end of an aptamer using an
appropriate linker molecule can also be used. Such photoreactive
functional groups include benzophenone, anthraquinone,
4-azido-2-nitro-aniline, psoralen, derivatives of any of these, and
the like.
[0089] A photoreactive functional group incorporated by a
photoaptamer may be activated by any suitable method. In one
embodiment, a photoaptamer containing a photoreactive functional
group can be crosslinked to its target by exposing the photoaptamer
and its bound target molecule to a source of electromagnetic
radiation. Suitable types of electromagnetic radiation include
ultraviolet light, visible light, X-rays, and gamma rays. Suitable
radiation sources include sources that utilize either monochromatic
light or filtered polychromatic light.
[0090] As used herein, the term "the affinity SELEX process" refers
to embodiments of the SELEX process in which non-photocrosslinking
aptamers to targets are generated. In some embodiments of the
affinity SELEX process, the target is immobilized on a solid
support either before or after the target is contacted with the
candidate mixture of nucleic acids. The association of the target
with the solid support allows nucleic acids in the candidate
mixture that have bound and in the case where a slow off-rate
enrichment process is used, stay bound to the target to be
partitioned from the remainder of the candidate mixture. The term
"bead affinity SELEX process" refers to particular embodiments of
the affinity SELEX process where the target is immobilized on a
bead, for example, before contact with the candidate mixture of
nucleic acids. In some embodiments, the beads are paramagnetic
beads. The term "filter affinity SELEX process" refers to
embodiments where nucleic acid target complexes are partitioned
from candidate mixture by virtue of their association with a
filter, such as a nitrocellulose filter. This includes embodiments
where the target and nucleic acids are initially contacted in
solution, and contacted with the filter, and also includes
embodiments where nucleic acids are contacted with target that is
pre-immobilized on the filter. The term "plate affinity SELEX
process" refers to embodiments where the target is immobilized on
the surface of a plate, such as, for example, a multi-well
microtiter plate. In some embodiments, the plate is comprised of
polystyrene. In some embodiments, the target is attached to the
plate in the plate affinity SELEX process through hydrophobic
interactions.
[0091] The present disclosure describes improved SELEX methods for
generating aptamers that are capable of binding to target
molecules. More specifically, the present disclosure describes
methods for identifying aptamers and/or photoaptamers having slower
rates of dissociation from their respective targeted molecules than
aptamers obtained with previous SELEX methods. The disclosure
further describes aptamers and/or photoaptamers obtained using the
methods described herein and methods of using the same.
[0092] In one embodiment, a method is provided for identifying an
aptamer having a slow rate of dissociation from its target
molecule, the method comprising (a) preparing a candidate mixture
of nucleic acid sequences; (b) contacting the candidate mixture
with a target molecule wherein nucleic acids with the highest
relative affinities to the target molecule preferentially bind the
target molecule, forming nucleic acid-target molecule complexes;
(c) applying a slow off-rate enrichment process to allow the
dissociation of nucleic acid-target molecule complexes with
relatively fast dissociation rates; (d) partitioning the remaining
nucleic acid-target molecule complexes from both free nucleic acids
and non-target molecules in the candidate mixture; and (e)
identifying an aptamer to the target molecule. The process may
further include the iterative step of amplifying the nucleic acids
that bind to the target molecule to yield a mixture of nucleic
acids enriched in sequences that are able to bind to the target
molecule yet produce nucleic acid-target molecule complexes having
slow dissociation rates. As defined above, the slow off-rate
enrichment process can be selected from (a) diluting the candidate
mixture containing the nucleic acid-target molecule complexes; (b)
adding at least one competitor to the candidate mixture containing
the nucleic acid-target molecule complexes, and diluting the
candidate mixture containing the nucleic acid-target molecule
complexes; (c) and adding at least one competitor to the candidate
mixture containing the nucleic acid-target molecule complexes.
[0093] In one embodiment, a method is provided for producing an
aptamer having a slow rate of dissociation from its target
molecule, the method comprising (a) preparing a candidate mixture
of nucleic acid sequences; (b) contacting the candidate mixture
with a target molecule wherein nucleic acids with the highest
relative affinities to the target molecule preferentially bind the
target molecule, forming nucleic acid-target molecule complexes;
(c) applying a slow off-rate enrichment process to allow the
dissociation of nucleic acid-target molecule complexes with
relatively fast dissociation rates; (d) partitioning the remaining
nucleic acid-target molecule complexes from both free nucleic acids
and non-target molecules in the candidate mixture; and (e)
producing an aptamer to the target molecule. The process may
further include the iterative step of amplifying the nucleic acids
that bind to the target molecule to yield a mixture of nucleic
acids enriched in sequences that are able to bind to the target
molecule yet produce nucleic acid-target molecule complexes having
slow dissociation rates. As defined above, the slow off-rate
enrichment process can be selected from (a) diluting the candidate
mixture containing the nucleic acid-target molecule complexes; (b)
adding at least one competitor to the candidate mixture containing
the nucleic acid-target molecule complexes, and diluting the
candidate mixture containing the nucleic acid-target molecule
complexes; (c) and adding at least one competitor to the candidate
mixture containing the nucleic acid-target molecule complexes.
[0094] In one embodiment, a method is provided for identifying an
aptamer having a slow rate of dissociation from its target
molecule, the method comprising: (a) preparing a candidate mixture
of nucleic acids; (b) contacting the candidate mixture with a
target molecule, wherein nucleic acids having an increased affinity
to the target molecule relative to other nucleic acids in the
candidate mixture bind the target molecule, forming nucleic
acid-target molecule complexes; (c) incubating the candidate
mixture and target molecule together for a period of time
sufficient to achieve equilibrium binding; (d) applying a slow
off-rate enrichment process to allow the dissociation of nucleic
acid-target molecule complexes with relatively fast dissociation
rates to the mixture of (c); (e) incubating the mixture of the
candidate mixture, the nucleic acid-target molecule complexes and
the competitor molecule from (d) for a predetermined period of
time; (f) partitioning the nucleic acid-target molecule complexes
from the candidate mixture; (g) dissociating the nucleic
acid-target molecule complexes to generate free nucleic acids; (h)
amplifying the free nucleic acids to yield a mixture of nucleic
acids enriched in nucleic acid sequences that are capable of
binding to the target molecule with increased affinity, whereby an
aptamer to the target molecule may be identified. As defined above,
the slow off-rate enrichment process can be selected from (a)
diluting the candidate mixture containing the nucleic acid-target
molecule complexes; (b) adding at least one competitor to the
candidate mixture containing the nucleic acid-target molecule
complexes, and diluting the candidate mixture containing the
nucleic acid-target molecule complexes; (c) and adding at least one
competitor to the candidate mixture containing the nucleic
acid-target molecule complexes.
[0095] In another embodiment, a method is provided for producing an
aptamer having a slow rate of dissociation from its target
molecule, the method comprising: (a) preparing a candidate mixture
of nucleic acids; (b) contacting the candidate mixture with a
target molecule, wherein nucleic acids having an increased affinity
to the target molecule relative to other nucleic acids in the
candidate mixture bind the target molecule, forming nucleic
acid-target molecule complexes; (c) incubating the candidate
mixture and target molecule together for a period of time
sufficient to achieve equilibrium binding; (d) applying a slow
off-rate enrichment process to allow the dissociation of nucleic
acid-target molecule complexes with relatively fast dissociation
rates to the mixture of (c); (e) incubating the mixture of the
candidate mixture, the nucleic acid-target molecule complexes and
the competitor molecule from (d) for a predetermined period of
time; (f) partitioning the nucleic acid-target molecule complexes
from the candidate mixture; (g) dissociating the nucleic
acid-target molecule complexes to generate free nucleic acids; (h)
amplifying the free nucleic acids to yield a mixture of nucleic
acids enriched in nucleic acid sequences that are capable of
binding to the target molecule with increased affinity, whereby an
aptamer to the target molecule may be produced. As defined above,
the slow off-rate enrichment process can be selected from (a)
diluting the candidate mixture containing the nucleic acid-target
molecule complexes; (b) adding at least one competitor to the
candidate mixture containing the nucleic acid-target molecule
complexes, and diluting the candidate mixture containing the
nucleic acid-target molecule complexes; (c) and adding at least one
competitor to the candidate mixture containing the nucleic
acid-target molecule complexes.
[0096] In another embodiment, a method is provided of identifying
an aptamer having a slow rate of dissociation from its target
molecule, the method comprising: (a) preparing a candidate mixture
of nucleic acids, wherein the candidate mixture comprises modified
nucleic acids in which one, several or all pyrimidines in at least
one, or each, nucleic acid of the candidate mixture is chemically
modified at the 5-position; (b) contacting the candidate mixture
with a target molecule, wherein nucleic acids having an increased
affinity to the target molecule relative to other nucleic acids in
the candidate mixture bind the target molecule, forming nucleic
acid-target molecule complexes; (c) partitioning the increased
affinity nucleic acids from the remainder of the candidate mixture;
and (d) amplifying the increased affinity nucleic acids to yield a
mixture of nucleic acids enriched in nucleic acid sequences that
are capable of binding to the target molecule with increased
affinity, whereby an aptamer to the target molecule may be
identified.
[0097] In another embodiment, a method is provided for producing an
aptamer having a slow rate of dissociation from its target
molecule, said method comprising preparing or synthesizing an
aptamer that includes a nucleic acid sequence identified by the
following process: (a) preparing a candidate mixture of nucleic
acids, wherein the candidate mixture comprises modified nucleic
acids in which one, several or all pyrimidines in at least one, or
each, nucleic acid of the candidate mixture is chemically modified
at the 5-position; (b) contacting the candidate mixture with a
target molecule, wherein nucleic acids having an increased affinity
to the target molecule relative to other nucleic acids in the
candidate mixture bind the target molecule, forming nucleic
acid-target molecule complexes; (c) partitioning the increased
affinity nucleic acids from the remainder of the candidate mixture;
and (d) amplifying the increased affinity nucleic acids to yield a
mixture of nucleic acids enriched in nucleic acid sequences that
are capable of binding to the target molecule with increased
affinity, whereby an aptamer to the target molecule is
identified.
[0098] In another embodiment, a non-covalent complex of an aptamer
and its target is provided, wherein the rate of dissociation
(t.sub.1/2) of the aptamer from the target is chosen from one of:
greater than or equal to about 30 minutes; between about 30 minutes
and about 240 minutes; about 30 minutes to about 60 minutes; about
60 minutes to about 90 minutes; about 90 minutes to about 120
minutes; about 120 minutes to about 150 minutes; about 150 minutes
to about 180 minutes; about 180 minutes to about 210 minutes; about
210 minutes to about 240 minutes.
[0099] In another embodiment, a non-covalent complex of an aptamer
and a target is provided, wherein the aptamer has a K.sub.d for the
target of about 100 nM or less, wherein the rate of dissociation
(t.sub.1/2) of the aptamer from the target is greater than or equal
to about 30 minutes, and wherein one, several or all pyrimidines in
the nucleic acid sequence of the aptamer are modified at the
5-position of the base. The modifications may be selected from the
group of compounds shown in FIG. 14, these modifications are
referred to as "base modified nucleotides". Aptamers may be
designed with any combination of the base modified pyrimidines
desired.
