Methods for generating aptamers with improved off-rates

Abstract

To provide novel aptamers, and methods for producing and using the aptamers.SOLUTION: Disclosed herein are 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. Particularly, the present disclosure describes methods for producing aptamers and photoaptamers having slower dissociation rate constants than those 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.SELECTED DRAWING: None

Description

[Technical Field]

[0001]

[0001] This disclosure generally relates to methods for the production of aptamers and photoaptamers with improved properties, and the improved aptamers and photoaptamers produced thereby. In particular, this disclosure describes slow off-rate aptamers that are highly specific for targets of interest. This disclosure describes compositions of these slow off-rate aptamers, as well as methods for their selection. Additionally, this disclosure describes aptamer constructs with improved functionality for detection methods. Furthermore, this disclosure describes applications enabled by these improved aptamers. [Background technology]

[0002]

[0002] The following discussion provides a summary of information relevant to the present disclosure and is not an admission that any of the information provided herein or the publications cited therein is prior art to the claimed invention.

[0003] The SELEX method is a method for the in vitro selection of nucleic acid molecules capable of binding highly specifically to a target molecule, and is described in U.S. Patent No. 5,475,096, entitled "Nucleic Acid Ligands," and U.S. Patent No. 5,270,163 (see also WO 91 / 19813), entitled "Nucleic Acid Ligands," each of which is specifically incorporated herein by reference. These patents, collectively referred to herein as the SELEX Patents, describe methods for generating aptamers to any desired target molecule.

[0004]

[0004] The basic SELEX method has been modified to achieve several specific objectives. For example, U.S. Patent No. 5,707,796, entitled "Method for Selecting Nucleic Acids on the Basis of Structure," describes the use of the SELEX method in combination with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. Patent No. 5,580,737, entitled "High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine," describes a method for identifying highly specific aptamers capable of discriminating between closely related molecules, called counter-SELEX. U.S. Patent No. 5,567,588, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Solution SELEX," describes a SELEX-based method that achieves highly efficient partitioning between oligonucleotides with high and low affinity for a target molecule. U.S. Patent No. 5,496,938, entitled "Nucleic Acid Ligands to HIV-RT and HIV-1 Rev," describes a method for obtaining improved aptamers after performing SELEX. U.S. Patent No. 5,705,337, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Chemi-SELEX" describes a method for covalently linking aptamers to their targets.

[0005]

[0005] The SELEX method involves the identification of high-affinity aptamers containing modified nucleotides that confer improved properties on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include modifications at the ribose and / or phosphate positions. Aptamers identified by the SELEX method containing modified nucleotides are described in U.S. Patent No. 5,660,985, entitled "High Resolution Image Download MS PowerPoint Slide," which is incorporated herein by reference. The patent is described in "Affinity Nucleic Acid Ligands Containing Modified Nucleotides," which describes oligonucleotides containing nucleotide derivatives chemically modified at the 5' and 2' positions of the pyrimidine. The above-mentioned U.S. Pat. No. 5,580,737 describes highly specific aptamers containing one or more nucleotides modified with 2'-amino (2'-NH2), 2'-fluoro (2'-F), and / or 2'-O-methyl (2'-OMe).

[0006] Further modifications of the SELEX method are described in U.S. Patent Nos. 5,763,177, 6,001,577, and 6,291,184, each entitled "Systematic Evolution of Nucleic Acid Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX"; see also, e.g., U.S. Patent 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 to, and / or photocrosslinking with, and / or photoinactivating target molecules. The resulting photoreactive aptamers are referred to as photocrosslinking aptamers or photoaptamers.

[0007] While these SELEX and photoSELEX methods are useful, there is a continuing need for processes leading to improved aptamer properties arising from in vitro selection techniques. For example, there is a need for aptamers that target molecules with binding affinities superior to those achieved using naturally occurring DNA or RNA nucleotides, as well as methods for producing such aptamers. For many applications, such as in vitro assays, diagnostics, therapeutics, or imaging applications, it is of interest to produce aptamers with slow off-rates from aptamer / target affinity complexes. Several techniques have been proposed to produce such reagents (see, e.g., WO 99 / 27133 and US 2005 / 0003362). However, these selection methods do not distinguish between the selection of reagents with fast target association kinetics (i.e., fast on-rates) and those with slow target dissociation kinetics (e.g., slow off-rates). Thus, there is a need for new processes and techniques that favor the selection of aptamers with slow off-rates while inhibiting the selection of aptamers that simply associate with targets quickly.

[0008] Finally, there is a need for aptamer constructs that contain different built-in functionalities, which may include tags for immobilization, labels for detection, means to facilitate or control separation, etc. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 5,475,096 [Patent Document 2] U.S. Patent No. 5,270,163 [Patent Document 3] WO 91 / 19813 [Patent Document 4] U.S. Patent No. 5,707,796 [Patent Document 5] U.S. Patent No. 5,580,737 [Patent Document 6] U.S. Patent No. 5,567,588 [Patent Document 7] U.S. Patent No. 5,496,938 [Patent Document 8] U.S. Patent No. 5,705,337 [Patent Document 9] U.S. Patent No. 5,660,985 [Patent Document 10] U.S. Patent No. 5,763,177 [Patent Document 11] U.S. Patent No. 6,001,577 [Patent Document 12] U.S. Patent No. 6,291,184 [Patent Document 13] U.S. Patent No. 6,458,539 [Patent Document 14] WO99 / 27133 [Patent Document 15] US2005 / 0003362 Summary of the Invention

[0010]

[0009] This disclosure describes novel aptamers and methods for producing and using such aptamers. In particular, this disclosure describes slow-off-rate (slow dissociation) aptamers, slow-off-rate aptamers containing C-5 modified pyrimidines, and processes for selecting slow-off-rate aptamers by dilution, by adding competitors, or by a combination of both approaches. Additionally, slow-off-rate aptamers for a variety of targets, such as proteins and peptides, are described. Slow-off-rate aptamers with unique structural features and melting points are also described. This disclosure also describes slow-off-rate aptamers with photoreactive functional groups, aptamers that are resistant to degradation in the presence of polyanionic substances, and selection processes for these aptamers, as well as aptamers constructed with a variety of other functionalities that improve their usefulness in a variety of applications.

[0011]

[0010] The present disclosure describes an improved SELEX method for generating aptamers capable of binding to target molecules. More specifically, the present disclosure describes methods for producing aptamers and / or photoaptamers that have a slower dissociation rate from their respective target molecules than aptamers and photoaptamers obtained by previous SELEX methods. Generally, after contacting a candidate mixture with a target molecule and allowing nucleic acid-target complex formation to occur, a slow off-rate enrichment process is introduced, in which nucleic acid-target complexes with fast off-rates dissociate and do not reform, while complexes with slow off-rates remain intact. Methods for introducing a slow off-rate enrichment process include, but are not limited to, adding a competitor molecule to a mixture of nucleic acid and target molecule, diluting the mixture of nucleic acid and target molecule, or a combination of both. The present disclosure further describes aptamers and photoaptamers obtained using these methods.

[0012] In one embodiment, the method includes the steps of preparing a candidate mixture of nucleic acids; contacting the candidate mixture with a target molecule, wherein nucleic acids with the highest relative affinity for the target molecule preferentially bind to the target molecule and form nucleic acid-target molecule complexes; introducing a slow off-rate enrichment process to induce dissociation of nucleic acid-target molecule complexes with relatively fast off-rates; partitioning the remaining bound nucleic acid-target molecule complexes from unbound (free) nucleic acids in the candidate mixture; and identifying the nucleic acids bound to the target molecule. The process may further include the repeated steps of amplifying the nucleic acids that bind to the target molecule to obtain a nucleic acid mixture enriched for nucleic acids that bind to the target molecule and produce nucleic acid-target molecule complexes with slow off-rates.

[0013] In another embodiment, the candidate mixture of nucleic acids includes nucleic acids containing modified nucleotide bases that may support the formation of modified nucleic acid-target complexes with slow dissociation rates. Improved methods for performing SELEX with modified nucleotides, including nucleotides containing photolabile groups or other functional groups, or nucleotides containing placeholders for photolabile groups, are incorporated herein by reference in their entirety, filed concurrently herewith. No. 12 / 175,388, filed July 17, 2008, entitled "Improved SELEX and PHOTOSELEX," which is incorporated herein by reference. Position-preserving nucleotides may also be used for the introduction of non-photoreactive modified nucleotides during or after SELEX.

[0014]

[0013] Using the various methods and processes described herein, it is possible to generate aptamers that are either (1) capable of binding to a target molecule or (2) capable of binding to a target molecule and subsequently forming a covalent bond with the target molecule when irradiated with light in the UV or visible spectrum.

[0015]

[0014] In another aspect, the various methods and processes described herein can be used to generate aptamers capable of modifying the biological activity of target molecules through binding and / or cross-linking to the target molecules. In one embodiment, aptamers are identified for unique target molecules implicated in or associated with a particular disease process. The aptamers can be used as diagnostic reagents either in vitro or in vivo. In another embodiment, aptamers for target molecules associated with a disease state can be administered to an individual and used to treat the disease in vivo. The aptamers and photoaptamers identified herein can be used, without limitation, in any diagnostic, imaging, high-throughput screening, or target validation technique or method or assay that can use aptamers, oligonucleotides, antibodies, and ligands. For example, the aptamers and photoaptamers identified herein can be used according to the methods described in detail in co-filed U.S. Application No. ____, entitled "Multiplexed Analyses of Test Samples," which is incorporated herein by reference in its entirety.

[0016] 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, and in nearly all of these uses, the slow off-rate aptamers will have improved performance compared to aptamers that have not been selected to have slow off-rate properties.

[0017] The aptamer Macugen® (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 function due to its specific affinity for VEGF. Other aptamers are being investigated and / or developed for use as therapeutic agents. Aptamers that are not selected to have slow off-rate properties also find use 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-throughput screening, and other applications. Slow off-rate aptamers of the present disclosure have also been used in sequencing (see, e.g., U.S. Pat. Nos. 6,329,145; 6,670,132; and 7,258,980, each of which is incorporated herein in its entirety) and PCR kits (see, e.g., U.S. Pat. Nos. 6,183,967; 6,020,130; 5,763,173; 5,874,557; and 5,693,502, each of which is incorporated herein in its entirety). The slow off-rate aptamers of the present disclosure may be used in any diagnostic, therapeutic, imaging, or any other use in which antibodies, aptamers, and ligand binding pairs have been used.

[0018]

[0017] In another aspect, the present disclosure provides aptamers and photoaptamers identified by the improved methods disclosed herein, diagnostic kits containing such aptamers and photoaptamers, and therapeutic and diagnostic uses of such aptamers and photoaptamers. Novel aptamers and photoaptamers with slow off-rates identified using the described methods can be used in a variety of assays, including assays using planar arrays, beads, and other types of solid supports. The assays may be used in a variety of contexts, including life science research applications, clinical diagnostic applications (e.g., diagnostic tests for disease, or "wellness" tests for preventive health care); ALONA and UPS assays; and in vivo imaging applications. For some applications, multiplexed assays using the described aptamers and photoaptamers may be used.

[0019] In some embodiments, the slow off-rate aptamers (or photoaptamers) described herein may be used as intravenous or oral contrast agents for CAT scans and other imaging applications. CAT scans are used to diagnose muscle and bone disorders, locate blood clots, detect internal bleeding, monitor diseases such as cancer, and the like. Slow off-rate aptamers may be labeled with a CAT scan-detectable moiety, such as iodine, barium, or gastrografin. In addition to carrying a detectable moiety, aptamers may be designed to direct the moiety to a specific tissue or desired target. Aptamers can act to concentrate or localize the detectable moiety, thus improving the signal-to-noise ratio by increasing the available signal. The off-rate of an aptamer may be sufficiently slow that the scan duration may be increased, improving the signal-to-noise ratio of the scan. The specificity of the aptamer for its target can also improve the signal-to-noise ratio in these imaging applications.

[0020]

[0019] In one embodiment, the slow-off aptamer is labeled with a diamagnetic or paramagnetic substance. In this embodiment, the labeled aptamer can also be used to improve the performance of magnetic resonance imaging (MRI). MRI is particularly well suited for imaging small, selected regions and tissues with high water content or for monitoring blood flow. The specificity of the slow-off aptamer can also improve the localization of MRI reagents to desired tissue regions. Similarly, for use in PET scans, the slow-off aptamer can be modified with substances such as fluorine, carbon-11, oxygen-15, or nitrogen-13. In another embodiment, the aptamer can be labeled with an IR-active substance that can be used for infrared imaging. It is also contemplated that the slow-off aptamer can be labeled for use in other imaging modalities.

[0021] In one embodiment, slow-off aptamers may be used as highly sensitive and specific reagents for incorporation into various in vitro diagnostic methods or kits. In some embodiments, slow-off aptamers are used as surrogates for antibodies in methods for detecting certain infectious or other types of diseases, where the aptamer for the target of interest includes a detectable substance and either or both of an immobilization or capture component. In these embodiments, various assay formats are available after mixing the aptamer from the kit with a clinical specimen. In one embodiment, the aptamer also includes a detectable label, such as a fluorophore. In other embodiments, assay formats may include fluorescence quenching, hybridization, flow cytometry, mass spectrometry, inhibition or competition, enzyme-linked oligonucleotide assays, SPR, evanescent light, and the like. In some embodiments, the aptamer is provided in solution in the kit. In other embodiments, the aptamer in the kit is immobilized on a solid support that is used in conjunction with an assay to test 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 for extracting the target of interest, for amplifying the aptamer, for performing washes, detection reagents, and the like.

[0022] In another embodiment, slow-off aptamers can be used in therapeutic imaging studies. During the development of new therapeutic compounds, it is often difficult to evaluate certain properties of the compound, such as biodistribution, washout rate, bioavailability, and in vivo drug / target interactions. In many cases, if the therapeutic compound is modified with an appropriate detectable substance, imaging studies can be used to evaluate all of these properties. While direct modification of a therapeutic compound often inhibits its ability to interact with the target and therefore reduces efficacy, the small size and customizable specificity of aptamers make them well-suited to react with therapeutic compounds (e.g., antibodies or other protein-based therapeutics) while potentially minimizing any undesirable effects on the compound's therapeutic efficacy. To evaluate such properties, such as biodistribution and washout rate, the aptamer / therapeutic complex can also be persisted for long periods of time. These types of studies can be simplified if the therapeutic compound is an aptamer with a slow off-rate. 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, such as nucleases and other sample or body fluid components, that may be present in a test sample or in vivo. [Brief explanation of the drawings]

[0023] [Figure 1A] FIG. 1A illustrates an exemplary SELEX method, and FIG. 1B illustrates an exemplary SELEX method that includes incorporating one or more slow off-rate enrichment processes. [Figure 1B]FIG. 1A illustrates an exemplary SELEX method, and FIG. 1B illustrates an exemplary SELEX method that includes incorporating one or more slow off-rate enrichment processes. [Figure 2] The figure illustrates representative aptamer template, primer, and complementary oligonucleotide sequences used in this disclosure. Oligonucleotides were prepared by standard solid-phase synthesis techniques. B = dT-biotin. [Figure 3]

[0024] Figure 3 illustrates histograms of dissociation rate constants for affinity aptamers selected without (A) and with (B) the slow off-rate enrichment process, as described in Example 2. [Figure 4A]

[0025] Figures 4A and 4B show the oligonucleotides used to prepare the candidate mixture or perform various steps in the selection process described in Examples 3 and 4. The oligonucleotides were prepared by standard solid-phase synthesis techniques. In this example, two candidate mixture sequences, designated 1 and 2, were used. B = dT-biotin. BrdU (5-bromo-dUTP), anthraquinone (AQ), and psoralen (Psor) chromophores were purchased as phosphoramidites and added to the 5' end of the forward primer during synthesis. 4-azido-2-nitro-aniline (ANA) was prepared as a para-nitro-phenylcarbonate derivative and coupled to the 5' hexylamine phosphoramidite after synthesis. In this example, two candidate mixture sequences, designated 1 and 2, were used. (A) Template 1 was used with the candidate mixture containing 5'-BrdU, AQ, and ANA, and (B) Template 2 was used with the candidate mixture containing 5'-Psor. [Figure 4B]

[0025] Figures 4A and 4B show the oligonucleotides used to prepare the candidate mixture or perform various steps in the selection process described in Examples 3 and 4. The oligonucleotides were prepared by standard solid-phase synthesis techniques. In this example, two candidate mixture sequences, designated 1 and 2, were used. B = dT-biotin. BrdU (5-bromo-dUTP), anthraquinone (AQ), and psoralen (Psor) chromophores were purchased as phosphoramidites and added to the 5' end of the forward primer during synthesis. 4-Azido-2-nitro-aniline (ANA) was prepared as a para-nitro-phenylcarbonate derivative and coupled to the 5' hexylamine phosphoramidite after synthesis. In this example, two candidate mixture sequences, designated 1 and 2, were used. (A) Template 1 was used with a candidate mixture containing 5'-BrdU, AQ, and ANA, and (B) template 2 was used with a candidate mixture containing 5'-Psor. [Figure 5-1]

[0026] FIG. 5 illustrates the chemical structure of a chromophore coupled to the 5′ end of a forward primer as illustrated in FIGS. 4A and 4B. [Figure 5-2] FIG. 5 illustrates the chemical structure of the chromophore coupled to the 5' end of the forward primer as illustrated in FIGS. 4A and 4B. [Figure 6]

[0027] Figure 6 illustrates PAGE analysis of the cross-linking activity of the TIMP-3 5' ANA / BzdU-enriched library using 5'-anchored photoSELEX as described in Example 3. The gel illustrates the separation of unbound aptamer (Af), intramolecularly cross-linked aptamer (Af*), and cross-linked protein:aptamer complexes (P:A). [Figure 7-1]

[0028] FIG. 7 is a chart of over 500 targets for which slow off-rate aptamers have been identified. [Figure 7-2]

[0028] Figure 7 is a chart of over 500 targets for which slow off-rate aptamers have been identified. [Figure 8]

[0029] 8A-8D illustrate aptamer constructs containing a variety of different and optional functionalities, including immobilization tags, labels, photocrosslinking moieties, spacers, and releasable moieties. [Figure 9A]

[0030] 9A-9F illustrate examples of aptamer constructs that include a cleavable or releasable element, a tag (eg, biotin), a spacer, and a label (eg, Cy3). [Figure 9B] 9A-9F illustrate examples of aptamer constructs that include a cleavable or releasable element, a tag (eg, biotin), a spacer, and a label (eg, Cy3). [Figure 9C] 9A-9F illustrate examples of aptamer constructs that include a cleavable or releasable element, a tag (eg, biotin), a spacer, and a label (eg, Cy3). [Figure 9D] 9A-9F illustrate examples of aptamer constructs that include a cleavable or releasable element, a tag (eg, biotin), a spacer, and a label (eg, Cy3). [Figure 9E] 9A-9F illustrate examples of aptamer constructs that include a cleavable or releasable element, a tag (eg, biotin), a spacer, and a label (eg, Cy3). [Figure 9F] 9A-9F illustrate examples of aptamer constructs that include a cleavable or releasable element, a tag (eg, biotin), a spacer, and a label (eg, Cy3). [Figure 10]

[0031] Figure 10 illustrates the aptamer and primer constructs described in this disclosure. Cy3 represents the cyanine 3 dye, PC represents the photocleavable linker, ANA represents the photoreactive crosslinking group, (AB)2 represents a pair of biotin residues separated by a dA residue, and (T)8 represents the poly-dT linker. The primer construct is complementary to the entire 3' anchored region of the aptamer construct. [Figure 11A]

[0032] 11A-11C illustrate dose-response curves of slow off-rate aptamers relative to traditional aptamers for three different targets. [Figure 11B]

[0032] Figures 11A-11C illustrate dose-response curves of slow off-rate aptamers relative to traditional aptamers for three different targets. [Figure 11C]

[0032] Figures 11A-11C illustrate dose-response curves of slow off-rate aptamers relative to traditional aptamers for three different targets. [Figure 12A]

[0033] 12A and 12B illustrate the performance curves of slow off-rate aptamers whose targets are peptides. [Figure 12B] 12A and 12B illustrate performance curves of slow off-rate aptamers whose targets are peptides. [Figure 13]

[0034] FIG. 13 illustrates a plot of the measured melting temperatures of several slow off-rate aptamers compared to the predicted melting temperatures. [Figure 14-1]

[0035] Figure 14 describes the base modifications of nucleotides included in this disclosure. The available R groups are listed, along with the nucleotide attachment point and available linkers (X) between the R groups. The modified position of the nucleotide is also shown. [Figure 14-2]

[0035] Figure 14 describes base modifications of nucleotides included in this disclosure. Available R groups are listed, along with available linkers (X) between the nucleotide attachment point and the R group. The position of the modification on the nucleotide is also shown. [Figure 15]

[0036] FIG. 15 illustrates the plot used in determining the binding constants of aptamers containing C-5 modified pyrimidines. DETAILED DESCRIPTION OF THE INVENTION

[0024]

[0037] The practice of the invention disclosed herein employs, unless otherwise indicated, conventional methods of chemistry, microbiology, molecular biology, and recombinant DNA technology, within the level of skill in the art. These techniques are fully explained in the literature. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (current edition); DNA Cloning: A Practical Approach, vols. I and II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., current edition); Nucleic Acid Hybridization (B. Hames and S. Higgins, eds., current edition); Transcription and Translation (B. Hames and S. Higgins, eds., current edition).

