Mirror selection of l-nucleic acid aptamers

Abstract

Disclosed are methods for screening L-nucleic acid aptamers for binding to a target molecule. The methods comprise: (a) contacting a plurality of L-nucleic acid aptamers with the target molecule under conditions that selectively capture target-binding L-nucleic acid aptamers from the plurality of L-nucleic acid aptamers; (b) amplifying L-nucleic acid aptamers of the target-binding L-nucleic acid aptamers to generate amplified double-stranded L-nucleic acid oligonucleotides; and (c) separating the amplified double-stranded L-nucleic acid oligonucleotides using an electrophoresis-based method, thereby screening the plurality of L-nucleic acid aptamers.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Application No. 63 / 306,139, filed February 3, 2022, and U.S. Application No. 63 / 311,092, filed February 17, 2022, which are incorporated herein by reference in their entirety.

[0003] Sequence list declaration

[0004] A file named 94498.xml, created on February 1, 2023, containing 12,923,440 bytes, which was filed concurrently with this application, is incorporated herein by reference.

[0005] Field and background of the invention

[0006] In some embodiments thereof, the present invention relates to a method for selecting L-nucleotide aptamers and a sequencing method thereof.

[0007] Aptamers are nucleic acid polymeric ligands that bind to specific target molecules via tertiary interactions, selected through phylogenetic evolution of exponentially enriched ligands (SELEX) or in vitro selection. Natural, unmodified aptamers are readily degraded by ubiquitous nucleases both in vitro and in vivo, significantly limiting their practical application as diagnostic and therapeutic tools. Although chemical modification and xeno nucleic acid (XNA) design have been shown to enhance aptamer stability, their discovery and production require designed, specialized nucleotides, and even then, nuclease degradation of non-natural nucleic acid aptamers cannot be completely avoided.

[0008] Chiral inverted L-DNA or L-RNA aptamers (mirror image aptamers), exhibiting excellent biostability both in vitro and in vivo, have been selected to bind natural target molecules. Their large-scale production can be readily achieved using commercially available L-deoxynucleosides or L-ribonucleoside phosphoramidide via automated oligonucleotide synthesizers, making them ideal for practical applications in diagnostics and therapy. Since their biochemical advantages were recognized over two decades ago, mirror image aptamers have primarily been selected via an indirect scheme known as 'selection-reflection': first, a mirror image of the target molecule is chemically synthesized to select the D-aptamer, followed by the synthesis of a mirror image aptamer with the same sequence to bind the corresponding natural target. However, the first step of chemically synthesizing mirror image target molecules often presents problems, particularly for proteins with large size, extensive post-translational modifications (PTMs), and low in vitro folding efficiency. In practice, based on current technology, most biologically important target molecules, such as large proteins, cannot be chemically synthesized and correctly folded. As a result, only a small number of mirror aptamers have been discovered through selection-reflection over the past two decades. All of these target small molecules, short peptides, short RNAs, and small proteins. The largest is a 12 kDa 110-amino acid (aa) ribonuclease from Bacillus amyloliquefaciens. Selection of mirror aptamers targeting the vast majority of biologically important but unsynthesized target molecules has not yet been achieved.

[0009] Background art includes U.S. Patent Application No. 20210332360, U.S. Patent No. 11,015,178, and U.S. Patent No. 10,975,370. Invention Overview

[0011] According to one aspect of the present invention, a method is provided for screening multiple L-nucleotide aptamers for targeting L-nucleotide aptamers with binding affinity to a target molecule, comprising:

[0012] (a) The plurality of L-nucleotide aptamers are brought into contact with the target molecule under conditions of selectively capturing the L-nucleotide aptamer bound to the target from the plurality of L-nucleotide aptamers;

[0013] (b) Amplifying the L-nucleotide aptamer of the target-bound L-nucleotide aptamer to generate amplified double-stranded L-nucleotide oligonucleotides; and

[0014] (c) Amplified double-stranded L-nucleotide oligonucleotides were isolated using an electrophoresis-based method, thereby screening for a variety of L-nucleotide aptamers.

[0015] According to another aspect of the present invention, a kit for identifying L-nucleotide aptamers comprises:

[0016] (i) Calf intestinal phosphatase (CIP);

[0017] (ii) L-deoxyribonucleotide triphosphates (L-dNTPs) or modified L-dNTPs; and / or

[0018] (iii) A polymerase capable of adding one or more L-nucleotides to the 3' end of a first L-nucleic acid.

[0019] According to another aspect of the present invention, a method for sequencing purified L-DNA molecules is provided, comprising:

[0020] (a) A sample containing purified L-DNA molecules was treated with a phosphatase under conditions where 3'-monophosphate was removed from the L-DNA molecules: and

[0021] (b) The sample was subjected to phosphate thioester sequencing, thereby sequencing the purified L-DNA molecules.

[0022] According to another aspect of the invention, isolated L-DNA aptamers that bind thrombin are provided, comprising sequences as shown in SEQ ID NO:10, 12, 14, 16, 27 or 28 or sequences having at least 80% identity with SEQ ID NO:10, 12, 14, 16, 27 or 28.

[0023] According to one embodiment of the present invention, the method further includes converting the amplified double-stranded L-nucleotide oligonucleotide into a single-stranded oligonucleotide after step (b) and before step (c).

[0024] According to one embodiment of the invention, steps (a) and (b) and the transformation step are repeated at least three times before isolation to enrich the L-nucleotide aptamer bound to the target.

[0025] According to one embodiment of the present invention, the method further includes monitoring the enrichment of L-nucleotide aptamers bound to the target.

[0026] According to one embodiment of the invention, the monitoring is achieved by electrophoretic mobility variation measurement (EMSA).

[0027] According to one embodiment of the present invention, the electrophoresis-based method is selected from non-denaturing PAGE; denaturing PAGE; denaturing gradient gel electrophoresis (DGGE); constant denaturing gel electrophoresis (CDGE) and time-temperature gradient gel electrophoresis (TTGE).

[0028] According to one embodiment of the present invention, the electrophoresis-based method includes DGGE.

[0029] According to one embodiment of the present invention, the target molecule is selected from peptides, polypeptides, small molecules, carbohydrates, and nucleic acid molecules.

[0030] According to one embodiment of the invention, the target molecule is contained in cells or tissues.

[0031] According to one embodiment of the present invention, the amplification utilizes D-amino acid polymerase.

[0032] According to one embodiment of the present invention, the D-amino acid polymerase is selected from D-ASFV pol X, D-Taq polymerase, D-Pfu polymerase, Sulfolobus and solfataricus P2 DNA polymerase IV (DPO4), fusion proteins containing said DPO4, and polymerases having an amino acid sequence having at least 80% identity with DPO4.

[0033] According to one embodiment of the invention, the polymerase has an amino acid sequence as shown in SEQ ID NO:38 or SEQ ID NO:40.

[0034] According to one embodiment of the present invention, the method further includes sequencing the isolated members after step (c) to obtain the sequence of the L-nucleotide aptamer having binding affinity for the target molecule.

[0035] According to one embodiment of the present invention, the sequencing is performed using a method selected from: L-DNA chemical sequencing; L-DNA phosphate thioester sequencing; L-DNA dideoxy sequencing; L-DNA ion-fluid sequencing; L-DNA Illumina sequencing; and L-DNA nanopore sequencing.

[0036] According to one embodiment of the present invention, the method is L-DNA phosphate thioester sequencing.

[0037] According to one embodiment of the present invention, the method further includes contacting the amplified double-stranded L-nucleotide oligonucleotide with a phosphatase prior to the sequencing.

[0038] According to one embodiment of the present invention, the phosphatase comprises calf intestinal phosphatase (CIP).

[0039] According to one embodiment of the present invention, each of the plurality of L-nucleotide aptamers has the same length.

[0040] According to one embodiment of the present invention, the plurality of L-nucleotide aptamers are libraries, and each member of the library has the same 5' and 3' nucleic acid sequences as well as different core sequences.

[0041] According to one embodiment of the invention, the method further includes constructing an additional aptamer library, wherein each member of the library has the same 5' and 3' nucleic acid sequences and is randomized by up to 60% compared to the sequences of isolated L-nucleotide aptamers.

[0042] According to one embodiment of the present invention, the method further includes synthesizing a variety of L-nucleotide aptamers prior to step (a).

[0043] According to one embodiment of the present invention, the synthesis includes error-prone PCR.

[0044] According to one embodiment of the present invention, the error-prone PCR includes the use of an error-prone polymerase.

[0045] According to one embodiment of the present invention, the core sequence comprises a random or semi-random sequence.

[0046] According to an embodiment of the invention, the polymerase comprises P2 DNA polymerase IV (DPO4) of sulfur-mineralized leaf fungus or a polymerase having an amino acid sequence that is at least 80% identical to that of DPO4.

[0047] According to an embodiment of the invention, the polymerase has an amino acid sequence as shown in SEQ ID NO:38 or SEQ ID NO:40.

[0048] According to an embodiment of the present invention, the thrombin-binding L-DNA aptamer comprises a sequence as shown in SEQ ID NO:10, 14 or 28 or a sequence having at least 80% identity with SEQ ID NO:10, 12, 14, 16, 27 or 28.

[0049] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While similar or equivalent methods and materials to those described herein may be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, this specification (including definitions) shall prevail. Furthermore, materials, methods, and / or embodiments are merely illustrative and are not intended to be limiting.

[0050] Brief descriptions of several attached figures

[0051] Some embodiments of the invention have been described herein by way of example only with reference to the accompanying drawings. Reference will now be made in detail to the drawings, with emphasis placed on the details shown as examples, and the embodiments of the invention are discussed in an illustrative manner. In this regard, the description with accompanying drawings makes it apparent to those skilled in the art how embodiments of the invention can be practiced.

[0052] In the attached diagram:

[0053] Figure 1A -B. Design of a mirror selection scheme. A, Schematic overview of mirror selection of L-DNA aptamers directly from a large randomized L-DNA library (color), which bypasses the need for chemical synthesis of mirror target molecules as in indirect, selection-reflection schemes (grey). PDB source: 1PPB (natural human thrombin). B, Schematic overview of the procedure in the mirror selection scheme: Selection begins with binding to immobilized protein targets, such as a large randomized L-DNA library of natural human thrombin (e.g., approximately 1 × 10⁻⁶ in this work). 14 (Different L-DNA sequences); the bound L-DNA was eluted and amplified by mirror PCR; the amplified L-DNA conjugate was separated into single-stranded L-DNA for the next round; after the final round of selection, the enriched L-DNA conjugate was analyzed by DGGE, separated, and sequenced using the phosphate thioester method via L-DNA sequencing-while-synthesizing.

[0054] Figure 2A -C. Mirror selection of L-DNA aptamers targeting natural human thrombin. A, The progress of mirror selection was monitored by EMSA using 200 nM of the corresponding L-DNA conjugate and 1 μM natural human thrombin or 1 μM streptavidin, analyzed by 8% non-denaturing PAGE, and stained with SYBR Green II. B, Gel quantification results of (A), where the binding fraction was determined by ImageJ software using the band intensity of the bound L-DNA conjugate relative to the total lane intensity. ND, (binding) not detected. C, Re-amplification by mirror PCR using D-Dpo4-5m with L-DNA primers, analyzed by 10% denaturing PAGE in 2.1 M to 4.2 M urea and 12% to 24% formamide, and analyzed by DGGE of the corresponding L-DNA conjugate stained with SYBR-Green II, as well as the separated bands L-9-1 and L-9-2.

