Aptamer discovery and selection techniques

Abstract

Coding aptamer candidate libraries having nucleotide modifications are described. The aptamer candidate comprises a first conserved primer region and a second conserved primer region. The aptamer candidate also includes a variable region disposed between the first conserved primer region and the second conserved primer region and including at least one modified nucleotide. The code region includes a nucleotide sequence that is unique to the modification type of the at least one modified nucleotide such that the sequence of each aptamer candidate can be used to identify the associated modification type.

Description

[0001] References to Electronic Sequence Listings

[0002] This application contains a sequence listing, which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. The .XML copy created on March 30, 2023 is named "IP-2332-PRV.xml" and is 11,047 bytes in size. The sequence listing contained in this .XML file is part of this specification and is hereby incorporated by reference in its entirety. Background Art

[0003] The disclosed technology generally relates to aptamer discovery, generation and / or selection technology. In particular, the disclosed technology relates to technology for use in aptamer libraries to select improved and more diverse aptamer candidates for use in aptamer-based assays or for aptamers for therapeutic applications.

[0004] The subject matter discussed in this section should not be considered to be prior art simply because it is mentioned in this section. Similarly, problems mentioned in this section or associated with the subject matter provided as background technology should not be considered to have been previously recognized in the prior art. The subject matter in this section merely represents different approaches, which themselves may also correspond to specific implementations of the technology protected by the claims.

[0005] Protein expression patterns help determine the identity and state of cells. RNA transcripts are often used as a surrogate for protein expression, but the relationship between the abundance of proteins and mRNA is not one-to-one. There are differences caused by post-transcriptional, translational, and protein degradation regulation. Therefore, direct nucleic acid sequencing of RNA transcripts may not provide an accurate estimate of protein expression.

[0006] Aptamers are nucleic acids that bind to molecular targets (such as proteins) with high affinity and specificity. Advances in aptamer selection and design include systematic evolution of ligands (SELEX) of exponential enrichment. In SELEX, high-affinity nucleic acids of different analytes of interest can be separated from combinatorial libraries, allowing high-throughput characterization of aptamer-target binding and multiple determination of analytes in complex biological samples. After aptamers are combined with analyte targets, binding events can be detected to characterize the presence and concentration of various analytes in biological samples. However, the aptamer group that can be used for protein detection is a function of the aptamer discovery process, and not all desired protein targets can produce aptamers with appropriate binding properties. Therefore, improved aptamer generation and discovery will be beneficial to expanding available protein targets. Summary of the invention

[0007] In one embodiment, the present disclosure provides a library of aptamer candidates. The library includes multiple at least partially single-stranded nucleic acids. A single at least partially single-stranded nucleic acid in the multiple at least partially single-stranded nucleic acids includes: a first conserved primer region, which is conserved in multiple at least partially single-stranded nucleic acids; a second conserved primer region, which is conserved in multiple at least partially single-stranded nucleic acids; a variable region, which is arranged between the first conserved primer region and the second conserved primer region, wherein the variable region is variable in multiple at least partially single-stranded nucleic acids, and wherein each variable region includes at least one modified nucleotide; and a code region, wherein the code region includes a unique nucleotide sequence for the modification type of at least one modified nucleotide.

[0008] In one embodiment, the present disclosure provides an aptamer selection method, which includes providing a library of aptamer candidates of different aptamer subgroups, wherein each aptamer candidate of a single subgroup in the different aptamer subgroups comprises: a first conserved primer region, which is conserved in the aptamer candidate; a second conserved primer region, which is conserved in the aptamer candidate; a variable region, which is arranged between the first conserved primer region and the second conserved primer region, wherein the variable region is variable in the aptamer candidate, and wherein the variable region includes at least one modified nucleotide; and a code region, wherein the code region includes a nucleotide sequence that is unique to the modification type of at least one modified nucleotide and uniquely identifies the modification type from different modification types of other subgroups in different subgroups. The method also includes: selecting aptamer candidates based on binding to a target molecule; amplifying the selected aptamer candidates using primers based on the first conserved primer region and the second conserved primer region; and

[0009] The amplified aptamer candidates are sequenced to determine the sequence of the variable region and identify the type of modification.

[0010] In one embodiment, the present disclosure provides an aptamer candidate library, which includes a plurality of partially double-stranded aptamer candidates, the plurality of partially double-stranded aptamer candidates comprising a first chain and a second chain. The first chain includes: a duplex portion, the duplex portion including a first complementary region, the first complementary region hybridizing with a second complementary region of the second chain; a single-stranded portion, the single-stranded portion including: a first conserved primer region, the first conserved primer region is conserved in the first chain and the second chain of the plurality of partially double-stranded aptamer candidates; a second conserved primer region, the second conserved primer region is conserved in the first chain and the second chain of the plurality of partially double-stranded aptamer candidates; a variable region, the variable region is disposed between the first conserved primer region and the second conserved primer region, wherein the variable region is variable in the first chain and the second chain of the plurality of partially double-stranded aptamer candidates, and the variable region includes at least one modified nucleotide; and a code region, wherein the code region includes a nucleotide sequence unique to the modification type of at least one modified nucleotide.