[0025]

[0038] 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 this invention pertains. All publications, published patent documents, and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication, published patent document, or patent application was specifically and individually indicated to be incorporated by reference herein.

[0026]

[0039] As used herein, 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 a mixture of aptamers, reference to "a probe" includes a mixture of probes, and so forth.

[0027]

[0040] As used herein, the term "about" indicates an insignificant modification or variation of a numerical value such that the basic function of the item to which the numerical value is associated remains unchanged.

[0041] As used herein, the terms "comprises," "comprising," "includes," "including," "contains," "containing," and any variations thereof, are intended to include non-exclusive inclusions, and thus the process, method, product of a process, or composition in question that includes, contains, or contains an element or list of elements does not include only those elements. Other elements, not included, but not expressly listed or essential to such process, method, product of a process, or composition in question, may also be included.

[0028]

[0042] As used herein, the terms "nucleic acid ligand," "aptamer," and "clone" are used interchangeably to refer to a non-naturally occurring nucleic acid that has or can have a desired effect on a target molecule. Desired effects include, but are not limited to, binding to the target, catalytically altering the target, reacting with the target in a manner that modifies or alters the target or its functional activity, covalently binding to the target (as in a suicide inhibitor), and facilitating a reaction between the target and another molecule. In one embodiment, the effect is specific binding affinity to a target molecule, where such a target molecule is a three-dimensional chemical structure other than a polynucleotide that binds to the aptamer primarily through a mechanism that does not rely on Watson / Crick base pairing or triple helix binding, and where the aptamer is not a nucleic acid with a known physiological function that binds to the target molecule. Aptamers include nucleic acids identified from a candidate mixture of nucleic acids by a method comprising: (a) contacting a target with a candidate mixture, wherein nucleic acids having increased affinity for the target relative to other nucleic acids in the candidate mixture may be partitioned from the remainder of the candidate mixture; (b) partitioning nucleic acids with increased affinity and / or slow off-rates from the remainder of the candidate mixture; and (c) amplifying the nucleic acids with increased affinity to obtain a ligand-enriched mixture of nucleic acids, thereby identifying aptamers for the target molecule, where the aptamer is a ligand for a given target. 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 with a generally much higher degree of affinity than it can bind to other non-target components in the mixture or sample. An "aptamer" or "nucleic acid ligand" (singular) is a set of copies of one type or species of nucleic acid molecule having a specific nucleotide sequence. Aptamers can include any suitable number of nucleotides. "Aptamers" refers to more than one such set of molecules. Different aptamers can have either the same or different numbers of nucleotides.Aptamers may be DNA or RNA, and may be single-stranded, double-stranded, or contain double-stranded regions.

[0029]

[0043] As used herein, "slow off-rate" or "slow rate of dissociation" or "slow off-rate" refers to the time it takes for an aptamer / target complex to begin to dissociate. This is also referred to as the half-life, t 1 / 2 t, or the time point at which 50% of the aptamer / target complex has dissociated. 1 / 2 The off-rate or dissociation rate of a slow off-rate aptamer, expressed as a value, may be about 30 minutes or more, 60 minutes or more, 90 minutes or more, 120 minutes or more, 150 minutes or more, 180 minutes or more, 210 minutes or more, and about 240 minutes or more.

[0030]

[0044] In one embodiment, a method for producing a synthetic library of nucleic acids comprises the steps of: 1) synthesizing nucleic acids; 2) deprotecting nucleic acids; 3) purifying nucleic acids; and 4) analyzing nucleic acids. In the synthesis step, a monomer mixture is prepared, in which the ratio of various nucleotides in the mixture is optimized to produce equal proportions of each nucleotide in the final product. One or more monomers in the mixture may contain modified nucleotides. In this method, an amidite protecting group is used, and in one embodiment, the monomer concentration is 0.1 M. During synthesis, the 5' protecting group is retained in the product nucleic acid. Synthesis is performed on a solid support (controlled pore glass, CPG), and at least about 80 cycles are completed to synthesize the final product.

[0031]

[0045] After the synthesis process, the nucleic acid product is deprotected. Apurinic base sites are cleaved using 1.0 M aqueous lysine buffer, pH 9.0, while the product is attached to a support (controlled pore glass, The truncated sequences are then retained on a CPG (Cell Probe Graphene Gel). These cleaved truncated sequences are washed twice with deionized water (dI). After the two washes, 500 μL of dI water is added in preparation for the deprotection step. This step involves treatment with 1.0 mL of t-butylamine:methanol:water (1:1:2) at 70°C for 5 hours, followed by freezing, filtration, and evaporation to dryness. The nucleic acid products are purified on a PRP-3 HPLC column (Hamilton) based on the hydrophobicity of the protecting groups. Appropriate column fractions are collected and pooled, desalted, and evaporated to dryness to remove the volatile elution buffer. The final products are washed with water by a centrifugation process and then resuspended. Finally, the resuspended material is processed to deprotect the final products. The final products are characterized by base composition, primer extension, and sequencing gel.

[0032]

[0046] Alternatively, a solid-phase enzymatic method can be used to generate a candidate mixture of nucleic acids. In one embodiment, this method includes the same basic steps as described above. In this case, the goal is to synthesize antisense libraries, and these libraries are produced with 5' biotin modifications. All remaining synthesis processes are as described above. Once the synthetic library is prepared, the nucleic acids can be used in a primer extension mixture containing one or more modified nucleotides to generate a final candidate mixture in a classical primer extension method.

[0033]

[0047] Aptamers can be synthesized using the same chemical reactions used to synthesize libraries. However, instead of a mixture of nucleotides, a single nucleotide is introduced at each step of the synthesis to adjust the final sequence produced by routine methods. Modified nucleotides can be introduced during the synthesis process at desired positions in the sequence. Known chemical modifications of nucleotides can also be used to introduce other functionalities as needed.

[0034]

[0048] As used herein, a "candidate mixture" refers to a mixture of nucleic acids with different sequences from which a desired ligand is to be selected. The source of the candidate mixture may be naturally occurring nucleic acids or fragments thereof, chemically synthesized nucleic acids, enzymatically synthesized nucleic acids, or nucleic acids produced by a combination of the aforementioned techniques. Modified nucleotides, such as nucleotides with photoreactive groups or other modifications, may be incorporated into the candidate mixture. Furthermore, a candidate mixture may be produced using SELEX; ​​i.e., a first SELEX experiment may be used to produce a ligand-enriched nucleic acid mixture, which may then be used as the candidate mixture in a second SELEX experiment. A candidate mixture may also contain nucleic acids with one or more common structural motifs. As used herein, a candidate mixture is sometimes also referred to as a "pool" or "library." For example, an "RNA pool" refers to a candidate mixture composed of RNA.

[0035]

[0049] In various embodiments, each nucleic acid in the candidate mixture may have fixed ends on either side of the randomized region to facilitate the amplification process. The nucleic acids in the candidate mixture of nucleic acids may further comprise fixed regions or "tail" sequences at their 5' and 3' ends, respectively, to prevent the formation of high molecular weight parasites during the amplification process.

[0036]

[0050] As used herein, "nucleic acid," "oligonucleotide," and "polynucleotide" are used interchangeably to refer to a polymer of nucleotides of any length, which 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.

[0037]

[0051] When present, chemical modifications of the nucleotide include, alone or in any combination, 2'-sugar modifications, 5-pyrimidine modifications (e.g., 5-(N-benzylcarboxymethyl)-N-methyl ...Amido)-2'-deoxyuridine, 5-(N-isobutylcarboxamido)-2'-deoxyuridine, 5-(N-[2-(1H-indol-3yl)ethyl]carboxamido)-2'-deoxyuridine, 5-(N-[1-(3-trimethylammonium)propyl]carboxamido)-2'-deoxyuridine chloride, 5-(N-naphthylcarboxamido)-2'-deoxyuridine, or 5-(N-[1-(2,3-dihydroxypropyl)]carboxamido)-2'-deoxyuridine), 8-position purine modifications, modifications with exocyclic amines, 4-thiouridine substitution, 5-bromo- or 5-iodouracil substitution, backbone modifications, methylation, unusual base pairing combinations such as isobases, isocytidine, and isoguanidine, etc. Modifications may also include 3' and 5' modifications such as capping or PEGylation. Other modifications may include substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications, such as modifications with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), and modifications with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), modifications with intercalating agents (e.g., acridine, psoralens, etc.), modifications containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), modifications containing alkylating agents, and modifications with modified linkages (e.g., alpha-anomeric nucleic acids, etc.). Additionally, any hydroxyl groups normally present on the sugar may be replaced with phosphonate or phosphate groups; protected with standard protecting groups; or activated for further linkage to additional nucleotides or solid supports. The 5' and 3' terminal OH groups may be phosphorylated or substituted with amines, organic capping group moieties of about 1 to about 20 carbon atoms, or organic capping group moieties of polyethylene glycol (PEG) polymers or other hydrophilic or hydrophobic biological or synthetic polymers of about 1 to about 20 carbon atoms. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The 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.

[0038]

[0052] Polynucleotides may also contain analogs of ribose or deoxyribose sugars commonly known in the art, including 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, 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 those in which the phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H, or a substituted or unsubstituted alkyl (1-20C) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. Similar types of substitutions of sugars, purines, and pyrimidines may be advantageous in the design of the final product, and alternative backbone structures, such as polyamide backbones, may also be advantageous.

[0039]

[0053] In one embodiment, the variable region of the aptamer includes nucleotides containing 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 with very slow off-rates from their respective targets while maintaining high affinity for the targets. In one embodiment, the C-5 position of the pyrimidine base may be modified. Aptamers containing nucleotides with modified bases have several properties that differ from those of standard aptamers containing only naturally occurring nucleotides (i.e., unmodified nucleotides). In one embodiment, the nucleotide modification method includes the addition of amino acids. Modifications include the use of modified linkages. However, other suitable modification methods may also be used. Surprisingly, it was observed that the structures of identified slow-off aptamers do not appear to completely match those predicted by standard base-pairing models. This observation is supported by the fact that the measured melting temperatures of slow-off aptamers do not match those predicted by the models. See Figure 13. As shown, there appears to be no correlation between the measured and predicted melting temperatures of slow-off aptamers. The calculated melting temperatures (Tm) are, on average, 6°C lower than the measured Tm. The measured melting temperatures indicate that slow-off aptamers containing these modified nucleotides are more stable than could be predicted and potentially possess novel secondary structures. These modified aptamers also have different circular dichroism spectra than corresponding aptamers containing only unmodified nucleotides. For many targets, slow-off aptamers to the target are more likely to be identified if modified nucleotides are used during the generation of the initial library or candidate mixture.

[0040]

[0054] Specific 5-position pyrimidine modifications include those described in US Pat. Nos. 5,719,273 and 5,945,527, and those illustrated in FIG.

[0055] As used herein, "modified nucleic acid" refers to a nucleic acid sequence that contains one or more modified nucleotides. In some embodiments, it may be desirable for modified nucleotides to be compatible with the SELEX methodology.

[0041]

[0056] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and / or may be interrupted by non-amino acids. The terms also include amino acid polymers that are modified, either naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or 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 may be single chains or associated chains.

[0042]

[0057] As used herein, the term "photoreactive nucleotide" refers to any modified nucleotide that can be photocrosslinked with a target, such as a protein, upon irradiation with light of a specific wavelength. For example, photoaptamers produced by the photoSELEX method include 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. The photoreactive group may be selected from the group consisting of 5-iodo-2'-deoxyuridine, 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. "Photoreactive pyrimidine" refers to any modified pyrimidine that is capable of photocrosslinking with a target upon irradiation with a specific wavelength. 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 light at a wavelength that is not absorbed by the target or the unmodified portion of the oligonucleotide.

[0043]

[0058] "SELEX" refers to a process that combines the selection of nucleic acids that interact with a target in a desired manner (e.g., bind to a protein) with the amplification of these selected nucleic acids. Optionally, repeated cycling of the selection / amplification steps allows for the selection of one or a few nucleic acids that interact most strongly with the target from a pool containing a much larger number of nucleic acids. The cycling of the selection / amplification process continues until the selected goal is achieved. SELEX methodologies are described in SELEX patents. In some embodiments of the SELEX method, aptamers that bind non-covalently to the target are generated. In other embodiments of the SELEX method, aptamers that bind covalently to the target are generated.

[0044]

[0059] As used herein, the term "amplification" or "amplifying" refers to any process or combination of process steps that increases the amount or copy number of a molecule or class of molecules.

[0045]

[0060] "SELEX target" or "target molecule" or "target," as used herein, refers to any compound with which a nucleic acid may act in a desired manner. SELEX target molecules can be, without limitation, proteins, peptides, nucleic acids, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, viruses, pathogens, toxic substances, substrates, metabolites, transition-state analogs, cofactors, inhibitors, drugs, dyes, nutrients, growth factors, cells, tissues, any portion or fragment of any of the foregoing, and the like. In one embodiment, SELEX targets do not include molecules known to bind to nucleic acids, such as known nucleic acid-binding proteins (e.g., transcription factors). Virtually any chemical or biological effector can be a suitable SELEX target. Molecules of any size can serve as SELEX targets. Targets can also be modified in certain ways to enhance the likelihood or strength of interaction between the target and nucleic acid. A target also includes any insignificant variation of a particular compound or molecule, e.g., in the case of a protein, may include, for example, insignificant variation of the amino acid sequence, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component, that 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 can bind to an aptamer. "Target molecules" or "targets" refer to more than one such set of molecules. An embodiment of the SELEX method in which the target is a peptide is described in U.S. Patent No. 6,376,190, entitled "Modified SELEX Processes Without Purified Protein," which is incorporated herein by reference in its entirety. Figure 7 lists over 500 targets for which aptamers have been produced, including various aptamers with slow off-rates.

[0046]

[0061] As used herein, the terms "competitor molecule" and "competitor" are used interchangeably and refer to any molecule that can form a non-specific complex with a non-target molecule. In this context, non-target molecules include unbound aptamers, and in this case, for example, a competitor can be used to inhibit the aptamer from non-specifically binding (rebinding) 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 of such sets 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, dNTP, and pyrophosphate). In various embodiments, a combination of one or more competitors may be used.

[0047]

[0062] As used herein, the term "non-specific complex" refers to a non-covalent bond between two or more molecules other than an aptamer and its target molecule. A non-specific complex corresponds to an interaction between molecular classes. Non-specific complexes include complexes formed between an aptamer and a non-target molecule, between a competitor and a non-target molecule, between a competitor and a target molecule, and between a target molecule and a non-target molecule.

[0048]

[0063] As used herein, the term "slow off-rate enrichment process" refers to a process that alters the relative concentrations of specific components of a candidate mixture such that the relative concentration of aptamer affinity complexes with slow dissociation rates is increased relative to the concentration of aptamer affinity complexes with 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, the solution-based slow off-rate enrichment process is performed in solution such that neither the target nor the nucleic acid that forms the aptamer affinity complex in the mixture is immobilized on a solid support during the slow off-rate enrichment process. In various embodiments, the slow off-rate enrichment process includes one or more steps, which may include adding and incubating a competitor molecule with the molecule, diluting the mixture, or a combination thereof (e.g., diluting the mixture in the presence of a competitor molecule). Because the effectiveness of a slow off-rate enrichment process generally depends on the different 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 to substantially reduce the number of aptamer affinity complexes with fast dissociation rates while retaining a high proportion of aptamer affinity complexes with slow dissociation rates. A slow off-rate enrichment process may be used in one or more cycles during the SELEX process. When dilution and competitor addition are used in combination, these may be performed simultaneously or sequentially in any order. A slow off-rate enrichment process may 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 may be diluted as much as practical, keeping in mind that nucleic acids retained by aptamers will be recovered for subsequent rounds of the SELEX process. In one embodiment, the slow off-rate enrichment process involves the use of a competitor as well as dilution, allowing the mixture to be diluted less than would be necessary without the use of the competitor.

[0049]

[0064] In one embodiment, the slow off-rate enrichment process includes the addition of a competitor, which is a polyanion (e.g., heparin or dextran sulfate (dextran)). Heparin or dextran has been used in previous SELEX selections to identify specific aptamers. However, in these methods, the heparin or dextran is present during the equilibrium step, during which the target and aptamer bind to form a complex. In these 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, high concentrations 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. In contrast, the method described here adds the competitor after target / aptamer complexes have been allowed to form and therefore does not affect the number of complexes formed. Addition of a competitor after equilibrium binding between target and aptamer creates a non-equilibrium state that evolves until a new equilibrium is reached containing fewer target / aptamer complexes. Because fast off-rate aptamers dissociate first, capturing target / aptamer complexes before the new equilibrium is reached enriches the sample for slow off-rates.

[0050]

[0065] In another embodiment, polyanionic competitors are used during the slow off-rate enrichment process. The use of a combination agent (e.g., dextran sulfate or another polyanionic substance) facilitates the identification of aptamers that are resistant to degradation in the presence of polyanions. In this context, a "polyanion-resistant aptamer" is an aptamer that can form an aptamer / target complex that also contains a polyanion-resistant substance, which is less likely to dissociate in solution than an aptamer / target complex containing a non-polyanion-resistant aptamer. In this manner, when the detection method involves the use of a polyanion-resistant substance (e.g., dextran sulfate) that the polyanion-resistant aptamer is resistant to degradation, the aptamer can also be used in performing an analytical method to detect the presence, amount, or concentration of a target in a sample.

[0051]

[0066] Thus, in one embodiment, a method for producing polyanionic degradable aptamers is provided. In this embodiment, a candidate mixture of nucleic acids is contacted with a target, and then the target and nucleic acids in the candidate mixture are allowed to reach equilibrium. A polyanionic competitor is introduced into the solution and allowed to incubate with the competitor for a period of time sufficient to ensure that the majority of fast-off-rate aptamers in the candidate mixture dissociate from the target molecule. Additionally, aptamers in the candidate mixture that can dissociate in the presence of the polyanionic competitor will be released from the target molecule. The mixture is partitioned to isolate high-affinity, slow-off-rate aptamers that remain associated with the target molecule, and any uncomplexed material is removed from the solution. The aptamers can then be released from the target molecule and isolated. The isolated aptamers can also be amplified and subjected to additional rounds of selection to increase the overall performance of the selected aptamers. If selection of aptamers with slow off-rates is not required for a particular application, this process can also be used with minimal incubation times.

[0052]

[0067] Therefore, in one embodiment, a modified SELEX method is provided for identifying or producing aptamers with slow (long) off-rates, wherein a target molecule and a candidate mixture are contacted and incubated together for a period sufficient for equilibrium binding between the target molecule and the nucleic acid contained in the candidate mixture to occur.After equilibrium binding, an excess of competitor molecules, such as a polyanion competitor, is added to the mixture and incubated with the excess of competitor molecules for a predetermined period.A significant proportion of aptamers with off-rates shorter than this predetermined incubation period will dissociate from the target during this predetermined incubation period.Since excess competitor molecules may nonspecifically bind to the target and occupy the target binding site, the re-association of these "fast" off-rate aptamers with the target is minimized.A significant proportion of aptamers with longer off-rates will remain complexed with the target during the predetermined incubation period. At the end of the incubation period, the nucleic acid-target complexes are partitioned from the remainder of the mixture, allowing for the separation of the population of slow-off-rate aptamers from those with fast off-rates. A dissociation step may be used to dissociate the slow-off-rate aptamers from the target, allowing for the isolation, identification, sequencing, synthesis, and amplification of slow-off-rate aptamers (either individual aptamers or groups of slow-off-rate aptamers) with high affinity and specificity for the target molecule. As with traditional SELEX, the aptamer sequences identified from one round of the modified SELEX method may be used to synthesize a new candidate mixture, and the steps of contacting, equilibrium binding, addition of competitor molecules, incubation with competitor molecules, and partitioning of slow-off-rate aptamers may then be repeated / repeated as many times as necessary.

[0053]

[0068] By combining the steps of allowing equilibrium binding of the target and candidate mixture before adding the competitor, followed by adding excess competitor and incubating with the competitor for a predetermined period of time, it becomes possible to select a population of aptamers with off-rates much longer than previously achieved.

[0054]

[0069] To achieve equilibrium binding, the candidate mixture may be incubated with the target for at least about 5 minutes, or for 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.

[0055]

[0070] The predetermined incubation period of the candidate mixture and the mixture of target molecules with the competitor molecule can be selected as needed, taking into consideration factors such as the nature of the target and the known off-rates (if any) of known aptamers to the target, etc. The predetermined incubation period can be selected 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, and at least about 6 hours.

[0056]

[0071] In other embodiments, dilution may be used as the off-rate enhancement process, and the diluted candidate mixture, target molecule / aptamer complex, may be incubated for a predetermined period of time, which may be selected 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, or at least about 6 hours.

[0057]

[0072] Aspects of the present disclosure relate to the identification, production, synthesis, and use of slow off-rate aptamers, which have dissociation rates from non-covalent aptamer-target complexes (t ) that are higher than those of aptamers typically obtained by conventional SELEX. 1 / 2 ) for a mixture containing a non-covalent complex of aptamer and target. 1 / 2corresponds to the time it takes for half of the aptamers to dissociate from the aptamer-target complex. The t of the slow dissociation aptamers described in this disclosure 1 / 2 is selected from one of: about 30 minutes or more; about 30 minutes to 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; and about 210 minutes to about 240 minutes.