[0100] Improved methods for performing SELEX with modified
nucleotides, including nucleotides which contain photoactive groups
or nucleotides which contain placeholders for photoactive groups
are disclosed in U.S. application Ser. No. 12/175,388, entitled
"Improved SELEX and PHOTOSELEX" which is being filed concurrently
with the instant application and which is incorporated herein by
reference in its entirety. In another embodiment, the candidate
mixture of nucleic acid molecules includes nucleic acids containing
modified nucleotide bases that may aid in the formation of modified
nucleic acid-target complexes with relatively slow dissociation
rates.
[0101] The various methods and steps described herein can be used
to generate an aptamer capable of either (1) binding to a target
molecule or (2) binding to a target molecule and subsequently
forming a covalent linkage with the target molecule upon
irradiation.
[0102] Aptamers identified according to the methods described
herein are useful in a range of diagnostic and therapeutic methods.
Slow off-rate aptamers will bind to the target for a longer
duration. This is useful in diagnostic methods where the binding of
an aptamer to the target may be used to detect the presence,
absence, amount or quantity of the target molecule and a prolonged
interaction of the aptamer and target facilitates such detection. A
similar advantage may be afforded where slow off-rate aptamers are
used in imaging methods, in vitro or in vivo. A prolonged
interaction of aptamer and target also provides for improved
therapeutic methods of treatment where the prolonged interaction
may allow for an improved therapeutic effect, e.g. owing to the
longer activation or inhibition of the target molecule or
downstream signaling cascade.
[0103] Accordingly, in various embodiments, slow off-rate aptamers
obtained, identified or produced by the described methods can be
used in a variety of methods of medical treatment or methods of
diagnosis (in vitro or in vivo). In one embodiment, slow off-rate
aptamers can be used in a method of treatment of disease. In one
embodiment, slow off-rate aptamers can be used in a method for
diagnosis of disease in vivo. In another embodiment, slow off-rate
aptamers can be used in vitro for the diagnosis of disease. In
another embodiment, a slow off-rate aptamer can be used in the
manufacture of a therapeutic (e.g. pharmaceutical composition) or
the manufacture of a diagnostic agent for use in a method of
treatment or diagnosis of disease. Diagnostic or therapeutic
applications of slow off-rate aptamers may involve a diagnostic or
therapeutic outcome that depends on the specific and/or high
affinity binding of the slow off-rate aptamer to its target. Slow
off-rate aptamers may also be used in target validation and high
throughput screening assays in the drug development process.
[0104] In one embodiment, slow off-rate aptamers are suitable
reagents for molecular imaging in vivo. In this embodiment, a slow
off-rate aptamer may be used in vivo to detect the presence of a
pathology, disease process, or other condition in the body of an
individual (e.g., a human or an animal), where the binding of the
aptamer to its target indicates the presence of the disease process
or other condition. For example, an aptamer to the VEGF receptor
may be used in vivo to detect the presence of cancer in a
particular area (e.g., a tissue, an organ, etc.) of the body of an
individual, as the VEGF receptor is abundantly expressed within
tumors and their neovasculature, or an aptamer to the EGF receptor
may be used in vivo to detect the presence of cancer in a
particular area (e.g., a tissue, an organ, etc.) of the body of an
individual, as the EGF receptor is often expressed at high levels
on tumor cells. That is, the molecular target will be the
extracellular domain (ECD) of an induced receptor, as such targets
are located outside of the cells and are accessible through the
vasculature. Additionally, the ECDs tend to be localized at the
site of pathology, even though some small fraction of the specific
ECD may be shed through biological processes, including cell
death.
[0105] The obvious candidates for molecular imaging, high affinity
monoclonal antibodies, have not become the reagent of choice for
this application. Molecular imaging reagents have precise
requirements. They must have high binding activity for their
intended target, and low binding activity for other targets in a
human or animal. Slow off-rate aptamers have unique advantages that
render them desirable for use in molecular imaging in vivo. On the
one hand, they are selected to have slow dissociation rate
constants, thus allowing residence in vivo on the intended target
for a substantial length of time (at least about 30 minutes). On
the other hand, slow off-rate aptamers are expected to have very
fast clearance from the vasculature. Slow dissociation rate
constants and fast clearance from the vasculature are two desired
properties for molecular imaging in vivo. From a kinetic
prospective, good in vivo molecular imaging reagents must stay
localized at the site of the pathology while the free reagent
concentration in the surrounding vasculature becomes low. This is a
signal-to-noise constraint. Suitable signal-to-noise ratios may be
obtained by accumulation of signal at the site of pathology in
excess of the signal in the vasculature, or may be obtained by
retention of a signal at the site of the pathology while the
vasculature concentration is diminished.
[0106] Aptamers that do not have slow off-rate properties, of about
the same molecular weight and net charge as slow off-rate aptamers,
have been studied in animals and humans for more than a decade.
Generally, it has been found that these aptamers clear from the
vasculature quickly, usually by entering the kidney and/or the
liver and then being further metabolized for excretion. Such
aptamers show so-called "first pass" clearance unless high
molecular weight adducts (such as, for example, PEG) are linked to
the aptamers. Experiments have been done with an aptamer whose
target is tenascin C, an extracellular protein (not an ECD) found
at high concentrations in some tumors. In those experiments, the
tenascin C-specific aptamer cleared quickly and was able to be
retained at the site of the tumor because the extracellular local
concentration of tenascin C is very high. Slow off-rate aptamers,
by contrast, will maintain the fast clearance rate of aptamers, but
offer a kinetic advantage due to their slow dissociation rates,
rendering them suitable for use with targets whose presence at the
site of interest (e.g., the site of pathology) may be somewhat
sparse (ECDs on tumors, for example).
[0107] Alternative reagents for molecular imaging do not share the
two slow off-rate aptamer properties (i.e., slow dissociation rate
and fast clearance from the body). Monoclonal antibodies often have
high affinity and specificity, and may have slow dissociation rate
constants; however, monoclonal antibodies have very slow clearance
rates from the vasculature. Short peptides, identified through, for
example, phage display, may have fast clearance but poor affinity
and specificity and fast dissociation rates from their intended
targets. Affibodies, a particular peptide version of an antibody
mimetic, may have reasonable affinity and specificity and may have
faster clearance than monoclonal antibodies, yet in order to
achieve slow dissociation rates from their targets, affibodies are
often made into dimers and higher order multimers, slowing their
clearance at the same time that their dissociation rates are
enhanced.
[0108] Slow off-rate aptamers may be used for molecular imaging in
vivo with one or more low molecular weight adducts to both protect
the slow off-rate aptamer from nucleases in the body and detect the
intended target once bound by the slow off-rate aptamer. For
example, slow off-rate aptamers may be attacked by nucleases in the
blood, typically exonucleases (for DNA) that are easily blocked by
using exonuclease refractive adducts at the 5' and 3' terminal
positions of the slow off-rate aptamer, or endonucleases (for RNA)
that are easily blocked by incorporating endonuclease refractive
pyrimidines (such as, for example, 2' fluoro nucleotides) in the
slow off-rate aptamer. Detection of the slow off-rate
aptamer-target complex may be achieved by attaching a detection
moiety to the slow off-rate aptamer. In some embodiments, the
detection moiety for these purposes may include cages for
radioactive molecules (e.g., technetium 99), clusters of iron for
magnetic resonance detection, isotopes of fluorine for PET imaging,
and the like. The modifications made to the slow off-rate aptamer
to protect the integrity of the slow off-rate aptamer in the body
and enable detection of the intended target should be designed such
that they do not interfere with the slow off-rate aptamer's
interaction with its target and do not cause the slow off-rate
aptamer to clear too slowly from the vasculature.
[0109] Diagnostic or assay devices, e.g. columns, test strips or
biochips, having one or more slow off-rate aptamers adhered to a
solid surface of the device are also provided. The aptamer(s) may
be positioned so as to be capable of binding target molecules that
are contacted with the solid surface to form aptamer-target
complexes that remain adhered to the surface of the device, thereby
capturing the target and enabling detection and optionally
quantitation of the target. An array of slow off-rate aptamers
(which may be the same or different) may be provided on such a
device.
[0110] In another embodiment, complexes including a slow off-rate
aptamer and a target molecule are provided. In other embodiments, a
class of aptamers characterized by having high affinity for their
corresponding target molecules and slow dissociation rates
(t.sub.1/2) from a non-covalent complex of the aptamer and target
is provided.
[0111] With reference to FIG. 1A, the basic SELEX process generally
begins with the preparation of a candidate mixture of nucleic acids
of differing sequence. The candidate mixture generally includes
nucleic acid sequences that include two fixed regions (i.e., each
of the members of the candidate mixture contains the same sequences
in the same location) and a variable region. Typically, the fixed
sequence regions are selected such that they assist in the
amplification steps described below, or enhance the potential of a
given structural arrangement of the nucleic acids in the candidate
mixture. The variable region typically provides the target binding
region of each nucleic acid in the candidate mixture, and this
variable region can be completely randomized (i.e., the probability
of finding a base at any position being one in four) or only
partially randomized (e.g., the probability of finding a base at
any location can be selected at any level between 0 and 100
percent). The prepared candidate mixture is contacted with the
selected target under conditions that are favorable for binding to
occur between the target and members of the candidate mixture.
Under these conditions, the interaction between the target and the
nucleic acids of the candidate mixture generally forms nucleic
acid-target pairs that have the strongest relative affinity between
members of the pair. The nucleic acids with the highest affinity
for the target are partitioned from those nucleic acids with lesser
affinity to the target. The partitioning process is conducted in a
manner that retains the maximum number of high affinity candidates.
Those nucleic acids selected during partitioning as having a
relatively high affinity to the target are amplified to create a
new candidate mixture that is enriched in nucleic acids having a
relatively high affinity for the target. By repeating the
partitioning and amplifying steps above, the newly formed candidate
mixture contains fewer and fewer unique sequences, and the average
degree of affinity of the nucleic acid mixture to the target will
generally increase. Taken to its extreme, the SELEX process will
yield a candidate mixture containing one or a very small number of
unique nucleic acids representing those nucleic acids from the
original candidate mixture that have the highest affinity to the
target molecule. However, this basic SELEX process does not select
for aptamers that have slow off-rates from their targets.
[0112] The SELEX Patents and the PhotoSELEX Patents describe and
elaborate on this process in great detail. These patents include
descriptions of the various targets that can be used in the
process; methods for the preparation of the initial candidate
mixture; methods for partitioning nucleic acids within a candidate
mixture; and methods for amplifying partitioned nucleic acids to
generate enriched candidate mixtures. The SELEX Patents also
describe aptamer solutions obtained to a number of different types
of target molecules, including protein targets wherein the protein
is and is not a nucleic acid binding protein.
[0113] With reference to FIG. 1B the modified SELEX process
disclosed herein includes the introduction of a slow off-rate
enrichment process following equilibration of the candidate mixture
of nucleic acids with the target or targets and a partitioning step
prior to subsequent steps in the SELEX process. Introduction of a
slow off-rate enrichment process to the basic SELEX process
provides a means for enrichment of aptamer affinity complexes with
slow dissociation rates from a set of nucleic acid-target complexes
that includes a variety of dissociation rates. Thus, the modified
SELEX process provides a method for identifying aptamers that bind
target molecules and, once bound, have relatively slow rates of
dissociation (also referred to herein as "off-rates") from the
target molecule.
[0114] As used herein "binding" generally refers to the formation
of a non-covalent association between the ligand and the target,
although such binding is not necessarily reversible. The terms
"nucleic acid-target complex" or "complex" or "affinity complex"
are used to refer to the product of such non-covalent binding
association.