[0055] Figure 3A -N. Characterization of the selected L-DNA aptamer. A, Secondary structure of the L-9-1 aptamer predicted by Mfold, wherein nucleotides derived from the randomized region are shown in blue (SEQ ID NO: 9). B, ITC analysis of the L-9-1 aptamer bound to native human thrombin, wherein Kd The measured value was 29 nM. C, Secondary structure of the L-9-1t (truncated version) aptamer predicted by Mfold, wherein nucleotides derived from the randomized region are shown in cyan (SEQ ID NO:10). D, ITC analysis of the binding of the L-9-1t aptamer to native human thrombin, wherein K d The measured value was 39 nM. E, EMSA of 200 nM Cy5-L-9-1t aptamer bound to 1 μM natural human thrombin or 1 μM streptavidin (without or with 50 units / ml DNase I) analyzed by 8% non-denaturing PAGE. F, EMSA of 35 nM Cy5-L-9-1t aptamer bound to various concentrations of natural human thrombin analyzed by 8% non-denaturing PAGE. G, Gel quantification results (f), where the binding fraction was determined by band intensity of the bound Cy5-L-9-1t aptamer relative to the total lane intensity using ImageJ software. H, Secondary structure of L-9-2 (SEQ ID NO:13) aptamer predicted by Mfold, where nucleotides derived from randomized regions are shown in red. I, ITC analysis of L-9-2 aptamer bound to natural human thrombin, where K d The measured value was 168 nM. J, Secondary structure of the L-9-2t (truncated version) aptamer (SEQ ID NO: 14) predicted by Mfold, wherein nucleotides derived from the randomized region are shown in light green. K, ITC analysis of the binding of the L-9-2t aptamer to native human thrombin, wherein K d The measured value was 251 nM. L, EMSA of 200 nM Cy5-L-9-2t aptamer bound to 1 μM natural human thrombin or 1 μM streptavidin (without or with 50 units / ml DNase I) analyzed by 8% non-denaturing PAGE. M, EMSA of 200 nM Cy5-L-9-2t aptamer bound to various concentrations of natural human thrombin analyzed by 8% non-denaturing PAGE. N, (M) gel quantification results, where the binding fraction was determined by the band intensity of the bound Cy5-L-9-2t aptamer relative to the total lane intensity using ImageJ software.

[0056] Figure 4A-I. Detection and inhibition of native human thrombin using selected L-DNA aptamers. A, Schematic overview of the detection of native human thrombin using an L-DNA aptamer sensor based on the L-9-1t aptamer. B, Relative fluorescence of the L-DNA aptamer sensor measured in physiological buffer alone or in physiological buffer containing 10% human serum incubated with 1 μM native human thrombin for up to 48 min, with excitation wavelength at 494 nm and emission wavelength at 518 nm, measured every 4 min. NC1, Negative control in physiological buffer alone. NC2, Negative control in physiological buffer containing 10% human serum. RFU, Relative fluorescence units. Data are expressed as mean ± SD (n = 3, independent measurements). C, Thrombin concentration measured by D-DNA and L-DNA aptamer sensors in physiological buffer alone or in physiological buffer containing 10% human serum or 50 units / ml DNase I incubated with 300 nM native human thrombin for 1 h or 4 h. Data are expressed as mean ± SD (n = 3, independent measurements). D. Schematic overview of the detection of natural human thrombin using L-DNA aptamer Western blotting. E. Natural human thrombin separated by 15% SDS-PAGE, transferred to a nitrocellulose membrane, incubated with 500 nM Cy5-L-13t aptamer, and scanned using an Amersham Typhoon Biomolecular Imager running in Cy5 mode. F. Streptoacidin separated by 15% SDS-PAGE, incubated with 500 nM Cy5-L-13t aptamer, and scanned using an Amersham Typhoon Biomolecular Imager running in Cy5 mode. G. Natural human thrombin separated by 15% SDS-PAGE, transferred to a nitrocellulose membrane, incubated with a monoclonal primary antibody targeting natural human thrombin and an Alexa Fluor 647-labeled polyclonal secondary antibody, and scanned using an Amersham Typhoon Biomolecular Imager running in Cy5 mode. M. Protein labeling. H, Schematic overview of the inhibition of native human thrombin enzymatic activity using L-DNA aptamers. I, Relative thrombin enzymatic activities of L-9-2 and L-9-2t aptamers in physiological buffer with 10 nM native human thrombin and 100 μM fluorescent substrate benzoyl-Phe-Val-Arg-AMC, where IC50... 50 The measurements were 317 ± 128 nM and 479 ± 65 nM, respectively. Data are expressed as mean ± SD (n = 3, independent measurements).

[0057] Figure 5A-C. Sequencing of DGGE-isolated L-DNA aptamers using the phosphate thioester method. A, Band L-9-1 was amplified using forward sequencing primers for D-Dpo4-5m with L-dNTPαS and 5'-FAM-labeled L-DNA, cleaved by 2-iodoethanol, and analyzed by 10% denaturing PAGE. B, Band L-9-1 (SEQ ID NO:9) was amplified using forward sequencing primers for D-Dpo4-5m with L-dNTPαS and 5'-FAM-labeled L-DNA, cleaved by 2-iodoethanol, treated by CIP, and analyzed by 10% denaturing PAGE. C, Band L-9-2 (SEQ ID NO:13) was amplified using forward sequencing primers for D-Dpo4-5m with L-dNTPαS and 5'-FAM-labeled L-DNA, cleaved by 2-iodoethanol, treated by CIP, and analyzed by 10% denaturing PAGE. Unspecified nucleotide positions are marked with an asterisk, which indicates the most likely substituted nucleotide (A and G) or deletion (-).

[0058] Figure 6A -B. Excluding incorrect sequences from L-9-2 band sequencing results using DGGE. A. Schematic overview of excluding incorrect sequences using DGGE, because the correct sequence should be the same as the T... m The band L-9-2 migrated together. B, the natural versions of the eight most likely L-DNA aptamer sequences in band L-9-2 (Table 1A) (DL-9-2-1 to DL-9-2-8, calculated T... m (Indications in parentheses) Amplification was performed by native PCR using FastPfu Fly DNA polymerase with D-DNA primers and L-DNA conjugates from R0 and R9, analyzed by 10% denaturing PAGE in 2.1M to 4.2M urea and 12% to 24% formamide, and stained with SYBR-Green II, where the co-migration of DL-9-2-7 and band L-9-2 is indicated by a blue dashed line.

[0059] Figure 7A-N. Reselection and optimization of L-DNA aptamers from a partially randomized L-DNA library. A, Schematic overview of the reselection and optimization of L-DNA aptamers from a partially randomized L-DNA library, where 34 nucleotides were partially randomized at a frequency of 10% based on the L-9-2 aptamer. B, The progress of mirror selection was monitored by EMSA using 200 nM of the corresponding L-DNA conjugate and 1 μM of natural human thrombin or 1 μM of streptoacidin, analyzed by 8% non-denaturing PAGE, and stained with SYBR Green II. C, Gel quantification results (b), where the binding fraction was determined by ImageJ software using the band intensity of the bound L-DNA conjugate relative to the total lane intensity. ND, (Binding) Not detected. D. Re-amplification by mirror PCR using D-Dpo4-5m and L-DNA primers, analyzed by 10% denaturing PAGE in 2.1M to 4.2M urea and 12% to 24% formamide, and DGGE analysis of the corresponding L-DNA conjugates and isolated band L-13 by SYBR-Green II staining. E. Sequencing chromatogram of band L-13 after natural CIP treatment using L-DNA sequencing primers labeled with L-dNTPαS and 5'-FAM using D-Dpo4-5m (with two mutations highlighted in yellow). F. Secondary structure of L-13 aptamer (SEQ ID NO: 27) predicted by Mfold, where reselected nucleotides are shown in red and two mutations (adenosine to cytidine) are shown. G. ITC analysis of L-13 aptamer bound to natural human thrombin, where K d The measured value was 22 nM. H, Secondary structure of the L-13t (truncated version) aptamer (SEQ ID NO:28) predicted by Mfold, wherein the reselected nucleotides are shown in pink and two mutations (adenosine to cytidine) are shown. I, ITC analysis of the L-13t aptamer bound to native human thrombin, wherein K d The measured value was 34 nM. J, EMSA analysis of the binding of 35 nM Cy5-L-13t aptamer to various concentrations of native human thrombin by 8% non-denaturing PAGE. K, Gel quantification results (J), where the binding fraction was determined by band intensity of the bound Cy5-L-13t aptamer relative to the total lane intensity using ImageJ software. L, Schematic overview of the inhibition of native human thrombin enzymatic activity using a reselected L-DNA aptamer. M, Relative thrombin enzymatic activities of L-13 and L-13t aptamers in physiological buffer incubated with 10 nM native human thrombin and 100 μM fluorescent substrate benzoyl-Phe-Val-Arg-AMC, where IC50 was the highest value. 50The values ​​were measured as 27 ± 3 nM and 46 ± 4 nM, respectively. Data are presented as mean ± SD (n = 3, independent measurements). N, a schematic overview of anticoagulation using L-DNA aptamers. Prothrombin time was measured with native versions of 2.5 μM L-9-1t, L-13t, and L-9-1t (DL-9-1t) aptamers in the presence of 50% (v / v) human plasma. NC, negative controls using separate physiological buffer. Data are presented as mean ± SD (n = 3, independent measurements, two-tailed unpaired Student's t-test).

[0060] Detailed Description of the Invention

[0061] In some embodiments, the present invention relates to methods for selecting L-nucleotide aptamers and sequencing methods thereof.

[0062] Before explaining at least one embodiment of the invention in detail, it should be understood that the invention is not necessarily limited to the details set forth in the following description or illustrated by examples. The invention can have other embodiments or can be practiced or implemented in various ways.

[0063] Mirror-image aptamers made from chiral inverted nucleic acids are nuclease-resistant and exceptionally biostable. Despite their diagnostic and therapeutic potential, only a small number of mirror-image aptamers have been selected via indirect selection schemes, such as 'selection-reflection' selection, primarily because the vast majority of biologically important target molecules, such as large proteins, cannot be chemically synthesized and correctly folded. The inventors have now developed a 'mirror-image selection' scheme for discovering L-DNA aptamers directly selected from large randomized L-DNA libraries using mirror-image molecular tools (see [link to invention]). Figure 1A The inventors performed an iterative cycle combining denaturing gradient gel electrophoresis (DGGE) for separation and sequencing-by-synthesis of L-DNA for determining the enriched L-DNA aptamer sequences. This enrichment and D-amino acid polymerase chain reaction (PCR) amplification of the L-DNA sequences binding to natural human thrombin identified several high-affinity L-DNA aptamers binding to thrombin (e.g., ...). Figure 1B (Examples provided).

[0064] While further putting this invention into practice, the inventors designed sensors and inhibitors based on selected L-DNA aptamers that function in physiologically relevant nuclease-rich environments, even in the presence of human serum that rapidly degrades D-DNA aptamers (e.g. Figure 4B (proven in -C, 4H-I and 7N).

[0065] The realization of direct, mirror-image selection schemes for L-DNA aptamers greatly expands the applications of mirror biology systems, fully unleashing their potential as biostable biosensors, therapeutic agents, and tools for basic research. The biostability of L-DNA conjugates and mirror-image molecular tools will also make the systems completely resistant to degradation by contaminating nucleases and proteases, particularly for low-purity target molecules and for cell- and tissue-based selection.

[0066] Therefore, according to a first aspect of the present invention, a method is provided for screening multiple L-nucleotide aptamers for L-nucleotide aptamers having binding affinity for a target molecule, comprising:

[0067] (a) The plurality of L-nucleotide aptamers are brought into contact with the target molecule under conditions of selectively capturing the L-nucleotide aptamer bound to the target from the plurality of L-nucleotide aptamers;

[0068] (b) Amplify the L-nucleotide aptamer of the target-bound L-nucleotide aptamer to generate amplified double-stranded L-nucleotide oligonucleotide;

[0069] (c) Amplified double-stranded L-nucleotide oligonucleotides were isolated using an electrophoresis-based method, thereby screening for a variety of L-nucleotide aptamers.

[0070] As used herein, the term "aptamer" refers to a nucleic acid molecule that exhibits a specific binding affinity for a target molecule, where the target is different from a polynucleotide that binds to the aptamer sequence through a mechanism that primarily relies on Watson / Crick base pairing.

[0071] The phrase "L-nucleotide aptamer" refers to an aptamer containing at least one L-deoxyribonucleotide or at least one L-ribonucleotide. According to one particular embodiment, at least 50% of the nucleotides in the L-nucleotide aptamer are L-nucleotides. In yet another embodiment, all the nucleotides in the L-nucleotide aptamer are L-nucleotides. Here, it is also intended that sugar components other than deoxyribose or ribose, which can form nucleotides, be used instead of the sugar component. Furthermore, this includes the use of nucleotides with further modifications at position 2', such as NH2, OMe, OBt, Oalkyl, NHalkyl, and the use of natural and non-natural nucleobases, such as, for example, isocytoside and isoguanosine.

[0072] L-nucleotide aptamers can be double-stranded or single-stranded. Typically, they are single-stranded L-nucleotides; however, due to their primary sequence, they can form defined secondary structures, and thus also tertiary structures. In these secondary structures, a large number of L-nucleotides possess double-stranded portions.

[0073] The target molecule may be a peptide (e.g., a naturally occurring or synthetic peptide), a protein (or a portion thereof), a sugar (e.g., a monosaccharide or polysaccharide), a lipid, a small molecule (e.g., less than 1500 Daltons), a mixture of cell membrane fragments, or a microorganism. In some embodiments, the target molecule does not include any nucleotide or polynucleotide molecules.