[0058]

[0073] A characteristic feature of aptamers identified by the SELEX method is their high affinity for their target. The aptamer has a dissociation constant (k) for its target selected from one of the following: less than about 1 μM, 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, or less than about 1 pM. d )

[0059]

[0074] "Tissue target" or "tissue" as used herein refers to a specific 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, which are typically isolated soluble molecules such as proteins. In some embodiments, tissues are insoluble macromolecules several orders of magnitude larger than simpler SELEX targets. Tissues are complex targets composed of many macromolecules, each with many potential epitopes. Different macromolecules containing many epitopes can be proteins, lipids, carbohydrates, etc., or combinations thereof. Tissues are generally physical arrays of macromolecules that can be fluid or rigid in terms of both structure and composition. The extracellular matrix is ​​an example of a more rigid tissue, while membrane bilayers are more fluid in structure and composition. Tissues are generally not soluble and remain in the solid phase, and therefore partitioning is relatively easy to achieve. Tissues include, but are not limited to, aggregates of cells, usually of a particular type, with intercellular substances forming one of the structural materials commonly used to describe the overall cellular infrastructure of a given organ, e.g., kidney tissue, brain tissue. Four general types of tissues are: Typical types are epithelial tissue, connective tissue, nervous tissue and muscle tissue.

[0060]

[0075] Examples of tissues that fall within this definition include, but are not limited to, heterogeneous aggregates of macromolecules, such as acellular fibrin clots; homogeneous or heterogeneous aggregates of cells; higher-order structures that have specific functions and contain cells, such as organs, tumors, lymph nodes, and arteries; and individual cells. Tissues or cells can be in their native environment, isolated, or in tissue culture. Tissues can be intact or modified. Modifications can include many changes, such as transformation, transfection, activation, and substructure isolation, e.g., isolation of cell membranes, nuclei, organelles, etc.

[0061]

[0076] Sources of tissues, cells or subcellular structures can be obtained from prokaryotes as well as eukaryotes, including human, animal, plant, bacterial, fungal, and viral structures.

[0062]

[0077] As used herein, the terms "labeling agent," "label," or "detectable moiety," or "detectable element," or "detectable component" refer to one or more reagents that can be used to detect a target molecule bound to an aptamer. A detectable moiety or label can be directly or indirectly detected. Generally, any detectable reporter molecule can be a label. Labels include, for example, (i) reporter molecules that are directly detectable by producing a signal; (ii) specific binding pair members that are indirectly detectable by subsequent binding to a cognate containing the reporter molecule; (iii) mass tags that are detectable by mass spectrometry; (iv) oligonucleotide primers that can provide templates for amplification or ligation; and (v) specific polynucleotide sequences or recognition sequences that can act as ligands for, for example, repressor proteins; in the latter two examples, the oligonucleotide primers or repressor proteins carry or could carry reporter molecules, etc. The reporter molecule may be a catalyst, such as an enzyme, a polynucleotide encoding the catalyst, a promoter, a dye, a fluorescent molecule, a quantum dot, a chemiluminescent molecule, a coenzyme, an enzyme substrate, a radioactive group, a small organic molecule, an amplifiable polynucleotide sequence, a particle such as latex or carbon particles, a metal sol, a microcrystal, a liposome, a cell, etc., which may or may not be further labeled with a dye, a catalyst or other detectable group, a mass tag that modifies the weight of a molecule, etc., that is conjugated to the molecule for mass spectrometry purposes. The label may be selected from electromagnetic or electrochemical. In one embodiment, the detectable label is a fluorescent dye. Based on the disclosure herein, other labels and labeling schemes will be apparent to those skilled in the art.

[0063]

[0078] Detectable moieties (elements or components) can include any of the reporter molecules listed above, as well as any other chemicals or components that can be used to generate a detectable signal in any manner. The detectable moiety may be detected via a fluorescent signal, a chemiluminescent signal, or any other detectable signal depending on the identity of the moiety. If the detectable moiety is an enzyme (e.g., alkaline phosphatase), the signal may be generated in the presence of an enzyme substrate and any additional factors required for enzymatic activity. If the detectable moiety is an enzyme substrate, the signal may be generated in the presence of the enzyme and any additional factors required for enzymatic activity. Reagent configurations suitable for attaching a detectable moiety to a target molecule include covalent binding of the detectable moiety to the target molecule, noncovalent binding of the detectable moiety to another labeling agent component covalently bound to the target molecule, and covalent binding of the detectable moiety to a labeling agent component noncovalently bound to the target molecule. Universal protein stains (UPS) are described in detail in U.S. Patent Application No. 10 / 504,696, filed August 12, 2004, entitled "Methods and Reagents for Detecting Target Binding by Nucleic Acid Ligands." It is described in.

[0064]

[0079] "Solid support," as used herein, refers to any support having a surface to which molecules can be attached, directly or indirectly, through either covalent or noncovalent bonds. Support materials may be naturally occurring, synthetic, or modified naturally occurring materials. Solid support materials may include silicon, graphite, mirrored surfaces, laminates, ceramics, plastics (e.g., poly(vinyl chloride), cycloolefin copolymers, polyacrylamide, polyacrylate, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), polytetrafluoroethylene (PTFE or Teflon), nylon, poly(vinyl butyrate)), germanium, gallium arsenide, gold, silver, etc., used alone or in combination with other materials. Additional robust materials, such as glass, including silica, and further including glass available as Bioglass, are also contemplated. Other materials that may be used include porous materials, such as controlled-pore glass beads. Any other material known in the art capable of having one or more functional groups incorporated onto its surface, such as any amino, carboxyl, thiol, or hydroxyl functional group, is also contemplated.

[0065]

[0080] Solid supports may take any of a variety of forms, ranging from simple to complex, and may have any one of several shapes, including strips, plates, disks, rods, particles including beads, test tubes, wells, etc. Surfaces may be relatively flat (e.g., slides), spherical (e.g., beads), cylindrical (e.g., columns), or grooved. Exemplary solid supports that can be used include microtiter wells, microscope slides, membranes, paramagnetic beads, charged paper, Langmuir-Blodgett membranes, silicon wafer chips, flow-through chips, and microbeads.

[0066]

[0081] As used herein, "partitioning" refers to any process that separates one or more components of a mixture from other components of the mixture. For example, aptamers bound to a target molecule may be partitioned from other nucleic acids not bound to the target molecule and from non-target molecules. More broadly, partitioning allows all nucleic acids in a candidate mixture to be separated into at least two pools based on their relative affinity to the target molecule and / or dissociation rate. Partitioning can be achieved by a variety of methods known in the art, including filtration, affinity chromatography, liquid-liquid partitioning, HPLC, etc. For example, nucleic acid-protein pairs can bind to a nitrocellulose filter, while unbound nucleic acids do not. Columns that specifically retain nucleic acid-target complexes can also be used for partitioning. For example, in the case of oligonucleotides capable of associating with a target molecule bound to a column, column chromatography can be used to separate and isolate the aptamer with the highest affinity. Beads to which target molecules are conjugated can also be used to partition aptamers in a mixture. If the beads are paramagnetic, partitioning can be achieved by applying a magnetic field. By immobilizing a target on a sensor chip and flowing a mixture over the chip, surface plasmon resonance technology can be used to partition the nucleic acids in the mixture, where nucleic acids with affinity for the target can bind to the target and the remaining nucleic acids can be washed away. Filtration gel retardation and density gradient centrifugation can be used in addition to liquid-liquid partitioning. Also, affinity tags on target molecules can be used to separate nucleic acid molecules bound to tagged targets from unbound aptamers in solution. For example, streptavidin paramagnetic beads can be used to separate biotinylated target molecules, along with aptamers bound to the molecules, from a solution of unbound nucleic acid sequences. During preparation, affinity tags can also be incorporated into aptamers. stomach.

[0067]

[0082] As used herein, "photoSELEX" is an acronym for Photochemical Systematic Evolution of Ligands by Exponential Enrichment and refers to an embodiment of the SELEX method that generates photocrosslinking aptamers. In one embodiment of the photoSELEX method, photoreactive nucleotides activated by absorption of light are incorporated into an RNA or ssDNA randomized oligonucleotide library in place of natural bases, and a mixture of nucleic acid target molecules is irradiated with light to crosslink some of the nucleic acids incorporated into the nucleic acid-target molecule complex to the target molecule via the photoreactive functional group, and the selection step is selection for photocrosslinking activity. The photoSELEX method is described in great detail in the PhotoSELEX patent.

[0068]

[0083] As used herein, the terms "photoaptamer," "photoreactive aptamer," and "photoreactive aptamer" are used interchangeably to refer to an aptamer containing one or more photoreactive functional groups that can covalently bind to or "crosslink" a target molecule. For example, naturally occurring nucleic acid residues can be modified to contain chemical functional groups that confer photoreactivity to the nucleic acid residue upon exposure to an appropriate wavelength of radiation. In some embodiments, a photoreactive aptamer is first identified. In other embodiments, an aptamer is first identified and 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 the aptamer either by substituting a photoreactive nucleic acid residue for one or more other nucleotides, such as one or more thymidine and / or cytidine nucleotides, or by modifying one or more nucleic acid residues to contain a photoreactive functional group.

[0069]

[0084] Exemplary photoreactive functional groups that may be incorporated into photoaptamers 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-azidoguanine, 8-isopropyl ... 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.

[0070]

[0085] In addition to these exemplary nucleoside-based photoreactive functional groups, other photoreactive functional groups that can be added to the termini of aptamers using appropriate linker molecules may also be used, including benzophenone, anthraquinone, 4-azido-2-nitroaniline, psoralen, derivatives of any of these, and the like.

[0071]

[0086] The photoreactive functional group incorporated into the photoaptamer can be activated by any suitable method.In one embodiment, the photoaptamer containing the photoreactive functional group can be crosslinked to its target by exposing the photoaptamer and its bound target molecule to an electromagnetic radiation source.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.

[0072]

[0087] As used herein, the term "affinity SELEX" refers to an embodiment of the SELEX method in which non-photocrosslinkable aptamers to a target are generated. In some affinity SELEX embodiments, the target is immobilized on a solid support either before or after contacting the target with a candidate mixture of nucleic acids. The association of the target with the solid support allows for the partitioning of nucleic acids in the candidate mixture that are bound, and, if a slow off-rate enrichment process is used, those nucleic acids that remain bound to the target, from the remainder of the candidate mixture. The term "bead affinity SELEX" refers to a specific embodiment of the affinity SELEX method in which the target is immobilized on beads, for example, before contacting the candidate mixture of nucleic acids. In some embodiments, the beads are paramagnetic beads. The term "filter affinity SELEX" refers to an embodiment in which the nucleic acid-target complex is partitioned from the candidate mixture by association with a filter, such as a nitrocellulose filter. This includes embodiments in which the target and nucleic acid are first contacted in solution and then contacted with a filter, and also includes embodiments in which the nucleic acid is contacted with a target pre-immobilized on a filter. The term "plate affinity SELEX" refers to an embodiment in which a target is immobilized on a plate surface, such as a multi-well microtiter plate. In some embodiments, the plate is made of polystyrene. In some embodiments, in plate affinity SELEX, the target is attached to the plate through hydrophobic interactions.

[0073]

[0088] The present disclosure describes improved SELEX methods for generating aptamers capable of binding to target molecules. More specifically, the present disclosure describes methods for identifying aptamers and / or photoaptamers that have slower dissociation rates from their respective target molecules than aptamers obtained by previous SELEX methods. The present disclosure further describes aptamers and / or photoaptamers obtained by using the methods described herein, as well as methods for using the aptamers.

[0074]

[0089] In one embodiment, there is provided a method for identifying aptamers that have a slow dissociation rate from their target molecules, the method comprising the steps of: (a) preparing a candidate mixture of nucleic acid sequences; (b) contacting the candidate mixture with target molecules, wherein the nucleic acids with the highest relative affinity for the target molecules preferentially bind to the target molecules and form nucleic acid-target molecule complexes; (c) applying a slow off-rate enrichment process to allow the nucleic acid-target molecule complexes with a relatively fast dissociation rate to dissociate; (d) separating the remaining nucleic acid-target molecule complexes from both unbound nucleic acids and non-target molecules in the candidate mixture; and (e) identifying aptamers for target molecules.The method may further comprise the repeated step of amplifying the nucleic acid that binds to target molecules to produce a nucleic acid mixture enriched with sequences that can bind to target molecules and produce nucleic acid-target molecule complexes with a slow dissociation rate. As defined above, the slow off-rate enrichment process may be selected from the steps of diluting the candidate mixture containing the nucleic acid-target molecule complexes, 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 and adding at least one competitor to the candidate mixture containing the nucleic acid-target molecule complexes.

[0075]

[0090] In one embodiment, a method for identifying an aptamer that has a slow dissociation rate from its target molecule comprises: (a) preparing a candidate mixture of nucleic acids; (b) contacting the candidate mixture with a target molecule, wherein nucleic acids in the candidate mixture that have increased affinity for the target molecule relative to other nucleic acids bind to the target molecule to form a nucleic acid-target molecule complex; (c) incubating the candidate mixture and the target molecule together for a period of time sufficient to achieve equilibrium binding; (d) adding at least one competitor molecule in excess to the mixture of (c); and (e) contacting the candidate mixture from (d), the nucleic acid-target molecule complex. (f) incubating the mixture of the complex and competitor molecule for a predetermined period of time; (g) dissociating the nucleic acid-target molecule complex from the candidate mixture; (h) amplifying the unbound nucleic acid to obtain a nucleic acid mixture enriched for nucleic acid sequences capable of binding to the target molecule with increased affinity, thereby enabling the identification of aptamers for the target molecule.

[0076]

[0091] In another embodiment, a method for producing an aptamer that has a slow dissociation rate from its target molecule comprises: (a) preparing a candidate mixture of nucleic acids; (b) contacting the candidate mixture with the target molecule, wherein nucleic acids in the candidate mixture that have increased affinity for the target molecule relative to other nucleic acids bind to the target molecule to form a nucleic acid-target molecule complex; (c) incubating the candidate mixture and the target molecule together for a period of time sufficient to achieve equilibrium binding; (d) adding at least one competitor molecule in excess to the mixture of (c); and (e) contacting the candidate mixture from (d). (f) incubating the mixture of the complement mixture, nucleic acid-target molecule, and competitor molecule for a predetermined period of time; (g) dissociating the nucleic acid-target molecule complex from the candidate mixture to generate unbound nucleic acids; and (h) amplifying the unbound nucleic acids to obtain a nucleic acid mixture enriched for nucleic acid sequences capable of binding to the target molecule with increased affinity, thereby preparing or synthesizing an aptamer comprising the nucleic acid sequence identified by the process of identifying an aptamer for the target molecule.

[0077]

[0092] In another embodiment, a method is provided for identifying an aptamer that has a slow dissociation rate from its target molecule, comprising the steps of: (a) preparing a candidate mixture of nucleic acids, wherein the candidate mixture comprises modified nucleic acids in which one, some, or all of the pyrimidines in at least one or each nucleic acid of the candidate mixture are chemically modified at the 5-position; (b) contacting the candidate mixture with a target molecule, wherein nucleic acids in the candidate mixture that have increased affinity for the target molecule relative to other nucleic acids bind to the target molecule and form nucleic acid-target molecule complexes; (c) partitioning the nucleic acids with increased affinity from the remainder of the candidate mixture; and (d) amplifying the nucleic acids with increased affinity to obtain a nucleic acid mixture enriched for nucleic acid sequences capable of binding to the target molecule with increased affinity, thereby enabling identification of aptamers for the target molecule.

[0078]

[0093] In another aspect, a method is provided for producing an aptamer that has a slow dissociation rate from its target molecule, the method comprising the steps of: (a) preparing a candidate mixture of nucleic acids, wherein the candidate mixture comprises modified nucleic acids in which one, some, or all of the pyrimidines in at least one or each nucleic acid of the candidate mixture are chemically modified at the 5-position; (b) contacting the candidate mixture with a target molecule, wherein nucleic acids in the candidate mixture that have increased affinity for the target molecule relative to other nucleic acids bind to the target molecule and form nucleic acid-target molecule complexes; (c) partitioning the nucleic acids with increased affinity from the remainder of the candidate mixture; and (d) amplifying the nucleic acids with increased affinity to obtain a nucleic acid mixture enriched for nucleic acid sequences capable of binding to the target molecule with increased affinity, thereby preparing or synthesizing an aptamer comprising the nucleic acid sequence identified by the process of identifying an aptamer for the target molecule.

[0079]

[0094] In another embodiment, the dissociation rate of the aptamer from the target (t 1 / 2) is: about 30 minutes or more; about 30 minutes to 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 A selected non-covalent complex of an aptamer and its target is provided.

[0080]

[0095] In another embodiment, the aptamer has a K of about 100 nM or less for the target. d and the dissociation rate of the aptamer from the target (t 1 / 2 ) for about 30 minutes or more, and one, some, or all of the pyrimidines in the nucleic acid sequence of the aptamer are modified at the 5-position of the base. Modifications may be selected from the group of compounds shown in Figure 14, and these modifications are referred to as "base-modified nucleotides." Aptamers may be designed using any combination of desired base-modified pyrimidines.

[0081]

[0096] Improved methods for performing SELEX with modified nucleotides, including nucleotides containing photolabile groups or nucleotides containing position-maintaining groups for photolabile groups, are disclosed in U.S. Application No. 12 / 175,388, entitled "Improved SELEX and PHOTOSELEX," filed concurrently with the present application and 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 can support the formation of modified nucleic acid-target complexes with relatively slow dissociation rates.

[0082]

[0097] The various methods and processes described herein can be used to generate aptamers that are either (1) capable of binding to a target molecule or (2) capable of binding to a target molecule and subsequently forming a covalent bond with the target molecule upon irradiation.

[0083]

[0098] Aptamers identified according to the methods described herein are useful in a range of diagnostic and therapeutic methods. Aptamers with slower off-rates will bind to targets for longer periods of time. This is useful in diagnostic methods that can detect the presence, absence, amount, or quantity of target molecules using the binding of aptamers to targets, and the extended interaction period between aptamers and targets facilitates such detection. Similar advantages can be provided when using aptamers with slower off-rates in in vitro or in vivo imaging methods. The extended interaction period between aptamers and targets can improve therapeutic efficacy, for example, due to longer activation or inhibition of target molecules or downstream signaling cascades.

[0084]

[0099] Thus, in various embodiments, slow off-rate aptamers obtained, identified, or produced by the described methods may be used in various medical therapeutic or diagnostic methods (in vitro or in vivo). In one embodiment, slow off-rate aptamers may be used in disease therapy. In one embodiment, slow off-rate aptamers may be used in in vivo disease diagnostic methods. In another embodiment, slow off-rate aptamers may be used in vitro for disease diagnosis. In another embodiment, slow off-rate aptamers may be used in the manufacture of therapeutic agents (e.g., pharmaceutical compositions) for use in disease therapy or diagnostic methods, or in the manufacture of diagnostic agents. Diagnostic or therapeutic applications of slow off-rate aptamers can involve diagnostic or therapeutic outcomes depending 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 during the drug development process.

[0085]

[0100] In one embodiment, slow off-rate aptamers are suitable reagents for in vivo molecular imaging. In this embodiment, slow off-rate aptamers can also be used in vivo to detect pathologies, disease processes, or other conditions within an individual (e.g., a human or animal), where binding of the aptamer to its target is important. The presence of a specific aptamer indicates the presence of a disease process or other condition. For example, because VEGF receptors are abundantly expressed in tumors and their neovasculature, aptamers to VEGF receptors can be used in vivo to detect the presence of cancer in specific regions (e.g., tissues, organs, etc.) of an individual's body. Or, because EGF receptors are often expressed at high levels on tumor cells, aptamers to EGF receptors can be used in vivo to detect the presence of cancer in specific regions (e.g., tissues, organs, etc.) of an individual's body. That is, the molecular target is the extracellular domain (ECD) of the induced receptor, because such targets are located outside the cell and accessible through the vasculature. Furthermore, even though a certain small percentage of ECDs may be shed through biological processes including cell death, ECDs tend to be localized at the site of the lesion.

[0086]

[0101] High-affinity monoclonal antibodies, obvious candidates for molecular imaging, are no longer the reagent of choice for this application. Molecular imaging reagents need to be precise. They must have high avidity for their intended target and low avidity for other targets in humans or animals. Slow-off aptamers have unique advantages that make them desirable for use in in vivo molecular imaging. On the one hand, they are selected to have slow dissociation rate constants, thus allowing them to persist on their intended target in vivo for significant periods of time (at least approximately 30 minutes). On the other hand, slow-off aptamers are expected to have very rapid clearance from the vasculature. Slow dissociation rate constants and rapid clearance from the vasculature are two desirable properties for in vivo molecular imaging. From a kinetic perspective, a good in vivo molecular imaging reagent must remain localized at the lesion site while maintaining a low concentration of unbound reagent in the surrounding vasculature. This is a signal-to-noise constraint. A suitable signal-to-noise ratio can be obtained by signal accumulation at the lesion site amid excess signal in the vasculature, or by signal retention at the lesion site while vasculature concentration is reduced.

[0087]

[0102] Aptamers without slow off-rate properties, with molecular weights and net charges similar to those of slow off-rate aptamers, have been studied in animals and humans for over a decade. These aptamers have generally been found to be rapidly cleared from the vascular system, typically by entering the kidney and / or liver and then being further metabolized for excretion. These aptamers exhibit so-called "first-pass" clearance unless a high-molecular-weight additive (e.g., PEG) is attached to the aptamer. Experiments have been conducted with aptamers targeting tenascin-C, an extracellular protein (not ECD) found at high concentrations in some tumors. In these experiments, tenascin-C-specific aptamers were rapidly cleared and were able to remain at tumor sites due to the very high local extracellular concentration of tenascin-C. In contrast, aptamers with slow off-rates maintain the fast clearance rate of the aptamer but have a slower dissociation rate, providing a kinetic advantage and making them suitable for use with targets (e.g., ECDs on tumors) whose presence at the site of interest (e.g., lesion site) may be somewhat sparse.