[0011] The foregoing description is presented to enable making and using the disclosed technology. Various modifications to the disclosed implementations will be apparent, and the general principles defined herein may be applied to other implementations and applications without departing from the spirit and scope of the disclosed technology. Therefore, the disclosed technology is not intended to be limited to the specific implementations shown, but to conform to the widest scope consistent with the principles and features disclosed herein. The scope of the disclosed technology is defined by the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] These and other features, aspects, and advantages of the disclosed embodiments will become better understood when the following detailed description is read with reference to the accompanying drawings, in which like characters refer to like parts throughout, and in which:

[0013] Figure 1 is a schematic diagram of the prior art SELEX method;

[0014] Figure 2 is a schematic diagram of a SELEX method for selecting aptamers from a candidate library with various modifications according to an embodiment of the present disclosure;

[0015] Figure 3 is a schematic diagram of amplification and characterization of selected aptamer candidates according to embodiments of the present disclosure;

[0016] Figure 4 is a schematic diagram of a SELEX method for aptamer selection to promote duplex formation according to an embodiment of the present disclosure;

[0017] Figure 5is a schematic diagram of amplification and characterization of selected aptamer duplex candidates according to embodiments of the present disclosure;

[0018] Figure 6 shows example uridine modifications that may be incorporated into aptamers according to embodiments of the present disclosure;

[0019] Figure 7 is a schematic diagram of conserved and variable nucleotide regions in an aptamer nucleotide sequence according to an embodiment of the present disclosure;

[0020] Figure 8 is a schematic diagram of a SELEX selection cycle with aptamer amplification according to an embodiment of the present disclosure;

[0021] Fig. 9 is a schematic diagram of a SELEX selection cycle with aptamer amplification according to an embodiment of the present disclosure; and

[0022] Fig.10 is a block diagram of a sequencing device configured to collect sequencing data according to the present technology. DETAILED DESCRIPTION

[0023] The following discussion is presented to enable any person skilled in the art to make and use the disclosed technology, and is provided in the context of a specific application and its requirements. Various modifications to the disclosed implementations will be apparent to those skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the spirit and scope of the disclosed technology. Therefore, the disclosed technology is not intended to be limited to the specific implementations shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.

[0024] Aptamers are short single-stranded nucleic acid molecules (ssDNA or ssRNA) or modified nucleic acids that can bind to their specific target molecules with high affinity. In addition to their use in high-throughput multi-omics applications, identifying aptamers with high specificity for molecules of interest can also be used therapeutically. For example, the PEGylated anti-vascular endothelial growth factor aptamer pegaptanib has been approved for clinical use. Aptamers are selected using a method called systematic evolution of ligands by exponential enrichment (SELEX). In SELEX, up to 10 nucleic acid molecules are synthesized. 15 However, conventional SELEX does not always produce aptamers with the desired specificity and affinity for the target molecule.

[0025] The generation of high-quality aptamers for relevant targets remains a major challenge. One of the biggest limitations is that the use of structures with limited chemical diversity makes the process of finding high-affinity specific aptamers more difficult. Unlike peptides, single-stranded DNA molecules do not have a wide range of chemical groups. The limited chemical diversity of nucleotide-based libraries limits the search for successful aptamers. Aptamers typically have four nucleotide building block components. In addition, oligonucleotides are negatively charged polymers, which also limits the chemical properties of candidate aptamers. In contrast, the benefit of antibodies is that 20 amino acids are combined in their sequence to achieve high selectivity and sensitivity.

[0026] To overcome this limited range of starting materials, several strategies have been reported in recent years, including LOOPER-SELEX, Click-SELEX, X-SELEX, post-SELEX modification, and the use of modified nucleic acid bases (i.e., SOMAmers) in the SELEX step. These aptamers with expanded chemical diversity show enhanced binding properties compared to their unmodified forms. Figure 1 An exemplary prior art method for aptamer selection is shown. The first step is to generate a single-stranded DNA (ssDNA) library. This may include ssDNA with one modified nucleotide, typically dUTP modified with an aromatic molecule. Most of the aptamers identified in this method are not selective enough for the target protein and need to be further modified with the help of computational and mutation studies. Therefore, after selecting the best aptamer sequence, a second step is performed, which involves computational modeling and creating a new library with new modifications on the selected sequence. Screening new libraries or successive new libraries is time-consuming and inefficient, and may typically involve 5-15 rounds of selection. Therefore, existing methods for developing aptamers with high selectivity and affinity are insufficient.

[0027] Provided herein is an aptamer generation technology that integrates encoded small organic molecules or other modifications into a single-stranded or partially single-stranded SELEX library. Two molecular evolution-based technologies: the combination of aptamer discovery and DNA encoding libraries promotes the generation of aptamers (such as small molecule-DNA hetero-suitables) with rich chemical diversity, overcoming the lack of chemical diversity in existing methods and one of the biggest challenges in aptamer discovery. Generating aptamer candidates with increased chemical diversity has the following advantages, i.e., generating aptamers with high selectivity and specificity for a given target molecule (i.e., protein). The use of DNA encoding allows each nucleotide modification type to be identified via its associated coding DNA sequence, so that the identified candidate aptamer can be characterized by its binding sequence and its modification type and / or modification position. The disclosed aptamer discovery technology utilizing a combination of DNA encoding libraries and SELEX processes can introduce up to millions of modified fragments in a single round of selection, thereby overcoming the limitations of traditional aptamers and providing the following advantages: high-throughput screening, expanded chemical space, easy synthesis, stability, fast generation time, low production cost, and high specificity and selectivity.

[0028] Figure 2 An example workflow for encoding aptamer selection according to the present technology is shown. In the illustrated embodiment, the aptamer candidate library 12 is formed by combining different aptamer candidate subsets 14 (shown as different subsets 14a, 14b, 14c, 14d, 14e, 14f, 14g, and collectively referred to as the aptamer candidate library 12), wherein the different subsets 14 have different types of nucleotide modifications relative to each other. It should be understood that this illustration is by way of example, and any number of different subsets 14 can be used to form the aptamer candidate library 12. Each subset 14 can be formed separately, and then the separate subsets 14 are merged with other subsets 14 to generate the aptamer library 12. In this way, a specific modification or modification group used to generate one subset 14 will not modify other subsets 14.