[0074] According to a particular implementation, the target molecule is a protein (or a portion thereof).

[0075] The binding affinity (Kd) of the aptamer to the target molecule is preferably less than 2000 nM, less than 1000 nM, less than 750 nM, less than 500 nM, less than 250 nM, less than 100 nM, and even less than 50 nM, as measured by EMSA (in the absence of serum).

[0076] As mentioned, the aptamer selected according to the methods described herein specifically (or selectively) binds to its target, meaning that the aptamer binds to the target molecule with an affinity of at least 10, 20, or even 50 times that to a non-target molecule of the same type. Thus, for example, if an aptamer selectively binds to a protein (e.g., human thrombin), it binds to thrombin with an affinity of at least 10 times that to a protein of similar size (e.g., bovine thrombin).

[0077] The method for selecting candidate aptamers begins by contacting multiple L-nucleotide aptamer candidates with a target molecule under conditions that selectively capture the L-nucleotide aptamer that binds to the target from multiple L-nucleotide aptamer candidates.

[0078] Synthesis of L-nucleotide aptamer candidates:

[0079] Multiple L-nucleotide aptamer candidates include any number of candidates, such as at least 10, at least 100, or at least 1000, each with a different sequence. Candidate L-nucleotide aptamers may all have the same length or may have different lengths. Exemplary lengths of L-nucleotide aptamers are 20-500 nucleotides, 20-400 nucleotides, 20-300 nucleotides, 20-200 nucleotides, and 20-100 nucleotides.

[0080] The chemical synthesis of L-nucleotide aptamers can be carried out via solid-phase synthesis using L-DNA phosphorous chemistry as known in the art. Candidate L-nucleotide aptamers can be purified post-synthesis using methods known in the art, including but not limited to non-denaturing polyacrylamide gel electrophoresis to remove readily aggregated L-nucleotide aptamers.

[0081] In one embodiment, the plurality of L-nucleotide aptamer candidates are members of a library, wherein each member of the library has the same 5' and 3' nucleotide sequences and a different (e.g., random) core sequence. The core sequence may be 10-100 nucleotides long, 10-80 nucleotides long, 10-70 nucleotides long, 10-60 nucleotides long, 10-50 nucleotides long, 10-40 nucleotides long, or 10-30 nucleotides long.

[0082] The preparation of such combinatorial libraries is described, for example, in Conrad, RC, Given, L., Tian, ​​Y. and Ellington, AD, 1996, Methods Enzymol., Vol. 267, 336-367.

[0083] To efficiently increase the number of library members with identical 5' and 3' sequences, chemically synthesized aptamers can be amplified via error-prone PCR, whereby the 5' and 3' ends (which are primer binding sites) remain constant thanks to the primers used during the PCR reaction, and the core is susceptible to error-prone PCR. In one embodiment, the error-prone PCR utilizes an error-prone polymerase (e.g., Dpo4 or Taq DNA polymerase). In another embodiment, a high-fidelity polymerase (e.g., Pfu DNA polymerase) is used, and amplification conditions that promote incorrect insertion are selected (e.g., the addition of Mn). 2+ ).

[0084] It should be understood that the amount of variation in L-DNA candidate merging can be controlled during chemical synthesis by doping each of the other three L-DNA nucleotides with a wild-type nucleotide.

[0085] Furthermore, in principle, if the T7 promoter is already included, an RNA library can also be generated from double-stranded DNA using a suitable DNA-dependent RNA polymerase, such as T7 RNA polymerase. Using this method, 10 15 A library of one or more DNA or RNA molecules. Each molecule from the library has a different sequence and therefore a different three-dimensional structure.

[0086] L-aptamers that capture target-binding

[0087] To differentiate between L-aptamers that bind to their targets with high affinity and those with low affinity, the target can be used as a decoy to capture the aptamer that binds to the target. This is used to enrich aggregates of L-aptamer aptamers that bind to the target.

[0088] To capture and bind aptamers to the target, the target molecule can be immobilized onto a solid support. Exemplary solid supports include, but are not limited to, laminated graphene, carbon nanotubes, fullerenes, and particles. Examples of materials that can be used to manufacture the particles include, but are not limited to, silica beads, polystyrene beads, latex beads, and may include metal colloids. According to one particular embodiment, the particles are magnetic particles. The target molecule can be immobilized on the surface of the solid support through hydrophobic interactions, electrostatic interactions, covalent bonds, coordination bonds, or non-covalent intermolecular interactions (such as biotin-streptavitin).

[0089] In other embodiments, the target molecule may be attached to a readable marker, such as a fluorescent marker, so that the signal from the aptamer-bound target molecule can be read and recorded using, for example, FACS. In other embodiments, the target molecule may not contain a readable marker. In such cases, the aptamers in the library to be screened may have certain scaffolds (e.g., hairpin scaffolds and substitutional chains) that alter their structure upon binding to the target molecule. The conformational change induced by target molecule binding can then generate a readable signal to be recorded (e.g., due to FRET interactions).

[0090] It should be understood that candidate pools can be pre-enriched through at least one round of negative selection (i.e., depletion of candidate pools that nonspecifically bind to non-target sequences) before capturing L-nucleotide aptamers that bind to target molecules. For example, selection can be performed on bead-immobilized human serum to reduce the number of nonspecifically binding non-target aptamers.

[0091] L-aptamers that amplify target-binding L-aptamers

[0092] Once the target-binding aptamer is separated from the non-target-binding aptamer, they can be amplified using a mirror PCR reaction.

[0093] As used in this article, the term "mirror image" refers to an isomer that is a mirror image of the natural material in terms of chirality.

[0094] The phrase "mirror PCR reaction" refers to a polymerase chain reaction that incorporates L-nucleotides into the amplified sequence.

[0095] Mirror-image PCR reactions typically use mirror-image polymerases, which are D-amino acid polymerases that are mirror images of the natural polymerase (i.e., L-type polymerase). The terms "mirror-image polymerase" are used interchangeably with "D-type polymerase" or "D-amino acid polymerase." For example, "D-Dpo4" refers to a D-type Dpo4 polymerase that is a mirror image of the natural L-type Dpo4 polymerase.

[0096] Polymerases particularly suitable for use in this invention include D-ASFV pol X, D-Dpo4, D-Taq polymerase, D-Pfu polymerase, and their functional variants.

[0097] Dpo4 (P2 DNA polymerase IV of *Lactobacillus sulfideus*) is a thermostable polymerase that can synthesize DNA even at 37°C. Its mismatch rate is 8 × 10⁻⁶. -3 Up to 3×10 -4 It is a polymerase that can replace Taq polymerase for multi-cycle PCR reactions. Its amino acid sequence length is within the range of current chemical synthesis techniques.

[0098] Taq polymerase is a thermostable polymerase that retains its activity at DNA denaturation temperatures. The optimal temperature for Taq is between 75°C and 80°C, and its half-life at 92.5°C is approximately 2 hours.

[0099] Pfu polymerase was discovered in *Pyrococcus furiosus*, and its function in microorganisms is to replicate DNA during cell division. Its superiority over Taq lies in its 3'-5' exonuclease activity, which allows it to cleave incorrectly added nucleotides on the extended strand during DNA synthesis. The mismatch rate of commercial Pfu is approximately one in 1.3 million.

[0100] As used herein, the term "functional variant" refers to a variant in which the amino acid sequence of a wild-type enzyme contains one or more (e.g., 1-5, 1-10, or 1-15, particularly, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or even more) amino acids through substitution, deletion, or addition, and said variant substantially retains the biology of the wild-type enzyme. For example, it retains 50%, 60%, 70%, 80%, or 90% or more of the biological activity of the wild-type enzyme. A "functional variant" can be a naturally occurring variant or an artificial variant, such as a variant obtained by site-directed mutagenesis or a variant produced by gene recombination methods.

[0101] In a preferred embodiment of the invention, the mirrored nucleic acid polymerase may include affinity tags to facilitate protein purification and reuse, such as multihistidine tags (His-tags or His tags), polyarginine tags, glutathione-S-transferase tags, etc.

[0102] A specific functional variant of the Dpo4 protein is Dpo4-5m, which contains amino acid mutations at five positions. In one embodiment, the Dpo4 protein comprises at least one, two, three, four, or each of the following mutations: C31S, S86C, Nl23A, S207A, and S313A.

[0103] The amino acid sequence of Dpo4-5m polymerase may include the sequence shown in SEQ ID NO:38.

[0104] In another embodiment, the Dpo4-5m polymerase includes an Sso7d domain fused to the C-terminus of Dpo4-5m (an exemplary sequence is shown in SEQ ID NO:40).

[0105] In one implementation, mirror PCR was performed in a buffer of 50 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 1 mM DTT and 50 mM KCl.

[0106] The present invention also provides D-ASFV pol X, the sequence of which is shown in SEQ ID NO:39, wherein all amino acids except for the achiral glycine are D-type amino acids.

[0107] In some implementations, the mirrored nucleic acid, mirrored nucleic acid template, mirrored nucleic acid primer, and mirrored dNTP / rNTP are L-type, and the mirrored nucleic acid polymerase is D-type.

[0108] In this paper, the nucleic acid replication reaction may be performed in only one cycle or in multiple cycles. This can be determined by those skilled in the art based on actual needs.

[0109] As used in this article, the term "multiple" means at least two. For example, "multiple cycles" means two or more cycles, such as three, four, or ten cycles.

[0110] As used herein, the term "replication" includes obtaining one or more copies of target DNA in the presence of a DNA template and dNTPs; and also one or more copies of target RNA in the presence of a DNA template and rNTPs (this process can also be referred to as RNA "transcription"). In nucleic acid replication, the template and primers are typically DNA. If the target nucleic acid is DNA, dNTPs should be added to the reaction system; if the target nucleic acid is RNA, rNTPs should be added to the reaction system.

[0111] In a particularly preferred embodiment, the reaction is carried out in a buffer solution of 50 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 1 mM MTT, and 50 mM KCl.

[0112] It should be understood that if the L-nucleotide aptamer is an RNA aptamer, then reverse transcription should be performed prior to the amplification step via polymerase chain reaction. The library enriched after the first round of selection can be used for a new round of selection, allowing molecules enriched in the first round to gain an advantage again through selection and amplification and enter the next round of selection with even more daughter molecules. The enriched pools emerge in this manner, and their members are then separated using electrophoresis-based methods, as further described below.

[0113] In the case of multiple rounds of selection, the amplified aptamer sequence (which is double-stranded) is converted into a single-stranded nucleic acid sequence before the target is added.

[0114] Methods for obtaining single-stranded nucleic acids are known in the art and are contemplated for use in this invention. In one particular embodiment, a spacer is used to interrupt the reverse primer (e.g., the Sp18 spacer) so that the PCR product contains two strands of unequal length. The two strands can then be separated using denaturing PAGE (see the Examples section below).

[0115] In another embodiment, the binding moiety is used to modify one of the reverse primers (e.g., biotin), and the double-stranded DNA is captured by a reagent (e.g., streptavidin-coated beads) that specifically binds to the binding moiety. Strains without the binding moiety can be eluted with NaOH, while strands with the binding moiety remain attached to the reagent.

[0116] This invention considers at least 3 rounds of selection, amplification, and conversion to single-stranded aptamers, at least 4 rounds of selection, amplification, and conversion to single-stranded aptamers, at least 5 rounds of selection, amplification, and conversion to single-stranded aptamers, and at least 6 rounds of selection, amplification, and conversion to single-stranded aptamers. In one embodiment, no more than 10 rounds of selection, amplification, and conversion to single-stranded aptamers are performed. In yet another embodiment, no more than 15 rounds of selection, amplification, and conversion to single-stranded aptamers are performed.

[0117] Enrichment of target-binding molecules in L-nucleotide aptamer complexes can be monitored using methods known in the art. Such methods include electromobility migration assay (EMSA).

[0118] As mentioned, after sufficient enrichment of the L-nucleotide aptamer conjugate, the resulting aptamers were further purified using an electrophoresis-based method, as described below.

[0119] Isolation and amplification of L-nucleotide oligonucleotides

[0120] Electrophoresis-based methods for separating aptamers that bind to targets include, but are not limited to, non-denaturing PAGE; denaturing PAGE; denaturing gradient gel electrophoresis (DGGE); constant denaturing gel electrophoresis (CDGE); capillary electrophoresis; and time-temperature gradient gel electrophoresis (TTGE).

[0121] According to a specific implementation, the electrophoresis-based method for separating candidate target-binding aptamers is DGGE.