[0088]

[0103] Other reagents for molecular imaging do not share the two properties of slow off-rate aptamers (i.e., slow dissociation rate and fast clearance from the body). Monoclonal antibodies often have high affinity and specificity and can have slow dissociation rate constants; however, monoclonal antibodies have very slow clearance rates from the vasculature. Short peptides identified, for example, through phage display, have fast clearance but may also have poor affinity and specificity and fast dissociation rates from their intended targets. A specific peptide type of antibody mimic, the afibo, has been shown to be a promising candidate for aptamers. Affibodies can have reasonable affinity and specificity and can have faster clearance rates than monoclonal antibodies, but to achieve slow dissociation rates from the target, affibodies often form dimers and higher order multimers, which simultaneously enhances dissociation rates and slows clearance.

[0089]

[0104] For in vivo molecular imaging, slow-off aptamers with one or more low molecular weight adducts can be used to protect the slow-off aptamers from nucleases in the body and detect the intended target once bound by the slow-off aptamers.For example, slow-off aptamers can be attacked by nucleases in blood, typically exonucleases (for DNA) that can be easily blocked by using exonuclease-resistant adducts at the 5' and 3' ends of the slow-off aptamers, or endonucleases (for RNA) that can be easily blocked by incorporating endonuclease-resistant pyrimidines (such as 2' fluoronucleotides) into the slow-off aptamers.Detection of slow-off aptamer-target complexes can be achieved by attaching a detection moiety to the slow-off aptamer. In some embodiments, detection moieties for these purposes may include cages for radioactive molecules (e.g., technetium-99), iron clusters for magnetic resonance detection, fluorine isotopes for PET imaging, etc. Modifications made to slow off-aptamers that protect the integrity of the slow off-aptamer in the body and allow detection of the intended target must be designed so that they do not interfere with the interaction of the slow off-aptamer with its target and prevent the slow off-aptamer from being cleared from the vasculature too slowly.

[0090]

[0105] Also provided is a diagnostic or assay device, such as a column, test strip or biochip, which has one or more slow-off aptamers attached to the solid surface of the device.A aptamer(s) can be arranged so that the aptamer binds to the target molecule that contacts the solid surface to form an aptamer-target complex, and the complex remains attached to the device surface, thereby capturing the target, and allowing the target to be detected and optionally quantified.An array of slow-off aptamers (can be the same or different) can be provided on such a device.

[0091]

[0106] In another embodiment, a complex comprising an aptamer and a target molecule is provided that has a slow off-rate. In other embodiments, a complex is provided that has a high affinity for the corresponding target molecule and a slow dissociation rate (t) of the aptamer and target from the non-covalent complex. 1 / 2 The present invention provides a class of aptamers characterized by having a

[0092]

[0107] With reference to FIG. 1A, the basic SELEX method generally begins with the preparation of a candidate mixture of nucleic acids of different sequences. The candidate mixture generally includes nucleic acid sequences comprising two fixed regions (i.e., each member of the candidate mixture contains the same sequence at the same location) and a variable region. The fixed sequence regions are typically selected so that they aid in the amplification process described below or enhance the potential for a given structural arrangement of the nucleic acids in the candidate mixture. The variable region typically provides a target binding region for each nucleic acid in the candidate mixture, and this variable region may be fully randomized (i.e., it is possible to find one base in four at any position) or only partially randomized (e.g., the probability of finding a base at any position can be selected to any level between 0 and 100 percent). The prepared candidate mixture is contacted with the selected target under conditions that support binding between the target and the candidate mixture members. Under these conditions, interactions between the target and the nucleic acids of the candidate mixture generally occur as paired members. The process involves forming nucleic acid-target pairs with the strongest relative affinity between the nucleic acids. Nucleic acids with the highest affinity for the target are partitioned from nucleic acids with lower affinity for the target. The partitioning process is carried out in a manner that retains the maximum number of high-affinity candidates. Nucleic acids selected during partitioning as having relatively high affinity for the target are amplified to generate a new candidate mixture enriched for nucleic acids with relatively high affinity for the target. By repeating the partitioning and amplification steps described above, the newly formed candidate mixture contains fewer unique sequences, and the average degree of affinity of the nucleic acid mixture for the target generally increases. In the extreme, the SELEX method will produce a candidate mixture containing one or very few unique nucleic acids corresponding to the nucleic acids from the original candidate mixture with the highest affinity for the target molecule. However, this basic SELEX method does not select for aptamers with slow off-rates from their targets.

[0093]

[0108] The SELEX and 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 method; how to prepare the initial candidate mixture; how to partition the nucleic acids within the candidate mixture; and how to amplify the partitioned nucleic acids to produce an enriched candidate mixture. The SELEX patents also describe aptamer solutions that can be obtained against several different types of target molecules, including protein targets where the protein is a nucleic acid-binding protein and protein targets where it is not.

[0094]

[0109] With reference to Figure 1B, the modified SELEX method disclosed herein includes the introduction of a slow off-rate enrichment process after equilibration of a candidate mixture of nucleic acids with one or more targets and a partitioning step prior to subsequent steps in the SELEX method. The introduction of a slow off-rate enrichment process to the basic SELEX method provides a means for enriching aptamer affinity complexes with slow off-rates from a set of nucleic acid-target complexes containing diverse off-rates. Thus, the modified SELEX method provides a method for identifying aptamers that bind to a target molecule and, once bound, have a relatively slow rate of dissociation from the target molecule (also referred to herein as "off-rates").

[0095]

[0110] As used herein, "binding" generally refers to the formation of a non-covalent bond between a ligand and a 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 a non-covalent association.

[0096]

[0111] In various embodiments, the slow-off aptamer may be a single-stranded or double-stranded RNA or DNA oligonucleotide. The aptamer may contain non-stranded or modified bases. Furthermore, the aptamer may contain any type of modification. As used herein, the term "modified base" may include relatively simple modifications to natural nucleic acid residues, which alter 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 such as benzyl, isobutyl, indole, or naphthyl, or substitution with hydrophilic groups such as quaternary amines or guanidinium, or more "neutral" groups such as imidazole. Further modifications may be present in the ribose ring, for example, the 2'-position, such as 2'-amino (2'-NH2) and 2'-fluoro (2'-F), or in the phosphodiester backbone, such as phosphorothioate or methylphosphonate.

[0097]

[0112] In various embodiments, a candidate mixture containing a randomized set of nucleic acid sequences containing modified nucleotide bases is mixed with an amount of target molecules, and the target Generally, only the proportion of nucleic acids that bind with high affinity to the target molecule will be efficiently distributed with the target.

[0098]

[0113] In various embodiments, the candidate mixture includes nucleic acid sequences having variable regions that contain modifying groups. The modifying groups may be modified nucleotide bases. The variable regions may contain completely or partially random sequences; they may also contain subportions of fixed sequences that are incorporated into the variable regions. The nucleotides in the fixed regions may also contain modified nucleotide bases or may contain a standard set of naturally occurring bases.

[0099]

[0114] In some embodiments, amplification occurs after the members of the test mixture are distributed, and it is the nucleic acids that are amplified. For example, amplification of RNA molecules can be achieved through a series of three reactions: creating cDNA copies of the selected RNA, increasing the copy number of each cDNA using the polymerase chain reaction, and transcribing the cDNA copies to obtain RNA molecules with the same sequence as the selected RNA. As will be recognized by those skilled in the art, any reaction or combination of reactions known in the art can be used as appropriate, including direct DNA replication, direct RNA amplification, etc. The amplification method can produce a ratio of the amplified mixture that is representative of the ratio of different sequences in the mixture before amplification. Many modifications to nucleic acids are known to be compatible with enzymatic amplification. If necessary, modifications that are not compatible with amplification can be made after each round of amplification.

[0100]

[0115] The nucleic acid candidate mixture may be modified in various ways to increase the likelihood that the nucleic acids will have facilitating or other desirable properties, particularly those that enhance the interaction between the nucleic acid and the target. Contemplated modifications include those that introduce other chemical groups with the correct charge, polarity, hydrogen bonding, or electrostatic interactions to enhance the desired ligand-target interaction. For example, modifications that can enhance binding properties, including nucleic acid affinity and / or dissociation rate, include hydrophilic moieties, hydrophobic moieties, rigid structures, and functional groups found in proteins, such as imidazole, primary alcohol, carboxylate, guanidinium group, amino group, and thiol. Modifications may also be used to increase the survival of aptamer-target complexes under stringent selection pressures, which can be applied to generate slow-off-rate aptamers for a wide range of targets. In one embodiment, Bz-dU (benzyl-dU) is used in generating the candidate mixture used to generate slow-off-rate aptamers, although other modified nucleotides are well suited for generating such aptamers. Other modified nucleotides are shown in FIG.

[0101]

[0116] For the purpose of this application, the modified nucleotide candidate mixture can be any RNA or DNA candidate mixture, including both naturally occurring and non-naturally occurring nucleotides.Suitable modifications include modifications on all residues of nucleic acids, on single residues of nucleic acids, on random residues, on all pyrimidines or all purines, on all occurrences of specific bases (i.e., G, C, A, T, or U) in nucleic acids, or any other modification scheme that may be suitable for a particular application.It is recognized that modification is not a requirement for the promoting activity or binding ability of aptamers.Aptamers can also include modified dUTP and dCTP residues.

[0102]

[0117] Candidate mixtures for slow off-rate aptamers may include sets of pyrimidines with different modifications at the C-5 base position. The C-5 modifications may be introduced directly through an amide linkage or indirectly through another type of linkage. These candidate mixtures are used in SELEX to identify slow off-rate aptamers. This process may also include the use of a slow off-rate enrichment process. Candidate mixtures may be produced enzymatically or synthetically.

[0103]

[0118] As mentioned above, nucleotides may be modified in several ways, including modifications at the ribose, phosphate, and / or base positions. Specific modifications are described in U.S. Patent No. 5,660,985, entitled "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides," U.S. Patent No. 5,428,149, entitled "Method for Palladium Catalyzed Carbon-Carbon Coupling and Products," and U.S. Patent No. 5,580,972, entitled "Purine Nucleoside Modifications by Palladium Catalyzed Methods," all of which are incorporated herein by reference. In one embodiment, the modification involves attaching another chemical group to the 5-position of the pyrimidine, the 8-position of the purine, or the 2'-position of the sugar. There is no limit to the type of other chemical group that can be incorporated onto an individual nucleotide. In some embodiments, the resulting modified nucleotides can be amplified or modified following the amplification step (see, e.g., U.S. Pat. No. 6,300,074, entitled "Systematic evolution of ligands by (See “Exponential Enrichment: Chemi-SELEX”)

[0104]

[0119] In yet another embodiment, specific nucleotides are modified for a method to produce an aptamer that binds and forms a covalent crosslink with its target molecule upon photoactivation of the affinity complex. This method involves an aptamer that binds to a target molecule, photocrosslinks with the molecule, and / or photoinactivates the molecule. In various embodiments, the aptamer contains a photoreactive group that can photocrosslink with the target molecule upon irradiation with light. In other embodiments, the aptamer can form a bond with the target in the absence of irradiation.

[0105]

[0120] The photoreactive group may be any chemical structure that contains a photochromophore and is photocrosslinkable with a target molecule. Although referred to herein as a photoreactive group, in some cases, as described below, irradiation is not necessary for covalent bonding between the aptamer and the target. In some embodiments, the photoreactive group will absorb light at a wavelength that is not absorbed by the target or the unmodified portion of the oligonucleotide. Photoreactive groups include 5-halo-uridine, 5-halo-cytosine, 7-halo-adenosine, 2-nitro-5-azidobenzoyl, diazirine, aryl azide, fluorinated aryl azide, benzophenone, amino-benzophenone, psoralen, anthraquinone, and the like.

[0106]

[0121] Photoreactive groups generally form bonds with targets upon irradiation of the associated nucleic acid-target pair. In some cases, irradiation is not required to form bonds. Typically, photocrosslinking occurs as the formation of covalent bonds between the associated aptamer and target. However, tight ionic interactions between the aptamer and target can also occur upon irradiation.

[0107]

[0122] In one embodiment, photocrosslinking occurs by exposure to electromagnetic radiation, including ultraviolet light, visible light, X-rays, and gamma rays.

[0123] In various other embodiments, limited selection of oligonucleotides using SELEX is followed by selection using photoSELEX. An initial round of SELEX selection is performed using oligonucleotides containing photoreactive groups. After several rounds of SELEX, photoSELEX is performed to select oligonucleotides capable of binding to the target molecule.

[0108]

[0124] In another embodiment, the aptamer sequence contains a cleavable or releasable section. The present invention describes the production of aptamers that include additional components (also referred to as elements or moieties). These additional components or moieties are structural elements or moieties that introduce additional functionality into the aptamer, and are therefore functional elements or moieties. Aptamers are further produced using one or more of the following additional components (also referred to as functional or structural elements or moieties, in any combination of these terms): labels or detectable components, spacer components, and specific binding tags or immobilization components or moieties.

[0109]

[0125] As described above, the present disclosure provides methods for identifying aptamers that bind to a target molecule and, once bound, exhibit slow dissociation or off-rates. The slow off-rates obtained by this method exceed a half-life of approximately one hour and extend for up to approximately 240 minutes, i.e., once a set of nucleic acid-target complexes is generated, half of the complexes in the set remain bound after one hour. Because the effectiveness of the slow off-rate enrichment process depends on the different dissociation rates of the aptamer-affinity complexes, the duration of the slow off-rate enrichment process is selected so that a high proportion of aptamer-affinity complexes with slow dissociation rates are retained while the number of aptamer-affinity complexes with fast dissociation rates is substantially reduced. For example, incubating the mixture for a relatively long period of time after imposing the slow off-rate enrichment process will select aptamers with longer dissociation rates than aptamers selected using a slow off-rate enrichment process with a shorter incubation period.

[0110]

[0126] In various embodiments, the candidate mixture is mixed with a certain amount of target molecules and allowed to establish binding equilibrium with the target molecules. Before partitioning the target-bound nucleic acids from those unbound in solution, a slow off-rate enrichment process is imposed to enrich the bound population for slow dissociation rates. As described above, the slow off-rate enrichment process may be applied by adding competitor molecules, by sample dilution, or by a combination of sample dilution in the presence of competitor molecules. Thus, in one embodiment, the slow off-rate enrichment process is applied by introducing competitor molecules into a mixture containing nucleic acid-target complexes and incubating the mixture for a period of time before partitioning the unbound from the bound nucleic acids. The amount of competitor molecules is generally at least one order of magnitude greater than the amount of nucleic acid molecules, and may be two or more orders of magnitude greater. In another embodiment, the slow off-rate enrichment process is applied by diluting the volume of the sample mixture of nucleic acid-target complexes several times (e.g., at least about one of 2x, 3x, 4x, or 5x) and incubating the mixture for a period of time before partitioning the unbound from the bound nucleic acids. The dilution volume is generally at least one order of magnitude larger than the original volume, and may be two or more orders of magnitude larger. In yet another embodiment, a slow off-rate enrichment process is applied using a combination of competitor molecules and dilution. In another embodiment, a candidate mixture that shows an increased frequency of aptamers with slow dissociation rates is used to select several candidate aptamers. These aptamers are screened to identify aptamers with slow off-rates.

[0111]

[0127] In another embodiment, slow off-rate aptamers are produced that contain a cleavable or releasable section in the fixed region of the aptamer. Aptamers may also be produced that contain one or more of the following additional components: a labeling component, a spacer component, and a specific binding tag. Any of these components may be incorporated into a single-stranded aptamer. In one embodiment, the component is incorporated at the 5' end of the aptamer. In another embodiment, one or more of these components are included by generating a partially double-stranded aptamer in which one strand contains, along with the desired diverse components, a sequence complementary to one of the fixed sequence sections of the second strand that contains the diverse target-binding region.

[0112]

[0128] A "releasable" or "cleavable" element or moiety or component is a "Releasable" refers to a functional group in which a specific bond in the functional group can be broken to produce two separate components. In various embodiments, the functional group can be cleaved by irradiating the functional group with an appropriate wavelength (photocleavable) or by treating with an appropriate chemical or enzymatic reagent. In another embodiment, the releasable component can be a disulfide bond, the bond of which can be broken by treatment with a reducing agent. The releasable component allows an aptamer / target affinity complex attached to a solid support to be separated from the solid support, such as by elution of the complex. The releasable component can be stable to the remaining assay conditions and can be releasable under conditions that do not disrupt the aptamer / target complex.

[0113]

[0129] As disclosed herein, aptamers may 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 attachment or immobilization of an aptamer (and any target molecule bound thereto) to a solid support. A "tag" is a set of copies of one type or species of component that can associate with a probe. "Tags" refers to more than one such set of components. Tags may be attached to or included in an aptamer by any suitable method. Generally, tags allow the aptamer to associate, either directly or indirectly, with a probe or receptor attached to a solid support. The probe may be highly specific in its interaction with the tag and may retain that association during all subsequent processing steps or procedures. Tags may allow the aptamer affinity complex (or, in some cases, a covalent aptamer affinity complex) to be localized to a spatially defined address on a solid support. Thus, different tags can allow different aptamer covalent complexes to be localized at different spatially defined addresses on a solid support. Tags can be polynucleotides, polypeptides, peptide nucleic acids, locked nucleic acids, oligosaccharides, polysaccharides, antibodies, affibodies, antibody mimics, cellular receptors, ligands, lipids, biotin, any fragments or derivatives of these structures, any combination of the foregoing, or any other structure to which a probe (or a linker molecule, as described below) can be designed or configured to specifically bind or otherwise associate. Generally, tags are configured so as not to interact intramolecularly with themselves or with the aptamer to which they are attached or of which they are a part. When SELEX is used to identify aptamers, tags can be added to the aptamer either before or after SELEX. After SELEX, a tag may be included on the 5' end of the aptamer, or after SELEX, a tag may be included on the 3' end of the aptamer, or tags may be included on both the 3' and 5' ends of the aptamer in a post-SELEX modification process.

[0114]

[0130] As illustrated in Figure 8D, a fluorescent dye (e.g., Cy3), photocleavable, and biotin moieties are all added to the termini of the aptamer. Due to 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 Figures 9A-9F. Functionality can be split between the 5' and 3' ends or combined at either end. In addition to the photocleavable moiety, 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 other than biotin (also referred to as immobilization or specific binding elements or components) 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).

[0115]

[0131] In one embodiment, nucleotide base modifications are used in the production of the variable region of the aptamer. These modified nucleotides have been shown to produce aptamers with very slow off-rates to their targets.

[0116]

[0132] In the disclosed methods, the candidate mixture may include modified nucleic acids in which one, some (e.g., 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 at least one) or all pyrimidines in at least one or each nucleic acid of the candidate mixture are 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.

[0117]

[0133] In another embodiment, the slow off-rate aptamer is mixed with or exposed to a sample. The slow off-rate aptamer is allowed to react with or bind to a specific target in the sample. 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 release 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 is desired for the detection of multiple targets. The methods of the present disclosure are generally illustrated in Examples 1-7.

[0118]

[0134] The methods of the present disclosure are generally illustrated in Examples 1-6. Example 1 describes a general affinity SELEX method using a candidate mixture composed of modified nucleotides. Example 2 describes a photoSELEX method using a candidate mixture composed of modified nucleotides and 5'-terminal photoreactive groups, as well as an improved SELEX method using dilution to provide a slow off-rate enrichment process for the equilibrated aptamer:target mixture. Example 3 extends the method described in Example 2 by adding a competitor to the dilution step. Example 4 illustrates the effectiveness of the slow off-rate enrichment process. The average dissociation half-life (t) value for aptamers using the modified nucleotides 5-benzyl-dUTP (BzdUTP), 5-isobutyl-dUTP (iBdUTP), or 5-tryptamino-dUTP, selected in the absence of a slow off-rate enrichment process, was 20 minutes, with some aptamers having t values ​​of up to 1 hour. This is substantially longer than that previously described with natural bases or other modified nucleotides. The average for aptamers selected using the slow off-rate enrichment process was greater than 85 minutes. More specifically, with reference to FIG. 3B, the introduction of the slow off-rate enrichment process resulted in t times of about 30 minutes or more, about 60 minutes or more, about 90 minutes or more, about 120 minutes or more, about 150 minutes or more, about 180 minutes or more, about 210 minutes or more, and about 240 minutes or more. 1 / 2 These dissociation rates for the aptamer:target complex are unprecedented.

[0119]

[0135] Example 5 describes the generation of slow off-rate aptamers using the NpdUTP candidate mixture.