[0029] In one example, the different types of modifications of each subset 14 may include different types of base modifications and / or different properties of the modified bases. For example, a specific type of modified uridine (dUTP) 22a may be used to generate the aptamer candidates 20a in the first subset 14a. Different types of modified uridine (dUTP) 22b may be used to generate the aptamer candidates 20b in the second subset 14b, such that the modified uridine 22a is chemically distinguishable from the modified uridine 22b. Modified adenosine 22c may be used to generate the aptamer candidates 20c in the third subset 14c, and so on. The identity of the modified nucleotides 22 (A, C, T, U, G) may be changed or combined to increase library diversity. A single subset 14 of a library 12 may be formed using a single type of modified nucleotide 22 or two or more types of modified nucleotides 22. The types of chemical modifications may include those discussed herein (see Figure 6 ), and may include modifications introduced via click chemistry.

[0030] Within each subset 14, aptamer candidates 20 may also typically differ from one another in terms of variable nucleotide regions (see Figure 3 ), the variable nucleotide region may be randomly generated in some cases. Therefore, the aptamer candidate 20a may include a mixture of different sequences, so that the aptamer candidates 20a of the subgroup 14a are at least partially different from each other, and so on. Because the individual aptamer candidates 20 within a specific subgroup 14 have at least different nucleotide sequences in the variable region, the incorporation of the modified nucleotide 22 (and any associated molecules) is also variable. That is, if the modified nucleotide 22 is uridine, one variable sequence of the aptamer candidate 20 may include 10 or more U (e.g., thymidine) sites, while another variable sequence may include 1, 2, 3 or 4 U sites. In addition, for different aptamer candidates 20, these sites may be located at different positions. In addition, in some cases, the incorporation may not be complete. Therefore, if the variable region has 10 available sites, additional diversity can be generated by incompletely incorporating the modified nucleotide 22 through a mixture with unmodified nucleotides during synthesis. However, the complete incorporation of the modified nucleotide 22 at all available sites can produce a more direct characterization of the candidate in subsequent steps.

[0031] Therefore, although a specific subgroup 14a can be generated using a limited subset of one type of modified nucleotide 22a or modified nucleotide 22a, the aptamer candidates 20a generated in the subgroup 14a are different from each other at least based on the diversity of the variable nucleotide sequences of the aptamer candidates 20a. For example, the first aptamer candidate 20a of the subgroup 14a may include the sequence AAAU*GC as part of the randomly generated variable region, wherein the modified nucleotide 22a dUTP is incorporated at the fourth position available. The second aptamer candidate 20a of the subgroup 14a may include the sequence U*GCGCU* as part of the randomly generated variable region, wherein the modified nucleotide 22a dUTP is incorporated at the first position and the sixth position available. It should be understood that these sequences are only examples. The structures of these different aptamer candidates 20a can be determined based on sequencing, and in one embodiment, it is assumed that the modified nucleotide 22a is incorporated at all available sites.

[0032] The disclosed embodiments advantageously improve the efficiency of candidate selection in a SELEX-type workflow by allowing different types of modifications to be screened together, where aptamer candidates 20 with desired binding properties are parsed and characterized by sequencing to determine which, if any, associated nucleotide modifications may contribute to the binding activity. Thus, combinatorial libraries 12 can be screened together, rather than just one subset at a time 14. Figure 2 As shown, the aptamer candidates 20 of the library are contacted with a target molecule 30 (e.g., a protein). Unbound aptamer candidates 20 can be separated from any bound aptamer candidates 20, and bound aptamer candidates 20 (in Figure 2 The aptamer candidate 20f shown in the figure as having a modified nucleotide 22f can be retained. For example, the target molecule 30 can be immobilized on a substrate, and the unbound aptamer candidate 20 can be washed away. Subsequently, the bound aptamer candidate 20 can be separated from the target molecule 30 (e.g., eluted based on a change in buffer conditions) for amplification and subsequent sequencing.

[0033] Any nucleotide modification is not retained after the amplification step. However, by including the unique code of each subgroup 14 uniquely associated with the modification type (e.g., the identity and type of modified nucleotides 22) of the entire subgroup 14, the nucleotide modification information is retained via the DNA sequence. Therefore, different subgroups 14 (e.g., different subgroups 14a, 14b, 14c, 14d, 14e, 14f, 14g) can be distinguished from each other using their unique codes. In the illustrated embodiment, the sequencing of the amplification product of any retained candidate aptamer (shown as aptamer candidate 20f by way of example) and the identification of both the unique code of the binding sequence and the associated modification type can be used as the input of one or more additional selection cycles. Additional cycles may include negative selections for binding activity with other target molecules. However, as discussed herein, the ability to distinguish different subgroups 14 and their associated modifications in a single library 12 via the encoded nucleic acid sequence allows more diverse aptamer candidates 20 to be screened together in the library 12, thereby improving the library selection efficiency and potentially reducing the number of selection cycles.

[0034] Figure 3 Shown in Figure 2 Example arrangement of ssDNA aptamer candidates 20f retained or selected during the selection cycle of Figure 2 As shown, selected aptamer candidates 20 can be isolated from library 12 based on their binding to target molecule 30. During the isolation stage, any bound aptamer candidates 20 may be uncharacterized. To identify features that contribute to binding, retained aptamer candidates 20f are amplified and sequenced.