[0115] In various embodiments, the slow off-rate aptamers can be
single- or double-stranded RNA or DNA oligonucleotides. The
aptamers can contain non-standard or modified bases. Further, the
aptamers can contain any type of modification. As used herein, a
"modified base" may include a relatively simple modification to a
natural nucleic acid residue, which modification confers a change
in the physical properties of the nucleic acid residue. Such
modifications include, but are not limited to, modifications at the
5-position of pyrimidines, substitution with hydrophobic groups,
e.g., benzyl, iso-butyl, indole, or naphthylmethyl, or substitution
with hydrophilic groups, e.g., quaternary amine or guanidinium, or
more "neutral" groups, e.g., imidazole and the like. Additional
modifications may be present in the ribose ring, e.g., 2'-position,
such as 2'-amino (2'-NH.sub.2) and 2'-fluoro (2'-F), or the
phosphodiester backbone, e.g., phosphorothioates or methyl
phosphonates.
[0116] In various embodiments, a candidate mixture containing a
randomized set of nucleic acid sequences containing modified
nucleotide bases is mixed with a quantity of the target molecule
and allowed to establish binding equilibrium with the target
molecule. Generally, only some of those nucleic acids that bind
with high affinity to the target molecule will efficiently
partition with the target.
[0117] In various embodiments, the candidate mixture includes
nucleic acid sequences having variable regions that include
modified groups. The modified groups can be modified nucleotide
bases. The variable region can contain fully or partially random
sequences; it can also contain subportions of a fixed sequence that
is incorporated within the variable region. The nucleotides within
the fixed regions can also contain modified nucleotide bases, or
they can contain the standard set of naturally occurring bases.
[0118] In some embodiments, amplification occurs after members of
the test mixture have been partitioned, and it is the nucleic acid
that is amplified. For example, amplifying RNA molecules can be
carried out by a sequence of three reactions: making cDNA copies of
selected RNAs, using the polymerase chain reaction to increase the
copy number of each cDNA, and transcribing the cDNA copies to
obtain RNA molecules having the same sequences as the selected
RNAs. Any reaction or combination of reactions known in the art can
be used as appropriate, including direct DNA replication, direct
RNA amplification and the like, as will be recognized by those
skilled in the art. The amplification method may result in the
proportions of the amplified mixture being representative of the
proportions of different sequences in the mixture prior to
amplification. It is known that many modifications to nucleic acids
are compatible with enzymatic amplification. Modifications that are
not compatible with amplification can be made after each round of
amplification, if necessary.
[0119] The nucleic acid candidate mixture can be modified in
various ways to enhance the probability of the nucleic acids having
facilitating properties or other desirable properties, particularly
those that enhance the interaction between the nucleic acid and the
target. Contemplated modifications include modifications that
introduce other chemical groups that have the correct charge,
polarizability, hydrogen bonding, or electrostatic interaction to
enhance the desired ligand-target interactions. The modifications
that may enhance the binding properties, including the affinity
and/or dissociation rates, of the nucleic acid, for example,
include hydrophilic moieties, hydrophobic moieties, rigid
structures, functional groups found in proteins such as imidazoles,
primary alcohols, carboxylates, guanidinium groups, amino groups,
thiols and the like. Modifications can also be used to increase the
survival of aptamer-target complexes under stringent selection
pressures that can be applied to produce slow off-rate aptamers to
a wide range of targets. In one embodiment, BndU
(5-(N-benzylcarboxyamide)-dU) is used in the generation of the
candidate mixtures used to produce slow off-rate aptamers, although
other modified nucleotides are well suited to the production of
such aptamers. Other modified nucleotides are shown in FIG. 14.
[0120] A modified nucleotide candidate mixture for the purpose of
this application is any RNA or DNA candidate mixture that includes
both naturally occurring and other than the naturally occurring
nucleotides. Suitable modifications include modifications on every
residue of the nucleic acid, on a single residue of the nucleic
acid, on random residues, on all pyrimidines or all purines, on all
occurrences of a specific base (i.e., G, C, A, T or U) in the
nucleic acid, or any other modification scheme that may be suitable
for a particular application. It is recognized that modification is
not a prerequisite for facilitating activity or binding ability of
the aptamers. Aptamers may include modified dUTP and dCTP
residues.
[0121] Candidate mixtures for slow off-rate aptamers may comprise a
set of pyrimidines having a different modification at the C-5 base
position. The C-5 modification may be introduced through an amide
linkage, directly, or indirectly, or through another type of
linkage. These candidate mixtures are used in a SELEX process to
identify slow off-rate aptamers. This process may be also include
the use of the slow off-rate enrichment process. Candidate mixtures
may be produced enzymatically or synthetically.
[0122] As described above, the nucleotides can be modified in any
number of ways, including modifications of the ribose and/or
phosphate and/or base positions. Certain modifications are
described in U.S. Pat. No. 5,660,985 entitled "High Affinity
Nucleic Acid Ligands Containing Modified Nucleotides," U.S. Pat.
No. 5,428,149 entitled "Method for Palladium Catalyzed
Carbon-Carbon Coupling and Products," U.S. Pat. No. 5,580,972
entitled "Purine Nucleoside Modifications by Palladium Catalyzed
Methods," all of which are incorporated by reference herein. In one
embodiment, modifications are those wherein another chemical group
is attached to the 5-position of a pyrimidine or the 2' position of
a sugar. There is no limitation on the type of other chemical group
that can be incorporated on the individual nucleotides. In some
embodiments, the resulting modified nucleotide is amplifiable or
can be modified subsequent to the amplification steps (see, e.g.,
U.S. Pat. No. 6,300,074 entitled "Systematic evolution of ligands
by exponential enrichment: Chemi-SELEX").
[0123] In yet other embodiments, certain nucleotides are modified
to produce aptamers that bind and form a covalent crosslink to
their target molecule upon photo-activation of the affinity
complex. This method encompasses aptamers that bind,
photocrosslink, and/or photoinactivate target molecules. In various
embodiments, the aptamers contain photoreactive groups that are
capable of photocrosslinking to the target molecule upon
irradiation with light. In other embodiments, the aptamers are
capable of bond formation with the target in the absence of
irradiation.
[0124] A photoreactive group can be any chemical structure that
contains a photochromophore and that is capable of
photocrosslinking with a target molecule. Although referred to
herein as a photoreactive group, in some cases, as described below,
irradiation is not necessary for covalent binding to occur between
the aptamer and the target. In some embodiments, the photoreactive
group will absorb light of a wavelength that is not absorbed by the
target or the non-modified portions of the oligonucleotide.
Photoreactive groups include 5-halo-uridines, 5-halo-cytosines,
7-halo-adenosines, 2-nitro-5-azidobenzoyls, diazirines, aryl
azides, fluorinated aryl azides, benzophenones,
amino-benzophenones, psoralens, anthraquinones, etc.
[0125] The photoreactive groups generally form bonds with the
target upon irradiation of the associated nucleic acid-target pair.
In some cases, irradiation is not required for bond formation to
occur. The photocrosslink that typically occurs will be the
formation of a covalent bond between the associated aptamer and the
target. However, a tight ionic interaction between the aptamer and
target may also occur upon irradiation.
[0126] In one embodiment, photocrosslinking occurs due to exposure
to electromagnetic radiation. Electromagnetic radiation includes
ultraviolet light, visible light, X-ray, and gamma ray.
[0127] In various other embodiments, a limited selection of
oligonucleotides using a SELEX method is followed by selection
using a photoSELEX method. The initial SELEX selection rounds are
conducted with oligonucleotides containing photoreactive groups.
After a number of SELEX rounds, photoSELEX is conducted to select
oligonucleotides capable of binding the target molecule.
[0128] In another embodiment, the production of an aptamer that
includes a cleavable or releasable section (also described as an
element or component) in the aptamer sequence is described. These
additional components or elements are structural elements or
components that introduce additional functionality into the aptamer
and are thus functional elements or components. The aptamer is
further produced with one or more of the following additional
components (also described as a functional or structural element or
component or moiety in any combination of these terms): a labeled
or detectable component, a spacer component, and a specific binding
tag or immobilization element or component.
[0129] As noted above, the present disclosure provides methods for
identifying aptamers that bind target molecules and once bound have
slow rates of dissociation or off-rates. The slow off-rates
obtained with this method can exceed a half-life of about one hour
and as much as about 240 minutes, that is, once a set of nucleic
acid-target complexes is generated, half of the complexes in the
set remain bound after one hour. Because the effect of a slow
off-rate enrichment process depends upon the differing dissociation
rates of aptamer affinity complexes, the duration of the slow
off-rate enrichment process is chosen so as to retain a high
proportion of aptamer affinity complexes with slow dissociation
rates while substantially reducing the number of aptamer affinity
complexes with fast dissociation rates. For example, incubating the
mixture for relatively longer periods of time after imposing the
slow off-rate enrichment process will select for aptamers with
longer dissociation rates than aptamers selected using slow
off-rate enrichment process having shorter incubation periods.
[0130] In various embodiments, the candidate mixture is mixed with
a quantity of the target molecule and allowed to establish binding
equilibrium with the target molecule. Prior to partitioning the
target bound nucleic acids from those free in solution, a slow
off-rate enrichment process is imposed to enrich the bound
population for slow dissociation rates. As noted above, the slow
off-rate enrichment process can be applied by the addition of a
competitor molecule, by sample dilution, by a combination of sample
dilution in the presence of a competitor molecule. Thus, in one
embodiment, the slow off-rate enrichment process is applied by
introducing competitor molecules into the mixture containing the
nucleic acid-target complexes and incubating the mixture for some
period of time before partitioning free from bound nucleic acids.
The amount of competitor molecules is generally at least one order
of magnitude higher than that of the nucleic acid molecules and may
be two or more orders of magnitude higher. In another embodiment,
the slow off-rate enrichment process is applied by diluting the
sample mixture of nucleic acid-target complexes several fold (e.g.
at least about one of 2.times., 3.times., 4.times., 5.times.) in
volume and incubating the mixture for some period of time before
partitioning free from bound nucleic acids. The dilution volume is
generally at least one order of magnitude higher, and may be about
two or more orders of magnitude higher, than the original volume.
In yet another embodiment, a combination of both competitor
molecules and dilution is used to apply the slow off-rate
enrichment process. In another embodiment, candidate mixtures that
have been shown to result in an increased frequency of slow
dissociation aptamers are used to select a number of candidate
aptamers. These aptamers are screened to identify slow dissociation
rate aptamers.
[0131] In another embodiment, a slow off-rate aptamer that includes
a cleavable or releasable section in the fixed region of the
aptamer is produced. The aptamer can also be produced with one or
more of the following additional components: a labeled component, a
spacer component, and a specific binding tag. Any or all of these
elements may be introduced into a single stranded aptamer. In one
embodiment, the element is introduced at the 5' end of the aptamer.
In another embodiment, one or more of these elements is included by
creating a partially double stranded aptamer, where one strand
contains the various elements desired as well as a sequence
complementary to one of the fixed sequence sections of the second
strand containing the variable target binding region.
[0132] A "releasable" or "cleavable" element or moiety or component
refers to a functional group where certain bonds in the functional
group can be broken to produce 2 separate components. In various
embodiments, the functional group can be cleaved by irradiating the
functional group (photocleavable) at the appropriate wavelength or
by treatment with the appropriate chemical or enzymatic reagents.
In another embodiment, the releasable element may be a disulfide
bond that can be treated with a reducing agent to disrupt the bond.