[0136] Example 6 describes the generation of slow off-rate aptamers against peptide targets. [Example]

[0120]

[0137] The following examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. It is not intended to limit the scope of the invention as defined in the paragraphs. Example 1 Incorporation of modified nucleotides into nucleic acid libraries leads to higher affinity enriched libraries in affinity SELEX

[0138] A. Preparation of the Candidate Mixture

[0139] Candidate mixtures were prepared using dATP, dGTP, 5-methyl-dCTP (MedCTP), and dTTP or one of three dUTP analogs: 5-benzyl-dUTP (BzdUTP), 5-isobutyl-dUTP (iBdUTP), or 5-tryptaamino-dUTP (TrpdUTP). Candidate mixtures were prepared by polymerase extension of primers annealed to biotinylated templates (Figure 2). For each candidate mixture composition, 4.8 nmol of forward PCR primer and 4 nmol of template were combined in 100 μL of 1× KOD DNA polymerase buffer (Novagen), heated to 95°C for 8 minutes, and chilled on ice. To a 400 μL extension reaction containing 1× KOD DNA polymerase buffer, 0.125 U / μL KOD DNA polymerase, and 0.5 mM each of dATP, MedCTP, dGTP, and dTTP or dUTP analogs, 100 μL of each primer:template mixture was added and incubated at 70°C for 30 minutes. 1 mL of streptavidin-coated magnetic beads (MagnaBind Streptavidin, Pierce, 1M The double-stranded product was captured via the template strand biotin by adding 0.75 mL SB1T buffer (40 mM HEPES, pH 7.5, 125 mM NaCl, 5 mM KCl, 1 mM MgCl, 1 mM ATP, 5 mg / 5 mL in NaCl + 0.05% TWEEN-20) and incubating with mixing for 10 minutes at 25°C. The beads were washed three times with 1.2 mL of 20 mM NaOH (0.05% TWEEN-20). The aptamer strands were eluted from the beads with 0.3 mL of 80 mM NaOH. The mixture was neutralized with HCl and buffered with 15 μL 1 M HEPES, pH 7.5. The candidate mixture was concentrated to approximately 0.2 mL with a Centricon-30 and quantified by UV absorption spectroscopy.

[0121]

[0140] B. Immobilization of target proteins

[0141] Purchase a target protein containing a poly-His tag, such as a (His)6 tag (R&D Systems), and +2 The target protein was immobilized on NTA paramagnetic beads (TALON, Invitrogen). The target protein was 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 at 25°C for 30 minutes and stored at 4°C until use. TALON beads coated with (His)6 peptide were also prepared and stored as described above. Before use, the beads were washed three times with B / W buffer, once with SB1T, and resuspended in SB1T.

[0122]

[0142] C. Aptamer Selection Scheme

[0143] Affinity selections were performed separately with each candidate mixture to compare binding between target protein beads (signal, S) and (His)6 beads (background, B). For each sample, a 0.5 μM candidate DNA mixture was prepared in 40 μL of SB1T. 1 μL of (His)6-complementary oligo (1 mM) (Figure 2) was added to the DNA along with 10 μL of protein competitor mixture (0.1% HSA, 10 μM casein, and 10 μM prothrombin in SB1T).

[0123]

[0144] The binding reaction was carried out by adding 50 μL of target protein-coated beads or (His)6-coated beads (5 mg / mL in SB1T) to the DNA mixture and incubating at 37° C. for 15 minutes with mixing. The DNA solution was removed, and the beads were washed five times at 37°C with SB1T containing 0.1 mg / mL herring sperm DNA (Sigma-Aldrich). Unless indicated, all washes were performed by resuspending the beads in 100 μL of 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 μL of SB1T + 2 M guanidine-HCl and incubating with mixing for 5 minutes at 37°C. After magnetic separation, the aptamer eluate was transferred to a new tube. After the first two selection rounds, the final two target bead washes were performed for 5 minutes instead of 30 seconds.

[0124]

[0145] Primer beads were prepared by immobilizing biotinylated reverse PCR primers on streptavidin-coated paramagnetic beads (MyOne-SA, Invitrogen). 5 mL of MyOne-SA beads (10 mg / mL) were washed once with NaClT (5 M NaCl, 0.01% TWEEN-20) and resuspended in 5 mL of biotinylated reverse PCR primer (5 μM in NaClT). Samples were incubated at 25°C for 15 minutes, washed twice with 5 mL of NaClT, resuspended in 12.5 mL of NaClT (4 mg / mL), and stored at 4°C.

[0125]

[0146] To 100 μL of aptamer solution in guanidine buffer, 25 μL of primer beads (4 mg / mL in NaClT) was added and incubated at 50°C for 15 minutes with mixing. The aptamer solution was removed, and the beads were washed five times with SB1T. The aptamer was eluted from the beads by adding 85 μL of 20 mM NaOH and incubating at 37°C for 1 minute with mixing. After magnetic separation, 80 μL of the aptamer eluate was transferred to a new tube, neutralized with 20 μL of 80 mM HCl, and buffered with 1 μL of 0.5 M Tris-HCl, pH 7.5.

[0126]

[0147] D. Aptamer Amplification and Purification

[0148] The selected aptamer DNA was amplified and quantified by QPCR. 48 μL DNA was added to 12 μL QPCR mixture (5X KOD DNA polymerase buffer, 25 mM MgCl2, 10 μM forward PCR primer, 10 μM biotinylated reverse PCR primer, 5X SYBR Green I, 0.125 U / μL KOD DNA polymerase, and 1 mM each of dATP, dCTP, dGTP, and dTTP) and thermally cycled in an ABI5700 QPCR instrument using the following protocol: 1 cycle of 99.9°C for 15 seconds, 55°C for 10 seconds, and 70°C for 30 minutes; 30 cycles of 99.9°C for 15 seconds and 72°C for 1 minute. Quantification was performed using the instrument software, and the copy numbers of DNA selected by target beads and (His)6 beads were compared to determine the signal-to-background ratio.

[0127]

[0149] After amplification, the PCR product was captured onto MyOne-SA beads via the biotinylated antisense strand. 1.25 mL of MyOne-SA beads (10 mg / mL) were washed twice with 0.5 mL of 20 mM NaOH, once with 0.5 mL of SB1T, resuspended in 2.5 mL of 3 M NaCl, and stored at 4°C. 25 μL of MyOne-SA beads (4 mg / mL in 3 M NaCl) were added to 50 μL of double-stranded QPCR product and incubated at 25°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 μL of 20 mM NaOH and incubating at 37°C for 1 minute with mixing. The eluted strand was discarded, and the beads were washed three times with SB1T and once with 16 mM NaCl.

[0128]

[0150] The aptamer sense strand was prepared with the appropriate nucleotide composition by primer extension from the immobilized antisense strand. 20 μL primer extension reaction mixture ( The beads were resuspended in 1X KOD DNA polymerase buffer, 1.5 mM MgCl2, 5 μM forward PCR primer, 0.125 U / μL KOD DNA polymerase, 0.5 mM each of dATP, MedCTP, dGTP, and either dTTP or dUTP analogs, and incubated at 68°C for 30 minutes with mixing. The beads were washed three times with SB1T, and the aptamer strands were eluted from the beads by adding 85 μL 20 mM NaOH and incubating at 37°C for 1 minute with mixing. After magnetic separation, 80 μL of the aptamer eluate was transferred to a new tube, and 20 μL Neutralized with 80 mM HCl and 5 μL 0.1 M HEPES, pH 7.5.

[0129]

[0151] E. Selection Stringency and Feedback

[0152] The relative target protein concentration in the selection step is decreased in each round according to the S / B ratio as follows, where signal S and background B are defined in section C above: If S / B<10, then [P](i+l)=[P]i If 10≦S / B<100, then [P](i+l)=[P]i / 3.2 If S / B≧100, then [P](i+l)=[P]i / 10 where [P] = protein concentration and i = current round number.

[0130]

[0153] The target protein concentration was reduced by adjusting the mass of the target protein beads (and (His)6 beads for background determination) added to the selection step.

[0131]

[0154] After each selection round, the convergence state of the enriched DNA mixture was determined. 5 μL of double-stranded QPCR product was diluted to 200 μL with 4 mM MgCl2 containing 1X SYBR Green I. 75 μL of silicone oil was overlaid onto the sample and analyzed for convergence using C0t analysis, which measures hybridization time for complex mixtures of double-stranded oligonucleotides. Samples were thermally cycled using the following protocol: 3 cycles of 98°C for 1 minute, 85°C for 1 minute; 1 cycle of 93°C for 1 minute, 85°C for 15 minutes. Fluorescence images were taken at 5-second intervals during the 15-minute treatment at 85°C. Fluorescence intensity was plotted as a function of logarithm (time) to assess sequence diversity.

[0132]

[0155] F. Measurement of equilibrium binding constant (Kd)

[0156] Equilibrium binding constants of the enriched libraries were measured using TALON bead partitioning. DNA was renatured by heating to 95°C and slowly cooling to 37°C. A low concentration of radiolabeled DNA (~1x10) was added in SB1 buffer (described above). -11 M) to a range of concentrations of target protein (1x10 -7 M~1x10 -12 Complexes were formed by mixing with 100 μL of 5000 ribonucleotides (M final) and incubating at 37°C. A portion of each reaction was transferred to a nylon membrane and dried to determine the total counts in each reaction. A small amount of 5 mg / mL MyOne TALON beads (Invitrogen) was added to the remainder of each reaction and mixed for 1 minute at 37°C. Aliquots were passed through a MultiScreen HV plate (Millipore) under vacuum to separate the protein-bound complexes from unbound DNA and washed with 100 μL SB1 buffer. The nylon membrane and MultiScreen HV plate were phosphorimaged, and the amount of radioactivity in each sample was quantified using a FUJI FLA-3000. The fraction of captured DNA was plotted as a function of protein concentration, and the equilibrium binding constant (K dTable 1 shows the K values ​​determined for each candidate mixture enriched against the target set. d NT indicates the extent to which the library enriched for a particular base composition is unchanged from the original candidate mixture as determined by C0t analysis (described above). and therefore indicates not tested (NT).

[0133]

[0157] Table 1 shows the equilibrium binding constants (K) for 15 different protein targets and four different DNA libraries: pools enriched for the naturally occurring bases (dT), benzyl (BzdU), isobutyl (iBdU), or tryptophan (TrpdU). d ) 1x10 -8 Less than K d Aptamers with a slow off-rate are desirable. The use of modified bases in SELEX yields a significantly higher percentage of the desired high-affinity aptamers. Only two of 14 aptamers produced with normal nucleotides were observed to have the desired slow off-rate. Slow off-rate aptamers produced using modified nucleotides were identified in 9 of 14, 7 of 14, and 14 of 14 for BzdUTP, iBdUTP, and TRPdUTP, respectively.

[0134]

[0158]

[0135] [Table 1] Table 1. Equilibrium binding constants (Kd) of enriched libraries selected with different modified nucleotides, reported in molar units. NT = not tested.

[0136] Example 2. Photoaptamer generation using 5'-anchored photoSELEX and a slow off-rate enrichment process by dilution

[0159] A. Preparation of the Candidate Mixture

[0160] Candidate mixtures containing dATP, dCTP, dGTP, and BzdUTP were prepared by polymerase extension of primers annealed to biotinylated templates (Figure 4A-B). Four different forward primers, each carrying a unique chromophore at its 5' end, were used for each template (Figure 5). For each candidate mixture, 11 nmol of forward primer (containing the 5' chromophore) and 10 nmol of template were combined in 250 μL of primer extension buffer (120 mM Tris-HCl, pH 7.8, 10 mM KCl, 6 mM (NH4)2SO4, 7 mM MgSO4, 0.1 mg / mL BSA, 0.1% Triton X-100), heated to 95°C for 5 minutes, and cooled on ice. Primer extension buffer, 0.125 U / μL KOD To a 1 mL extension reaction containing DNA polymerase and 0.5 mM each of dATP, dCTP, dGTP, and BzdUTP, 125 μL of each primer:template mixture was added and incubated at 70°C for 30 min. Each 1 mL reaction was divided into four 250 μL aliquots and chilled on ice. 1 mL of streptavidin-coated magnetic beads (MagnaBind Streptavidin, Pierce) was added. The double-stranded product was captured via the template strand biotin by adding 5 mg / 5 mL of SB17T buffer (5 mg / 5 mL in 1 M NaCl + 0.05% TWEEN-20) to each 250 μL aliquot and incubating at 25°C for 60 minutes with mixing. The beads were washed three times with 0.5 mL of SB17T buffer (40 mM HEPES, pH 7.5, 125 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA, 0.05% TWEEN-20). The aptamer strand was eluted from the beads with 1 mL of 20 mM NaOH, neutralized with 0.25 mL of 80 mM HCl, and buffered with 10 μL of 1 M HEPES, pH 7.5. The candidate mixture was concentrated to approximately 0.2 mL on a Centricon-30 and quantified by UV absorption spectroscopy.

[0137]

[0161] B. Target Protein Preparation

[0162] Untagged target proteins were biotinylated by covalently attaching NHS-PEO4-biotin (Pierce) to lysine residues. Protein (300 pmol in 50 μL) was exchanged into SB17T using a Sephadex G-25 microspin column. NHS-PEO4-biotin was added to 1.5 mM, and the reaction was incubated at 4°C for 16 hours. Unreacted NHS-PEO4-biotin was removed using a Sephadex G-25 microspin column.

[0138]

[0163] C. Aptamer selection with slow off-rate enrichment process and photocrosslinking

[0164] Separate selections were performed with each candidate mixture to compare binding between samples with target protein (signal S) and without target protein (background B). The first three rounds were performed with affinity selection (no photocrosslinking); the second and third rounds included a slow off-rate enrichment process. Rounds 4-8 included both a slow off-rate enrichment process and photocrosslinking.

[0139]

[0165] For each sample, a 90 μL DNA mixture was prepared in SB17T with 10–20 pmol of candidate mixture (100 pmol for the first round) and 100 pmol of reverse primer. The sample was heated to 95°C for 3 min and cooled to 37°C at a rate of 0.1°C / sec. The sample was combined with 10 μL of protein competitor mixture (0.1% HSA, 10 μM casein, and 10 μM prothrombin in SB17T), added to 0.5 mg Dynal MyOne streptavidin beads (prewashed twice with 20 mM NaOH and once with SB17T), and incubated at 37°C for 5 min with mixing. The beads were removed by magnetic separation.

[0140]

[0166] Binding reactions were carried out by adding 10 μL target protein (0.5 μM in SB17T) or SB17T to 40 μL of the DNA mixture and incubating at 37° C. for 30 minutes.

[0141]

[0167] When using the slow off-rate enrichment process, samples were diluted 20X by adding 950 μL SB17T (preheated to 37° C.) and incubated at 37° C. for 30 minutes before capturing the complex.

[0142]

[0168] Complexes were captured onto SA beads via the protein biotin by adding 0.25 mg MyOne-SA beads (Invitrogen) and incubating at 37°C for 15 minutes with mixing. Unbound DNA was removed by washing the beads five times with SB17T. Unless indicated, all washes were performed by resuspending the beads in 100 μL wash solution, mixing at 25°C for 30 seconds, separating the beads with a magnet, and removing the wash solution. The aptamer strands were eluted from the beads by adding 85 μL 20 mM NaOH and incubating at 37°C for 1 minute with mixing. After magnetic separation, 80 μL of the aptamer eluate was transferred to a new tube. The mixture was transferred to a 1000 ml buffer, neutralized with 20 μL 80 mM HCl, and buffered with 1 μL 0.5 M Tris-HCl, pH 7.5.

[0143]

[0169] When photoselection was used, 50 μL of the binding reaction from above (or 1 mL of the binding reaction, optionally after a slow off-rate concentration process by dilution) was illuminated with a high-pressure mercury lamp (Optical Associates, Inc. Model 0131-0003-01, 500 W, 310 nm mirror set). Candidate mixtures bearing the BrdU chromophore were illuminated for 37 seconds, those bearing the ANA chromophore for 60 seconds, and those bearing the AQ or psoralen chromophore for 10 minutes. Additional filters (5 mm plate glass) were used for the ANA, AQ, and psoralen chromophores to filter out unwanted, but potentially harmful, wavelengths below 320 nm. Complexes were captured as described above, and uncrosslinked DNA was removed by washing the beads once with 4 M guanidine-HCl + 0.05% TWEEN-20 for 10 min at 50° C., once with 20 mM NaOH for 2 min at 25° C., twice with SB17T, and once with 16 mM NaCl. Crosslinked DNA was not removed from the bead surface for the amplification step.

[0144]

[0170] D. Aptamer Amplification and Purification

[0171] The selected aptamer DNA was amplified and quantified by QPCR. 48 μL DNA was added to 12 μL QPCR mixture (5× KOD DNA polymerase buffer, 25 mM MgCl2, 10 μM forward PCR primer, 10 μM biotinylated reverse PCR primer, 5× SYBR Green I, 0.125 U / μL KOD DNA polymerase, and 1 mM each of dATP, dCTP, dGTP, and dTTP) and thermally cycled in a Bio-Rad MyIQ QPCR instrument using the following protocol: 1 cycle of 99.9°C for 15 seconds, 55°C for 10 seconds, and 68°C for 30 minutes; 30 cycles of 99.9°C for 15 seconds and 72°C for 1 minute. For the photoSELEX round, the initial 30-minute incubation was performed, and quantification was performed using the instrument software. The copy numbers of DNA selected with and without the target protein were compared to determine the signal-to-background ratio.

[0145]

[0172] When using photoselection, cDNA copies of the selected DNA were prepared by primer extension on the bead surface. Washed beads were resuspended in 20 μL cDNA extension mixture (primer extension buffer containing 5 μM reverse PCR primer, 0.5 mM each of dATP, dCTP, dGTP, and dTTP, and 0.125 U / μL KOD DNA polymerase) and incubated at 68°C for 30 minutes with mixing. The beads were washed three times with SB17T, and the aptamer strands were eluted from the beads by adding 85 μL 20 mM NaOH and incubating at 37°C for 1 minute with mixing. After magnetic separation, 80 μL of the aptamer eluate was transferred to a new tube, neutralized with 20 μL 80 mM HCl, and buffered with 1 μL 0.5 M Tris-HCl, pH 7.5. cDNA was amplified and quantified by QPCR as described above for 30 cycles of 99.9°C for 15 seconds and 72°C for 1 minute.

[0146]

[0173] After amplification, the PCR product was captured onto MyOne-SA beads via the biotinylated antisense strand. 1.25 mL MyOne-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°C. 25 μL MyOne-SA beads (10 mg / mL in 3 M NaClT) were added to 50 μL of double-stranded QPCR product and incubated at 25°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 μL 20 mM NaOH and incubating at 37°C for 1 minute with mixing. The eluted strand was discarded, and the beads were resuspended in SB17T. The mixture was washed three times with 16 mM NaCl and once with 16 mM NaCl.

[0147]

[0174] Aptamer sense strands containing the appropriate chromophore were prepared by primer extension from the immobilized antisense strand. The beads were resuspended in 20 μL primer extension reaction mixture (1× primer extension buffer, 1.5 mM MgCl, 5 μM forward PCR primer containing the appropriate 5′ chromophore, 0.5 mM each of dATP, dCTP, dGTP, and BzdUTP, and 0.125 U / μL KOD DNA polymerase) and incubated at 68°C for 30 minutes with mixing. The beads were washed three times with SB17T, and the aptamer strands were eluted from the beads by adding 85 μL 20 mM NaOH and incubating at 37°C for 1 minute with mixing. After magnetic separation, 80 μL of the aptamer eluate was transferred to a new tube, neutralized with 20 μL 80 mM HCl, and buffered with 5 μL 0.1 M HEPES, pH 7.5.

[0148]

[0175] E. Selection Stringency and Feedback

[0176] In each round, the target protein was adjusted as described in Example 1. After each selection round, the convergence state of the enriched pool was determined as described in Example 1.

[0149]

[0177] F. Equilibrium binding constants of the enriched library

[0178] Binding affinities were determined as described above in Example 1, but using MyOne-SA capture beads. The following table, Table 2, summarizes the equilibrium binding constants (Kd) obtained using the photoSELEX protocol with a slow off-rate enrichment process.

[0150] [Table 2] Table 2. Equilibrium binding constants (Kd) of enriched libraries selected with different chromophores, reported in molar units. No measurements were performed on libraries that failed to converge (indicated by an x).

[0151]

[0179] G. Cross-linking Activity Assay

[0180] The cross-linking yield of the enriched library was determined by measuring the percent of DNA cross-linked to protein under saturating protein and light conditions. Radiolabeled DNA (50 pM) was mixed with the reverse primer (16 nM) in SB17T, heated to 95°C for 3 minutes, and cooled to 37°C at 0.1°C / sec. Target protein was added to the DNA mixture to a final concentration of 10 nM and incubated at 37°C for 30 minutes. A control sample without protein was prepared simultaneously. Samples were cross-linked as above, but at saturating doses (6 minutes for BrdU, 10 minutes for ANA, and 30 minutes for AQ and Psor) under the chromophore-specific conditions listed above. Samples were analyzed by denaturing PAGE (Figure 6) and quantified, and the results are tabulated in Table 3.

[0152] [Table 3] Table 3. Cross-linking yields of enriched libraries selected with different chromophores, reported as percent of total DNA cross-linked to protein. No measurements were performed on libraries that failed to converge (indicated by an x).