[0035] The aptamer candidate 20f includes a variable region 100 and a code region 102. The variable region 100 and the code region 102 are flanked by primer regions 110, 112. Because the aptamer candidate 20 is at least partially single-stranded, the first primer region 110 may represent a primer binding site that is reverse complementary to the first primer 120, and the second primer region 112 may correspond to a sequence of the second primer 122 that binds to the amplified strand generated by the first primer 120. In one embodiment, the length of the aptamer candidate 20 is between 50 nucleotides and 200 nucleotides. In one embodiment, the length of the variable region 100 involved in target binding may be 20-120 nucleotides. In one embodiment, the length of the code region 102 may be between 5 nucleotides and 30 nucleotides. In one embodiment, the length of the primer regions 110, 112 may be between 10 nucleotides and 30 nucleotides. To prevent overlap between the randomly generated variable region 100 and the code region 102, the code region 102 may be formed of only a subset of available nucleotides (eg, T, C or A, T, C).

[0036] The amplified product is sequenced to generate a variable region nucleic acid sequence 130 and a unique code sequence 132. The unique code sequence 132 can be used to identify a subset 14f of the aptamer candidate 20f and its corresponding modified nucleotide type. That is, in one embodiment, the code region 102 can be retained for all members of the subset 14. In the illustrated embodiment, the modified nucleotide 22f may include a mixture of different modifications, shown as modified nucleotides 22f', 22f". The candidate aptamer 22f (including its modifications) is able to generate sufficient amplification products, and the presence of the modifications does not hinder amplification. As shown, primer regions 110, 112 are 5' and 3' of the variable region 100 and the code region 102. The code region 102 can be 5' or 3' of the variable region 100. In addition, as discussed herein, the code region 102 may include two or more different code regions 102, each encoding a different aspect of the modification.

[0037] like Figure 4 As shown, by allowing DNA duplexes to form between aptamer candidates 20, the aptamer candidate library 12 can be further expanded exponentially. In one embodiment, individual aptamer candidates 20 partially hybridize to each other to form partial duplex aptamer candidates 150. A single aptamer candidate 20 will be able to be combined with a second aptamer 20 having a different, similar or identical modification, or no modification at all, thereby increasing the diversity of the library. 2 An advantage of this approach is that it can be extended to more structures showing more than two variable sites. Furthermore, while the illustrated embodiment shows a partially duplex aptamer candidate 150 formed from two different aptamer candidates 20, additional and / or more complex structures can be formed. For example, three, four, five or more chains of aptamer candidates 20 can be hybridized and joined in a single structure via complementary regions.

[0038] In one embodiment, partially duplex candidate aptamers 150 are formed by combining different library subsets (e.g., subsets 14a, 14b, 14c) with each other and allowing complementary regions on each aptamer candidate 20 to hybridize to form duplex regions 154. Thus, in an embodiment, aptamer candidates 20a may each include complementary regions designed to be complementary to corresponding regions on aptamer candidate 20b and / or on aptamer candidate 20c. Complementary regions may be designed to avoid self-complementarity, thereby promoting partial duplex region formation between subsets 14, rather than within-subgroup combinations. However, it should be understood that within-subgroup combinations are also encompassed within the present disclosure.

[0039] In one embodiment, the library 12 may include all or some potential combinations of two or more subsets 14. In another embodiment, the library 12 may include a combination of one or more subsets 14 into which modified nucleotides 22 have been incorporated and an unmodified subset 14. In addition, while the depicted arrangement includes a partially duplexed candidate aptamer 150 having a terminal duplex region 154 from which a corresponding single-stranded branch 156 extends, other arrangements are also contemplated. In one embodiment, the duplex region 154 is an internal region having a single-stranded branch 156 having a split variable region extending in both the 5' and 3' directions. In addition, one or more corresponding single-stranded branches 156 may be flanked by different duplex regions 154 linked to additional chains.

[0040] Once formed, such as Figure 2 As generally discussed, the library 12 can be used to select partial duplex candidate aptamers 150 having binding activity or specific affinity for a specific target molecule 30 of interest. For example, a single partial duplex candidate aptamer 150ab formed by partially hybridizing aptamer candidates 20a, 20b binds to the target molecule 30 and is retained during selection. As shown, the binding activity may be mediated by a single strand branch 156, and the duplex region 154ab may not directly participate in the binding. Therefore, region 154ab may be conserved in multiple partial duplex candidate aptamers 150 formed by different aptamer candidates 20a, 20b. After selection, the partial duplex candidate aptamer 150ab is unwound and sequenced.

[0041] To maintain the identity of the duplex candidate aptamer 150 formed from two different aptamer candidates 20, chemical ligation can be performed to link the component oligonucleotides together, which can then be identified by sequencing the combined construct after PCR amplification. The chemical ligation can be click chemistry ligation or enzyme-mediated ligation.

[0042] Figure 5 Shown in Figure 4 Example arrangement of partial duplex candidate aptamers 150ab retained or selected during the selection cycle of Figure 4 As noted in , selected partial duplex candidate aptamers 150ab can be isolated from library 12 based on their binding to target molecule 30. To identify features that contribute to binding, the retained partial duplex candidate aptamers 150ab are unwound or denatured to separate the strands of component aptamer candidates 20a, 20b, and the strands of the component aptamer candidates 20a, 20b are amplified and sequenced.

[0043] The component aptamer candidates 20a, 20b include a variable region 100 and a coding region 102 flanked by primer regions 110, 112, as described with respect to Figure 3Generally discussed. Primer regions 110, 112 may be conserved between component aptamer candidates 20a, 20b, so that a single set of primers can be used to amplify two chains 20a, 20b or any selected chain of a partial duplex candidate aptamer 150. In addition, the component aptamer candidates 20 for partial duplex formation also include complementary regions 160 that hybridize to each other (e.g., are complementary to each other). In one embodiment, the complementary region is located outside the amplification region so that only the active single-stranded portion of the aptamer candidate 20 is amplified to obtain efficiency. However, other arrangements are also conceivable, and one or both of the primer regions 110, 112 may be part of the complementary region 160. In the partial duplex candidate aptamer 150ab, the variable region 100a of the first chain aptamer 20a may be different from the variable region 100b of the second chain aptamer 20b. In addition, the corresponding code regions 102a, 102b are different, reflecting different types of modifications. Finally, the complementary regions 160a, 160b are reverse complementary to promote partial duplex formation.