The releasable element allows an aptamer/target affinity complex
that is attached to a solid support to be separated from the solid
support, such as by elution of the complex. The releasable element
may be stable to the conditions of the rest of the assay and may be
releasable under conditions that will not disrupt the
aptamer/target complex.
[0133] As disclosed herein, an aptamer can further comprise a "tag"
or "immobilization component or element" or "specific binding
component or element" which refers to a component that provides a
means for attaching or immobilizing an aptamer (and any target
molecule that is bound to it) to a solid support. A "tag" is a set
of copies of one type or species of component that is capable of
associating with a probe. "Tags" refers to more than one such set
of components. The tag can be attached to or included in the
aptamer by any suitable method. Generally, the tag allows the
aptamer to associate, either directly or indirectly, with a probe
or receptor that is attached to the solid support. The probe may be
highly specific in its interaction with the tag and retain that
association during all subsequent processing steps or procedures. A
tag can enable the localization of an aptamer affinity complex (or
optional covalent aptamer affinity complex) to a spatially defined
address on a solid support. Different tags, therefore, can enable
the localization of different aptamer covalent complexes to
different spatially defined addresses on a solid support. A tag can
be a polynucleotide, a polypeptide, a peptide nucleic acid, a
locked nucleic acid, an oligosaccharide, a polysaccharide, an
antibody, an affybody, an antibody mimic, a cell receptor, a
ligand, a lipid, biotin, any fragment or derivative of these
structures, any combination of the foregoing, or any other
structure with which a probe (or linker molecule, as described
below) can be designed or configured to bind or otherwise associate
with specificity. Generally, a tag is configured such that it does
not interact intramolecularly with either itself or the aptamer to
which it is attached or of which it is a part. If SELEX is used to
identify an aptamer, the tag may be added to the aptamer either
pre- or post-SELEX. The tag is included on the 5'-end of the
aptamer post-SELEX, or the tag is included on the 3'-end of the
aptamer post-SELEX, or the tags may be included on both the 3' and
5' ends of the aptamers in a post-SELEX process.
[0134] As illustrated in FIG. 8D, a fluorescent dye (such as Cy3),
the photocleavable and biotin moieties are all added to the end of
the aptamer. Because of potential interactions between the
photocleavable moiety and the dye, a spacer is inserted between
these two moieties. All constructs can be synthesized using
standard phosphoramidite chemistry. Representative aptamer
constructs are shown in FIG. 9A through FIG. 9F. The functionality
can be split between the 5' and 3' end or combined on either end.
In addition to photocleavable moieties, other cleavable moieties
can be used, including chemically or enzymatically cleavable
moieties. A variety of spacer moieties can be used and one or more
biotin moieties can be included. Tags (also referred to as
immobilization or specific binding elements or components) other
than biotin can also be incorporated. Suitable construction
reagents include biotin phosphoramidite, PC Linker (Glen Research
PN 10-4920-02); PC biotin phosphoramidite (Glen Research PN
10-4950-02); dSpacer CE phosphoramidite (Glen Research PN
10-1914-02); Cy3 phosphoramidite (Glen Research PN 10-5913-02); and
Arm26-Ach Spacer Amidite (Fidelity Systems PN SP26Ach-05).
[0135] In one embodiment, base modifications of the nucleotides are
used in the production of the variable region of the aptamer. These
modified nucleotides have been shown to produce aptamers that have
very slow off-rates from their targets.
[0136] In the methods of the present disclosure the candidate
mixture may comprise modified nucleic acids in which one, several
(e.g. one of, or at least one of, 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) or all pyrimidines in at least one, or each, nucleic acid
of the candidate mixture is chemically modified at the 5-position.
Optionally, all C residues in the nucleic acids of the candidate
mixture are chemically modified at the 5-position. Optionally, all
T residues in the nucleic acids of the candidate mixture are
chemically modified at the 5-position. Optionally, all U residues
in the nucleic acids of the candidate mixture are chemically
modified at the 5-position.
[0137] In another embodiment, the slow off-rate aptamers are mixed
or exposed to a sample. The slow off-rate aptamer is allowed to
react with, or bind to, its specific target in the sample to form a
complex. A variety of methods may be used to detect either the
target or the aptamer. The target may be detected in the complex or
upon liberation from the complex. The aptamer may be detected in
the complex or upon liberation from the complex. The aptamer/target
complex may be used to isolate the specific target from other
components in the test sample. Multiple aptamers may be used when a
multiplexed assay for the detection of a variety of targets is
desired.
[0138] The method of the instant disclosure is illustrated
generally in Examples 1-8. Example 1 describes the general affinity
SELEX method using a candidate mixture comprised of modified
nucleotides. Example 2 describes a photo SELEX method using a
candidate mixture comprised of modified nucleotides and a
5'-terminal photoreactive group, and the improved SELEX method in
which dilution is used to provide the slow off-rate enrichment
process to the equilibrated aptamer:target mixture. Example 3
extends the method described in Example 2 by the addition of a
competitor to the dilution step. Example 4 illustrates the
effectiveness of the slow off-rate enrichment process. The average
dissociation half-life value (t.sub.1/2) for aptamers using the
modified nucleotides 5-(N-benzylcarboxyamide)-dUTP (BndUTP),
5-(N-isobutylcarboxyamide)-dUTP (iBudUTP), or
5-(N-tryptaminocarboxyamide)-dUTP selected in the absence of a slow
off-rate enrichment process was 20 minutes with some aptamers
having a t.sub.1/2 value of up to one hour (FIG. 3A). This is
substantially longer than what has been previously described with
natural bases or other modified nucleotides. The average for
aptamers selected with a slow off-rate enrichment process was over
85 minutes. More specifically, with reference to FIG. 3B, it can be
seen that introduction of a slow off-rate enrichment process
produced aptamers with t.sub.1/2 values of about .gtoreq.30 min.,
.gtoreq.about 60 min., .gtoreq.about 90 min., .gtoreq.about 120
min., .gtoreq.about 150 min., .gtoreq.about 180 min., .gtoreq.about
210 min., and .gtoreq.about 240 min. These dissociation rates for
aptamer:target complexes are unprecedented.
[0139] Example 5 describes the generation of slow off-rate aptamers
using a NapdU (5-(N-naphthylmethylcarboxyamide)-dUP) candidate
mixture.
[0140] Example 6 describes the generation of a slow off-rate
aptamer to a peptide target.
[0141] Example 7 illustrates the utility of slow off-rate aptamers
relative to conventional aptamers.
[0142] Example 8 further illustrates the generation of slow
off-rate aptamers using a BndU candidate mixture.
EXAMPLES
[0143] The following examples are provided for illustrative
purposes only and are not intended to limit the scope of the
invention as defined in the appended claims.
Example 1
Incorporation of Modified Nucleotides in Nucleic Acid Libraries
Leads to Higher Affinity Enriched Libraries in Affinity SELEX
[0144] A. Preparation of Candidate Mixtures
[0145] Candidate mixtures were prepared with dATP, dGTP,
5-methyl-dCTP (MedCTP) and either dTTP or one of three dUTP
analogs: 5-(N-benzylcarboxyamide)-dUTP (BndUTP),
5-(N-isobutylcarboxyamide)-dUTP (iBudUTP), or
5-(N-tryptaminocarboxyamide)-dUTP (TrpdUTP). Candidate mixtures
were prepared by polymerase extension of a primer annealed to a
biotinylated template (FIG. 2). For each candidate mixture
composition, 4.8 nmol forward PCR primer and 4 nmol template were
combined in 100 .mu.L 1.times. KOD DNA Polymerase Buffer (Novagen),
heated to 95.degree. C. for 8 minutes, and cooled on ice. Each 100
.mu.L primer:template mixture was added to a 400 .mu.L extension
reaction containing 1.times. KOD DNA Polymerase Buffer, 0.125
U/.mu.L KOD XL DNA Polymerase, and 0.5 mM each dATP, MedCTP, dGTP,
and dTTP or dUTP analog, and incubated at 70.degree. C. for 30
minutes. Double-stranded product was captured via the template
strand biotins by adding 1 mL streptavidin-coated magnetic beads
(MagnaBind Streptavidin, Pierce, 5 mg/mL in 1M NaCl+0.05% TWEEN-20)
and incubating at 25.degree. C. for 10 minutes with mixing. Beads
were washed three times with 0.75 mL SB1T Buffer (40 mM HEPES, pH
7.5, 125 mM NaCl, 5 mM KCl, 1 mM MgCl.sub.2, 1 mM CaCl.sub.2, 0.05%
TWEEN-20). The aptamer strand was eluted from the beads with 1.2 mL
20 mM NaOH, neutralized with 0.3 mL 80 mM HCl, and buffered with 15
.mu.L 1 M HEPES, pH 7.5. Candidate mixtures were concentrated with
a Centricon-30 to approximately 0.2 mL, and quantified by UV
absorbance spectroscopy.
[0146] B. Immobilization of Target Proteins
[0147] Target proteins were purchased with poly His tags, such as,
(His).sub.6 tags (R&D Systems) and immobilized on Co.sup.+2-NTA
paramagnetic beads (MyOne TALON, Invitrogen, or hereinafter
referred to as Talon beads). Target proteins were diluted to 0.2
mg/mL in 0.5 mL B/W Buffer (50 mM Na-phosphate, pH 8.0, 300 mM
NaCl, 0.01% TWEEN-20), and added to 0.5 mL TALON beads (pre-washed
three times with B/W Buffer and resuspended to 10 mg/mL in B/W
Buffer). The mixture was rotated for 30 minutes at 25.degree. C.
and stored at 4.degree. C. until use. TALON beads coated with
(His).sub.6 peptide were also prepared and stored as above. Prior
to use, beads were washed 3 times with B/W Buffer, once with SB1T,
and resuspended in SB1T.
[0148] C. Aptamer Selection Scheme
[0149] Affinity selections were performed separately with each
candidate mixture, comparing binding between target protein beads
(signal, S) and (His).sub.6 beads (background, B). For each sample,
a 0.5 .mu.M candidate DNA mixture was prepared in 40 .mu.L SB1T. 1
.mu.L (His).sub.6-complement oligo (1 mM) (FIG. 2) was added to the
DNA, along with 10 .mu.L of a protein competitor mixture (0.1% HSA,
10 .mu.M casein, and 10 .mu.M prothrombin in SB1T).
[0150] Binding reactions were performed by adding 50 .mu.L target
protein-coated beads or (His).sub.6-coated beads (5 mg/mL in SB1T)
to the DNA mixture and incubating 37.degree. C. for 15 minutes with
mixing. The DNA solution was removed and the beads were washed 5
times at 37.degree. C. with SB1T containing 0.1 mg/mL herring sperm
DNA (Sigma-Aldrich). Unless indicated, all washes were performed by
re-suspending the beads in 100 .mu.L wash solution, mixing for 30
seconds, separating the beads with a magnet, and removing the wash
solution. Bound aptamers were eluted from the beads by adding 100
.mu.L SB1T+2 M Guanidine-HCl and incubating at 37.degree. C. for 5
minutes with mixing. The aptamer eluate was transferred to a new
tube after magnetic separation. After the first two selection
rounds, the final two of five target beads washes were done for 5
minutes instead of 30 seconds.
[0151] Primer beads were prepared by immobilizing biotinylated
reverse PCR primer to streptavidin-coated paramagnetic beads
(MyOne-Streptavidin C1 (SA beads), Invitrogen). 5 mL SA beads (10
mg/mL) were washed once with NaClT (5 M NaCl, 0.01% TWEEN-20), and
resuspended in 5 mL biotinylated reverse PCR primer (5 .mu.M in
NaClT). The sample was incubated at 25.degree. C. for 15 minutes,
washed twice with 5 mL NaClT, resuspended in 12.5 mL NaClT (4
mg/mL), and stored at 4.degree. C.