[0153] Example 3. Generation of slow off-rate aptamers using a competitive slow off-rate enrichment process

[0181] A. Preparation of the Candidate Mixture

[0182] For 94 protein targets, a candidate mixture containing dATP, dCTP, dGTP, and BzdUTP was prepared by polymerase extension of a primer annealed to a biotinylated template. 55 nmol of forward primer (containing the 5'ANA chromophore) and 55 nmol of template were combined in 0.5 mL of primer extension buffer (120 mM Tris-HCl, pH 7.8, 10 mM KCl, 6 mM (NH4)2SO4, 7 mM MgSO4, 0.1 mg / mL BSA, 0.1% Triton X-100), heated to 95°C for 5 min, 70°C for 5 min, and 48°C for 5 min, and then chilled on ice. The primer:template mixture was added to a 5.5 mL extension reaction containing primer extension buffer, 0.125 U / μL KOD DNA polymerase, and 0.5 mM each of dATP, dCTP, dGTP, and BzdUTP, and incubated at 70°C for 60 min. After completion of the extension reaction, the solution was cooled on ice. 25 mL of streptavidin-coated magnetic beads (MagnaBind Streptavidin, Pierce, 5 mg / 5 mL in 1 M NaCl + 0.05% TWEEN-20) were added to the primer extension product and the double-stranded product was captured via the template strand biotin by incubating at 25°C for 15 min with rotation. The beads were washed three times with 40 mL SB17T buffer (40 mM HEPES, pH 7.5, 125 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA, 0.05% TWEEN-20). The aptamer strands were eluted from the beads with 35.2 mL 20 mM NaOH for 5 minutes with shaking. The eluted strands were neutralized with 8.8 mL 80 mM HCl and buffered with 400 μL 1 M HEPES, pH 7.3. The candidate mixture was concentrated to approximately 0.7 mL using a Centricon-30 and quantified by UV absorption spectroscopy.

[0154]

[0183] B. Target Protein Preparation

[0184] Untagged target proteins were biotinylated as described in Example 2.

[0155]

[0185] C. Aptamer selection with slow off-rate enrichment process and photocrosslinking

[0186] During the slow off-rate enrichment process in rounds 6-9, 10 mM dextran sulfate was added as a competitor for aptamer rebinding, as described in Example 2. Selections were made separately.

[0156]

[0187] Three different formats of the slow off-rate enrichment process were used. In rounds 2 and 3, samples were diluted 20X by adding 950 μL SB17T (preheated to 37°C) and incubated at 37°C for 30 minutes before complex capture. In rounds 4 and 5, samples were diluted 20X by adding 950 μL SB17T (preheated to 37°C) and incubated at 37°C for 30 minutes before crosslinking. In rounds 6 and 7, samples were diluted 20X by adding 950 μL SB17T (preheated to 37°C). 50 μL of each diluted sample was diluted again by transferring to 950 μL SB17T + 10 mM 5000K dextran sulfate (preheated to 37°C) for a total dilution of 400X and incubated at 37°C for 60 minutes before crosslinking. In rounds 8 and 9, samples were diluted 20X by adding 950 μL SB17T (preheated to 37°C) and diluted again by transferring 50 μL of each sample to 950 μL SB17T (preheated to 37°C) to give a 400X dilution. Finally, 50 μL of each 400X diluted sample was diluted again by transferring 50 μL to 950 μL SB17T + 10 mM 5000K dextran sulfate (preheated to 37°C) to give a total 8000X dilution and incubated at 37°C for 60 minutes before crosslinking. Complexes were captured and washed as described in Example 2. When photocrosslinking was used, 1 mL binding reactions after the slow off-rate concentration process were illuminated for 60 seconds with a 470 nm LED array before complex capture as in Example 2.

[0157]

[0188] D. Aptamer Amplification and Purification

[0189] Amplification and purification were carried out as in Example 2.

[0190] E. Selection Stringency and Feedback

[0191] The target protein was adjusted in each round as described in Example 1, except for rounds 6 and 8. To maximize the signal after these large dilutions, the target protein was increased to 100 nM for rounds 6 and 8. After each selection round, the convergence state of the enriched pool was determined as described in Example 1.

[0158]

[0192] F. Dissociation Rate Constant Determination Protocol

[0193] The rate constant for aptamer:protein complex dissociation (koff) was determined for each aptamer by measuring the fraction of preformed aptamer:protein complexes that remained bound after dilution as a function of time. dRadiolabeled aptamer (50 pM) was equilibrated at 37°C in SB17T-0.002 (SB17T with TWEEN-20 reduced to 0.002%) with a protein concentration 10X higher than the reference value. Samples were diluted 100X in SB17T-0.002 at 37°C, and aliquots were removed and distributed at various time points to separate unbound aptamer from the protein:aptamer complex. Distribution was completed by adding ZORBAX resin (Agilent) to the sample, capturing the complex on the resin, passing the sample through a DuraPore membrane under vacuum, and washing the resin with SB17T-0.002. Because proteins are not efficiently captured by ZORBAX resin, assays were performed with biotinylated proteins in SB17T, and distribution was completed by capturing the complex with Dyanal MyOne-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 dissociation rate constant (koff) and dissociation half-life value (t) were determined by plotting the fraction of complex as a function of time and fitting the data to analytical expressions for biomolecular dissociation kinetics using nonlinear regression. 1 / 2 ) was decided.

[0159]

[0194] G. Kinetic Properties of Some Aptamers

[0195] The following table, Table 4, shows the dissociation half-life values ​​(t) obtained for aptamers selected against 10 targets using this protocol. 1 / 2 ) is summarized below.

[0160] [Table 4] Table 4. Dissociation half-life values ​​(t) of aptamers using a slow off-rate enrichment step protocol including competitors. 1 / 2 )

[0196] Example 4: A slow off-rate enrichment process increases the dissociation half-life of selected aptamers.

[0161]

[0197] Dissociation half-life values ​​(t ) were obtained for 65 aptamers selected by either the affinity SELEX method described in Example 1 or the photoSELEX method described in U.S. Pat. No. 6,458,539, entitled "Photoselection of Nucleic Acid Ligands," without the slow off-rate enrichment process. 1 / 2 ) was measured and plotted (FIG. 3A). For the 72 aptamers selected by the slow off-rate enrichment process described in Example 2, involving the slow off-rate enrichment process by dilution or dilution with competitor, t 1 / 2 The average t values ​​were measured and plotted (Figure 3B). The average t values ​​for aptamers using the modified nucleotides 5-benzyl-dUTP (BzdUTP), 5-isobutyl-dUTP (iBdUTP), or 5-tryptamino-dUTP, selected in the absence of a slow off-rate enrichment process, were plotted. 1 / 2 The value is 20 minutes, and some aptamers have t values ​​up to 1 hour. 1 / 2 This is substantially longer than previously described for natural bases or other modified nucleotides. The average for aptamers selected using the slow off-rate enrichment process was over 85 minutes, with some aptamers exceeding 4 hours. 1 / 2 had value.

[0162]

[0198] Example 5: Generation of aptamers from NpdUTP random libraries

[0199] A. Preparation of the Candidate Mixture

[0200] A candidate mixture containing dATP, dCTP, dGTP, and NpdUTP was prepared as described in Example 3, but without the 5'-ANA photoreactive group.

[0163]

[0201] B. Immobilization of target proteins As described in Example 1, the target protein contained a (His)6 tag, and Co+2-NTA beads were used to capture the protein.

[0164]

[0202] C. Aptamer selection using a slow off-rate enrichment process

[0203] Aptamer selection was performed as described in Example 3, but without photocrosslinking.

[0165]

[0204] D. Aptamer Amplification and Purification

[0205] Amplification and purification were carried out as described in Example 3.

[0206] E. Selection Stringency and Feedback

[0207] Selection stringency and feedback were performed as described in Example 3.

[0166]

[0208] F. Aptamer Properties

[0209] The equilibrium binding constants (Kd) of the four aptamers derived from this selection are listed in Table 5.

[0167] Table 5. Equilibrium binding constant (Kd) of NpdUTP aptamer

[0168] [Table 5]

[0210] Example 6. Generation of slow off-rate aptamers for peptide targets using a slow off-rate enrichment process with a competitor

[0211] A. Preparation of the Candidate Mixture

[0212] A candidate mixture containing dATP, dCTP, dGTP, and BzdUTP was prepared by polymerase extension of a primer containing a 5' ANA chromophore, as described in Example 3, and purified.

[0169]

[0213] B. Aptamer selection using a slow off-rate enrichment process and photocrosslinking

[0214] Aptamer selection was performed as described in Example 3 using the 29 amino acid biotinylated target peptide SMAP29 (sheep bone marrow antibacterial peptide MAP-29, Anaspec).

[0170]

[0215] C. Aptamer Amplification and Purification

[0216] Amplification and purification were carried out as described in Example 3.

[0217] D. Selection Stringency and Feedback

[0218] Selection stringency and feedback were performed as described in Example 3.

[0171]

[0219] E. Aptamer Properties

[0220] The equilibrium binding constant (Kd) of the aptamer derived from this selection was 1.2e-8 M (measured according to the protocol described in Example 1). The dissociation half-life (t) of this aptamer was 69 minutes (measured according to the protocol described in Example 3). The results are shown in Figures 12A and 12B.

[0172]

[0221] Example 7: Affinity bead hybrid capture

[0222] Step 1: Example 7: Protein measurement in test samples is enabled by slow off-rate aptamers.

[0173]

[0223] Step 1: Preparation of aptamer / primer mixtures and test samples Aptamers containing biotin Cy3 detection labels (4 nM each) are mixed with a 3X excess of capture probes (oligonucleotides complementary to the 3' anchored region of the aptamer containing a biotin tag and a photocleavable element) in 1X SB17T and heated at 95°C for 4 minutes, then at 37°C for 13 minutes, and diluted 1:4 in 1X SB17T. 55 μL of the aptamer / primer mixture is added to a microtiter plate (Hybaid #AB-0407) and sealed with foil. Test samples are prepared in the microtiter plate by mixing with known concentrations of protein analyte in SB17T and serially diluting in SB17T.

[0174]

[0224] Step 2: Sample equilibration

[0225] 55 μL of the aptamer / primer mixture is added to 55 μL of test sample and incubated in a foil-sealed microtiter plate at 37° C. for 15 minutes. The final concentration of each aptamer in the equilibration mixture is 0.5 nM. After equilibration, all subsequent steps of the method are performed at room temperature unless otherwise noted.

[0175]

[0226] Step 3: Aptamer capture and unbound protein removal

[0227] A DuraPore filtration plate (Millipore HV catalog #MAHVN4550) is washed once with 100 μL 1XSB17T by vacuum filtration, 133 μL 7.5% streptavidin-agarose resin (Pierce) is added to each well, and washed twice with 200 μL 1XSB17T. 100 μL of the equilibrated sample is transferred to the Durapore plate containing the streptavidin-agarose resin and incubated for 5 minutes at 800 rpm on a thermal mixer (Eppendorf). The resin is washed once with 200 μL 1XSB17T + 100 μM biotin and once with 200 μL 1XSB17T.

[0176]

[0228] Step 4: Protein tagging with biotin

[0229] 100 μL of 1.2 mM NHS-PEO4-biotin in SB17T, prepared immediately before use, is added to the resin along with the capture aptamer and aptamer:protein complex and incubated for 20 minutes at 800 rpm on a thermal mixer. Wash the resin five times with 200 μL 1X SB17T by vacuum filtration.

[0177]

[0230] Step 5: Slow off-rate condensation process and photocleavage

[0231] Remove the drip director from the bottom of the Durapore plate and place the plate on a 1 mL microtiter collection plate. Wash the resin once with 200 μL 1XSB17T by centrifugation at 1000 x g for 30 seconds. Add 80 μL of 1XSB17T + 10 mM DxSO4 to the resin and irradiate with a BlackRay mercury lamp on a thermal mixer at 800 rpm for 10 minutes. Transfer the DuraPore plate to a new 1 mL deep-well plate and centrifuge at 1000 x g for 30 seconds to collect the photocleaved aptamer and protein:aptamer complexes.

[0178]

[0232] Step 6: Protein capture and removal of unbound aptamer

[0233] Add 50 μL of MyOne Streptavidin C1 Paramagnetic Beads (Invitrogen) (10 mg / mL in 1XSB17T) to the microtiter plate. Separate the beads with a magnet for 60 seconds and remove the supernatant. Add 225 μL of photocleavage mixture to the beads and mix for 5 minutes. Wash the beads four times with 200 μL 1XSB17T by separating the magnetic beads and replacing the wash buffer. Remove the final wash buffer.

[0179]

[0234] Step 7: Aptamer elution

[0235] 100 μL of sodium phosphate elution buffer (10 mM NaHPO, pH 11) is added to the beads and mixed for 5 minutes. 90 μL of the eluate is transferred to a microtiter plate and neutralized with 10 μL of sodium phosphate neutralization buffer (10 mM NaHPO, pH 5).

[0180]

[0236] Step 8: Aptamer hybridization to microarrays

[0237] DNA arrays were prepared containing oligonucleotide capture probes composed of complementary sequences of the variable regions of each aptamer immobilized on custom microscope slide supports. Multiple arrays (subarrays) were present on each slide, and the subarrays were physically separated by the installation of gaskets (Grace) for sample application. The arrays were pretreated with 100 μL of blocking buffer and incubated at 65°C for 15 minutes on a thermal mixer. 30 μL of high-salt hybridization buffer was added to 90 μL of neutralized aptamer eluate in a microtiter plate, incubated at 95°C for 5 minutes in a thermal cycler, and cooled to 65°C at 0.1°C / s. The blocking buffer was removed from the array, and 110 μL of aptamer sample was added to the array and incubated at 65°C for 20 hours in a humidified chamber.

[0181]

[0238] Step 9: Array washing

[0239] The aptamer sample is removed from the array, and the array is washed once with 200 μL of sodium phosphate Tween-20 wash buffer at 65°C with the gasket in place, and three times with 25 mL of sodium phosphate Tween-20 wash buffer in a pap bottle at 65°C with the gasket removed. The array is dried with a nitrogen gun.

[0182]

[0240] Step 10: Quantifying signals on the array

[0241] The array slides are scanned on a TECAN LS300 Reloaded in the appropriate channel for Cy3 detection, and the Cy3 signal on each array feature is quantified.

[0183]

[0242] result:

[0243] Aptamers specific for 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 was generated using 5-position modified nucleotides and selected for very slow off-rates against each target. Aptamers generated using the traditional process measured off-rates of less than 5 minutes. Aptamers generated using the slow off-rate enrichment process, including modified nucleotides during selection, had off-rates longer than 20 minutes. Two sets of aptamers were generated for each target by two different methods, resulting in a total of four distinct aptamer populations for each target. The ability of these aptamer populations to measure analyte concentrations in test samples was evaluated over a range of target concentrations, as described above. Relative signals from DNA chip detection were plotted against input target concentration. See Figures 11A-11C. The response curves of traditional aptamers are very flat, and the detection sensitivity is quite low. The sensitivity of detection for each target using the slow off-rate aptamers is excellent, and this data supports the need to use slow off-rate aptamers to maximize analytical performance.

[0184]

[0244] Example 8 Generation of a high-affinity BzdU aptamer against human thrombin

[0245] A. Preparation of the Candidate Mixture

[0246] As described in Example 3, the polynucleotides of the primers containing the 5'ANA chromophore were A candidate mixture containing dATP, dCTP, dGTP, and BzdUTP was prepared by dimerization and purified.

[0185]

[0247] B. Target Protein Preparation

[0248] Human thrombin was tagged with biotin as described in Example 2.

[0186]

[0249] C. Aptamer selection using a slow off-rate enrichment process and photocrosslinking

[0250] Aptamer selection was performed as described in Example 3 using biotinylated human thrombin as the target.

[0187]

[0251] D. Aptamer Amplification and Purification

[0252] Amplification and purification were carried out as described in Example 3.

[0253] E. Selection Stringency and Feedback

[0254] Selection stringency and feedback were performed as described in Example 3.

[0188]

[0255] F. Aptamer Properties

[0256] As shown in Figure 15, the equilibrium binding constant (Kd) of aptamer 2336-17 derived from this selection was 4.4e-11 M (measured according to the protocol described in Example 1).

[0189]

[0257] Single-stranded DNA aptamers for human thrombin were selected from a library composed of natural dA, dC, dG, and dT nucleotides (Bock et al., 1992). The binding affinities of the aptamers had Kd values ​​ranging from 2.5e-8M to 2.0e-7M. Using a similar protocol with a library composed of natural dA, dC, dG, and modified 5-(1-pentynyl)-dUTP, aptamers with Kd values ​​ranging from 4e-7M to 1e-6M were selected (Latham et al., 1994). Example 7 describes the discovery of a very high-affinity aptamer for human thrombin selected from a library composed of dA, dC, dG, and modified BzdU. The highest-affinity aptamer from this library had a Kd value of 4.4e-11M.

[0190]

[0258] Several patents, patent application publications, and scientific publications are cited throughout the description and / or listed at the end. Each of these is incorporated herein by reference in its entirety. Similarly, all publications mentioned within the incorporated publications are incorporated herein by reference in their entirety.

[0191]

[0259] Examples of the cited publications and limitations associated therewith are intended to be illustrative and not exclusive. Other limitations of the cited publications will be apparent to those skilled in the art upon reading the specification and studying the figures.

[0192]

[0260] The words "comprise", "comprises" and "comprising" shall be construed as inclusive and not exclusive.