[0044] The amplification products of both component aptamer candidates 20a, 20b are sequenced to generate a first strand variable region nucleic acid sequence 162, a first strand unique code sequence 164, a second strand variable region nucleic acid sequence 162, and a second strand unique code sequence 168. The unique code sequences 164, 168 can be used to identify the subsets 14a, 14b of the aptamer candidates 20a, 20b and the corresponding modified nucleotide types, shown as 22a'a" and 22b. That is, in one embodiment, the code region 102 can be retained for all members of the subset 14.

[0045] Figure 6 Functional groups that can be added to the modified nucleotide 22 are shown. It should be understood that these are examples only, and other modifications are also conceivable, which are within the scope of the disclosed embodiments. For example, the modified nucleotide 22 may include a phosphate modification: methylphosphonate (neutral) thiophosphate (anion) guanidinopropylaminophosphorazone (cation). Additionally or alternatively, the modified nucleotide 22 may include a sugar modification: 2'-F; 2'-amino; 2'-OMe; 2'-azido; conformationally locked sugar (LNA) X=O, LNA; X=NR, amino-LNA; X=S, thio-LNA. Additionally or alternatively, the modified nucleotide 22 may include a phosphate replacement: triazole (neutral); guanidinium (cation). Additionally or alternatively, the modified nucleotide 22 may include a purine modification: 2,6-diaminopurine; 3-deaza-adenine; 7-deaza-guanine; 8-azido-adenine. Additionally or alternatively, the modified nucleotide 22 may include pyrimidine modifications: 2-thio-thymidine; 5-carboxamido-uracil 5-methyl-cytosine; 5-ethynyl-uracil (CLICK chemical site).

[0046] As provided herein, modified nucleotides may include non-standard functional groups incorporated by click chemistry by introducing azide modifications. The non-standard functional groups have the following properties: based on fragment small molecules, for example, fragments known to bind weakly to target proteins, and their positions are encoded in the DNA sequence in a binary code based on T (0), C (1) to avoid G-quadruplexes, such as Figure 7 General discussion.

[0047] In one embodiment, modified nucleotides may include modifications with virtually any azide-bearing functional group that can be conjugated to an alkyne-modified (at the C5 position) dU via a facile copper-catalyzed azide-alkyne cycloaddition (CuAAC) or "click chemistry." In one embodiment, modifications may be generated based on starting 5-ethynyluridine or 5-ethynyluracil (Jena Bioscience) that include an alkyne that can subsequently be attached to an azide-containing molecule via click ligation.

[0048] Primer extension was shown to work on modified templates to obtain good to excellent yields of full-length DNA. Thus, selection rounds as in traditional SELEX (see Figure 8 In addition, an adaptive SELEX method involving the assembly of ssDNA into duplexes can also be implemented (see Figure 4 ), thereby further increasing the chemical space of the SELEX library.

[0049] As provided herein, aptamer candidate technology can be combined with two molecular evolution techniques for finding binding molecules with high specificity and selectivity for target molecules of interest by merging small organic ligands encoded by DNA with ssDNA SELEX. In the first step, the synthesis and design of hybrid small molecule-DNA libraries and the selection of target molecules are performed. Standard methods using phosphoramidite chemistry will be used to synthesize hybrid small molecule-DNA libraries. Two strategies are used to study the attachment of small organic molecules or fragments. The first method utilizes clickSELEX after modification. In this method, chemical groups are introduced into the DNA library via click chemistry before the selection step and then removed during the amplification step. By doing so, the enzyme incompatibility problem associated with larger nucleobase modifications is avoided. Therefore, the disclosed embodiment allows easy implementation of a variety of different chemical functionalities adapted to the applied requirements. Mayer et al. have reported a fast and cost-effective scheme to generate nucleic acid modified by nucleobases on a large scale with high fidelity. After the solid phase synthesis of EdU containing DNA, a click reaction is performed with the DNA still attached to the solid phase, followed by deprotection and purification according to standard procedures. 5-ethynyl-2'-deoxyuridine (EdU) will be used as a linker for ssDNA and small molecules.

[0050] Figure 7 Example aptamer candidate 20 is shown. The position of "clickable" nucleotides is encoded in the DNA sequence using a binary code based on TC bases to avoid G-quadruplexes. The design of the library members will be as follows: regions a and a' correspond to conservative primer sequences 110, 112 for PCR amplification. Region b corresponds to the first part 170 of the code region 102 with a unique short DNA sequence (TC encoding) of 6 nucleotides, to identify the position of clickable nucleotides (e.g., modified nucleotides 22) in region c (variable region 100), which will be attached to the molecular fragment-"small molecule". Region c can be a random sequence of 40-50 nucleotides, including non-clickable and clickable functionalized nucleotides. Region d corresponds to the second part 172 of the code region 102, which contains codes (encoded as ATCG bases) for identifying aromatic non-clickable nucleotides and clickable nucleotides.

[0051] Figure 8 The results show that the template DNA strand library (about 10 14 An example selection cycle begins with chemical synthesis of a ssDNA library consisting of a random sequence flanked by fixed sequence regions required for PCR. PCR amplification of the template library in the presence of a 3'-primer containing a biotin capture molecule at its 5'-end produces biotinylated dsDNA that can be captured using a reaction with streptavidin. Note that this step helps ensure that the sequence entering the selection is amplifiable. The dsDNA is then treated with streptavidin immobilized on the beads, and the complementary strands are removed from the beads by heating or increasing the pH to generate an ssDNA library in solution. CE-SELEX works in a different way and does not require biotin-functionalized primers.