[0152] 25 .mu.L primer beads (4 mg/mL in NaClT) were added to the
100 .mu.L aptamer solution in Guanidine Buffer and incubated at
50.degree. C. for 15 minutes with mixing. The aptamer solution was
removed, and the beads were washed 5 times with SB1T. Aptamer was
eluted from the beads by adding 85 .mu.L 20 mM NaOH, and incubating
at 37.degree. C. for 1 minute with mixing. 80 .mu.L aptamer eluate
was transferred to a new tube after magnetic separation,
neutralized with 20 .mu.L 80 mM HCl, and buffered with 1 .mu.L 0.5M
Tris-HCl, pH 7.5.
[0153] D. Aptamer Amplification and Purification
[0154] Selected aptamer DNA was amplified and quantified by QPCR.
48 .mu.L DNA was added to 12 .mu.L QPCR Mix (5.times. KOD DNA
Polymerase Buffer, 25 mM MgCl.sub.2, 10 .mu.M forward PCR primer,
10 .mu.M biotinylated reverse PCR primer, 5.times. SYBR Green I,
0.125 U/.mu.L KOD XL DNA Polymerase, and 1 mM each dATP, dCTP,
dGTP, and dTTP) and thermal cycled in an ABI5700 QPCR instrument
with the following protocol: 1 cycle of 99.9.degree. C., 15
seconds, 55.degree. C., 10 seconds, 70.degree. C., 30 minutes; 30
cycles of 99.9.degree. C., 15 seconds, 72.degree. C., 1 minute.
Quantification was done with the instrument software and the number
of copies of DNA selected with target beads and (His).sub.6 beads
were compared to determine signal/background ratios.
[0155] Following amplification, the PCR product was captured on SA
beads via the biotinylated antisense strand. 1.25 mL SA beads (10
mg/mL) were washed twice with 0.5 mL 20 mM NaOH, once with 0.5 mL
SB1T, resuspended in 2.5 mL 3 M NaCl, and stored at 4.degree. C. 25
.mu.L SA beads (4 mg/mL in 3 M NaCl) were added to 50 .mu.L
double-stranded QPCR product and incubated at 25.degree. C. for 5
minutes with mixing. The beads were washed once with SB1T, and the
"sense" strand was eluted from the beads by adding 200 .mu.L 20 mM
NaOH, and incubating at 37.degree. C. for 1 minute with mixing. The
eluted strand was discarded and the beads were washed 3 times with
SB1T and once with 16 mM NaCl.
[0156] Aptamer sense strand was prepared with the appropriate
nucleotide composition by primer extension from the immobilized
antisense strand. The beads were resuspended in 20 .mu.L primer
extension reaction mix (1.times. Primer Extension Buffer (120 mM
Tris-HCl, pH 7.8 @ 20, 10 mM KCl, 7 mM MgSO.sub.4, 6 mM
(NH.sub.4).sub.2SO.sub.4, 0.001% BSA, and 0.01% Triton X100), 5
.mu.M forward PCR primer, 0.125 U/.mu.L KOD XL DNA Polymerase, 0.5
mM each dATP, MedCTP, dGTP, and either dTTP or dUTP analog) and
incubated at 68.degree. C. for 30 minutes with mixing. The beads
were washed 3 times with SB1T, and the aptamer strand was eluted
from the beads by adding 85 .mu.L 20 mM NaOH, and incubating at
37.degree. C. for 1 minute with mixing. 80 .mu.L aptamer eluate was
transferred to a new tube after magnetic separation, neutralized
with 20 .mu.L 80 mM HCl, and buffered with 5 .mu.L 0.1 M HEPES, pH
7.5.
[0157] E. Selection Stringency and Feedback
[0158] The relative target protein concentration of the selection
step was lowered each round in response to the S/B ratio as
follows, where signal S and background B are defined in Section C
above:
if S/B<10, [P](i+1)=[P]i
if 10.ltoreq.S/B<100, [P](i+1)=[P]i/3.2
if S/B.gtoreq.100, [P](i+1)=[P]i/10
where [P]=protein concentration and i=current round number.
[0159] Target protein concentration was lowered by adjusting the
mass of target protein beads (and (His).sub.6 beads for background
determination) added to the selection step.
[0160] After each selection round, the convergence state of the
enriched DNA mixture was determined. 5 .mu.L double-stranded QPCR
product was diluted to 200 .mu.L with 4 mM MgCl.sub.2 containing
1.times. SYBR Green I. Samples were overlaid with 75 .mu.L silicon
oil and analyzed for convergence using a Cot analysis which
measures the hybridization time for complex mixtures of double
stranded oligonucleotides. The sample was thermal cycled with the
following protocol: 3 cycles of 98.degree. C., 1 minute, 85.degree.
C., 1 minute; 1 cycle of 93.degree. C., 1 minute, 85.degree. C., 15
minutes. During the 15 minutes at 85.degree. C., fluorescent images
were measured at 5-second intervals. The fluorescence intensity was
plotted as a function of log (time) to evaluate the diversity of
the sequences.
[0161] F. Measurement of Equilibrium Binding Constant (Kd)
[0162] Equilibrium binding constants of the enriched libraries were
measured using TALON bead partitioning. DNA was renatured by
heating to 95.degree. C. and slowly cooling to 37.degree. C.
Complexes were formed by mixing a low concentration of radiolabled
DNA (.about.1.times.10.sup.-11 M) with a range of concentrations of
target protein (1.times.10.sup.-7 M to 1.times.10.sup.-12 M final)
in SB1 Buffer, and incubating at 37.degree. C. A portion of each
reaction was transferred to a nylon membrane and dried to determine
total counts in each reaction. A small amount of 5 mg/mL TALON
beads was added to the remainder of each reaction and mixed at
37.degree. C. for one minute. A portion was passed through a
MultiScreen HV Plate (Millipore) under vacuum to separate
protein-bound complexes from unbound DNA and washed with 100 .mu.L
SB1 Buffer. The nylon membranes and MultiScreen HV Plates were
phosphorimaged and the amount of radioactivity in each sample
quantified using a FUJI FLA-3000. The fraction of captured DNA was
plotted as a function of protein concentration and a non-linear
curve-fitting algorithm was used to extract equilibrium binding
constants (K.sub.d values) from the data. Table 1 shows the K.sub.d
values determined for each enriched candidate mixture to a set of
targets. NT indicates that the enriched library for a particular
base composition did not appear to have changed from the original
candidate mixture, as determined by C.sub.0t analysis, and was
therefore Not Tested (NT).
[0163] Table 1 shows the equilibrium binding constants (K.sub.d)
for enriched pools to fifteen different protein targets and four
different DNA libraries: naturally occurring bases (dT),
5-(N-benzylcarboxyamide) (BndU), 5-(N-isobutylcarboxyamide) (iBudU)
or 5-(N-tryptaminocarboxyamide) (TrpdU). An aptamer with a K.sub.d
of less than 1.times.10.sup.-8 is desirable. The use of modified
bases in the SELEX process produces a significantly higher
percentage of desirable high affinity aptamers. It was observed
that only 2 of the 14 aptamers produced with the normal nucleotides
have the desired slow dissociation rates. Slow off-rate aptamers
produced with the modified nucleotides were identified 9 of 14, 7
of 14, and 14 of 14 for BndUTP, iBudUTP, and TrpdUTP,
respectively.
TABLE-US-00001 TABLE 1 Equilibrium binding constants (K.sub.d) of
the enriched libraries selected with different modified
nucleotides, reported in units of molarity. Target Protein dTTP
BndUTP iBudUTP TrpdUTP 4-1BB >1.0 .times. 10.sup.-7 5.6. .times.
10.sup.-9 >1.0. .times. 10.sup.-7 3.9. .times. 10.sup.-9 B7
>1.0. .times. 10.sup.-7 1.1. .times. 10.sup.-8 NT 7.2. .times.
10.sup.-9 B7-2 >1.0. .times. 10.sup.-7 NT >1.0. .times.
10.sup.-7 5.7. .times. 10.sup.-9 CTLA-4 >1.0. .times. 10.sup.-7
NT NT 1.4. .times. 10.sup.-9 E-Selectin >1.0. .times. 10.sup.-7
>1.0. .times. 10.sup.-7 >1.0. .times. 10.sup.-7 1.9. .times.
10.sup.-9 Fractalkine NT >1.0. .times. 10.sup.-7 NT .sup. 5.1.
.times. 10.sup.-11 GA733-1 protein 8.9. .times. 10.sup.-9 2.8.
.times. 10.sup.-9 4.7. .times. 10.sup.-9 .sup. 4.5. .times.
10.sup.-10 Gp130 >1.0. .times. 10.sup.-7 5.9. .times. 10.sup.-9
2.2. .times. 10.sup.-8 1.2. .times. 10.sup.-9 HMG-1 >1.0.
.times. 10.sup.-7 NT 2.2. .times. 10.sup.-8 4.9. .times. 10.sup.-9
IR >1.0. .times. 10.sup.-7 1.9. .times. 10.sup.-9 1.2. .times.
10.sup.-8 .sup. 2.2. .times. 10.sup.-10 OPG 3.7. .times. 10.sup.-8
4.6. .times. 10.sup.-9 9.5. .times. 10.sup.-9 .sup. 1.7. .times.
10.sup.-10 PAI-1 >1.0. .times. 10.sup.-7 .sup. 3.7. .times.
10.sup.-10 .sup. 9.1. .times. 10.sup.-10 .sup. 4.3. .times.
10.sup.-10 P-Cadherin >1.0. .times. 10.sup.-7 3.5. .times.
10.sup.-9 5.2. .times. 10.sup.-9 2.7. .times. 10.sup.-9 sLeptin R
>1.0. .times. 10.sup.-7 2.3. .times. 10.sup.-9 NT .sup. 4.6.
.times. 10.sup.-10 NT = not tested.
Example 2
Generation of PhotoAptamers Using 5'-Fixed PhotoSELEX and Slow
Off-Rate Enrichment Process by Dilution
[0164] A. Preparation of Candidate Mixtures
[0165] Candidate mixtures containing dATP, dCTP, dGTP, and BndUTP
were prepared by polymerase extension of a primer annealed to a
biotinylated template (FIG. 4A-B). For each template, four
different forward primers were used, each possessing a unique
chromophore at the 5' terminus (see FIG. 5 for the chromophore
structures). For each candidate mixture, 11 nmol forward primer
(with 5' chromophore) and 10 nmol template were combined in 250
.mu.L Primer Extension Buffer (120 mM Tris-HCl, pH 7.8, 10 mM KCl,
6 mM (NH.sub.4).sub.2SO.sub.4, 7 mM MgSO.sub.4, 0.1 mg/mL BSA, 0.1%
Triton X-100), heated to 95.degree. C. for 5 minutes, and cooled on
ice. 125 .mu.L each primer:template mixture was added to a 1 mL
extension reaction containing Primer Extension Buffer, 0.125
U/.mu.L KOD XL DNA Polymerase, and 0.5 mM each dATP, dCTP, dGTP,
and BndUTP, and incubated at 70.degree. C. for 30 minutes. Each 1
mL reaction was split into four 250 .mu.L aliquots and chilled on
ice. Double-stranded product was captured via the template strand
biotins by adding 1 mL streptavidin-coated magnetic beads
(MagnaBind-Streptavidin, Pierce, 5 mg/mL in 1M NaCl+0.05% TWEEN-20)
to each 250 .mu.L aliquot and incubating at 25.degree. C. for 60
minutes with mixing. Beads were washed three times with 0.5 mL
SB17T Buffer (40 mM HEPES, pH 7.5, 125 mM NaCl, 5 mM KCl, 5 mM
MgCl.sub.2, 1 mM EDTA, 0.05% TWEEN-20). The aptamer strand was
eluted from the beads with 1 mL 20 mM NaOH, neutralized with 0.25
mL 80 mM HCl, and buffered with 10 .mu.L 1 M HEPES, pH 7.5.