[0122] Denaturing / Temperature Gradient Gel Electrophoresis (DGGE / TGGE): This method relies on detecting changes in electrophoretic mobility in response to small sequence variations. One of these methods, called "denaturing gradient gel electrophoresis" (DGGE), is based on the observation that slightly different sequences will exhibit different local unwinding patterns when electrophoretically resolved on a gradient gel. In this way, variants can be distinguished because the difference in unwinding properties of homoduplexes relative to heteroduplexes at the single nucleotide level can detect the presence of SNPs in the target sequence, as their electrophoretic mobilities change accordingly. The fragment to be analyzed, typically a PCR product, is "clamped" at one end by a long GC base pair (30-80) to completely denature the target sequence without completely disintegrating it. The attachment of the GC "clamp" to the DNA fragment increases the mutation sites that can be recognized by DGGE (Abrams et al., Genomics 7:463-475, 1990). Attaching a GC clip to a primer is crucial to ensuring a low dissociation temperature for the amplified sequence (Sheffield et al., Proc. Natl. Acad. Sci., 86:232-236, 1989; and Lerman and Silverstein, Meth. Enzymol., 155:482-501, 1987). Variations of this technique have been developed using temperature gradients (Wartell et al., Nucl. Acids Res., 18:2699-2701, 1990), and the method can also be applied to RNA:RNA duplexes (Smith et al., Genomics 3:217-223, 1988).

[0123] Limitations of DGGE include the requirement to optimize denaturation conditions for each type of DNA being tested. Furthermore, the method requires specialized equipment to prepare the gel and maintain the necessary high temperatures during electrophoresis. The associated cost of synthesizing pincers on an oligonucleotide for each sequence being tested is also a major consideration. Additionally, DGGE requires long run times. The long run time of DGGE is shortened in a variant called constant denaturing gel electrophoresis (CDGE) (Borrensen et al., Proc. Natl. Acad. Sci. USA 88:8405, 1991). CDGE requires gelation under different denaturation conditions to achieve high efficiency in detecting SNPs.

[0124] A technique similar to DGGE, called temperature gradient gel electrophoresis (TGGE), uses a thermal gradient instead of a chemical denaturation gradient (Scholz, et al., Hum. Mol. Genet. 2:2155, 1993). TGGE requires specialized equipment that generates a temperature gradient oriented perpendicular to an electric field. TGGE can detect mutations in relatively small DNA fragments; therefore, scanning large gene regions requires the use of multiple PCR products prior to gel running.

[0125] After separation by electrophoresis, the isolated L-DNA aptamers can be sequenced using methods known in the art.

[0126] Exemplary methods for sequencing L-DNA aptamers include, but are not limited to, L-DNA chemical sequencing; L-DNA phosphate thioester sequencing; L-DNA dideoxy sequencing; L-DNA ion-fluid sequencing; L-DNA Illumina sequencing; and L-DNA nanopore sequencing.

[0127] High-throughput methods can include techniques for rapid sequencing of large numbers of nucleic acids, including next-generation technologies such as massively parallel characterization sequencing (MPSS); Polony sequencing; 454 pyrosequencing; Illumina (Solexa) sequencing; SOLiD sequencing; ion-fluidic semiconductor sequencing; DNA nanosphere sequencing; Heliscope single-molecule sequencing; single-molecule real-time (SMRT) sequencing; or other methods such as nanopore DNA sequencing; tunneling current DNA sequencing; hybridization-while-sequencing; sequencing-accompanied mass spectrometry; microfluidic Sanger sequencing; microscopy-based techniques; RNAP sequencing; and in vitro viral high-throughput sequencing.

[0128] Automated dideoxy-terminant sequencing of isolated L-nucleotide aptamers was performed using either dye-terminant (unlabeled primers and labeled dideoxynucleotides) or dye-primer (labeled primers and unlabeled dideoxynucleotides) cyclic sequencing protocols. For dye-terminant reactions, PCR was performed using unlabeled PCR primers, followed by sequencing in the presence of one of the primers, a dideoxynucleotide, and a labeled dideoxynucleotide mixture. For dye-primer reactions, PCR was performed using PCR primers conjugated to universal or reverse primers (one for each direction), followed by sequencing in the presence of four separate mixtures (corresponding to A, G, C, and T nucleotides), each containing a labeled primer specific to the universal or reverse sequence and the corresponding unlabeled dideoxynucleotide.

[0129] Pyrosequencing TMAnalysis (Pyrosequencing, Inc., Westborough, MA, USA): This technique is based on the hybridization of sequencing primers with a single-stranded, PCR-amplified DNA template in the presence of DNA polymerase, ATP sulfurylase, luciferase, and adenosine triphosphate diphosphatase, as well as adenosine 5'-phosphate (APS) and a luciferin substrate. In the second step, the first of four deoxynucleoside triphosphates (dNTPs) is added to the reaction, and if the dNTP is complementary to a base in the template strand, the DNA polymerase catalyzes its incorporation into the DNA strand. Each incorporation event is accompanied by the release of pyrophosphate (PPi) in an equimolar amount of the incorporated nucleotide. In the final step, ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5'-phosphate. This ATP drives the luciferase-mediated conversion of luciferin to oxidative luciferin, which produces visible light in a proportionate amount to the amount of ATP. The light generated in the luciferase-catalyzed reaction is detected by a charge-coupled device (CCD) camera and plotted on a pyrogram. TM The peak is considered to be in the middle. Each optical signal is proportional to the number of incorporated nucleotides.

[0130] Phosphothioester sequencing: Phosphothioester sequencing can be performed by mirror PCR (e.g., using D-Dpo4-5m) in which one of the L-dNTPs is replaced by the corresponding L-dNTPαS. The product is mixed with a solution containing 2-iodoethanol. In one embodiment, the 3'-monophosphate is first removed from the 2-iodoethanol-cleaved DNA fragment using a phosphatase (e.g., calf intestinal phosphatase-CIP) before running on a denaturing sequencing gel. More information on phosphothioester sequencing can be found in Fan, C., et al. Nat. Biotechnol. 39:1548-1555 (2021) (the contents of which are incorporated herein by reference).

[0131] Once the sequence is obtained, L-DNA aptamers can be chemically synthesized, and their binding activity to their corresponding targets can be verified.

[0132] Once a lead candidate sequence is obtained, it can be used as a starting point for creating new libraries and thus identifying additional candidates with improved affinity / specificity. The lead sequence can be partially randomized (e.g., 1-60% randomized). In one embodiment, the lead candidate sequence is mutated with 10% randomization (approximately 3.3% for each base other than the original base), so the doping rate can be between 1% and 60%.

[0133] If needed, reagents for isolating the L-DNA aptamers of this invention can be provided in the kit. Instructions for use may be included with the kit.

[0134] According to one particular implementation, the kit comprises:

[0135] (i) Calf intestinal phosphatase (CIP);

[0136] (ii) L-deoxyribonucleotide triphosphates (L-dNTPs) or modified L-dNTPs; and / or

[0137] (iii)(iii) A polymerase capable of adding one or more L-nucleotides to the 3' end of a first L-nucleic acid.

[0138] The modified L-dNTPs include L-deoxynucleotide α-thiotriphosphate.

[0139] The aptamers of the present invention can be used in various methods to assess the presence or level of biomarkers in biological samples, such as target biological entities, like proteins, sugars, cells, or microvesicles. The aptamers function as binding agents to assess the presence or level of homologous target molecules. Therefore, in various embodiments of the invention relating to diagnostic, prognostic, or therapeutic diagnostics, one or more aptamers of the present invention are configured in ligand-target-based assays, wherein one or more aptamers of the present invention are contacted with a selected biological sample, wherein the one or more aptamers are associated with or bound to their target molecules. The aptamers of the present invention are used to identify candidate biomarkers based on the biological sample being evaluated and the biomarkers being detected.

[0140] The L-nucleotide aptamers discovered by the method of the present invention include those having a nucleic acid sequence as shown in SEQ ID NO: 10, 12, 14, 16, 27, or 28. In one embodiment, the L-nucleotide aptamer has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10, 14, or 28. In another embodiment, the L-nucleotide aptamer has a sequence as shown in SEQ ID NO: 10, 12, 14, 16, 27, or 28, wherein up to 10 nucleotides of the sequence are mutated, and the location of the mutation is a single-stranded region of the aptamer (as predicted by computational analysis such as Mfold). In one embodiment, the single-stranded region is Figure 3A , 3C The single-chain region shown in 3H, 3J, 7F or 7H.

[0141] The aptamers described in this article can be attached to detectable parts or markers.

[0142] Suitable markers include, but are not limited to, magnetic markers, fluorescent components, enzymes, chemiluminescent probes, metal particles, non-metallic colloidal particles, polymeric dye particles, pigment molecules, pigment particles, electrochemically active substances, semiconductor nanocrystals or other nanoparticles (including quantum dots or gold particles), fluorophores, quantum dots, or radioactive markers. Protein markers include green fluorescent protein (GFP) and its variants (e.g., cyan fluorescent protein and yellow fluorescent protein); and luminescent proteins, such as luciferase, as described below. Radioactive markers include, but are not limited to, radioisotopes (radionucleases), such as 3H, 11C, 14C, 18F, 32P, 35S, 64Cu, 68Ga, 86Y, 99Tc, 111In, 123I, 124I, 125I, 131I, 133Xe, 77Lu, 211At, or 213Bi. Fluorescent labels include, but are not limited to, rare earth chelates (e.g., europium chelates), rhodamine; fluoresceins, including but not limited to FITC, 5-carboxyfluorescein, 6-carboxyfluorescein; rhodamine derivatives, including but not limited to TAMRA; dansyl; lissamine; anthocyanins; phycoerythrin; Texas red; Cy3, Cy5, dapoxyl, NBD, Cadcade yellow, dansyl, PyMPO, pyrene, 7-diethylaminocoumarin 3-carboxylic acid and other coumarin derivatives, Marina Blue. TM PacificBlue TM Cascade Blue TM 2-Anthracenesulfonyl, PyMPO, 3,4,9,10-dinaphthalene-tetracarboxylic acid, 2,7-difluorofluorescein (Oregon Green) TM 488-X), 5-carboxyfluorescein, Texas Red TM-X, Alexa Fluor 430, 5-carboxytetramethylrhodamine (5-TAMRA), 6-carboxytetramethylrhodamine (6-TAMRA), BODIPY FL, bimane, and Alexa Fluor 350, 405, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, and 750, and their derivatives, as well as others. See, for example, "The Handbook—A Guide to Fluorescent Probes and Labeling Technologies," 10th edition, available at probes(dot)invitrogen(dot)com / handbook. The fluorescent marker may be one or more of FAM, dRHO, 5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET, dTAMRA, TAMRA, NED, dROX, PET, BHQ, Gold540, and LIZ.

[0143] Using conventional techniques, L-nucleotide aptamers can be directly or indirectly labeled, for example, by attaching a label to the aptamer via biotin-streptavitin (e.g., synthesizing a biotinylated aptamer that is then capable of binding itself to a streptavitin molecule conjugated with a detectable label; a non-limiting example is phycoerythrin-conjugated streptavitin (SAPE)). Chemical conjugation methods using multi-step procedures include conjugation with biotinylation, trinitrophenol (TNP), or digoxigenin, using succinimide esters of, for example, these compounds. Biotinylation can be performed, for example, using D-biotinyl-N-hydroxysuccinimide. The succinimide group reacts efficiently with amino groups at pH values ​​above 7, and preferably between about pH 8.0 and about pH 8.5. Alternatively, the aptamer may not be labeled, but a labeled secondary antibody may be contacted after the primary antibody binds to the target antigen.

[0144] Various enzyme-substrate markers can also be used to bind to L-nucleotide aptamers. Such enzyme-substrate markers are commercially available (e.g., U.S. Patent No. 4,275,149). The enzyme typically catalyzes a chemical change in a chromogenic substrate, which can be measured using various techniques. For example, the enzyme can catalyze a color change in the substrate, which can be measured spectrophotometrically. Alternatively, the enzyme can alter the fluorescence or chemiluminescence of the substrate. Examples of enzymatic markers include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, glucosylamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc. Examples of enzyme-substrate combinations include, but are not limited to: horseradish peroxidase (HRP) using catalase as a substrate, wherein catalase oxidizes dye precursors (e.g., o-phenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB)); alkaline phosphatase (AP) using p-nitrophenyl phosphate as a chromogenic substrate; and β-D-galactosidase (β-D-Gal) using a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) or a fluorescent substrate 4-methylumbellatus-β-D-galactosidase.