[0193] The present invention also relates to the following: [Claim 1] 1. A method for identifying an aptamer, comprising: (a) preparing a candidate mixture of nucleic acids; (b) contacting the target molecule with the candidate mixture, wherein the target molecule is a nucleic acid that is present in the candidate mixture relative to other nucleic acids; the nucleic acid having increased affinity for the target molecule compared to the nucleic acid binds to the target molecule, forming a nucleic acid-target molecule complex; (c) exposing the nucleic acid-target molecule complex to a slow off-rate concentration process; (d) partitioning the nucleic acid-target molecule complexes from the candidate mixture; (e) dissociating the nucleic acid-target molecule complex to generate free nucleic acid; (f) amplifying the unbound nucleic acids to obtain a nucleic acid mixture enriched for sequences capable of binding to the target molecule with increased affinity, thereby enabling identification of aptamers to the target molecule; (g) Repeat steps (b) through (f) as necessary; and (h) identifying at least one aptamer for the target molecule; The method comprises the steps of: [Claim 2] The method of claim 1, wherein the candidate mixture is single-stranded or double-stranded nucleic acids. [Claim 3] The method of claim 1 , wherein the candidate mixture comprises DNA or RNA. [Claim 4] The method of claim 1 , wherein the candidate mixture comprises at least one chemical modification. [Claim 5] 5. The method of claim 4, wherein the chemically modified nucleic acid comprises chemical substitutions at one or more positions independently selected from the group consisting of ribose positions, deoxyribose positions, phosphate positions, and base positions. [Claim 6] The method of claim 4, wherein the chemically modified nucleic acid is independently selected from the group consisting of a 2' sugar modification, 2'-amino (2'-NH2), 2'-fluoro (2'-F), 2'-O-methyl (2'-OMe), a 5-pyrimidine modification, a modification with an exocyclic amine of cytosine, a 5-bromouracil substitution, a 5-bromodeoxyuridine substitution, a 5-bromodeoxycytidine substitution, a backbone modification, a methylation, a 3' cap, and a 5' cap. [Claim 7] 5. The method of claim 4, wherein the chemically modified nucleic acids are independently selected from the group consisting of 5-(N-benzylcarboxamido)-2'-deoxyuridine, 5-(N-isobutylcarboxamido)-2'-deoxyuridine, 5-(N-[2-(1H-indol-3yl)ethyl]carboxamido)-2'-deoxyuridine, 5-(N-[1-(3-trimethylammonium)propyl]carboxamido)-2'-deoxyuridine chloride, 5-(N-naphthylcarboxamido)-2'-deoxyuridine, and 5-(N-[1-(2,3-dihydroxypropyl)]carboxamido)-2'-deoxyuridine. [Claim 8] 2. The method of claim 1, wherein the slow off-rate enrichment process comprises diluting the candidate mixture containing the nucleic acid-target molecule complex. [Claim 9] 2. The method of claim 1, wherein the slow off-rate enrichment process comprises adding at least one competitor to the candidate mixture containing the nucleic acid-target molecule complex. [Claim 10] The method of claim 1, wherein the slow off-rate enrichment process comprises diluting the candidate mixture containing the nucleic acid-target molecule complex and adding at least one competitor to the candidate mixture containing the nucleic acid-target molecule complex. [Claim 11] The target molecule is selected from the group consisting of proteins, carbohydrates, polysaccharides, glycoproteins, hormones, receptors, peptides, antigens, antibodies, viruses, substrates, metabolites, transition state analogs, cofactors, inhibitors, drugs, dyes, nutrients, growth factors, tissues, and regulatory substances. The method of claim 1. [Claim 12] At least one aptamer has a slow dissociation rate (t 1 / 2 10. The method of claim 1, comprising: [Claim 13] The dissociation rate (t 1 / 2 13. The method of claim 12, wherein the heating time is about 30 minutes or more. [Claim 14] The dissociation rate (t 1 / 2 13. The method of claim 12, wherein the heating time is between about 30 minutes and about 240 minutes. [Claim 15] The dissociation rate (t 1 / 2 13. The method of claim 12, wherein the time period for which the stimuli are present is selected from the group consisting of about 30 minutes or more, about 60 minutes or more, about 90 minutes or more, about 120 minutes or more, about 150 minutes or more, about 180 minutes or more, about 210 minutes or more, and about 240 minutes or more. [Claim 16] 10. The method of claim 9, wherein said at least one competitor molecule is independently selected from the group consisting of an oligonucleotide, a polyanion, an abasic phosphodiester polymer, a dNTP, and a pyrophosphate. [Claim 17] 17. The method of claim 16, wherein the polyanion is selected from the group consisting of heparin, herring sperm DNA, salmon sperm DNA, dextran sulfate, and polydextran. [Claim 18] 10. An aptamer identified according to the method of claim 1. [Claim 19] Its slow dissociation rate (t 1 / 2 19. The aptamer of claim 18, having the following structure: [Claim 20] Its dissociation rate (t 1 / 2 19. The aptamer of claim 18, having the following structure: [Claim 21] The dissociation rate (t 1 / 2 19. The aptamer of claim 18, having the following structure: [Claim 22] The dissociation rate (t) from its target molecule is selected from the group consisting of about 30 minutes or more, about 60 minutes or more, about 90 minutes or more, about 120 minutes or more, about 150 minutes or more, about 180 minutes or more, about 210 minutes or more, and about 240 minutes or more. 1 / 2 19. The aptamer of claim 18, having the following structure: [Claim 23] The slow off-rate enrichment process is: (i) allowing a candidate mixture containing nucleic acid-target molecule complexes to achieve equilibrium binding; (ii) after (i), diluting the candidate mixture containing the nucleic acid-target molecule complex; and (iii) After (ii), incubating the candidate mixture containing the nucleic acid-target molecule complex. The method of claim 1, comprising the steps of: [Claim 24] The slow off-rate enrichment process is: (i) allowing a candidate mixture containing nucleic acid-target molecule complexes to achieve equilibrium binding; (ii) After (i), the candidate mixture containing the nucleic acid-target molecule complex is subjected to at least and mixing with one more competitor molecule; (ii) After (ii), incubating the candidate mixture containing the competitor and the nucleic acid-target molecule complex. The method of claim 1, comprising the steps of: [Claim 25] The slow off-rate enrichment process is: (i) allowing a candidate mixture containing nucleic acid-target molecule complexes to achieve equilibrium binding; (ii) after (i), mixing the candidate mixture containing the nucleic acid-target molecule complex with at least one competitor molecule; (iii) after (i), diluting the candidate mixture containing the nucleic acid-target molecule complex; and (iv) incubating the diluted candidate mixture containing the competitor and the nucleic acid-target molecule complex; The method of claim 1, comprising the steps of: [Claim 26] The method of claim 1, wherein the candidate mixture of nucleic acids comprises photoreactive nucleotides. [Claim 27] An aptamer having a slow dissociation rate from a target, 1 / 2 ) for about 30 minutes or more. [Claim 28] The dissociation rate (t 1 / 2 28. The aptamer of claim 27, wherein the time period is between about 30 minutes and about 240 minutes. [Claim 29] a dissociation rate (t) selected from the group consisting of about 30 minutes or more, about 60 minutes or more, about 90 minutes or more, about 120 minutes or more, about 150 minutes or more, about 180 minutes or more, about 210 minutes or more, and about 240 minutes or more 1 / 2 28. The aptamer of claim 27, having the following structure: [Claim 30] 27. The aptamer of claim 26, wherein the target is selected from the group consisting of the targets listed in Figure 7. [Claim 31] The aptamer of claim 26, wherein the target is a protein. [Claim 32] The aptamer of claim 26, wherein the target is a peptide. [Claim 33] i) a cuttable element; ii) a detectable element; iii) a spacer element, and iv) Tags 27. The aptamer of claim 26, comprising at least one element independently selected from the group consisting of: [Claim 34] An aptamer that specifically binds to a target, the aptamer comprising at least one base-modified pyrimidine independently selected from the group consisting of the base-modified pyrimidines shown in Figure 14. [Claim 35] 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 35. The aptamer of claim 34, further comprising at least one photoreactive nucleotide independently selected from the group consisting of 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. [Claim 36] 34. The aptamer of claim 33, further comprising at least one photoreactive group independently selected from the group consisting of anthraquinone (AQ), psoralen (Psor), 4-azido-2-nitro-aniline (ANA), and 5-bromo-dUTP. [Claim 37] 1. A method for identifying an aptamer that has a slow dissociation rate from its target molecule, comprising: (a) preparing a candidate mixture of nucleic acids; (b) contacting the target molecule with the candidate mixture, wherein nucleic acids in the candidate mixture that have increased affinity for the target molecule relative to other nucleic acids bind to the target molecule to form nucleic acid-target molecule complexes; (c) exposing the nucleic acid-target molecule complex to an off-rate enrichment process; (d) partitioning the nucleic acid-target molecule complexes from the candidate mixture; (e) dissociating the nucleic acid-target molecule complex to generate unbound nucleic acid; (f) amplifying the unbound nucleic acids to obtain a nucleic acid mixture enriched for sequences capable of binding to the target molecule with increased affinity, thereby enabling identification of aptamers to the target molecule; (g) Repeat steps (b) through (f) as necessary; and (h) identifying at least one aptamer to a target molecule, wherein the aptamer has a slow off-rate from the target molecule; The method comprises the steps of: [Claim 38] 38. The method of claim 37, wherein the candidate mixture is single-stranded or double-stranded nucleic acids. [Claim 39] 38. The method of claim 37, wherein the candidate mixture comprises DNA or RNA. [Claim 40] 38. The method of claim 37, wherein the candidate mixture comprises chemically modified nucleic acids. [Claim 41] 41. The method of claim 40, wherein the chemically modified nucleic acid comprises chemical substitutions at one or more positions independently selected from the group consisting of ribose positions, deoxyribose positions, phosphate positions, and base positions. [Claim 42] 41. The method of claim 40, wherein the chemically modified nucleic acid is independently selected from the group consisting of a 2' sugar modification, 2'-amino (2'-NH2), 2'-fluoro (2'-F), 2'-O-methyl (2'-OMe), a 5-pyrimidine modification, a modification with an exocyclic amine of cytosine, a 5-bromouracil substitution, a 5-bromodeoxyuridine substitution, a 5-bromodeoxycytidine substitution, a backbone modification, a methylation, a 3' cap, and a 5' cap. [Claim 43] The at least one chemical modification may be selected from the group consisting of 5-(N-benzylcarboxamido)-2'-deoxyuridine, 5-(N-isobutylcarboxamido)-2'-deoxyuridine, 5-(N-[2-(1H-indol-3yl)ethyl]carboxamido)-2'-deoxyuridine, 5-(N-[1-(3-trimethylammonium)propyl]carboxamido)-2'-deoxyuridine, ...isobutylcarboxamido)-2'- 41. The method of claim 40, wherein the hydroxyl groups are independently selected from the group consisting of 5-(N-[1-(2,3-dihydroxypropyl)]carboxamido)-2'-deoxyuridine chloride, 5-(N-naphthylcarboxamido)-2'-deoxyuridine, and 5-(N-[1-(2,3-dihydroxypropyl)]carboxamido)-2'-deoxyuridine. [Claim 44] 38. The method of claim 37, wherein said slow off-rate enrichment process comprises diluting the candidate mixture containing the nucleic acid-target molecule complex. [Claim 45] 38. The method of claim 37, wherein said slow off-rate enrichment process comprises adding at least one competitor to the candidate mixture containing the nucleic acid-target molecule complexes. [Claim 46] 38. The method of claim 37, wherein the slow off-rate enrichment process comprises diluting the candidate mixture containing the nucleic acid-target molecule complexes and adding at least one competitor to the candidate mixture containing the nucleic acid-target molecule complexes. [Claim 47] 38. The method of claim 37, wherein the target molecule is selected from the group consisting of a protein, a carbohydrate, a polysaccharide, a glycoprotein, a hormone, a receptor, a peptide, an antigen, an antibody, a virus, a substrate, a metabolite, a transition state analog, a cofactor, an inhibitor, a drug, a dye, a nutrient, a growth factor, a tissue, and a regulatory substance. [Claim 48] The dissociation rate (t 1 / 2 38. The method of claim 37, wherein the heating time is about 30 minutes or more. [Claim 49] The dissociation rate (t 1 / 2 38. The method of claim 37, wherein the heating time is between about 30 minutes and about 240 minutes. [Claim 50] The dissociation rate (t 1 / 2 38. The method of claim 37, wherein the time period is selected from the group consisting of about 30 minutes or more, about 60 minutes or more, about 90 minutes or more, about 120 minutes or more, about 150 minutes or more, about 180 minutes or more, about 210 minutes or more, and about 240 minutes or more. [Claim 51] 46. ​​The method of claim 45, wherein said at least one competitor molecule is independently selected from the group consisting of an oligonucleotide, a polyanion, an abasic phosphodiester polymer, a dNTP, and a pyrophosphate. [Claim 52] 52. The method of claim 51, wherein the polyanion is selected from the group consisting of heparin, herring sperm DNA, salmon sperm DNA, dextran sulfate, and polydextran. [Claim 53] 38. An aptamer identified according to the method of claim 37. [Claim 54] Its dissociation rate (t 1 / 2 54. The aptamer of claim 53, having the following structure: [Claim 55] The dissociation rate (t 1 / 2 54. The aptamer of claim 53, having the following structure: [Claim 56] The dissociation rate (t) from its target molecule is selected from the group consisting of about 30 minutes or more, about 60 minutes or more, about 90 minutes or more, about 120 minutes or more, about 150 minutes or more, about 180 minutes or more, about 210 minutes or more, and about 240 minutes or more. 1 / 2 54. The aptamer of claim 53, having the following structure: [Claim 57] The slow off-rate enrichment process is: (i) allowing a candidate mixture containing nucleic acid-target molecule complexes to achieve equilibrium binding; (ii) after (i), diluting the candidate mixture containing the nucleic acid-target molecule complex; and (iii) After (ii), incubating the candidate mixture containing the nucleic acid-target molecule complex. 38. The method of claim 37, comprising the steps of: [Claim 58] The slow off-rate enrichment process is: (i) allowing a candidate mixture containing nucleic acid-target molecule complexes to achieve equilibrium binding; (ii) after (i), mixing the candidate mixture containing the nucleic acid-target molecule complex with at least one competitor molecule; (ii) After (ii), incubating the candidate mixture containing the competitor and the nucleic acid-target molecule complex. 38. The method of claim 37, comprising the steps of: [Claim 59] The slow off-rate enrichment process is: (i) allowing a candidate mixture containing nucleic acid-target molecule complexes to achieve equilibrium binding; (ii) after (i), mixing the candidate mixture containing the nucleic acid-target molecule complex with at least one competitor molecule; (iii) after (i), diluting the candidate mixture containing the nucleic acid-target molecule complex; and (iv) incubating the diluted candidate mixture containing the competitor and the nucleic acid-target molecule complex; 38. The method of claim 37, comprising the steps of: [Claim 60] 38. The method of claim 37, wherein the candidate mixture of nucleic acids comprises photoreactive nucleotides. [Claim 61] An aptamer having a slow dissociation rate from a target, 1 / 2 ) for about 30 minutes or more. [Claim 62] The dissociation rate (t 1 / 2 62. The aptamer of claim 61, wherein the time period is between about 30 minutes and about 240 minutes. [Claim 63] The dissociation rate (t 1 / 2 62. The aptamer of claim 61, wherein the time period is selected from the group consisting of about 30 minutes or more, about 60 minutes or more, about 90 minutes or more, about 120 minutes or more, about 150 minutes or more, about 180 minutes or more, about 210 minutes or more, and about 240 minutes or more. [Claim 64] 62. The aptamer of claim 61, wherein the target is selected from the group consisting of the targets listed in Figure 7. [Claim 65] The aptamer of claim 61, wherein the target is a protein. [Claim 66] 62. The aptamer of claim 61, wherein the target is a peptide. [Claim 67] i) a cuttable element; ii) a detectable element; iii) a spacer element, and iv) Tags 62. The aptamer of claim 61, comprising at least one element independently selected from the group consisting of: [Claim 68] A photoaptamer that specifically binds to a target, the photoaptamer comprising at least one base-modified pyrimidine independently selected from the group consisting of base-modified pyrimidines shown in Figure 14. [Claim 69] 69. The photoaptamer of claim 68, wherein the at least one base-modified pyrimidine is independently selected from the group consisting of 5-(N-benzylcarboxamido)-2'-deoxyuridine, 5-(N-isobutylcarboxamido)-2'-deoxyuridine, 5-(N-[2-(1H-indol-3yl)ethyl]carboxamido)-2'-deoxyuridine, 5-(N-[1-(3-trimethylammonium)propyl]carboxamido)-2'-deoxyuridine chloride, 5-(N-naphthylcarboxamido)-2'-deoxyuridine, and 5-(N-[1-(2,3-dihydroxypropyl)]carboxamido)-2'-deoxyuridine. [Claim 70] 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 69. The photoaptamer of claim 68, further comprising at least one photoreactive nucleotide independently selected from the group consisting of anthine, 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. [Claim 71] 69. The photoaptamer of claim 68, further comprising at least one photoreactive group independently selected from the group consisting of anthraquinone (AQ), psoralen (Psor), 4-azido-2-nitro-aniline (ANA), and 5-bromo-dUTP. [Claim 72] A diagnostic kit containing an aptamer that specifically binds to a target, wherein the aptamer comprises at least one base-modified pyrimidine independently selected from the group consisting of base-modified pyrimidines shown in Figure 14. [Claim 73] The aptamer is selected from the group consisting of 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-azi ... Xanthine, 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-iodoguanine. 73. The diagnostic kit of claim 72, further comprising at least one photoreactive nucleotide independently selected from the group consisting of aza-7-bromoguanine. [Claim 74] 73. The diagnostic kit of claim 72, wherein the aptamer further comprises at least one photoreactive group independently selected from the group consisting of anthraquinone (AQ), psoralen (Psor), 4-azido-2-nitro-aniline (ANA), and 5-bromo-dUTP. [Claim 75] 1. A method for identifying a photoaptamer that has a slow dissociation rate from its target molecule, comprising: (a) preparing a candidate mixture of nucleic acids; wherein each nucleic acid of said candidate mixture contains at least one photoreactive moiety; (b) contacting the target molecule with the candidate mixture, wherein nucleic acids in the candidate mixture that have increased affinity for the target molecule relative to other nucleic acids bind to the target molecule to form nucleic acid-target molecule complexes; (c) exposing the nucleic acid-target molecule complex formed in (b) to a slow off-rate concentration process; (d) irradiating the nucleic acid-target molecule complex, wherein the nucleic acid-target molecule is photocrosslinked; (e) distributing the photocrosslinked nucleic acid-target molecule complexes from the candidate mixture; (f) amplifying the photocrosslinked nucleic acids to obtain a nucleic acid mixture enriched for sequences capable of binding to the target molecule with increased affinity, thereby enabling identification of photoaptamers for the target molecule; (g) Repeat steps (b) through (f) as necessary; and (h) identifying at least one photoaptamer to a target molecule, wherein the photoaptamer has a slow dissociation rate from its target molecule. The method comprises the steps of: [Claim 76] 76. The method of claim 75, wherein the photoaptamer is a single-stranded nucleic acid or a double-stranded nucleic acid. [Claim 77] 77. The method of claim 76, wherein the photoaptamer comprises DNA or RNA. [Claim 78] The photoaptamer is selected from the group consisting of 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, 77. The method of claim 76, further comprising at least one photoreactive nucleotide independently selected from the group consisting of 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. [Claim 79] 76. The method of claim 75, wherein the photoaptamer comprises at least one photoreactive group independently selected from the group consisting of anthraquinone (AQ), psoralen (Psor), 4-azido-2-nitro-aniline (ANA), and 5-bromo-dUTP. [Claim 80] The slow off-rate enrichment process described above is carried out to obtain candidate mixtures containing nucleic acid-target molecule complexes. 76. The method of claim 75, further comprising the step of diluting the mixture. [Claim 81] 76. The method of claim 75, wherein said slow off-rate enrichment process comprises adding at least one competitor to the candidate mixture containing the nucleic acid-target molecule complexes. [Claim 82] 76. The method of claim 75, wherein the slow off-rate enrichment process comprises diluting the candidate mixture containing the nucleic acid-target molecule complexes and adding at least one competitor to the candidate mixture containing the nucleic acid-target molecule complexes. [Claim 83] 82. The method of claim 81, wherein said at least one competitor is independently selected from the group consisting of an oligonucleotide, a polyanion, an abasic phosphodiester polymer, a dNTP, and a pyrophosphate. [Claim 84] 84. The method of claim 83, wherein the polyanion is selected from the group consisting of heparin, herring sperm DNA, salmon sperm DNA, and dextran sulfate. [Claim 85] The dissociation rate (t 1 / 2 76. The method of claim 75, wherein the time period is about 30 minutes or more. [Claim 86] The dissociation rate (t 1 / 2 76. The method of claim 75, wherein the heating time is between about 30 minutes and about 240 minutes. [Claim 87] The dissociation rate (t 1 / 2 76. The method of claim 75, wherein the time period is selected from the group consisting of about 30 minutes or more, about 60 minutes or more, about 90 minutes or more, about 120 minutes or more, about 150 minutes or more, about 180 minutes or more, about 210 minutes or more, and about 240 minutes or more. [Claim 88] 76. A photoaptamer produced according to the method of claim 75. [Claim 89] The dissociation rate (t 1 / 2 89. The photoaptamer of claim 88, having the following structure: [Claim 90] The dissociation rate (t 1 / 2 89. The photoaptamer of claim 88, having the following structure: [Claim 91] 89. The optical aptamer of claim 88, having a dissociation rate from its target selected from the group consisting of times of about 30 minutes or more, about 60 minutes or more, about 90 minutes or more, about 120 minutes or more, about 150 minutes or more, about 180 minutes or more, about 210 minutes or more, and about 240 minutes or more. [Claim 92] 1. A method for identifying a polyanion-refractory aptamer, comprising: a) contacting a candidate mixture of nucleic acids with a target; b) allowing the mixture to reach equilibrium, where nucleic acid-target complexes are formed; c) adding a polyanionic substance to the mixture and incubating the mixture; d) partitioning nucleic acid-target molecule complexes from the candidate mixture; (e) dissociating the nucleic acid-target molecule complex to generate unbound nucleic acid; (f) amplifying the unbound nucleic acids to obtain a nucleic acid mixture enriched for sequences capable of binding to the target molecule with increased affinity, thereby enabling identification of polyanion-refractory aptamers to the target molecule; (g) Repeat steps (b) through (f) as necessary; and (h) identifying at least one polyanion-resistant aptamer for a target molecule; The method comprises the steps of: [Claim 93] An aptamer-target complex, wherein the aptamer is resistant to degradation in the presence of polyanionic substances. [Claim 94] A biochip comprising the aptamer of claim 53. [Claim 95] A diagnostic device comprising the aptamer of claim 53. [Claim 96] A therapeutic agent comprising the aptamer of claim 53. [Claim 97] 54. An imaging reagent comprising the aptamer of claim 53. [Claim 98] A histological reagent comprising the aptamer of claim 53. [Claim 99] A pathology reagent comprising the aptamer of claim 53. [Claim 100] A cytology reagent comprising the aptamer of claim 53. [Claim 101] 54. An affinity separation reagent comprising the aptamer of claim 53. [Claim 102] A biosensor comprising the aptamer of claim 53. [Claim 103] 55. An ALONA device comprising the aptamer of claim 54. [Claim 104] Dissociation rate (t 1 / 2 ) Aptamer-target complexes having [Claim 105] Dissociation rate (t 1 / 2 105. The aptamer-target complex of claim 104, having [Claim 106] The aptamer-target complex of claim 105, having a dissociation rate selected from the group consisting of about 30 minutes or more, about 60 minutes or more, about 90 minutes or more, about 120 minutes or more, about 150 minutes or more, about 180 minutes or more, about 210 minutes or more, and about 240 minutes or more. [Claim 107] 106. The aptamer-target complex of claim 105, wherein the aptamer is a photoaptamer. [Claim 108] 1. A method for identifying an aptamer comprising at least one base-modified pyrimidine, comprising: (a) preparing a candidate mixture of nucleic acids, wherein the nucleic acids each contain at least one base-modified pyrimidine independently selected from the group consisting of the base-modified pyrimidines shown in Figure 14; (b) contacting the target molecule with the candidate mixture, wherein nucleic acids in the candidate mixture that have increased affinity for the target molecule relative to other nucleic acids bind to the target molecule to form nucleic acid-target molecule complexes; (c) partitioning nucleic acid-target molecule complexes from the candidate mixture; (d) dissociating the nucleic acid-target molecule complex to generate unbound nucleic acid; (e) amplifying the unbound nucleic acids to obtain a nucleic acid mixture enriched for sequences capable of binding to the target molecule with increased affinity, thereby enabling identification of aptamers to the target molecule; (f) Repeat steps (b) through (e) as necessary; and (g) identifying at least one aptamer to the target molecule, wherein the aptamer comprises at least one base-modified pyrimidine; The method comprises the steps of: [Claim 109] 109. The method of claim 108, wherein the aptamer further comprises at least one additional chemical modification. [Claim 110] 109. The method of claim 108, wherein said at least one additional chemical modification is a chemical substitution at one or more positions independently selected from the group consisting of ribose positions, deoxyribose positions, phosphate positions, and base positions. [Claim 111] 109. The method of claim 108, wherein the at least one additional chemical modification is independently selected from the group consisting of a 2' sugar modification, a 2'-amino (2'-NH2), a 2'-fluoro (2'-F), a 2'-O-methyl (2'-OMe), a 5-pyrimidine modification, a modification with an exocyclic amine of cytosine, a 5-bromouracil substitution, a 5-bromodeoxyuridine substitution, a 5-bromodeoxycytidine substitution, a backbone modification, a methylation, a 3' cap, and a 5' cap. [Claim 112] 109. The method of claim 108, wherein the at least one base-modified pyrimidine is independently selected from the group consisting of 5-(N-benzylcarboxamido)-2'-deoxyuridine, 5-(N-isobutylcarboxamido)-2'-deoxyuridine, 5-(N-[2-(1H-indol-3yl)ethyl]carboxamido)-2'-deoxyuridine, 5-(N-[1-(3-trimethylammonium)propyl]carboxamido)-2'-deoxyuridine chloride, 5-(N-naphthylcarboxamido)-2'-deoxyuridine, and 5-(N-[1-(2,3-dihydroxypropyl)]carboxamido)-2'-deoxyuridine. [Claim 113] 109. The method of claim 108, wherein the target molecule is selected from the group consisting of a protein, a carbohydrate, a polysaccharide, a glycoprotein, a hormone, a receptor, a peptide, an antigen, an antibody, a virus, a substrate, a metabolite, a transition state analog, a cofactor, an inhibitor, a drug, a dye, a nutrient, a growth factor, a tissue, and a regulatory substance. [Claim 114] Aptamers have a slow dissociation rate (t 1 / 2 109. The method of claim 108, comprising: [Claim 115] The dissociation rate (t 1 / 2 115. The method of claim 114, wherein the time period is about 30 minutes or more. [Claim 116] The dissociation rate (t 1 / 2 116. The method of claim 115, wherein the heating time is between about 30 minutes and about 240 minutes. [Claim 117] The dissociation rate (t 1 / 2 117. The method of claim 116, wherein the time period is selected from the group consisting of about 30 minutes or more, about 60 minutes or more, about 90 minutes or more, about 120 minutes or more, about 150 minutes or more, about 180 minutes or more, about 210 minutes or more, and about 240 minutes or more. [Claim 118] 118. An aptamer identified according to the method of claim 117. [Claim 119] Its slow dissociation rate (t 1 / 2 119. The aptamer of claim 118, having the following structure: [Claim 120] Its dissociation rate (t 1 / 2 ) claim 1 18 aptamers. [Claim 121] The dissociation rate (t 1 / 2 119. The aptamer of claim 118, having the following structure: [Claim 122] The dissociation rate (t) from its target molecule is selected from the group consisting of about 30 minutes or more, about 60 minutes or more, about 90 minutes or more, about 120 minutes or more, about 150 minutes or more, about 180 minutes or more, about 210 minutes or more, and about 240 minutes or more. 1 / 2 119. The aptamer of claim 118, having the following structure: [Claim 123] 119. The method of claim 118, wherein the candidate mixture of nucleic acids comprises photoreactive nucleotides. [Claim 124] 1. A method for identifying or producing an aptamer that has a slow dissociation rate from its target molecule, comprising: (a) preparing a candidate mixture of nucleic acids; (b) contacting the target molecule with the candidate mixture, wherein nucleic acids in the candidate mixture that have increased affinity for the target molecule relative to other nucleic acids bind to the target molecule to form nucleic acid-target molecule complexes; (c) incubating the candidate mixture and the target molecule together for a period of time sufficient to achieve equilibrium binding; (d) adding at least one competitor molecule in excess to the mixture of (c); (e) incubating the mixture of candidate mixture, target molecule / aptamer complex and competitor molecule from (d) for a predetermined period of time; (f) distributing nucleic acid-target molecule complexes from the candidate mixture; (g) dissociating the nucleic acid-target molecule complex to generate unbound nucleic acid; (h) amplifying the unbound nucleic acids to obtain a nucleic acid mixture enriched for nucleic acid sequences capable of binding to the target molecule with increased affinity; The method comprises a step of: [Claim 125] (i) repeating steps (b) through (h) as necessary; and (h) identifying at least one aptamer to the target molecule, wherein the aptamer has a relatively slow dissociation rate from its target molecule; 125. The method of claim 124, further comprising the step of: [Claim 126] (e) The predetermined period is: (i) for at least 10 minutes; (ii) for at least 20 minutes; (iii) for at least 30 minutes; (iv) for at least 45 minutes; (v) at least one hour; (vi) at least two hours; (vii) at least three hours; (viii) at least 4 hours; (ix) at least 5 hours; (x) At least 6 hours 126. The method of claim 124 or 125, selected from: [Claim 127] Any of claims 124 to 126, further comprising the step of sequencing the unbound nucleic acids of (g) or nucleic acids from the mixture of (h), thereby identifying an aptamer for the target. How to do it. [Claim 128] The method of any one of claims 124 to 126, further comprising the step of preparing an aptamer based on the aptamer thus identified. [Claim 129] 129. A method for producing an aptamer that has a slow dissociation rate from its target molecule, comprising preparing the aptamer based on an aptamer identified by the method of claim 128. [Claim 130] 1. A method for producing an aptamer that has a slow dissociation rate from its target molecule, comprising: (a) preparing a candidate mixture of nucleic acids; (b) contacting the target molecule with the candidate mixture, wherein nucleic acids in the candidate mixture that have increased affinity for the target molecule relative to other nucleic acids bind to the target molecule to form nucleic acid-target molecule complexes; (c) incubating the candidate mixture and the target molecule together for a period of time sufficient to achieve equilibrium binding; (d) adding at least one competitor molecule in excess to the mixture of (c); (e) incubating the mixture of candidate mixture, target molecule / aptamer complex and competitor molecule from (d) for a predetermined period of time; (f) distributing nucleic acid-target molecule complexes from the candidate mixture; (g) dissociating the nucleic acid-target molecule complex to generate unbound nucleic acid; (h) amplifying the unbound nucleic acids to obtain a nucleic acid mixture enriched for nucleic acid sequences capable of binding to the target molecule with increased affinity; preparing or synthesizing an aptamer based on a nucleic acid sequence identified by the process, thereby identifying an aptamer for the target molecule. [Claim 131] (i) repeating steps (b) through (h) as necessary; and (h) identifying at least one aptamer to the target molecule, wherein the aptamer has a relatively slow dissociation rate from its target molecule; 131. The method of claim 130, further comprising the step of: [Claim 132] 10. The method of any one of the preceding claims, wherein the candidate mixture comprises modified nucleic acids, in which one, some or all of the pyrimidines in at least one or each nucleic acid are chemically modified at the 5-position. [Claim 133] 133. The method of claim 132, wherein all C residues in the nucleic acids of the candidate mixture are chemically modified at the 5 position. [Claim 134] 134. The method of claim 132 or 133, wherein all T residues in the nucleic acids of the candidate mixture are chemically modified at the 5 position. [Claim 135] 135. The method of claim 132 or 134, wherein all U residues in the nucleic acids of the candidate mixture are chemically modified at the 5 position. [Claim 136] Chemical modification at position 5: 5-(N-benzylcarboxamido)-2'-deoxyuridine, 5-(N-isobutylcarboxamido)-2'-deoxyuridine, 5-(N-[2-(1H-indol-3yl)ethyl]carboxamido)-2'-deoxyuridine, 5-(N-[1-(3-trimethylammonium)propyl]carboxamido)-2'-deoxyuridine 136. The method of any one of claims 132-135, wherein the uridine-containing compound is selected from the group consisting of 5-(N-naphthylcarboxamido)-2'-deoxyuridine chloride, 5-(N-naphthylcarboxamido)-2'-deoxyuridine, and 5-(N-[1-(2,3-dihydroxypropyl)]carboxamido)-2'-deoxyuridine. [Claim 137] 136. The method of any one of claims 132 to 135, wherein the 5-position chemical modifications are independently selected from the group of chemical modifications shown in Figure 14. [Claim 138] 1. A method for identifying or producing an aptamer that has a slow dissociation rate from its target molecule, comprising: (a) preparing a candidate mixture of nucleic acids, wherein the candidate mixture comprises modified nucleic acids, wherein one, some, or all of the pyrimidines in at least one, or each, nucleic acid of the candidate mixture are chemically modified at the 5-position; (b) contacting the target molecule with the candidate mixture, wherein nucleic acids in the candidate mixture that have increased affinity for the target molecule relative to other nucleic acids bind to the target molecule to form nucleic acid-target molecule complexes; (c) partitioning the nucleic acids with increased affinity from the remainder of the candidate mixture; and (d) amplifying the nucleic acids with increased affinity to obtain a nucleic acid mixture enriched for nucleic acid sequences capable of binding to the target molecule with increased affinity; The method comprises a step of: [Claim 139] Chemical modification at position 5: 139. The method of claim 138, wherein the uridine is selected from 5-(N-benzylcarboxamido)-2'-deoxyuridine, 5-(N-isobutylcarboxamido)-2'-deoxyuridine, 5-(N-[2-(1H-indol-3yl)ethyl]carboxamido)-2'-deoxyuridine, 5-(N-[1-(3-trimethylammonium)propyl]carboxamido)-2'-deoxyuridine chloride, 5-(N-naphthylcarboxamido)-2'-deoxyuridine, and 5-(N-[1-(2,3-dihydroxypropyl)]carboxamido)-2'-deoxyuridine. [Claim 140] 139. The method of claim 138, wherein the 5-position chemical modifications are independently selected from the group of chemical modifications shown in Figure 14. [Claim 141] 141. The method of any one of claims 138 to 140, wherein all C residues in the nucleic acids of the candidate mixture are chemically modified at the 5 position. [Claim 142] 142. The method of any one of claims 138 to 141, wherein all T residues in the nucleic acids of the candidate mixture are chemically modified at position 5. [Claim 143] The method of claims 138 to 141, wherein all U residues in the nucleic acids of the candidate mixture are chemically modified at the 5 position. [Claim 144] 143. The method of any one of claims 138 to 142, further comprising the step of sequencing nucleic acids from the mixture of (d), thereby identifying an aptamer for the target. [Claim 145] The method of any one of claims 138 to 143, further comprising the step of preparing an aptamer based on the aptamer thus identified. [Claim 146] 144. A method for producing an aptamer that has a slow dissociation rate from its target molecule, comprising: The method further comprises the step of preparing a mer. [Claim 147] 1. A method for producing an aptamer that has a slow dissociation rate from its target molecule, comprising: (a) preparing a candidate mixture of nucleic acids, wherein the candidate mixture comprises modified nucleic acids, wherein one, some, or all of the pyrimidines in at least one, or each, nucleic acid of the candidate mixture are chemically modified at the 5-position; (b) contacting the target molecule with the candidate mixture, wherein nucleic acids in the candidate mixture that have increased affinity for the target molecule relative to other nucleic acids bind to the target molecule to form nucleic acid-target molecule complexes; (c) partitioning the nucleic acids with increased affinity from the remainder of the candidate mixture; and (d) amplifying the nucleic acids with increased affinity to obtain a nucleic acid mixture enriched for nucleic acid sequences capable of binding to the target molecule with increased affinity; preparing or synthesizing an aptamer based on a nucleic acid sequence identified by the process, thereby identifying an aptamer for the target molecule. [Claim 148] Chemical modification at position 5: 148. The method of claim 147, wherein the uridine is selected from 5-(N-benzylcarboxamido)-2'-deoxyuridine, 5-(N-isobutylcarboxamido)-2'-deoxyuridine, 5-(N-[2-(1H-indol-3yl)ethyl]carboxamido)-2'-deoxyuridine, 5-(N-[1-(3-trimethylammonium)propyl]carboxamido)-2'-deoxyuridine chloride, 5-(N-naphthylcarboxamido)-2'-deoxyuridine, and 5-(N-[1-(2,3-dihydroxypropyl)]carboxamido)-2'-deoxyuridine. [Claim 149] 148. The method of claim 147, wherein the 5-position chemical modifications are independently selected from the group of chemical modifications shown in Figure 14. [Claim 150] 149. The method of any one of claims 147 to 148, wherein all C residues in the nucleic acids of the candidate mixture are chemically modified at the 5 position. [Claim 151] 151. The method of any one of claims 147 to 150, wherein all T residues in the nucleic acids of the candidate mixture are chemically modified at position 5. [Claim 152] 151. The method of any one of claims 147 to 150, wherein all U residues in the nucleic acids of the candidate mixture are chemically modified at the 5 position. [Claim 153] The dissociation rate (t 1 / 2 )but: (i) ≥30 minutes; (ii) about 30 minutes to about 240 minutes; (iii) 30 to 60 minutes (iv) 60 to 90 minutes (v) 90 to 120 minutes (vi) 120 to 150 minutes (vii) 150 to 180 minutes (viii) 180 to 210 minutes (ix) Between 210 and 240 minutes 10. The method of any one of the preceding claims, selected from: [Claim 154] (i) ≥30 minutes; (ii) about 30 minutes to about 240 minutes; (iii) 30 to 60 minutes (iv) 60 to 90 minutes (v) 90 to 120 minutes (vi) 120 to 150 minutes (vii) 150 to 180 minutes (viii) 180 to 210 minutes (ix) Between 210 and 240 minutes the dissociation rate (t) of the target and aptamer from the non-covalent complex, selected from one of 1 / 2 ) an aptamer having [Claim 155] A non-covalent complex of an aptamer and its target, where the dissociation rate of the aptamer from the target (t 1 / 2 )but: (i) ≥30 minutes; (ii) about 30 minutes to about 240 minutes; (iii) 30 to 60 minutes (iv) 60 to 90 minutes (v) 90 to 120 minutes (vi) 120 to 150 minutes (vii) 150 to 180 minutes (viii) 180 to 210 minutes (ix) Between 210 and 240 minutes The complex is selected from one of the following: [Claim 156] 156. The aptamer of claim 155, wherein one, some or all of the pyrimidines in the nucleic acid sequence of the aptamer are modified at the 5-position. [Claim 157] Chemical modification at position 5: 157. The aptamer of claim 156, wherein the aptamer is selected from 5-(N-benzylcarboxamido)-2'-deoxyuridine, 5-(N-isobutylcarboxamido)-2'-deoxyuridine, 5-(N-[2-(1H-indol-3yl)ethyl]carboxamido)-2'-deoxyuridine, 5-(N-[1-(3-trimethylammonium)propyl]carboxamido)-2'-deoxyuridine chloride, 5-(N-naphthylcarboxamido)-2'-deoxyuridine, and 5-(N-[1-(2,3-dihydroxypropyl)]carboxamido)-2'-deoxyuridine. [Claim 158] The aptamer of claim 157, wherein the 5-position chemical modifications are independently selected from the group of chemical modifications shown in Figure 14. [Claim 159] 159. The aptamer of any one of claims 156 to 158, wherein all C residues in the nucleic acid sequence of the aptamer are chemically modified at the 5-position. [Claim 160] 160. The aptamer of any one of claims 156 to 159, wherein all T residues in the nucleic acid sequence of the aptamer are chemically modified at the 5-position. [Claim 161] 161. The aptamer of any one of claims 156 to 160, wherein all U residues in the nucleic acid sequence of the aptamer are chemically modified at the 5-position. [Claim 162] A non-covalent complex of an aptamer and a target, wherein the aptamer binds to the target. K below 100 nM d and the dissociation rate of the aptamer from the target (t 1 / 2 ) is 30 minutes or longer, and one, some, or all of the pyrimidines in the nucleic acid sequence of the aptamer are modified at the 5-position, and the modifications are selected from: 5-(N-benzylcarboxamido)-2'-deoxyuridine, 5-(N-isobutylcarboxamido)-2'-deoxyuridine, 5-(N-[2-(1H-indol-3yl)ethyl]carboxamido)-2'-deoxyuridine, 5-(N-[1-(3-trimethylammonium)propyl]carboxamido)-2'-deoxyuridine chloride, 5-(N-naphthylcarboxamido)-2'-deoxyuridine, and 5-(N-[1-(2,3-dihydroxypropyl)]carboxamido)-2'-deoxyuridine, or independently selected from the group of chemical modifications shown in FIG. 14.