[0052] Fig. 9 Shown is a selection cycle starting from a DNA template library prepared by automated DNA synthesis. The amide bond of the modified dUTP derivative is stable to alkaline conditions for an extended time at ambient temperature. The modified ssDNA library can then undergo a selection step for binding to a protein of interest or any other target. The selected sequence will then be subjected to PCR with TTP, dATP, dCTP, and dGTP, avoiding the use of modified dUTP for PCR amplification. Using the same 3'-primer containing 5'-biotin will produce an enriched library ready to start the next selection cycle. When additional small molecule fragments are added, an additional next generation sequencing (NGS) step can be performed at the end of the selection.

[0053] Small fragments can be selected so that they can contribute to the binding affinity between the aptamer and its target through hydrogen bonding, structural compatibility, aromatic ring stacking, electrostatic and hydrophobic interactions, and van der Waals forces. For this reason, the first choice is to add ultra-small molecules as fragments. The selection of small molecules will take into account their commercial availability and hydrophobicity. The library will be composed of 1000 different fragments combined with random sequences of nucleotides containing modified bases dUTP, thereby forming a hybridization library.

[0054] In the second step, the optimal conditions for amplifying the library by PCR are identified. Positive and negative two rounds of selection will be used to identify hybrid aptamers. High-throughput sequencing SELEX (HTS-SELEX) will be used because the library is allowed to be sequenced in all selection rounds. The identification of molecular fragments encoded by DNA will be carried out in each round, so the enriched sequence will be visible at an earlier stage, making the process more time-efficient. The alkyne-modified DNA library synthesized by functionalization with azide fragments by CuAAC-click chemistry. After selecting and removing unbound library members, the bound sequences are separated and amplified by PCR, with alkyne-modified triphosphates replacing thymidine (alternatively azide modification can also be used). Therefore, the modification in the extended chain is removed, and the alkyne part is reintroduced. After PCR, single-stranded DNA is prepared by λ-acidase exonucleolytic digestion of 5'-phosphorylated antisense strands. Then the modification is reintroduced by CuAAC-click chemistry, and the obtained library is used for the next SELEX cycle.

[0055] In the third step, the identification of the best binding molecules is performed. The DNA sequence of the aptamer will be determined using standard NGS sequencing technology. The identity of the small molecule fragment will be revealed by its unique DNA code. Since HTS-SELEX will be used for each round of selection, it is expected that the best binding aptamer will be identified as early as possible.

[0056] In the fourth step, the binding properties of the hybrid aptamers are characterized. ITC (isothermal calorimetry), fluorescence polarization spectroscopy and SPR (surface plasmon resonance) will be used to measure their binding affinity to the selected aptamer. ITC measurements will give information related to the thermodynamics of the binding event between the ligand and the target molecule. SPR will be used to determine the kinetics (Kon / Koff) of the binding event. Finally, fluorescence polarization spectroscopy can be performed by introducing a fluorophore into the aptamer structure.

[0057] In some embodiments, the disclosed techniques are used to generate sequence data from amplified aptamer candidates 20 . Fig.10is a schematic diagram of a sequencing device 200 that can be used in conjunction with the disclosed embodiments for collecting sequencing data from candidate aptamers provided herein. The sequencing device 200 can be implemented according to any sequencing technology, such as those including the sequencing by synthesis methods described in the following patents: U.S. Patent Publication Nos. 2007 / 0166705, 2006 / 0188901, 2006 / 0240439, 2006 / 0281109, 2005 / 0100900, U.S. Patent No. 7,057,026, WO 05 / 065814, WO 06 / 064199, WO 07 / 010,251, the disclosures of which are incorporated herein by reference in their entirety. Alternatively, sequencing by ligation technology can be used in the sequencing device 200. Such techniques use DNA ligase to incorporate oligonucleotides and recognize the incorporation of such oligonucleotides, and are described in US Pat. No. 6,969,488; US Pat. No. 6,172,218; and US Pat. No. 6,306,597; the disclosures of which are incorporated herein by reference in their entireties.

[0058] In the depicted embodiment, the sequencing device 200 includes a separate sample substrate 202, such as a flow cell or a sequencing box, and an associated computer 204. However, as described above, these can be implemented as a single device. In the depicted embodiment, the biological sample can be loaded into the substrate 210, which is imaged to generate sequence data. For example, the reagent interacting with the biological sample responds to the excitation beam generated by the imaging module 212 and fluoresces at a specific wavelength, and the return radiation is used for imaging. For example, the fluorescent component can be generated by a fluorescently labeled nucleic acid, which is hybridized with a complementary molecule of the component or with a fluorescently labeled nucleotide incorporated into an oligonucleotide using a polymerase. As will be understood by those skilled in the art, the wavelength of the dye that excites the sample and the wavelength at which it fluoresces will depend on the absorption and emission spectra of the specific dye. Such returned radiation can be propagated back by guiding optics. The backward beam can usually be directed to the detection optics of the imaging module 212, which can be a camera or other optical detector.

[0059] The imaging module detection optics can be based on any suitable technology, and can be, for example, a charged coupled device (CCD) sensor that generates pixelated image data based on photons that affect the position in the device. However, it should be understood that any of a variety of other detectors may also be used, including but not limited to a detector array configured for time delay integration (TDI) operation, a complementary metal oxide semiconductor (CMOS) detector, an avalanche photodiode (APD) detector, a Geiger mode photon counter, or any other suitable detector. TDI mode detection can be coupled with line scanning, as described in U.S. Patent No. 7,329,860, which is incorporated herein by reference. Other available detectors are described in, for example, the references previously provided herein in the context of various nucleic acid sequencing methods.