[0145] L-nucleotide aptamers can be attached to a substrate, such as a planar substrate. Planar arrays typically contain addressable sites (e.g., pads, addresses, or micro-sites) of biomolecules in array form. The size of the array will depend on its composition and end use. Arrays containing two to thousands of different molecules can be fabricated. Typically, the arrays contain two to up to 100,000 or more molecules, depending on the end use and fabrication method. Microarrays used with this invention comprise at least one biomolecule that identifies or captures a biomarker present in a target biofeature, such as microRNAs or other biomolecules or vesicles constituting the biofeature. In some arrays, multiple substrates with different or identical compositions are used. Therefore, a planar array can contain multiple smaller substrates.

[0146] The inventors have demonstrated that the use of calf intestinal phosphatase (CIP) after iodoethanol cleavage of DNA fragments facilitates the sequencing of oligonucleotides containing L-deoxynucleotide α-thiotriphosphate.

[0147] Therefore, according to yet another aspect of the present invention, a method for sequencing purified L-DNA molecules is provided, comprising:

[0148] (a) Treating a sample containing purified L-DNA molecules with a phosphatase (e.g., CIP) under conditions where 3'-monophosphate has been removed from L-DNA molecules: and

[0149] (b) The sample was subjected to phosphate thioester sequencing, thereby sequencing the purified L-DNA molecules.

[0150] As used in this article, the term “about” means ±10%.

[0151] The terms “comprises”, “comprising”, “includes”, “including”, “have”, and their inflections mean “including but not limited to”.

[0152] The term "composed of" means "including and limited to".

[0153] The term "consistently made up of" means that a composition, method, or structure may include additional ingredients, steps, and / or portions, provided that such additional ingredients, steps, and / or portions do not substantially alter the fundamental and novel characteristics of the claimed composition, method, or structure.

[0154] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural references. For example, the term “compound” or “at least one compound” can include a variety of compounds, including mixtures thereof.

[0155] Throughout this application, various embodiments of the invention may be presented in a scope format. It should be understood that the scope format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Therefore, the scope description should be considered to have specifically disclosed all possible sub-scopes and individual numerical values ​​within those scopes. For example, a scope such as 1-6 should be considered to have specifically disclosed sub-scopes such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., and individual numbers within those scopes such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the scope.

[0156] Whenever a range of numbers is indicated herein, it means that any of the numbers (fractions or integers) indicated within the range shown are included. The phrases “range between the first indicated number and the second indicated number” and “range from the first indicated number to the second indicated number” are used interchangeably herein and mean that the first and second indicated numbers are included, as well as all fractions and integers in between.

[0157] As used herein, the term "method" means the manner, means, techniques and procedures used to accomplish a given task, including but not limited to those manner, means, techniques and procedures known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine, or those that can be readily developed by said practitioners from known manner, means, techniques and procedures.

[0158] When referring to a particular sequence listing, such reference should be understood to also include sequences that substantially correspond to their complementary sequences while including small sequence variations caused by, for example, sequencing errors, cloning errors, or other alterations resulting in base substitutions, deletions, or additions, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5000 nucleotides, alternatively, less than 1 in 10000 nucleotides.

[0159] It should be understood that any sequence number (SEQ ID NO) disclosed in this application may refer to a DNA sequence or an RNA sequence, depending on the context in which the SEQ ID NO is mentioned, even if the SEQ ID NO is represented only in DNA sequence format or RNA sequence format. Similarly, although some sequences are represented in RNA sequence format (e.g., U for uracil is listed), depending on the actual type of molecule described, it may refer to a sequence of an RNA molecule containing dsRNA, or a sequence of a DNA molecule corresponding to the RNA sequence shown. In any case, both DNA and RNA molecules having the disclosed sequences, as well as any alternatives, are contemplated.

[0160] It should be understood that certain features of the invention described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for brevity may also be provided individually or in any suitable sub-combination or, where appropriate, in any other described embodiment of the invention. Certain features described in the context of individual embodiments should not be considered essential features of those embodiments unless the embodiments are invalid without those elements.

[0161] The various embodiments and aspects of the invention described above and claimed in the appended claims are supported by the experimental results of the following examples. Example

[0162] The following embodiments, together with the foregoing description, illustrate some implementations of the invention in a non-limiting manner.

[0163] The nomenclature used herein and the laboratory procedures employed in this invention typically include molecular, biochemical, microbiological, and recombinant DNA technologies. These technologies are fully explained in the literature. General references are provided throughout this document.

[0164] Material

[0165] All L-DNA oligonucleotides (Tables 1A and 1B below) were synthesized on an H-8 oligonucleotide synthesizer (K&A Laborgeraete, Germany). All D-DNA oligonucleotides (Tables 1A-B below) were ordered from Genewiz (Jiangsu, China). L-deoxynucleoside phosphoramide was purchased from ChemGenes (MA, US). Hexaethylene glycol spacer (Sp18) phosphoramide was purchased from Glen Research (VA, US). Fluorescein (FAM) and cyanine 5 (Cy5) phosphoramide, as well as 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL) and monophosphate controlled-porosity glass (CPG) were purchased from Ruibiotech (Beijing, China). All D-DNA and L-DNA oligonucleotides were purified by HPLC or denaturing PAGE prior to use. L-deoxynucleoside triphosphates (L-dNTPs) and L-deoxynucleoside α-thiotriphosphates (L-dNTPαS) are synthesized from L-deoxynucleosides (ChemGenes, MA, US). 1 D-dNTPαS was purchased from TriLink Biotechnologies Inc. (CA, US). L-Dpo4-5m with an N-terminal His6 tag was expressed in Escherichia coli strain BL21 and as described in the literature. 2 Purification was performed as described above. FastPfu Fly DNA polymerase was purchased from TransGen Biotech (Beijing, China). D-Dpo4-5m was synthesized and folded according to previously reported methods, except that an automated peptide synthesizer was used and methionine (Met) was substituted for leucine (Nle). 2,32-Iodoethanol was purchased from Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). Natural human α-thrombin and natural bovine α-thrombin derived from plasma were purchased from Haematologic Technologies (VT, US). Streptomycin, calf intestinal alkaline phosphatase (CIP), and DNase I were purchased from New England Biolabs (MA, US). Human serum was purchased from Zhongke Chenyu Biotech (Beijing, China). Monoclonal primary antibody targeting natural human thrombin and Alexa Fluor 647-labeled polyclonal secondary antibody were purchased from Abcam (UK). ExRed was purchased from Beijing Zoman Biotech (Beijing, China). NHS-activated magnetic beads and SYBR-Green II were purchased from Thermo Fisher Scientific (MA, US). Benzoyl-Phe-Val-Arg-AMC (AMC, 7-amino-4-methylcoumarin) was purchased from Sigma-Aldrich (MO, US).

[0166] Table 1A – DNA Sequence

[0167]

[0168]

[0169] Table 1B - LDNA sequences used for reselection from partially randomized L-DNA libraries

[0170]

[0171] L-DNA library preparation

[0172] A molar ratio of 1.5:1.25:1.15:1 for D- or L-phosphoramide, dA, dC, dG, and dT was used to synthesize a 30-nt randomized region from a 65-nt total length D-DNA or L-DNA library to achieve approximately equal coupling efficiencies. 4 For example, in the literature 5As described, non-denaturing polyacrylamide gel electrophoresis (PAGE) purification was performed to remove easily aggregated DNA. Briefly, 5 nmol of synthetic D-DNA or L-DNA libraries were loaded onto a 1 mm × 200 mm × 550 mm plate and separated by PAGE. The PAGE consisted of a denaturing top portion (1 mm × 200 mm × 50 mm) and a non-denaturing bottom portion (1 mm × 200 mm × 500 mm). The denaturing top portion contained 7 M urea and 8% acrylamide in 0.5 × Tris-borate-EDTA (TBE), and the non-denaturing bottom portion contained 10% acrylamide and 10 mM Mg(OAc)₂ in 0.5 × TBE. The gel was run at 10 W (constant power) for 6 h and stained with SYBR-Green II. The fastest migrating bands were separated by a 'crush and soak' method. 6 Purification. Using L-Dpo4-5m or D-Dpo4-5m and the D-DNA or L-DNA primers listed in Table 1A above, amplify approximately 165 pmol of a non-denaturing-PAGE-purified library (with approximately 1 × 10⁻⁶ DNA) by native or mirror PCR. 14 (Multiple different sequences), where the reverse primer contains poly(A) modified by Sp18. 20 The tail is used to generate PCR products with chains of different lengths, which are then processed by denaturing PAGE. 7 Strand separation was performed. The native and mirror PCR program was set to 86°C for 3 min (initial denaturation); 86°C for 30 sec, 50°C for 1 min, and 65°C for 2 min, for 15 cycles; 65°C for 5 min (final extension). The 65 nt forward strand was separated from the 85 nt Sp18-modified reverse strand by 10% denaturing PAGE in 7M urea and used as the starting D-DNA or L-DNA library for aptamer selection. Selection of D-DNA or L-DNA aptamers targeting native human thrombin.

[0173] Magnetic beads coupled to natural human thrombin were prepared from N-hydroxysuccinimide (NHS) activated magnetic beads according to the manufacturer's instructions (Thermo Fisher Scientific, MA, US). Briefly, 300 μl of natural human thrombin at a concentration of 0.1 mg / ml was mixed with 3 mg of NHS-activated magnetic beads in a coupling buffer (20 mM HEPES-NaOH, 150 mM NaCl, 5% glycerol, pH 7.4). The coupling reaction was carried out at room temperature for 2 h, followed by quenching with 3 M ethanolamine at pH 9.0. After coupling, the beads were resuspended in 300 μl of a selection buffer (20 mM HEPES-NaOH, 150 mM NaCl, 5 mM KCl, 2 mM MgCl2, 1 mM CaCl2, 0.05% (v / v) Tween-20, pH 7.4). For round 1 (R1), approximately 600 pmol (with approximately 1 × 10⁻⁶ pmol) in a 250 μl volume will be used. 14 Approximately 3.6 × 10⁻⁶ different sequences 14A library of D-DNA or L-DNA (number of molecules) was heated to 85°C for 5 minutes in selection buffer and then slowly cooled to 25°C over 10 minutes. 50 μl of protein-free NHS-activated magnetic beads were then added, and the mixture was incubated at room temperature with gentle rotation for 1 hour. In each selection round, a negative selection step was performed using 50 μl of protein-free NHS-activated magnetic beads. The supernatant was mixed with 100 μl of magnetic beads conjugated with natural human thrombin, for a total volume of 400 μl, and incubated at room temperature with gentle rotation for 1 hour. The beads were then separated from the supernatant using a DynaMag-2 magnet (ThermoFisher Scientific, MA, US), and briefly washed three times (10 seconds each) with 400 μl of selection buffer. The bound DNA was eluted from the beads with 25 mM NaOH and 5 mM EDTA and precipitated with ethanol. The recovered D-DNA or L-DNA was used as a template for native or mirror PCR amplification via L-Dpo4-5m or D-Dpo4-5m to generate D-DNA or L-DNA conjugates for the next round. The number of native or mirror PCR cycles per selection round was determined based on the results of 10 μl-scale PCR. As shown in Tables 2 and 3, the amount of DNA conjugate was gradually reduced from approximately 600 pmol in R1 to approximately 50 pmol in R6 (for D-DNA conjugates), and from approximately 600 pmol in R1 to approximately 30 pmol in R9 (for L-DNA conjugates). The volume of magnetic beads coupled with native human thrombin was gradually reduced from 100 μl in R1 to 10 μl in R6 (for D-DNA conjugates), and from 100 μl in R1 to 3 μl in R9 (for L-DNA conjugates). The washing steps gradually increased from three 10-second washes in R1 to six 10-minute washes in R6 (for D-DNA conjugates), and from three 10-second washes in R1 to eight 10-minute washes in R9 (for L-DNA conjugates).

[0174] Table 2 – Conditions for D-DNA aptamer selection

[0175]

[0176]

[0177] Table 3 – Conditions for L-DNA aptamer selection

[0178]

[0179] Electrophoretic mobility variation measurement (EMSA)

[0180] D-DNA or L-DNA conjugates and D-DNA or L-DNA aptamers were heated to 85°C for 5 minutes in selection buffer and then slowly cooled to 25°C over 10 minutes. They were then mixed with natural human thrombin or streptoacidin in selection buffer containing 10% (v / v) glycerol. The mixture was incubated at room temperature for 30 minutes and analyzed by 8% non-denaturing PAGE (for D-DNA or L-DNA conjugates, D-6, Cy5-L-9-1t, and Cy5-L-13t aptamers) in 1× run buffer (20 mM HEPES-NaOH, 50 mM NaOAc, 5 mM KOAc, 2 mM Mg(OAc)2, 1 mM CaCl2, pH 7.4) or by 10% non-denaturing PAGE (for Cy5-L-9-2t aptamers) in 1× run buffer where 5% (v / v) glycerol was added to both the gel and run buffer. Gels were run at 150V (constant voltage) for 1–2 h, stained with SYBR-Green II, and scanned using an Amersham Typhoon Biomolecular Imager (Cytiva, US) running in Cy2 mode (for D-DNA or L-DNA conjugates and D-6 aptamers) or Cy5 mode (for Cy5-labeled L-DNA aptamers). Gel quantification was performed using ImageJ software, where the dissociation constant (K0) was used. d The combination score was calculated by fitting the sigmoidal model using KaleidaGraph software (Synergy Software, PA, US).