Candidate mixtures were concentrated with a Centricon-30 to
approximately 0.2 mL, and quantified by UV absorbance
spectroscopy.
[0166] B. Preparation of Target Proteins
[0167] Untagged target proteins were biotinylated by covalent
coupling of NHS-PEO4-biotin (Pierce) to lysines residues. Proteins
(300 pmol in 50 .mu.L) were exchanged into SB17T with a Sephadex
G-25 microspin column. NHS-PEO4-biotin was added to 1.5 mM and the
reaction was incubated at 4.degree. C. for 16 hours. Unreacted
NHS-PEO4-biotin was removed with a Sephadex G-25 microspin
column.
[0168] C. Aptamer Selection with Slow Off-Rate Enrichment Process
and Photocrosslinking
[0169] Selections were performed separately with each candidate
mixture, comparing binding between samples with target protein
(signal S) and samples without target protein (background B). The
first three rounds were performed with selection for affinity (no
photocrosslinking); the second and third included slow off-rate
enrichment process. Rounds four through eight included both slow
off-rate enrichment process and photocrosslinking.
[0170] For each sample, a 90 .mu.L DNA mixture was prepared in
SB17T with 10-20 pmoles candidate mixture (100 pmoles in the first
round) and 100 pmoles reverse primer. Samples were heated to
95.degree. C. for 3 minutes and cooled to 37.degree. C. at a rate
of 0.1 C/second. Samples were combined with 10 82 L protein
competitor mixture (0.1% HSA, 10 .mu.M casein, and 10 .mu.M
prothrombin in SB17T), added to 0.5 mg SA beads (pre-washed twice
with 20 mM NaOH and once with SB17T), and incubated at 37.degree.
C. for 5 minutes with mixing. Beads were removed by magnetic
separation.
[0171] Binding reactions were performed by adding 10 .mu.L target
protein (0.5 .mu.M in SB17T) or SB17T to 40 .mu.L DNA mixture and
incubating at 37.degree. C. for 30 minutes.
[0172] When slow off-rate enrichment process was employed, samples
were diluted 20.times. by adding 950 .parallel.L SB17T (preheated
to 37.degree. C.), and incubated at 37.degree. C. for 30 minutes
prior to capturing complexes.
[0173] Complexes were captured on SA beads via protein biotins by
adding 0.25 mg MyOne-SA beads (Invitrogen) and incubating at
37.degree. C. for 15 minutes with mixing. Free DNA was removed by
washing the beads five times with SB17T. Unless indicated, all
washes were performed by resuspending the beads in 100 .mu.L wash
solution, mixing for 30 seconds at 25.degree. C., separating the
beads with a magnet, and removing the wash solution. The aptamer
strand was eluted from the beads by adding 85 .mu.L 20 mM NaOH, and
incubating at 37.degree. C. for 1 minute with mixing. 80 .mu.L
aptamer eluate was transferred to a new tube after magnetic
separation, neutralized with 20 .mu.L 80 mM HCl, and buffered with
1 .mu.L 0.5 M Tris-HCl, pH 7.5.
[0174] When photo-selection was employed, the 50 .mu.L binding
reactions, (or 1 mL binding reactions after optional slow off-rate
enrichment process by dilution) were irradiated from above with a
high-pressure mercury lamp (Optical Associates, Inc. model
0131-0003-01, 500W, with 310 nm mirror set). Candidate mixtures
possessing a BrdU chromophore were irradiated for 37 seconds, those
possessing an ANA chromophore were irradiated for 60 seconds, and
those possessing an AQ or psoralen chromophore were irradiated for
10 minutes. An additional filter (5 mm plate glass) was used for
the ANA, AQ and psoralen chromophores to eliminate unnecessary, but
potentially damaging wavelengths below 320 nm. Complexes were
captured as above, and non-crosslinked DNA was removed by washing
the beads once with 4 M guanidine-HCl+0.05% TWEEN-20 at 50.degree.
C. for 10 minutes, once with 20 mM NaOH at 25.degree. C. for 2
minutes, twice with SB17T, and once with 16 mM NaCl. Crosslinked
DNA was not removed from the bead surface for the amplification
steps.
[0175] D. Aptamer Amplification and Purification
[0176] Selected aptamer DNA was amplified and quantified by QPCR.
48 .mu.L DNA was added to 12 .mu.L QPCR Mix (5.times. KOD DNA
Polymerase Buffer, 25 mM MgCl.sub.2, 10 .mu.M forward PCR primer,
10 .mu.M biotinylated reverse PCR primer, 5.times. SYBR Green I,
0.125 U/.mu.L KOD XL DNA Polymerase, and 1 mM each dATP, dCTP,
dGTP, and dTTP) and thermal cycled in an a Bio-Rad MyIQ QPCR
instrument with the following protocol: 1 cycle of 99.9.degree. C.,
15 sec, 55.degree. C., 10 sec, 68.degree. C., 30 min, 30 cycles of
99.9.degree. C., 15 seconds, 72.degree. C., 1 minute.
Quantification was done with the instrument software and the number
of copies of DNA selected with and without target protein were
compared to determine signal/background ratios.
[0177] When photo-selection was employed, a cDNA copy of the
selected DNA was prepared by primer extension on the bead surface.
Washed beads were resuspended in 20 .mu.L cDNA extension mix
(Primer Extension Buffer containing 5 .mu.M reverse PCR primer, 0.5
mM each dATP, dCTP, dGTP, and dTTP, and 0.125 U/.mu.L KOD XL DNA
Polymerase) and incubated at 68.degree. C. for 30 minutes with
mixing. The beads were washed 3 times with SB17T, and the aptamer
strand was eluted by from the beads by adding 85 .mu.L 20 mM NaOH,
and incubating at 37.degree. C. for 1 minute with mixing. 80 .mu.L
aptamer eluate was transferred to a new tube after magnetic
separation, neutralized with 20 .mu.L 80 mM HCl, and buffered with
1 .mu.L 0.5 M Tris-HCl, pH 7.5. The cDNA was amplified and
quantified by QPCR as above for the 30 cycles of 99.9.degree. C.,
15 seconds, 72.degree. C., 1 minute.
[0178] Following amplification, the PCR product was captured on SA
beads via the biotinylated antisense strand. 1.25 mL SA beads (10
mg/mL) were washed twice with 0.5 mL 20 mM NaOH, once with 0.5 mL
SB17T, resuspended in 1.25 mL 3 M NaCl+0.05% Tween, and stored at
4.degree. C. 25 .mu.L SA beads (10 mg/mL in 3 M NaClT) were added
to 50 .mu.L double-stranded QPCR product and incubated at
25.degree. C. for 5 minutes with mixing. The beads were washed once
with SB17T, and the "sense" strand was eluted from the beads by
adding 200 .mu.L 20 mM NaOH, and incubating at 37.degree. C. for 1
minute with mixing. The eluted strand was discarded and the beads
were washed 3 times with SB17T and once with 16 mM NaCl.
[0179] Aptamer sense strand was prepared with the appropriate
chromophore by primer extension from the immobilized antisense
strand. The beads were resuspended in 20 .mu.L primer extension
reaction mixture (1.times. Primer Extension Buffer, 1.5 mM
MgCl.sub.2, 5 .mu.M forward primer with appropriate 5' chromophore,
0.5 mM each dATP, dCTP, dGTP, and BndUTP, and 0.125 U/.mu.L KOD XL
DNA Polymerase) and incubated at 68.degree. C. for 30 minutes with
mixing. The beads were washed 3 times with SB17T, and the aptamer
strand was eluted from the beads by adding 85 .mu.L 20 mM NaOH, and
incubating at 37.degree. C. for 1 minute with mixing. 80 .mu.L
aptamer eluate was transferred to a new tube after magnetic
separation, neutralized with 20 .mu.L 80 mM HCl, and buffered with
5 .mu.L 0.1 M HEPES, pH 7.5.
[0180] E. Selection Stringency and Feedback
[0181] Target protein was adjusted at each round as described in
Example 1. After each round of selection, the convergence state of
the enriched pool was determined as described in Example 1.
[0182] F. Equilibrium Binding Constants of Enriched Libraries
[0183] The binding affinity was determined as described in Example
1 above, but with SA capture beads. The following table, Table 2,
summarizes the equilibrium binding constants (K.sub.d) obtained
using the photoSELEX protocol with slow off-rate enrichment
process.
TABLE-US-00002 TABLE 2 Equilibrium binding constants (K.sub.d) of
the enriched libraries selected with different chromophores,
reported in units of molarity. Measurements were not made on
libraries that failed to converge (indicated with an x). Target
Protein BrdU AQ ANA Psor .beta.-catenin 2.7. .times. 10.sup.-8 3.6.
.times. 10.sup.-9 1.1. .times. 10.sup.-9 1.6. .times. 10.sup.-9
bFGF 3.1. .times. 10.sup.-8 .sup. 5.7. .times. 10.sup.-10 7.1.
.times. 10.sup.-10 .sup. 5.1. .times. 10.sup.-10 CMP-SAS x 6.2.
.times. 10.sup.-9 7.3. .times. 10.sup.-9 4.9. .times. 10.sup.-8
endostatin 1.3. .times. 10.sup.-9 .sup. 8.7. .times. 10.sup.-10
8.8. .times. 10.sup.-10 1.3. .times. 10.sup.-9 IL-6 1.0. .times.
10.sup.-9 .sup. 5.4. .times. 10.sup.-10 4.0. .times. 10.sup.-10 x
myeloperoxidase .sup. 6.0. .times. 10.sup.-10 .sup. 2.8. .times.
10.sup.-10 5.0. .times. 10.sup.-10 .sup. 1.5. .times. 10.sup.-10
SDF-1.beta. .sup. 8.1. .times. 10.sup.-10 .sup. 5.7. .times.
10.sup.-10 3.8. .times. 10.sup.-10 x TIMP-1 5.2. .times. 10.sup.-9
7.3. .times. 10.sup.-9 8.9. .times. 10.sup.-9 x VEGF .sup. 7.2.
.times. 10.sup.-10 4.2. .times. 10.sup.-9 5.5. .times. 10.sup.-10 x
vWF 2.6. .times. 10.sup.-8 8.8. .times. 10.sup.-9 8.1. .times.
10.sup.-9 x
[0184] G. Crosslink Activity Assay
[0185] The crosslink yield of enriched libraries was determined by
measuring the percent of DNA crosslinked to protein under
conditions of saturating protein and light. Radiolabeled DNA (50
pM) was mixed with reverse primer (16 nM) in SB17T, heated to
95.degree. C. for 3 minutes, and cooled to 37.degree. C. at
0.1.degree. C./second. Target protein was added to the DNA mix to a
final concentration of 10 nM and incubated at 37.degree. C. for 30
minutes. Control samples with no protein were simultaneously
prepared. Samples were crosslinked with the chromophore-specific
conditions described above, but with a saturating dose (6 minutes
for BrdU, 10 minutes for ANA, and 30 minutes for AQ and Psor).