[0060] The imaging module 212 may be under processor control (e.g., via the processor 214), and may also include an I / O control 216, an internal bus 218, non-volatile memory 220, RAM 222, and any other memory structure that enables the memory to store executable instructions, and may be similar to that described with respect to Fig.10 In addition, the associated computer 204 may also include a processor 224, an I / O control 226, a communication module 234, and a memory architecture including a RAM 228 and a non-volatile memory 230, such that the memory architecture can store executable instructions 232. The hardware components may be linked by an internal bus 194, which may also be linked to a display 236. In embodiments where the sequencing device 200 is implemented as an integrated device, certain redundant hardware elements may be eliminated.

[0061] Processor 214 can be programmed to assign individual sequencing reads to subsets 14 based on one or more associated unique code sequences according to the techniques provided herein. Each sequence read can include both a variable region nucleic acid sequence and a unique code sequence. The sequencing data includes base calls for each base of the sequencing read.

[0062] As used herein, an aptamer may refer to a non-naturally occurring nucleic acid with a specific binding affinity to a target molecule. In certain embodiments, an aptamer candidate 20 is provided, which is a nucleic acid with unknown binding ability, which can be determined to have enough binding affinity to be selected as an aptamer for a specific application after screening and selection. In some cases, aptamer candidates 20 and aptamers can be used interchangeably in the present disclosure. The binding of an aptamer to a target molecule can result in catalytically changing the target molecule, thereby reacting with the target molecule in a manner that modifies or changes the functional activity of the target molecule or the target molecule, covalently attaching to the target molecule (such as in a suicide inhibitor), and promoting the reaction between the target molecule and another molecule. In one embodiment, the target molecule is a three-dimensional chemical structure other than a polynucleotide, which is combined with an aptamer by a mechanism that is mainly independent of Watson / Crick base pairing or triple helix binding. In one embodiment, an aptamer is not a nucleic acid with a known physiological function that is bound by a target molecule.

[0063] Appamers include nucleic acids identified from a candidate mixture of nucleic acids (e.g., aptamer candidates). In addition, the library of aptamer candidates may include one or more aptamers of interest for a specific target molecule. By contacting the candidate mixture with the target, an aptamer can be identified as a ligand of a given target molecule from the aptamer candidates, wherein the nucleic acid with increased affinity for the target relative to other nucleic acids in the candidate mixture can be separated from the remainder of the candidate mixture. The specific binding affinity of an aptamer to its target may refer to a much higher affinity of the combination of an aptamer and its target than the combination of other non-target components in the mixture or sample. Different aptamers may have the same or different numbers of nucleotides. An aptamer may be a DNA or RNA, and may be single-stranded, double-stranded, or contain a double-stranded region.

[0064] The disclosed aptamers and / or aptamer candidates can be used to generate aptamers that can change the biological activity of a target by binding to and / or cross-linking with the target. In one embodiment, aptamers for unique targets associated with or related to a specific disease process are identified. Such aptamers can be used as diagnostic reagents in vitro or in vivo. In another embodiment, aptamers for targets associated with a disease state can be administered to an individual and used to treat the disease in vivo. The aptamers identified herein can be used in any diagnostic, imaging, high throughput screening or target validation technology or procedure or assay that can use aptamers, oligonucleotides, antibodies and ligands (but not limited thereto).

[0065] In certain embodiments of the present disclosure, the aptamer candidate 20 may include one or more conserved regions, such as conserved primer regions, for example, a first conserved primer region and a second conserved primer region. The conserved region is conserved between at least some other aptamer candidates 20, so that the conserved region has the same or similar nucleotide sequence compared to the aptamer candidate 20. In one embodiment, the conserved region is conserved in both the nucleotide sequence and position between different aptamer candidates 20. In one embodiment, the conserved region has the same sequence, so that two or more different aptamer candidates 20 have the same sequence in the conserved region. In one embodiment, the conserved region has less than two different nucleotides between different aptamer candidates 20 with the conserved region. In one embodiment, the conserved region can be a universal region. For example, for a given library 12, all aptamer candidates in the library 12 can have the same first conserved primer region and the second conserved primer region. In this way, primers based on the first conserved primer region and the second conserved primer region can be used to amplify any selected aptamer candidate 20 of the library 12. In one embodiment, the conserved region may be conserved only within a specific subset 14, such that aptamer candidates 20 of a specific subset 14 all have a conserved code region or a conserved complementary region, but other aptamer candidates 20 of different subsets 14 have different code regions and / or complementary regions.

[0066] Conserved primer regions may include The capture primer sequence is in the region or The region to which the capture primer specifically hybridizes. Capture primers include, for example, P5 5'-AATGATACGGCGACCACCGA-3' ((SEQ ID NO: 1)) or P7 (5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 2)) or fragments thereof. The region to which the capture primer specifically hybridizes may include, for example, The reverse complementary sequence of capture primer P5 ("anti-P5": 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO: 3) or P7 ("anti-P7": 5'-TCGTATGCCGTCTTCTGCTTG-3' (SEQ ID NO: 4)) or fragments thereof.