Claims

1. A non-covalent complex of an aptamer and a target, wherein the aptamer comprises at least one modified pyrimidine nucleotide, the modified pyrimidine nucleotide having the following structure: 【Chemical 1】 Z is in addition to R (CH 2 ) n is a linking group, wherein n is 1, 2, or 3; and R is: 【Chemistry 2】 * indicates that the R group is (CH 2 ) n indicates the point of attachment of the ribose to the phosphate backbone; and the dissociation rate of the aptamer from the target (t １／２ ) for 30 minutes or more, and the aptamer has a dissociation constant (K ｄ ) The complex.

2. 2. The non-covalent complex of claim 1, wherein all C, all T or all U residues in the nucleic acid sequence of the aptamer are chemically modified at the 5-position.

3. 2. The non-covalent complex of claim 1, wherein the C-5 modification of the nucleotide uses an amide bond.

4. 2. The non-covalent complex of claim 1, wherein the aptamer comprises the modified nucleotide 5-benzyl-dUTP, 5-isobutyl-dUTP, or 5-tryptaamino-dUTP.

5. The dissociation rate of the aptamer from the target (t １／２ )but, (i) Between 30 and 240 minutes; (ii) 30 minutes to 60 minutes; (iii) 60 to 90 minutes; (iv) 90 minutes to 120 minutes; (v) 120 minutes to 150 minutes; (vi) 150 minutes to 180 minutes; (vii) 180 minutes to 210 minutes; (viii) 210 minutes to 240 minutes; A non-covalent complex of an aptamer and a target according to any one of claims 1 to 4, selected from one of:

6. The aptamer has a dissociation constant (K) for its target selected from one of: less than 100 nM, less than 10 nM, less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM. ｄ 6. A non-covalent complex of an aptamer and a target according to any one of claims 1 to 5, comprising:

7. A non-covalent complex of an aptamer and a target described in any one of claims 1 to 6, wherein the aptamer comprises at least one base-modified pyrimidine independently selected from the group consisting of base-modified pyrimidines shown in Figure 14 below. 【Chemistry 3-1】 【Chemistry 3-2】

8. 8. The non-covalent complex of claim 7, wherein the specific 5-position pyrimidine modification comprises the linker (X) shown in Figure 14 below. 【Chemistry 4-1】 【Chemistry 4-2】

9. A non-covalent complex of an aptamer and a target according to any one of claims 1 to 8, wherein position 5 of the aptamer is chemically modified with a group selected from 5-(N-benzylcarboxamido)-2'-deoxyuridine, 5-(N-isobutylcarboxamido)-2'-deoxyuridine, 5-(N-[2-(1H-indol-3yl)ethyl]carboxamido)-2'-deoxyuridine, and 5-(N-naphthylcarboxamido)-2'-deoxyuridine, or a group independently selected from the groups shown in Figure 14 below, wherein R is selected from benzyl, isobutyl, indole, or naphthyl. 【Chemistry 5-1】 【Chemistry 5-2】

10. Aptamers may be 2'-O-methyl (2'-OMe), 2'-O-allyl, 2'-fluoro (2'-F), 2'-amino (2'-NH ２ 10. The non-covalent complex of claim 1, wherein the sugar modification is selected from the group consisting of 2'-azide and 2'-diaminodiphenyl ether.

11. 11. A non-covalent complex of aptamer and target according to any of claims 1-10, wherein the aptamer comprises an epimeric sugar moiety selected from the group consisting of arabinose, xylose and lyxose.

12. Aptamers may be P(O)S ("thioate"), P(S)S ("dithioate"), P(O)NR ２ ("amidates"), P(O)R, P(O)OR', CO and CH ２ 12. The non-covalent complex of any of claims 1-11, comprising at least one additional modification comprising a backbone modification of the phosphate moiety selected from the group consisting of: wherein R and R' are independently H, aryl, alkenyl, cycloalkyl, cycloalkenyl, aralkyl, and C1-20 alkyl, and optionally have an ether (-O-) linkage.

13. The aptamer may be 5-bromo-1-uracilyl, 5-iodo-1-uracilyl, 5-bromovinyl-1-uracilyl, 5-iodovinyl-1-uracilyl, 5-azido-1-uracilyl, 4-thio-1-uracilyl, 4-thio-1-cytosinyl, 5-bromo-1-cytosinyl, 5-iodo-1-cytosinyl, 5-bromovinyl-1-cytosinyl, 5-iodovinyl-1-cytosinyl, 5-azido-1-cytosinyl, 8-azido-9-adeninyl, 8-bromo-9- Adeninyl, 8-iodo-9-adeninyl, 8-azido-9-guaninyl, 8-bromo-9-guaninyl, 8-iodo-9-guaninyl, 8-azidohypoxanthinyl, 8-bromohypoxanthinyl, 8-iodohypoxanthinyl, 8-azido-9-xanthinyl, 8-bromo-9-xanthinyl, 8-iodo-9-xanthinyl, 5-[(4-azidophenacyl)thio]-1-cytosinyl, 5-[(4-azidophenacyl)thio]-2-uracilyl, 5-(N- benzylcarboxamido)-1-uracilyl, 5-(N-isobutylcarboxamido)-1-uracilyl, 5-(N-tryptoaminocarboxamido)-1-uracilyl, 5-(N-[2-(1H-indol-3-yl)ethyl]carboxamido)-1-uracilyl, 5-(N-[1-(3-trimethylammonium)propyl]carboxamido)-1-uracil chloride, 5-(N-naphthylmethylcarboxamido)-1-uracilyl, 5-(N-[ 13. The non-covalent complex of any of claims 1-12, having at least one additional modified base selected from the group consisting of 1-(2,3-dihydroxypropyl)]carboxamido)-1-uracilyl, 7-deaza-7-iodo-9-adeninyl, 7-deaza-7-iodo-9-guaninyl, 7-deaza-7-bromo-9-adeninyl, 7-deaza-7-bromo-9-guaninyl, 1-isocytidinyl, and 9-isoguaninyl.

14. A non-covalent complex of an aptamer and a target according to any one of claims 1 to 13, wherein the target is a protein or peptide. Request 15 One, some, or all of the pyrimidines in the nucleic acid sequence of the aptamer are modified at the 5-position, the modification including substitution with benzyl, isobutyl, indole, or naphthyl, and the dissociation rate of the aptamer from the target (t １／２ ) for 30 minutes or more, and the aptamer has a dissociation constant (K ｄ ) an aptamer having

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