Samples were analyzed by denaturing PAGE, FIG. 6, and quantified
and the results are tabulated in Table 3.
TABLE-US-00003 TABLE 3 Crosslink yields of the enriched libraries
selected with different chromophores, reported in units of percent
of total DNA crosslinked to protein. Measurements were not made on
libraries that failed to converge (indicated with an x). Target
Protein BrdU AQ ANA Psor .beta.-catenin 15 9 8 1 bFGF 4 9 15 4
CMP-SAS x 3 5 2 Endostatin 2 1 18 3 IL-6 0 5 9 Myeloperoxidase 4 13
9 8 SDF-1.beta. 8 10 17 x TIMP-1 1 4 2 x VEGF 1 1 4 x vWF 2 2 7
x
Example 3
Generation of Slow Off-Rate Aptamers Using a Slow Off-Rate
Enrichment Process with a Competitor
[0186] A. Preparation of Candidate Mixtures
[0187] Candidate mixtures containing dATP, dCTP, dGTP, and BndUTP
were prepared by polymerase extension of a primer annealed to a
biotinylated template for 94 protein targets. 55 nmol forward
primer (with 5' ANA chromophore) and 55 nmol template were combined
in 0.5 mL Primer Extension Buffer (120 mM Tris-HCl, pH 7.8, 10 mM
KCl, 6 mM (NH.sub.4).sub.2SO.sub.4, 7 mM MgSO.sub.4, 0.1 mg/mL BSA,
0.1% Triton X-100), heated to 95.degree. C. for 5 minutes,
70.degree. C. for 5 minutes, 48.degree. C. for 5 minutes, and
cooled on ice. The primer:template mixture was added to a 5.5 mL
extension reaction containing Primer Extension Buffer, 0.125
U/.mu.L KOD XL DNA Polymerase, and 0.5 mM each dATP, dCTP, dGTP,
and BndUTP, and incubated at 70.degree. C. for 60 minutes. After
completion of the extension reaction, the solution was chilled on
ice. Double-stranded product was captured via the template strand
biotins by adding 25 mL streptavidin-coated magnetic beads
(MagnaBind-Streptavidin, Pierce, 5 mg/mL in 1 M NaCl+0.05%
TWEEN-20) to the primer extension product and incubating 25.degree.
C. for 15 minutes with rotating. Beads were washed three times with
40 mL SB17T Buffer (40 mM HEPES, pH 7.5, 125 mM NaCl, 5 mM KCl, 5
mM MgCl.sub.2, 1 mM EDTA, 0.05% TWEEN-20). The aptamer strand was
eluted from the beads with 35.2 mL 20 mM NaOH for 5 minutes with
shaking. The eluted strand was neutralized with 8.8 mL 80 mM HCl,
and buffered with 400 .mu.L 1 M HEPES, pH 7.3. Candidate mixtures
were concentrated with a Centricon-30 to approximately 0.7 mL, and
quantified by UV absorbance spectroscopy.
[0188] B. Preparation of Target Proteins
[0189] Untagged target proteins were biotinylated as described in
Example 2.
[0190] C. Aptamer Selection with Slow Off-Rate Enrichment Process
and Photocrosslinking
[0191] Selections were performed separately as described in Example
2, with the addition of 10 mM dextran sulfate as a competitor for
aptamer rebinding during the slow off-rate enrichment process in
rounds six through nine.
[0192] The slow off-rate enrichment process was employed in three
different ways. In rounds two and three, samples were diluted
20.times. by adding 950 .mu.L SB17T (preheated to 37.degree. C.),
and incubated at 37.degree. C. for 30 minutes prior to capturing
complexes. In rounds four and five, samples were diluted 20.times.
by adding 950 .mu.L SB17T (preheated to 37.degree. C.), and
incubated at 37.degree. C. for 30 minutes prior to crosslinking. In
rounds six and seven, samples were diluted 20.times. by adding 950
.mu.L SB17T (preheated to 37.degree. C.). 50 .mu.L of each diluted
sample was diluted again by transferring to 950 .mu.L SB17T+10 mM
5000K dextran sulfate (preheated to 37.degree. C.) to give an
overall 400.times. dilution, and incubated at 37.degree. C. for 60
minutes prior to crosslinking. In rounds eight and nine, samples
were diluted 20.times. by adding 950 .mu.L SB17T (preheated to
37.degree. C.), and 50 .mu.L of each sample was diluted again by
transferring to 950 .mu.L SB17T (preheated to 37.degree. C.) to
give 400.times. dilution. Finally, 50 .mu.L of each 400.times.
diluted sample was diluted again by transferring to 950 .mu.L
SB17T+10 mM 5000K dextran sulfate (preheated to 37.degree. C.) to
give an overall 8000.times. dilution, and incubated at 37.degree.
C. for 60 minutes prior to crosslinking. Complexes were captured
and washed as described in Example 2. When photo-crosslinking was
employed, the 1 mL binding reactions after the slow off-rate
enrichment process were irradiated from above with an array of 470
nm LEDs for 60 seconds prior to complex capture as in Example
2.
[0193] D. Aptamer Amplification and Purification
[0194] Amplification and purification were performed as in Example
2.
[0195] E. Selection Stringency and Feedback
[0196] Target protein was adjusted at each round as described in
Example 1, except in rounds six and eight. In order to maximize
signal after these large dilutions, the target protein was
increased to 100 nM for rounds six and eight. After each round of
selection, the convergence state of the enriched pool was
determined as described in Example 1.
[0197] F. Dissociation Rate Constant Determination Protocol.
[0198] The rate constant for aptamer:protein complex dissociation
(koff) was determined for each aptamer by measuring the fraction of
pre-formed aptamer:protein complexes that remain bound after
dilution as a function of time. Radiolabeled aptamer (50 pM) was
equilibrated in SB17T-0.002 (SB17T with TWEEN-20 reduced to 0.002%)
at 37.degree. C. with protein at a concentration 10.times. greater
than the measured K.sub.d value. Samples were diluted 100.times.
with SB17T-0.002 at 37.degree. C. and aliquots were removed at
various time points and partitioned to separate free aptamer from
protein:aptamer complexes. Partitioning was accomplished by adding
ZORBAX resin (Agilent) to the sample, capturing complexes on the
resin, passing the sample through a DuraPore membrane under vacuum,
and washing the resin with SB17T-0.002. For proteins not
efficiently captured with ZORBAX resin, the assay was performed
with biotinylated protein in SB17T and partitioning was
accomplished by capturing complexes with SA beads. The amount of
complex remaining at each time point was determined by quantifying
the radiolabeled aptamer on the resin with a FUJI FLA-3000
phosphorimager. The fraction of complex was plotted as a function
of time and the dissociation rate constant (koff) and dissociation
half-life value (t.sub.1/2) was determined by fitting the data to
an analytic expression for bimolecular dissociation kinetics using
non-linear regression.
[0199] G. Kinetic Properties of Some Aptamers
[0200] The following table, Table 4, summarizes the dissociation
half-life values (t.sub.1/2) obtained for aptamers selected against
10 targets using this protocol.
TABLE-US-00004 TABLE 4 Dissociation half-life values (t.sub.1/2) of
aptamers using the competitor slow off-rate enrichment step
protocol. Target Protein t.sub.1/2 (min) bFGF R 66 C3 164 catalase
58 FGF-17 91 group IB phospholipase A2 40 HB-EGF 49 HCC-4 143 IL-6
sR.alpha. 114 SAP 186 uPA 85
Example 4
The Slow Off-Rate Enrichment Process Increases the Dissociation
Half-Life of Selected Aptamers
[0201] Dissociation half-life values (t.sub.1/2) were measured and
plotted for 65 aptamers that were selected by either the affinity
SELEX method described in Example 1 or photo SELEX methods
described in U.S. Pat. No. 6,458,539, entitled "Photoselection of
Nucleic Acid Ligands" without a slow off-rate enrichment process
(FIG. 3A). t.sub.1/2 values were also measured and plotted for 72
aptamers that were selected by the slow off-rate enrichment process
described in Example 2 with a slow off-rate enrichment process by
dilution or dilution with competitor (FIG. 3B). The average
t.sub.1/2 value for aptamers using the modified nucleotides
5-(N-benzylcarboxyamide)-dUTP (BndUTP),
5-(N-isobutylcarboxyamide)-dUTP (iBudUTP), or
5-(N-tryptaminocarboxyamide)-dUTP (TrpdUTP) selected in the absence
of a slow off-rate enrichment process was 20 minutes with some
aptamers having a t.sub.1/2 value of up to one hour. This is
substantially longer than what has been previously described with
natural bases or other modified nucleotides. The average for
aptamers selected with a slow off-rate enrichment process was over
85 minutes, with some aptamers having a t.sub.1/2 value in excess
of four hours.
Example 5
Generation of Aptamers from a NapdU Random Library
[0202] A. Preparation of Candidate Mixtures
[0203] Candidate mixtures containing dATP, dCTP, dGTP, and NapdUTP
were prepared as described in Example 3 but without the 5'-ANA
photoreactive group.
[0204] B. Immobilization of Target Proteins
[0205] Target proteins contained a (His).sub.6 tag and were
captured with Talon beads as described in Example 1.
[0206] C. Aptamer Selection with Slow Off-Rate Enrichment
Process
[0207] Aptamer selection was performed as described in Example 3,
but without photocros slinking.
[0208] D. Aptamer Amplification and Purification
[0209] Amplification and purification were performed as described
in Example 3.
[0210] E. Selection Stringency and Feedback
[0211] Selection stringency and feedback were performed as
described in Example 3.
[0212] F. Aptamer Properties
[0213] The equilibrium binding constant (K.sub.d) of four aptamers
from this selection are listed in Table 5.
TABLE-US-00005 TABLE 5 Equilibrium binding constants (Kd) of
NapdUTP aptamers. Target Protein K.sub.d (M) bFGF 1.1. .times.
10.sup.-9 Endostatin 2.0. .times. 10.sup.-10 TIMP-3 1.5. .times.
10.sup.-10 VEGF 7.2. .times. 10.sup.-10
Example 6
Generation of Slow-Off-Rate Aptamers for a Peptide Target Using a
Slow Off-Rate Enrichment Process with a Competitor
[0214] A. Preparation of Candidate Mixtures
[0215] Candidate mixtures containing dATP, dCTP, dGTP, and BndUTP
were prepared by polymerase extension of a primer with a 5' ANA
chromophore and purified as described in Example 3.
[0216] B. Aptamer Selection with Slow Off-Rate Enrichment Process
and Photocrosslinking
[0217] Aptamer selection was performed as described in Example 3
with the 29 amino acid biotinylated target peptide SMAP29 (Sheep
Myeloid Antibacterial Peptide MAP-29, Anaspec).
[0218] C. Aptamer Amplification and Purification
[0219] Amplification and purification were performed as described
in Example 3.
[0220] D. Selection Stringency and Feedback
[0221] Selection stringency and feedback were performed as
described in Example 3.
[0222] E. Aptamer Properties
[0223] The equilibrium binding constant (K.sub.d) of an aptamer
from this selection was 1.2.times.10.sup.-8 M (measured according
to the protocol described in Example 1). The dissociation half-life
(t.sub.1/2) of this aptamer was 69 minutes (measured according to
the protocol described in Example 3). Results are shown in FIG. 12A
and FIG. 12B.