[0181] Denaturing gradient gel electrophoresis (DGGE)

[0182] D-DNA or L-DNA conjugates and D-DNA or L-DNA aptamers were amplified by native or mirror PCR using L-Dpo4-5m or D-Dpo4-5m and the D-DNA or L-DNA primers listed in Table 1A, wherein the forward primer contained a GC-rich region (GC-clamp) to prevent complete unwinding of the double-stranded PCR product during DGGE8. 8Native or mirror-image PCR products were purified by 3% sieve agarose gel electrophoresis and mixed with 2× loading buffer (100 mM Tris-HCl, 10 mM EDTA, 30% glycerol, pH 7.0) and separated by a 7.5% polyacrylamide gel (for D-DNA conjugates) or a 10% polyacrylamide gel (for L-DNA conjugates) (which consists of a linear denaturant gradient of 2.1 M urea, 12% (v / v) formamide (top) to 4.2 M urea and 24% (v / v) formamide (bottom) in 1× Tris-acetate-EDTA (TAE)). The gels were run at 100 V for 6 h at 60 °C (isotropic) (for D-DNA conjugates) or at 75 V for 13 h at 60 °C (for L-DNA conjugates). For DGGE separation of D-DNA or L-DNA aptamer sequences, 500 ng of native or mirror-image PCR products were separated by DGGE, followed by SYBR-Green II staining, separation by cutting the gel on a 254 nm UV transilluminator, and separation by 'crushing and soaking' method. 6 Purified and re-amplified using L-Dpo4-5m or D-Dpo4-5m and the D-DNA or L-DNA primers listed in Table 1A by native or mirror PCR. To exclude erroneous sequences from the L-9-2 band sequencing results, the native versions of the eight most likely L-DNA aptamer sequences in band L-9-2 (DL-9-2-1 to DL-9-2-8, ...) were used. Figure 6B (As shown in Table 4 below) Amplification was performed using FastPfu Fly DNA polymerase and D-DNA primers via native PCR, followed by separation via DGGE, SYBR-Green II staining, and scanning using an Amersham Typhoon Biomolecular Imager running in Cy2 mode. Melting temperature (T) m Using the nearest neighbor thermodynamic model via OligoCalc 9 The default parameters are calculated.

[0183] Table 4 - D-DNA oligonucleotides used for DGGE analysis and calculated melting temperatures (T) m )

[0184]

[0185]

[0186] High-throughput sequencing of selected D-DNA aptamers

[0187] Using L-Dpo4-5m and the D-DNA primers listed in Table 1A, R6 D-DNA conjugates and D-6 bands separated by DGGE were amplified by native PCR. PCR products were purified by 2.5% agarose and sequenced on an Illumina HiSeq system (Illumina, CA, US). Raw Illumina reads were processed and sorted by abundance using the Galaxy server (www.usegalaxy.org).

[0188] Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS)

[0189] MALDI-TOF MS was used to analyze L-DNA dephosphorylation via CIP. Approximately 100 ng of 3'-monophosphate-labeled L-DNA oligonucleotides (Table 1A) were treated with 20 units of CIP, incubated at 37°C in 1×CutSmart buffer (New England Biolabs, MA, US) for 1 h, desalted via a C18 column (Thermo Fisher Scientific, MA, US), and analyzed by MALDI-TOF MS (Applied Biosystems 4800plus, CA, US) in positive linear mode.

[0190] L-DNA aptamer sequencing

[0191] Using the 5'-FAM-labeled forward sequencing primers and unlabeled reverse primers listed in Table 1A, four separate PCR reactions were performed (where one of the L-dNTPs was coupled to the corresponding L-dNTPαS). 10 (Alternatively) L-DNA aptamers isolated via DGGE were amplified by mirror PCR using D-Dpo4-5m. The 5'-FAM-labeled PCR product was purified by 10% denaturing PAGE in 7M urea and dissolved in 10mM Tris-HCl at pH 7.4 to a final concentration of approximately 20 ng / μl. For each sequencing reaction, 5 μl of 5'-FAM-labeled L-DNA was mixed with 5 μl of lysis solution containing 2% (v / v) 2-iodoethanol in ddH2O, followed by heating to 95°C for 3 min and then rapidly placed on ice. To remove 3'-monophosphate from the 2-iodoethanol-lysed DNA fragments, each sequencing reaction was treated with 5 units of CIP, incubated at 37°C in 1×CutSmart buffer for 1 h, and then mixed with 10 μl of 2× loading buffer containing 95% formamide and 10 mM EDTA. The sample was mounted on a 0.4 mm × 340 mm × 300 mm plate and processed according to the previously reported method. 10Analysis was performed using 10% denaturing PAGE in 7M urea.

[0192] Isothermal titration calorimetry (ITC)

[0193] Natural human thrombin, natural bovine thrombin, and streptoacidin in the storage buffer were dialyzed against physiological buffer (20 mM HEPES-NaOH, 150 mM NaCl, 5 mM KCl, 2 mM MgCl2, 1 mM CaCl2, pH 7.4) at 4 °C for 16 h. D-DNA and L-DNA aptamers were equilibrated in physiological buffer by ultrafiltration, then heated to 85 °C for 5 minutes and slowly cooled to 25 °C over 10 minutes. MicroCal iTC was used for further analysis. 200 ITC was performed using a microcalorimeter (GE Healthcare, UK) with 7 μM to 20 μM of natural human thrombin, natural bovine thrombin, or streptoacidin in the reaction chamber and 70 μM to 200 μM of D-DNA or L-DNA aptamers in the syringe, under stirring at 25°C and 750 rpm. To measure the heat of dilution, 70 μM to 200 μM of D-DNA or L-DNA aptamers were injected into protein-free physiological buffer. Data were fitted using MicroCal Origin software (GE Healthcare, UK).

[0194] L-DNA Aptamer Sensor

[0195] D-DNA or L-DNA aptamer sensors containing 250 nM 5'-FAM-labeled fluorophore strand, 750 nM 3'-DABCYL-labeled quencher strand, and 500 nM D-6 or L-9-1t-based aptamer strand (Table 1A) were incubated with 300 nM natural human thrombin at 37 °C for 1 h or 4 h in physiological buffer alone or in physiological buffer containing 10% (v / v) human serum. Relative fluorescence was measured using a Varioskan Flash system (Thermo Fisher Scientific, MA, US) with excitation at 494 nm and emission at 518 nm. Standard curves were plotted using 0, 125, 250, 500, or 1000 nM of natural human thrombin, and relative fluorescence was measured after incubation at 37 °C in physiological buffer for 1 h. The change in relative fluorescence units (ΔRFU) relative to background (RFU measured with D-DNA or L-DNA aptamer sensors in separate physiological buffers) was used for data fitting. For measurements in physiological buffers containing 10% (v / v) human serum, standard curves were plotted using 0, 250, 500, 1000, or 2000 nM natural human thrombin, and relative fluorescence was measured after incubation at 37°C in physiological buffers containing 10% human serum for 1 h. To evaluate the biostability of the D-DNA and L-DNA aptamer sensors, the sensors were incubated at 37°C in physiological buffers containing 10% human serum for up to 24 h (for D-DNA aptamer sensors), or at 37°C in physiological buffers containing 83% (v / v) human serum for up to 24 h (for D-DNA aptamer sensors) or up to 30 days (720 h) (for L-DNA aptamer sensors). The sample was mixed with 2× loading buffer containing 95% formamide and 10 mM EDTA and rapidly incubated at -20°C, then analyzed by 10% denaturing PAGE in 7 M urea. Gel quantification was performed using ImageJ software, where the half-life (t) was... 1 / 2 The relative strip intensity was calculated by fitting it to an exponential decay model using KaleidaGraph software (Synergy Software, PA, US).

[0196] L-DNA aptamer protein blot

[0197] Cy5-L-13t aptamers were heated to 85°C for 5 minutes in physiological buffer and then slowly cooled to 25°C over 10 minutes. Natural human thrombin was separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane in 1× transfer buffer (25 mM Tris, 192 mM glycine, 20% (v / v) methanol, pH 8.3). The membrane was incubated for 1 h at room temperature in 1× blocking buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 25 mg / ml bovine serum albumin, 0.05% (v / v) Tween-20, pH 7.4) and then incubated for 1 h at room temperature with 500 nM Cy5-L-13t aptamers in selection buffer. After incubation, the membrane was washed five times with selection buffer (5 minutes each time) and scanned using an Amersham Typhoon Biomolecular Imager running in Cy5 mode. Conventional Western blotting was performed using antibodies according to the manufacturer's instructions (Abcam, UK).

[0198] L-DNA aptamer enzyme inhibitors

[0199] L-DNA aptamers were heated to 85°C for 5 minutes in physiological buffer, followed by slow cooling to 25°C over 10 minutes, with natural human thrombin added to a final concentration of 10 nM. The mixture was incubated in physiological buffer at room temperature for 30 minutes, followed by the addition of 100 μM of the fluorescent substrate benzoyl-Phe-Val-Arg-AMC. Relative fluorescence was measured using a Varioskan Flash system with excitation at 350 nm and emission at 450 nm. Relative thrombin enzymatic activity was determined, with ΔRFU at 0 minutes set to 0, and ΔRFU of the negative control in physiological buffer set to 100%. ΔRFU at 16 minutes was used to calculate relative thrombin enzymatic activity. The half-maximal inhibitory concentration (IC50) was determined. 50 The relative thrombin enzymatic activity was calculated by fitting it to the sigmoidal model using the KaleidaGraph software.

[0200] L-DNA Aptamer Coagulation Assay

[0201] Human plasma was obtained from healthy volunteers. D-DNA and L-DNA aptamers were heated to 85°C for 5 minutes in 180 μl of physiological buffer, followed by slow cooling to 25°C for annealing over 10 minutes, and then incubated at room temperature with 180 μl of human plasma (to a final concentration of 2.5 μM for both D-DNA and L-DNA aptamers) for up to 10 minutes. Prothrombin time was measured according to the manufacturer's instructions using a Stago STAR Max automated coagulation analyzer (Stago, France).

[0202] result

[0203] Validation and optimization of selection schemes for identifying L-DNA aptamers directly from large randomized L-DNA libraries

[0204] Although Dpo4-5m has been shown to effectively amplify short DNA sequences 7,8 However, it has not yet been tested in the amplification of large randomized DNA libraries. Here, approximately 1 × 10⁻⁶ oligonucleotides were prepared via solid-phase oligonucleotide synthesis. 14 Large randomized D-DNA libraries with different sequences were generated, in which 30 randomized nucleotides were side-joined by two constant regions for primer binding. The ability of L-Dpo4-5m to amplify large randomized D-DNA libraries was demonstrated, and the targeting of commercially available plasma-purified natural human thrombin (Materials and Methods) (high-affinity D-DNA aptamers had previously been selected for it) was also demonstrated. 30,31 D-DNA aptamers were selected in iterative rounds. The selection process was monitored by electrophoretic mobility shift assay (EMSA), which assessed the overall binding fraction of the sequence merged sequences during each selection round. 32 After six rounds of selection, approximately 70% of the D-DNA conjugates bound to 1 μM natural human thrombin but not to 1 μM streptavidin. The sixth round (R6) D-DNA conjugates were then sequenced using high-throughput sequencing, revealing enrichment of several DNA sequences, although the most abundant sequences comprised only about 1.1% of the total reads.