[0067] The conserved primer region may additionally or alternatively include The region of the sequence of the sequencing primer or its fragment, or The region to which a sequencing primer or its fragment specifically hybridizes. Sequencing primers include, for example, SBS3 (5'-ACACTCTTTCCCTACACGACGCTCT TCCGATCT-3' (SEQ ID NO: 5)) or SBS8 (5'-CGGTCTCGGCATTCCTGC TGAACCGCTCTTCCGATCT-3' (SEQ ID NO: 6)). The region to which a sequencing primer or fragment thereof specifically hybridizes may include, for example Sequencing primers SBS3 ("anti-SBS3": 5'-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT-3' (SEQ ID NO: 7)) or SBS8 ("anti-SBS8":

[0068] 5'-AGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCG-3'

[0069] (SEQ ID NO: 8)) or the reverse complement of a fragment thereof. Incorporation of a sequencing primer sequence into a candidate aptamer can be performed directly or via subsequent amplification, ligation or other sequencing library preparation steps.

[0070] This written description uses examples to enable any person skilled in the art to practice the disclosed embodiments, including making and using any devices or systems and performing any incorporated methods. The patentable scope is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal language of the claims.

Claims

1. An aptamer candidate library, comprising: a plurality of at least partially single-stranded nucleic acids, wherein a single at least partially single-stranded nucleic acid of the plurality of at least partially single-stranded nucleic acids comprises: a first conserved primer region, wherein the first conserved primer region is conserved among the plurality of at least partially single-stranded nucleic acids; a second conserved primer region, wherein the second conserved primer region is conserved among the plurality of at least partially single-stranded nucleic acids; a variable region disposed between the first conserved primer region and the second conserved primer region, wherein the variable region is variable in the plurality of at least partially single-stranded nucleic acids, and wherein each variable region comprises at least one modified nucleotide; and A code region, wherein the code region comprises a nucleotide sequence that is unique to the modification type of the at least one modified nucleotide.

2. The library of claim 1, wherein the plurality of at least partially single-stranded nucleic acids comprises a plurality of modification types, each modification type corresponding to a different code region having a corresponding different nucleotide sequence.

3. The library of claim 2, wherein the variable region of the single at least partially single-stranded nucleic acid of the plurality of at least partially single-stranded nucleic acids comprises only a single modification type of the at least one modified nucleotide.

4. The library of claim 3, wherein the variable region comprises two modified nucleotides of more of the at least one modified nucleotide, the two modified nucleotides of the more having the single modification type of the at least one modified nucleotide.

5. The library of claim 1, wherein the modification types include modifications with molecular fragments.

6. The library of claim 1, wherein the modification types include chemically modified uridine.

7. The library of claim 1, wherein the type of modification comprises modification via a click chemistry reaction.

8. The library of claim 7, wherein the at least one modified nucleotide is modified with an alkyne.

9. The library of claim 1, wherein the single at least partially single-stranded nucleic acid in the plurality of at least partially single-stranded nucleic acids further comprises: a complementary region that forms a duplex region with another at least partially single-stranded nucleic acid in the plurality of at least partially single-stranded nucleic acids.

10. The library of claim 9, wherein the complementary region is not between the first conserved primer region and the second conserved primer region.

11. The library of claim 1, wherein the code region is located between the first conserved primer region and the second conserved primer region.

12. The library of claim 1, wherein the code region comprises a first portion and a second portion, wherein the first portion identifies the modification of the modified nucleotide and the second portion identifies the position within the variable region of the modified nucleotide.

13. The library of claim 12, wherein the second portion comprises a code sequence encoded by only two different nucleotides.

14. The library of claim 12, wherein the first portion and the second portion are located on both sides of the variable region and between the first conserved primer region and the second conserved primer region.

15. A method for selecting an aptamer, comprising: A library of aptamer candidates of different aptamer subsets is provided, wherein each aptamer candidate of a single subset of the different aptamer subsets comprises: a first conserved primer region, wherein the first conserved primer region is conserved among the aptamer candidates; a second conserved primer region, wherein the second conserved primer region is conserved among the aptamer candidates; a variable region disposed between the first conserved primer region and the second conserved primer region, wherein the variable region is variable among the aptamer candidates, and wherein the variable region comprises at least one modified nucleotide; and a code region, wherein the code region comprises a nucleotide sequence that is unique to a modification type of the at least one modified nucleotide and that uniquely identifies the modification type from different modification types of other subsets in the different subsets; Aptamer candidates are selected based on binding to the target molecule; amplifying the selected aptamer candidate using primers based on the first conserved primer region and the second conserved primer region; as well as The amplified aptamer candidates are sequenced to determine the sequence of the variable region and identify the modification type.

16. The method of claim 15, wherein the code region is conserved within the single subset, such that the aptamer candidates of the single subset all have the same code region.

17. The method of claim 15, comprising using capture molecules to separate amplified aptamer candidates.

18. An aptamer candidate library, comprising: A plurality of partially duplex aptamer candidates, the plurality of partially duplex aptamer candidates comprising a first strand and a second strand, the first strand comprising: a duplex portion comprising a first complementary region that hybridizes to a second complementary region of the second strand; A single-stranded portion, the single-stranded portion comprising: a first conserved primer region that is conserved among the first and second strands of the plurality of partially duplex aptamer candidates; a second conserved primer region that is conserved among the first strand and the second strand of the plurality of partially duplex aptamer candidates; a variable region disposed between the first conserved primer region and the second conserved primer region, wherein the variable region is variable in the first strand and the second strand of the plurality of partially duplex aptamer candidates, and the variable region comprises at least one modified nucleotide; and A code region, wherein the code region comprises a nucleotide sequence that is unique to the modification type of the at least one modified nucleotide.

19. The library of claim 18, wherein the second strand comprises: a second strand single-stranded portion, wherein the second strand single-stranded portion comprises the first conserved primer region and the second conserved primer region; a second chain variable region, the second chain variable region being different from the variable region of the first chain; and A second chain code region, the second chain code region being different from the code region of the second chain.

20. The library of claim 19, wherein the second strand comprises a second modification type that is different from the modification type of the first strand.

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