Example 7
Protein Measurements in Test Samples Were Enabled by Aptamers with
Slow Off-Rates
[0224] A. Preparation of Aptamer/Primer Mixtures and Test
Samples
[0225] Aptamers with a biotin Cy3 detection label (4 nM each) were
mixed with a 3.times. excess of capture probe (oligonucleotide
complementary to the 3' fixed region of the aptamer containing a
biotin tag and photocleavable element) in 1.times. SB17T and heated
at 95.degree. C. for 4 minutes, then 37.degree. C. for 13 minutes,
and diluted 1:4 in 1.times. SB17T. 55 uL of aptamer/primer mix was
added to a microtiter plate (Hybaid #AB-0407) and sealed with foil.
Test samples were prepared in a microtiter plate by mixing known
concentrations of protein analytes in SB17T and diluting serially
with SB17T.
[0226] B. Sample Equilibration
[0227] 55 uL of aptamer/primer mix was added to 55 uL of test
sample and incubated at 37.degree. C. for 15 minutes in a
foil-sealed microtiter plate. The final concentration of each
aptamer in the equilibration mixture was 0.5 nM. After
equilibration, all subsequent steps of this method were performed
at room temperature unless otherwise noted.
[0228] C. Aptamer Capture and Free Protein Removal
[0229] A DuraPore filtration plate (Millipore HV cat #MAHVN4550)
was washed once with 100 uL 1.times. SB17T by vacuum filtration.
133.3 uL 7.5% Streptavidin-agarose resin (Pierce) was added to each
well and washed twice with 200 uL 1.times. SB17T. 100 uL of
equilibrated samples was transferred to the Durapore plate
containing the Streptavidin-agarose resin and incubated on a
thermomixer (Eppendorf) at 800 rpm for 5 minutes. The resin was
washed once with 200 uL 1.times. SB17T+100 uM biotin and once with
200 uL 1.times. SB17T.
[0230] D. Protein Tagging with Biotin
[0231] 100 uL of 1.2 mM NHS-PEO4-biotin in SB17T, prepared
immediately before use, was added to the resin with captured
aptamer and aptamer:protein complexes and incubated on a
thermomixer at 800 rpm for 20 minutes. The resin was washed five
times with 200 uL 133 SB17T by vacuum filtration.
[0232] E. Slow Off-Rate Enrichment Process & Photocleavage
[0233] The drip director was removed from underside of the DuraPore
plate and the plate was placed over a 1 mL microtiter collection
plate. The resin was washed once with 200 uL 1.times. SB17T by
centrifugation at 1000.times.g for 30 sec. 80 uL of 1.times.
SB17T+10 mM dextran sulfate was added to the resin and irradiated
with a BlackRay Mercury Lamp on a thermomixer at 800 rpm for 10
minutes. The DuraPore plate was transferred to a new 1 mL deepwell
plate and centrifuged at 1000.times.g for 30 seconds to collect the
photocleaved aptamer and protein:aptamer complexes.
[0234] F. Protein Capture and Free Aptamer Removal
[0235] 50 uL of MyOne-streptavidin C1 paramagnetic beads
(Invitrogen) (10 mg/mL in 1.times. SB17T) was added to a microtiter
plate. The beads were separated with a magnet for 60 seconds and
the supernatant was removed. 225 uL of photocleavage mixture was
added to the beads and mixed for 5 minutes. The beads were washed
four times with 200 uL 1.times. SB17T by separating the magnetic
beads and replacing the wash buffer. The final wash buffer was
removed.
[0236] G. Aptamer Elution
[0237] 100 uL Sodium Phosphate Elution Buffer (10 mM
Na.sub.2HPO.sub.4, pH 11) was added to the beads and mixed for 5
minutes. 90 uL of eluate was transferred to a microtiter plate and
neutralized with 10 uL Sodium Phosphate Neutralization Buffer (10
mM NaH.sub.2PO.sub.4, pH 5).
[0238] H. Aptamer Hybridization to Microarrays
[0239] DNA arrays were prepared with oligonucleotide capture probes
comprised of the complementary sequence of the variable region of
each aptamer immobilized on a custom microscope slide support.
Multiple arrays (subarrays) existed on each slide, and subarrays
were physically separated by affixing a gasket (Grace) for sample
application. Arrays were pretreated with 100 uL Blocking Buffer and
incubated for 15 minutes at 65.degree. C. on a thermomixer. 30 uL
of high salt Hybridization Buffer was added to 90 uL of neutralized
aptamer eluate in a microtiter plate, incubated at 95.degree. C.
for 5 minutes in a thermalcycler, and cooled to 65.degree. C. at
0.1.degree. C./second. Blocking Buffer was removed from the arrays
and 110 uL of aptamer sample was added to the arrays and incubate
in a humid chamber at 65.degree. C. for 20 hours.
[0240] I. Array Washing
[0241] Aptamer sample was removed from the arrays, and the arrays
were washed once with 200 uL of sodium phosphate Tween-20 wash
buffer at 65.degree. C., with the gasket in place, and three times
with 25 mL sodium phosphate, Tween-20 wash buffer at 65.degree. C.
in a pap jar with the gasket removed. Arrays were dried with a
nitrogen gun.
[0242] J. Quantitate Signal on Arrays
[0243] Array slides were scanned on a TECAN LS300 in an appropriate
channel for Cy3 detection and Cy3 signal on each array feature was
quantified.
[0244] Results:
[0245] Apatmers specific to three different targets (bFGF, VEGF,
and Myeloperoxidase) were produced using traditional SELEX methods
and materials. A second set of aptamers specific to the same set of
targets were made using 5-position modified nucleotides and
selected for very slow off-rates for their respective targets.
Aptamers made in the traditional process had measured off rates on
the order of less than 5 minutes. Aptamers made with the modified
nucleotides and using slow off-rate enrichment process during
selection had off rates of greater than 20 minutes. Two sets of
aptamers were made for each target by the two different methods for
a total of 4 different aptamer populations for each target. The
ability of these aptamer populations to measure analyte
concentrations in test samples was evaluated as described above
over a range of target concentrations. Relative signal from the DNA
chip detection was plotted against the input target concentration.
See FIGS. 11A to 11C. The response curve of the traditional
aptamers was very flat and the sensitivity of the detection was
fairly low. The sensitivity of detection of the respective targets
with the slow off-rate aptamers was excellent. The data supports
the need to use the slow off-rate aptamers for maximum analytic
performance.
Example 8
Generation of High Affinity BndU Aptamers to Human Thrombin
[0246] A. Preparation of Candidate Mixture
[0247] A candidate mixture containing dATP, dCTP, dGTP, and BndUTP
was prepared by polymerase extension of a primer with a 5' ANA
chromophore and purified as described in Example 3.
[0248] B. Preparation of Target Protein
[0249] Human thrombin was tagged with biotin as describe in Example
2.
[0250] C. Aptamer Selection with Slow Off-Rate Enrichment and
Photocrosslinking
[0251] Aptamer selection was performed as described in Example 3
with biotinylated human thrombin as the target.
[0252] D. Aptamer Amplification and Purification
[0253] Amplification and purification were performed as described
in Example 3.
[0254] E. Selection Stringency and Feedback
[0255] Selection stringency and feedback were performed as
described in Example 3.
[0256] F. Aptamer Properties
[0257] The equilibrium binding constant (K.sub.d) of aptamer
2336-17 from this selection with a modified BndU was
4.4.times.10.sup.-11 M (measured according to the protocol
described in Example 1) as demonstrated in FIG. 15. In the art,
single-stranded DNA aptamers to human thrombin were selected from a
library comprised of natural dA, dC, dG, and dT nucleotides (Bock,
et al., Selection of Single-Stranded DNA Molecules that Bind and
Inhibit Human Thrombin, Nature 1992 355: 564-566). The binding
affinities of the aptamers had K.sub.d values ranging from
2.5.times.10.sup.-8 M to 2.0.times.10.sup.-7 M. Using a similar
protocol with a library comprised of natural dA, dC, dG, and
modified 5-(1-pentynyl)-dUTP, aptamers were selected with K.sub.d
values ranging from 4.times.10.sup.-7 M to 1.times.10.sup.-6 M
(Latham, et al., The Application of a Modified Nucleotide in
Aptamer Selection: Novel Thrombin Aptamers Containing
5-(1-Pentynyl)-2'-Deoxyuridine, Nucleic Acid Research 1994 22(14):
2817-2822).
[0258] A number of patents, patent application publications, and
scientific publications are cited throughout and/or listed at the
end of the description. Each of these is incorporated herein by
reference in their entirety. Likewise, all publications mentioned
in an incorporated publication are incorporated by reference in
their entirety.
[0259] Examples in cited publications and limitations related
therewith are intended to be illustrative and not exclusive. Other
limitations of the cited publications will become apparent to those
of skill in the art upon a reading of the specification and a study
of the drawings.
[0260] The words "comprise", "comprises", and "comprising" are to
be interpreted inclusively rather than exclusively.
Sequence CWU 1
1
23179DNAArtificialSyntheticmisc_feature(23)..(62)n is a, c, g, or t
1ababgtcttc ttgtcgtttc gcnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
60nnggtggagt gtggtgagg 79225DNAArtificialSynthetic 2atatatatcc
tcaccacact ccacc 25330DNAArtificialSynthetic 3ababtttttt ttgtcttctt
gtcgtttcgc 30478DNAArtificialSyntheticmisc_feature(22)..(61)n is a,
c, g, or t 4ababccgtcc tcctctccgt cnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 60ngggacactg ggtgcagg 78525DNAArtificialSynthetic
5atatatatcc tgcacccagt gtccc 25629DNAArtificialSynthetic
6ababtttttt ttccgtcctc ctctccgtc 29718DNAArtificialSynthetic
7gtcttcttgt cgtttcgc
18876DNAArtificialSyntheticmisc_feature(20)..(59)n is a, c, g, or t
8ababcccgct cgtcgtctgn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnc
60aggcagacgg tcactc 76925DNAArtificialSynthetic 9atatatatga
gtgaccgtct gcctg 251025DNAArtificialSynthetic 10atatatatga
gtgaccgtct gcctg 251125DNAArtificialSynthetic 11atatatatga
gtgaccgtct gcctg 251225DNAArtificialSynthetic 12atatatatga
gtgaccgtct gcctg 251325DNAArtificialSynthetic 13atatatatga
gtgaccgtct gcctg 251423DNAArtificialSynthetic 14ttttttttcc
cgctcgtcgt ctg 231527DNAArtificialSynthetic 15ababtttttt ttcccgctcg
tcgtctg 271679DNAArtificialSyntheticmisc_feature(23)..(62)n is a,
c, g, or t 16ababgtgtct gtctgtgtcc tcnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 60nnggtggagt gtggtgagg 791725DNAArtificialSynthetic
17atatatatcc tcaccacact ccacc 251825DNAArtificialSynthetic
18atatatatcc tcaccacact ccacc 251925DNAArtificialSynthetic
19atatatatcc tcaccacact ccacc 252025DNAArtificialSynthetic
20atatatatcc tcaccacact ccacc 252125DNAArtificialSynthetic
21atatatatcc tcaccacact ccacc 252226DNAArtificialSynthetic
22ttttttttgt gtctgtctgt gtcctc 262330DNAArtificialSynthetic
23ababtttttt ttgtgtctgt ctgtgtcctc 30
* * * * *