[0205] To determine whether D-DNA sequences of similar length could be separated based on different melting temperatures, DGGE was performed to analyze native PCR products from R4 through R6, as well as from R0 prior to selection. Although no clear bands were observed in R0 and R4, single bands began to appear in R5, with both the number and intensity of bands increasing in R6. Next, a single band (D-6) was isolated from R6, accounting for approximately 1.7% of the total lane fluorescence intensity. Band D-6 was amplified by native PCR using L-Dpo4-5m and D-DNA primers, and the PCR products were analyzed by another DGGE, revealing a major band accounting for approximately 35% of the total lane fluorescence intensity. This band was recovered from the DGGE and its composition was analyzed by high-throughput sequencing, revealing a single sequence comprising approximately 45% of the total reads (249,272 out of 554,081 reads). In fact, the same sequence (D-6) was also found in the R6 merge before DGGE isolation, but it only accounted for about 0.8% of the R6 reads (ranked 4th). Therefore, although the D-6 sequence was quite rare in the R6 merge (about 1.7% revealed by DGGE and about 0.8% revealed by high-throughput sequencing), it became predominant after DGGE isolation and amplification by L-Dpo4-5m PCR (about 35% revealed by DGGE and about 45% revealed by high-throughput sequencing).

[0206] Next, the thiophosphate method using D-deoxynucleoside α-thiotriphosphate (D-dNTPαS) and cleavage via 2-iodoethanol were employed. 33 (It has recently been used for L-DNA sequencing-by-synthesis) 13 Sequencing was performed on the D-6 band. Band doubling (a phenomenon primarily attributed to the presence of 3'-hydroxyl and 3'-monophosphate groups in the cleaved DNA fragments) was observed. 34 The sequencing results were quite blurry. To address this issue, the DNA fragments cleaved from 2-iodine ethanol were treated with calf intestinal alkaline phosphatase (CIP). Most band doubling disappeared after CIP treatment, likely due to the removal of 3'-monophosphates from the cleaved DNA fragments, thus making the sequence of band D-6 easily identifiable. The sequence was then determined via Mfold. 35 Prediction of the secondary structure of the D-6 aptamer revealed that it contains a loop region that conforms to the common sequence of previously identified D-DNA aptamers targeting natural human thrombin. 30 Finally, the D-DNA aptamer D-6 was prepared via solid-phase oligonucleotide synthesis with a dissociation constant of 27 nM (K0). dThe D-6 aptamer binds to native human thrombin, as determined by isothermal titration calorimetry (ITC) in physiological buffer (20 mM HEPES-NaOH, 150 mM NaCl, 5 mM KCl, 2 mM MgCl2, 1 mM CaCl2, pH 7.4). Furthermore, the D-6 aptamer forms a stable complex with native human thrombin, as revealed by EMSA, which, as expected, can be digested by DNase I.

[0207] Mirror selection of L-DNA aptamers targeting natural human thrombin

[0208] Approximately 1 × 10⁻⁶ oligonucleotides were prepared via solid-phase oligonucleotide synthesis. 14 Large, randomized L-DNA libraries with distinct sequences, each containing 30 randomized nucleotides side-mounted by two constant regions for primer binding, similar to D-DNA libraries. The L-DNA libraries were amplified using mirror PCR with D-Dpo4-5m and L-DNA primers. As with the natural system, the mirror selection process was monitored using EMSA. Figure 2A After nine rounds of selection, approximately 70% of the L-DNA conjugates bound to 1 μM natural human thrombin, but not to 1 μM streptavidin. Figure 2A B). DGGE was performed to analyze mirror PCR products from R5 to R9, as well as from R0 prior to selection. Figure 2C Although no clear bands were observed in R0 and R5, single bands began to appear in R6, with the number and intensity of bands increasing from R7 to R9. Figure 2C Two bands (L-9-1 and L-9-2) were separated from R9, accounting for approximately 1.7% and 1.6% of the total fluorescence intensity of R9 lanes, respectively. Figure 2C In two separate reactions, bands were amplified by mirror PCR using D-Dpo4-5m and L-DNA primers, and the mirror PCR products were analyzed by another DGGE. Both revealed major bands, which accounted for approximately 18% and 12% of the corresponding total lane fluorescence intensity, respectively. Figure 2C ).

[0209] To sequence the enriched L-DNA aptamers, the isolated band L-9-1 was used via a phosphate thioester method with L-deoxynucleoside α-thiotriphosphate (L-dNTPαS) and cleavage by 2-iodoethanol. 13 L-DNA sequencing was performed simultaneously with synthesis. Similar to sequencing results of phosphate thioesters in natural systems, the sequencing results became blurred again due to band doubling. Figure 5A L-DNA fragments cleaved by 2-iodine ethanol were subjected to CIP, and unexpectedly, CIP treatment significantly improved L-DNA sequencing results. Figure 5BThis is likely due to the removal of 3'-monophosphate from L-DNA via the previously unreported cross-chiral dephosphorylation activity of CIP. Therefore, the sequence of band L-9-1 was readily determined.

[0210] Furthermore, the L-9-2 band was sequenced using the thiophosphate method, and it was observed that even after CIP treatment, three nucleotide positions in the central region of the sequenced aptamer caused read blurring (possibly due to sequence contamination) and yielded eight most probable L-DNA aptamer sequences. Figure 5C (See Table 4). There is reason to believe that incorrect sequences can be eliminated using DGGE by comparing the migration of potential aptamer sequences, since the correct sequence should co-migrate with band L-9-2, which has the same melting temperature. Figure 6A Therefore, the natural versions of the eight most likely L-DNA aptamer sequences (DL-9-2-1 to DL-9-2-8, Table 4) in band L-9-2 were screened by DGGE (to save costs and for mirror enzymes) to exclude incorrect sequences. Only the DL-9-2-7 sequence was observed to co-migrate with band L-9-2. Figure 6B This indicates that DL-9-2-7 and band L-9-2 may share the same sequence. Therefore, the sequence of band L-9-2 is determined by the first DGGE (dimethylformaldehyde) used for separation. Figure 2C The method uses a combination of L-DNA sequencing-while-synthesizing using the phosphate thioester method and a second DGGE to determine the sequence and exclude incorrect sequences. Figure 6B ).

[0211] Characterizing the selected L-DNA aptamer

[0212] To assess the binding affinity of the sequenced L-DNA aptamer to natural human thrombin, L-DNA aptamer L-9-1 was prepared via solid-phase oligonucleotide synthesis. Figure 3A ), which has a K of 29 nM d Combined with natural human thrombin, as measured by ITC in physiological buffer ( Figure 3B This is related to the K of the D-DNA aptamer D-6. d (27nM) equivalent. The L-9-1 aptamer, based on predictions made via Mfold, has its secondary structure truncated from 65nt to 36nt. Figure 3C Furthermore, it was observed that the truncated aptamer (L-9-1t) exhibited only a slightly reduced affinity (K). d =39nM, Figure 3DThe L-9-1t aptamer binds to natural human thrombin. Meanwhile, no binding was detected between the L-9-1t aptamer and streptavidin, nor between the natural version of L-9-1t (DL-9-1t) and natural human thrombin, indicating that the binding between the L-9-1t aptamer and natural human thrombin is target-specific and chiral-specific. Further shortening the L-9-1t aptamer from 36 nt to 32 nt by truncating a portion of the stem region resulted in an approximately 3-fold decrease in affinity (K0.05). d =111 nM, which may be due to the destabilization of the aptamer's secondary structure. Furthermore, the 5'-cyanin 5(Cy5)-labeled L-9-1t (Cy5-L-9-1t) aptamer has a K0.05 of 21 nM. d It forms a stable complex with natural human thrombin, as determined by EMSA, and exhibits resistance to DNase I digestion as expected. Figure 3E -G).

[0213] L-DNA aptamer L-9-2 was also prepared via solid-phase oligonucleotide synthesis. Figure 3H ), which has a K of 168 nM d Combined with natural human thrombin, as measured by ITC in physiological buffer ( Figure 3I Then, based on its secondary structure predicted by Mfold, the L-9-2 aptamer was truncated from 65 nt to 38 nt. Figure 3J ), and observed that the truncated aptamer (L-9-2t) had only a slightly reduced affinity (K). d =251nM, Figure 3K The aptamer binds to natural human thrombin. Conversely, no binding was detected between the L-9-2t aptamer and streptavidin, nor between the natural version of L-9-2t (DL-9-2t) and natural human thrombin, indicating that the binding between the L-9-2t aptamer and natural human thrombin is target-specific and chiral-specific. Furthermore, the 5'-Cy5-labeled L-9-2t (Cy5-L-9-2t) aptamer binds to natural human thrombin at 355 nM K. d Combined with natural human thrombin, as measured by EMSA ( Figure 3L -N), and as expected, it is resistant to DNase I digestion ( Figure 3L Furthermore, based on the DGGE-predicted contamination sequence (DL-9-2-1) from R9, a truncated version of L-9-2-1 (L-9-2-1t) aptamer was prepared via solid-phase oligonucleotide synthesis, and it was observed that the binding affinity of the L-9-2-1t aptamer to natural human thrombin was approximately 1 / 5 (K) of that of the L-9-2t aptamer. d =1337nM).

[0214] To further evaluate the target specificity of L-DNA aptamers, the binding affinity of the L-9-1t and L-9-2t aptamers to native bovine thrombin was measured. Bovine thrombin exhibited approximately 85% sequence identity with native human thrombin. 38 Observations show that the L-9-1t and L-9-2t aptamers have K+ values ​​of 1027 nM and 426 nM, respectively. d When bound to natural bovine thrombin, the binding affinity was reduced to approximately 1 / 26 and 1 / 1.7 compared to natural human thrombin (39 nM and 251 nM, respectively). These results indicate that the L-9-1t aptamer binds more tightly to natural human thrombin than to natural bovine thrombin, while the L-9-2t aptamer binds to both with similar affinity.

[0215] L-DNA Aptamer Sensor

[0216] To demonstrate the potential practical applications of thrombin-binding L-DNA aptamers, the high-affinity thrombin-binding L-DNA aptamer L-9-1t was hybridized with an L-DNA fluorophore chain labeled with 5'-fluorescein (FAM) and an L-DNA quencher chain labeled with 3'-4-(4-dimethylaminophenylazo)benzoic acid (DABCYL) (both were hybridized with the L-9-1t aptamer to form a stable L-DNA duplex). 39 ) combination, synthesis of structure-converting L-DNA aptamer sensor ( Figure 4A Upon binding to natural human thrombin, the L-9-1t aptamer undergoes a structural transition, releasing a quencher chain and resulting in an increase in relative fluorescence, with a linear response in the range of approximately 125–1000 nM. Figure 4B In contrast, the L-DNA aptamer sensor did not respond to the addition of 1 μM streptavidin or 1 μM natural bovine thrombin, consistent with the ITC results.

[0217] To evaluate the effect of serum enzymes on the biostability and thrombin-sensing ability of the L-DNA aptamer sensor, the L-DNA aptamer sensor was incubated in physiological buffer containing 10% (v / v) human serum (which provides a physiologically relevant nuclease-rich environment). The L-DNA aptamer sensor responded to the addition of native human thrombin in physiological buffer containing 10% human serum, with a linear response in the range of approximately 250–2000 nM. Figure 4B Simultaneously, a natural structure transition sensor (D-DNA aptamer sensor) was constructed based on the D-DNA aptamer D-6. Next, 300 nM (final concentration) of natural human thrombin was added to a sensor containing either D-DNA or L-DNA aptamer, along with 10% human serum or 50 units / ml DNase I (one of the major nucleases in serum). 41After incubation for 1 hour in physiological buffer containing 10% human serum, the D-DNA and L-DNA aptamer sensors measured thrombin concentrations of 416±62 nM and 457±72 nM, respectively, compared with those measured in physiological buffer alone (334±59 nM and 299±12 nM, respectively). Figure 4C Similar to the results. However, after incubation for 4 hours in physiological buffer containing 10% human serum, the D-DNA aptamer sensor measured a thrombin concentration of 784 ± 91 nM, while the L-DNA aptamer sensor measured a thrombin concentration of 375 ± 54 nM. Figure 4C Furthermore, after incubation in physiological buffer containing 50 units / ml DNase I for 1 h and 4 h, the D-DNA aptamer sensor measured thrombin concentrations of 1219±57 nM and 984±52 nM, respectively, while the L-DNA aptamer sensor measured thrombin concentrations of 334±58 nM and 251±34 nM, respectively. Figure 4C ).

[0218] The error-prone measurement of D-DNA aptamer sensors, rather than L-DNA aptamer sensors, may be attributed to the increased relative fluorescence caused by degradation of the D-DNA aptamer sensor by serum enzymes or DNase I (which causes premature release of the FAM fluorophore and DABCYL quencher). This contrasts with the estimated half-life (t) of approximately 1.7 h for D-DNA aptamer sensors incubated in physiological buffer containing 10% human serum, as determined by denaturing polyacrylamide gel electrophoresis (PAGE). 1 / 2 The results were largely consistent. To further validate the biostability of the L-DNA aptamer sensor, the sensor was incubated in physiological buffer containing 83% human serum. After an incubation period of up to 30 days (720 h), no significant degradation of the L-DNA aptamer sensor was observed by denaturing PAGE, while the D-DNA aptamer sensor showed an estimated degradation of approximately 2.1 h. 1 / 2 Rapid degradation, which is largely consistent with previous findings on other D-DNA aptamers in human serum. 3,42 .

[0219] L-DNA aptamer protein blot

[0220] To further explore the potential practical applications of L-DNA aptamers that bind thrombin, the L-13t aptamer (selected and optimized in the section "Reselection and Optimization of L-DNA Aptamers from a Partially Randomized L-DNA Library" described below) was applied to a proof-of-concept Western blot experiment based on L-DNA aptamers to detect natural human thrombin immobilized on a nitrocellulose membrane. Figure 4DNatural human thrombin, ranging from 6 ng to 180 ng, was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and subsequently transferred to a nitrocellulose membrane and incubated with 500 nM Cy5-L-13t aptamer at room temperature for 1 h. Figure 4E A fluorescent band with a molecular weight consistent with that of natural human thrombin (approximately 36 kDa) was detected, with a detection limit of <6 ng ( ). Figure 4F In the control experiment, we analyzed streptavidin from 6 ng to 180 ng by SDS-PAGE. The streptavidin was transferred to a nitrocellulose membrane and incubated with 500 nM Cy5-L-13t aptamer. No clear band was observed at the expected molecular weight (approximately 18 kDa) of streptavidin. Figure 4F In contrast, using a mouse monoclonal primary antibody targeting natural human thrombin and an Alexa Fluor 647-labeled goat anti-mouse IgG polyclonal secondary antibody (with excitation and emission wavelengths similar to Cy5), conventional Western blot analysis of 6 ng to 180 ng of natural human thrombin detected fluorescent bands consistent with the molecular weight (approximately 36 kDa) of natural human thrombin. Figure 4G ).

[0221] L-DNA aptamer enzyme inhibitors

[0222] Next, in a solution containing 100 μM benzoyl-Phe-Val-Arg-7-amino-4-methylcoumarin (AMC) (which is a fluorescent substrate for thrombin) 44 The inhibition of thrombin enzymatic activity by the L-DNA aptamers L-9-1 and L-9-2, which bind to thrombin, was tested in physiological buffer. Figure 4H L-9-2 aptamer inhibits thrombin enzymatic activity, with a half-maximal inhibitory concentration (IC50) of 1,500. 50 The measurement was 317±128 ( Figure 4I ), and its K as determined by ITC d (168nM, Figure 3I The results were largely consistent. In contrast, the previously selected R0 L-DNA conjugate did not inhibit thrombin enzymatic activity at concentrations up to 8 μM. The inhibition of thrombin enzymatic activity by the truncated aptamer (L-9-2t) was also measured. A slightly higher IC50 was recorded. 50 479±65nM ( Figure 4I This is consistent with its K value determined by ITC. d (251nM, Figure 3K The results were largely consistent. However, L-9-1 and L-9-1t aptamers did not inhibit thrombin enzymatic activity at concentrations up to 8 μM, despite their higher binding affinity (K). dThe concentrations were 29 nM and 39 nM, respectively, indicating that the binding sites of natural human thrombin targeted by the L-9-1 and L-9-2 aptamers are different. Furthermore, the inhibition of thrombin enzymatic activity by the L-9-2t aptamer showed chiral specificity, as the natural version of the L-9-2t aptamer (DL-9-2t) did not inhibit thrombin enzymatic activity at concentrations up to 8 μM.

[0223] L-DNA aptamers were reselected and optimized from a partially randomized L-DNA library.

[0224] The second-best L-9-2 aptamer binds to natural human thrombin (of which K... d The measured value was 168 nM) and the inhibition of thrombin enzymatic activity (of which IC50 was 168 nM). 50 The measured value was 317 ± 128 nM, which promotes further improvement and optimization of L-DNA aptamers targeting both binding and inhibition properties.

[0225] To reselect and optimize the L-9-2 aptamer, approximately 1 × 10⁻⁶ nucleotides were synthesized via solid-phase oligonucleotide synthesis. 11 Partially randomized L-DNA libraries (R10) with distinct sequences, in which 34 nucleotides based on the L-9-2 aptamer are partially randomized at a frequency of 10%, and are flanked by two constant regions for primer binding. Next, mirror selection is performed on the partially randomized L-DNA library targeting natural human thrombin. Figure 7A After 3 rounds of enrichment and mirror PCR amplification ( Figure 7B (C) DGGE was used to separate a single band (L-13) from R13, which accounted for approximately 0.2% of the total fluorescence intensity of the R13 lane. Figure 7D Band L-13 was amplified by mirror PCR using D-Dpo4-5m and L-DNA primers, and the mirror PCR products were analyzed by another DGGE assay, revealing a major band accounting for approximately 13% of the total fluorescence intensity in the corresponding lane. Figure 7D L-DNA was synthesized and sequenced simultaneously using a phosphate thioester method to determine enriched L-DNA aptamer sequences, and the mutant sequence of the L-9-2 aptamer was identified, in which two adenosines in the partially randomized region were mutated to cytosine. Figure 7E This reselected L-DNA aptamer (L-13) was produced at 22 nM K. d Combined with natural human thrombin, as measured by ITC in physiological buffer ( Figure 7F The L-13 aptamer (G) exhibited approximately 8-fold improved binding affinity to natural human thrombin compared to its parent aptamer L-9-2. Based on its secondary structure predicted via Mfold, the L-13 aptamer was truncated from 68 nt to 38 nt. Figure 7H Furthermore, the truncated aptamer (L-13t) exhibits only a slightly reduced affinity (K).d =34nM, Figure 7I ) binds to natural human thrombin. Furthermore, the 5'-Cy5-labeled L-13t (Cy5-L-13t) aptamer was found to bind at 28 nM K. d It forms a stable complex with natural human thrombin, as measured by EMSA ( Figure 7J K).

[0226] The inhibition of thrombin enzymatic activity by the reselected L-13 and L-13t aptamers was tested in physiological buffer containing 100 μM benzoyl-Phe-Val-Arg-AMC. Figure 7L L-13 aptamer measured at 27 ± 3 nM IC. 50 Inhibit thrombin enzymatic activity ( Figure 7M ), and its K as determined by ITC d (22nM, Figure 7G The inhibitory activity of the aptamer was largely consistent with that of its parent aptamer L-9-2, showing an approximately 12-fold improvement in thrombin activity inhibition. In contrast, the R10 partially randomized L-DNA conjugate prior to reselection did not inhibit thrombin activity at concentrations up to 1.4 μM. The inhibition of thrombin activity by the truncated aptamer (L-13t) was also measured, and a slightly higher IC50 was observed. 50 46±4nM ( Figure 7M ), and its K as determined by ITC d (34nM, Figure 7I They are largely consistent.

[0227] As a final test of the selected L-DNA aptamers and to demonstrate their clinical potential, an in vitro coagulation assay was performed on human plasma. After the addition of 2.5 μM L-9-1t and L-13t aptamers, the prothrombin time was measured to be approximately 4 times and approximately 2 times, respectively, that of the control without L-DNA aptamers or the prothrombin time of the natural version of L-9-1t (DL-9-1t). Figure 7N ).

[0228] Although the invention has been described in conjunction with its specific embodiments, it will be apparent to those skilled in the art that many alternatives, modifications, and variations will be readily apparent. Therefore, it is intended to include all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0229] The applicant intends that all publications, patents, and patent applications mentioned in this specification be incorporated herein by reference in their entirety, just as each individual publication, patent, or patent application specifically and separately indicates its inclusion by reference herein. Furthermore, any reference or designation of any reference in this application should not be construed as an admission that such reference is available as prior art to the invention. With regard to the use of partial headings, they should not be construed as necessarily limiting.

[0230] In addition, any priority documents of this application are incorporated herein by reference in their entirety.

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Claims

1. A method for screening multiple L-nucleotide aptamers that have binding affinity for target molecules, comprising: (a) Under conditions where the target-bound L-nucleotide aptamers are selectively captured from the plurality of L-nucleotide aptamers, the plurality of L-nucleotide aptamers are brought into contact with the target molecule; (b) Amplifying the L-nucleotide aptamer of the target-bound L-nucleotide aptamer to generate amplified double-stranded L-nucleotide oligonucleotides; and (c) Using an electrophoresis-based method to separate amplified double-stranded L-nucleotide oligonucleotides, thereby screening for a variety of L-nucleotide aptamers, wherein the electrophoresis-based method includes denaturing gradient gel electrophoresis (DGGE). The method further includes sequencing the isolated member after step (c) to obtain a sequence of an L-nucleotide aptamer having binding affinity for the target molecule; wherein the sequencing is performed using an L-DNA phosphate thioester sequencing method, which further includes contacting the amplified double-stranded L-nucleotide oligonucleotide with a phosphatase prior to the sequencing, wherein the phosphatase includes calf intestinal phosphatase (CIP).

2. The method of claim 1, further comprising converting the amplified double-stranded L-nucleotide oligonucleotide into a single-stranded oligonucleotide after step (b) and before step (c).

3. The method of claim 2, wherein steps (a) and (b) and the transformation step are repeated at least three times prior to the separation to enrich the L-nucleotide aptamer bound to the target.

4. The method of claim 3, further comprising monitoring the enrichment of L-nucleotide aptamers bound to the target.

5. The method of claim 4, wherein the monitoring is performed by electrophoretic mobility variation measurement (EMSA).

6. The method of any one of claims 1-4, wherein the electrophoresis-based method is selected from non-denaturing PAGE; denaturing PAGE; denaturing gradient gel electrophoresis (DGGE); constant denaturing gel electrophoresis (CDGE); and time-temperature gradient gel electrophoresis (TTGE).

7. The method of any one of claims 1-6, wherein the target molecule is selected from peptides, polypeptides, small molecules, carbohydrates, and nucleic acid molecules.

8. The method of any one of claims 1-7, wherein the target molecule is contained in a cell or tissue.

9. The method of any one of claims 1-8, wherein the amplification utilizes D-amino acid polymerase.

10. The method of claim 9, wherein the D-amino acid polymerase is selected from D-ASFV pol X, D-Taq polymerase, D-Pfu polymerase, sulfur leaf bacterium P2 DNA polymerase IV (DPO4), and fusion proteins containing said DPO4.

11. The method of claim 10, wherein the polymerase is shown by the amino acid sequence of SEQ ID NO:38 or SEQ ID NO:

40.

12. The method of any one of claims 1-11, wherein each of the plurality of L-nucleotide aptamers has the same length.

13. The method of any one of claims 1-12, wherein the plurality of L-nucleotide aptamers are libraries, and each member of the library has the same 5' and 3' nucleic acid sequences and different core sequences.

14. The method of claim 1, further comprising constructing an additional aptamer library, wherein each member of the library has the same 5' and 3' nucleic acid sequences and is randomized by up to 60% compared to the sequences of isolated L-nucleotide aptamers.

15. The method of any one of claims 1-13, further comprising synthesizing the plurality of L-nucleotide aptamers prior to step (a).

16. The method of claim 15, wherein the synthesis comprises error-prone PCR.

17. The method of claim 16, wherein the error-prone PCR comprises using an error-prone polymerase.

18. The method of claim 13, wherein the core sequence comprises a random or semi-random sequence.

19. A kit for carrying out the method of claim 9, comprising: (i) Calf intestinal phosphatase (CIP); (ii) L-deoxyribonucleotide triphosphates (L-dNTPs) or modified L-dNTPs; and (iii) D-amino acid polymerase.

20. The kit of claim 19, wherein the polymerase comprises *Sulphurobryctomyces sulfidei* P2 DNA polymerase IV.

21. The kit of claim 20, wherein the polymerase is as shown in the amino acid sequence of SEQ ID NO:38 or SEQ ID NO:

40.

22. A method for sequencing purified L-DNA molecules, comprising: (a) A sample containing the purified L-DNA molecule is treated with a phosphatase, wherein the phosphatase comprises calf intestinal phosphatase (CIP), under conditions in which 3'-monophosphate is removed from the L-DNA molecule; and (b) The sample was subjected to phosphate thioester sequencing, thereby sequencing the purified L-DNA molecules.

23. An isolated L-DNA aptamer bound to thrombin, the sequence of which is shown in SEQ ID NO:10, 12, 14, 27 or 28.

24. An inhibitor of thrombin activity, which is an L-DNA aptamer with the sequence shown in SEQ ID NO:10, 14 or 28.

Images