Mirror-image selection of L-nucleic acid aptamers

Abstract

A method for screening L-nucleic acid aptamers that bind to a target molecule is disclosed. The method includes the steps of (a) contacting a plurality of L-nucleic acid aptamers with a target molecule under conditions that selectively capture the target-binding L-nucleic acid aptamers from among the plurality of L-nucleic acid aptamers, (b) amplifying the L-nucleic acid aptamers of the target-binding L-nucleic acid aptamers to generate amplified double-stranded L-nucleic acid oligonucleotides, and (c) isolating the amplified double-stranded L-nucleic acid oligonucleotides using an electrophoresis-based method, thereby screening a plurality of L-nucleic acid aptamers.

Description

[Technical field]

[0001] Related Applications 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, the contents of which are incorporated by reference in their entireties herein.

[0002] Description of sequence listing A file entitled 94498.xml, consisting of 12,923,440 bytes, created on Feb. 1, 2023, which was submitted concurrently with the filing of this application, is hereby incorporated by reference.

[0003] The present invention in some embodiments relates to methods for selecting L-nucleotide aptamers and for sequencing them. [Background technology]

[0004] Aptamers are nucleic acid polymer ligands that bind to specific target molecules through tertiary interactions and are selected by systematic evolution of ligands by exponential enrichment (SELEX) or in vitro selection methods. Natural, unmodified aptamers are susceptible to degradation by ubiquitous nucleases in vitro and in vivo, severely limiting their practical application as diagnostic and therapeutic tools. Chemical modifications and xeno-nucleic acid (XNA) design have been shown to increase aptamer stability, but their discovery and production require engineered specialized nucleotides, which may still not completely avoid nuclease degradation of non-natural nucleic acid aptamers.

[0005] Chiral inverted L-DNA or L-RNA aptamers (mirror-image aptamers) have excellent biological stability both in vitro and in vivo and have been selected to bind natural target molecules. Their large-scale production can be easily performed on an automated oligosynthesizer using commercially available L-deoxynucleoside or L-ribonucleoside phosphoramidites, making them ideal for practical applications in diagnostics and therapeutics. Since their biochemical advantages were recognized more than 20 years ago, mirror-image aptamers have been mainly selected by an indirect scheme known as "selection-reflection". For the selection of D-aptamers, a mirror-image version of the target molecule is first chemically synthesized, and then a mirror-image aptamer with the same sequence is synthesized to bind to the corresponding natural target. However, the first step of chemically synthesizing the mirror-image target molecule is often problematic, especially for proteins that are large, have extensive post-translational modifications (PTMs), and have low folding efficiency in vitro. In fact, the majority of biologically important target molecules, such as large proteins, cannot be chemically synthesized or folded properly with current technology. As a result, only a few mirror-image aptamers have been found by selection-mirror mirroring in over two decades, all of which target small molecules, short peptides, short RNAs, and small proteins, the largest of which is the 12 kDa, 110 amino acid (aa) ribonuclease (barnase) from Bacillus amyloliquefaciens, whereas the selection of mirror-image aptamers targeting the majority of biologically important but non-synthetic target molecules remains elusive.

[0006] Background art includes U.S. Patent Application No. 20210332360, U.S. Patent No. 11,015,178, and U.S. Patent No. 10,975,370. Summary of the Invention

[0007] According to one aspect of the present invention, there is provided a method for screening a plurality of L-nucleic acid aptamers to obtain an L-nucleic acid aptamer having a binding affinity for a target molecule, comprising: (a) contacting a plurality of L-nucleic acid aptamers with a target molecule under conditions that selectively capture target-binding L-nucleic acid aptamers from among the plurality of L-nucleic acid aptamers; (b) amplifying the L-nucleic acid aptamer of the target-binding L-nucleic acid aptamer to produce an amplified double-stranded L-nucleic acid oligonucleotide; (c) isolating the amplified double-stranded L-nucleic acid oligonucleotide using an electrophoresis-based method; and by these steps a method for screening a plurality of L-nucleic acid aptamers is provided.

[0008] According to another aspect of the invention, a kit for identifying L-nucleic acid aptamers comprises: (i) calf intestinal phosphatase (CIP), (ii) L-deoxyribonucleotide triphosphates (L-dNTPs) or modified L-dNTPs, and / or (iii) a polymerase capable of adding one or more L-nucleotides to the 3' end of a first L-nucleic acid, Includes.

[0009] According to another aspect of the invention there is provided a method for sequencing purified L-DNA molecules, comprising the steps of: (a) treating a sample containing purified L-DNA molecules with a phosphatase under conditions that remove the 3'-monophosphate from the L-DNA molecules; (b) subjecting the sample to phosphorothioate sequencing, thereby sequencing the purified L-DNA molecules; A method is provided, comprising:

[0010] According to another aspect of the present invention, there is provided an isolated thrombin-binding L-DNA aptamer comprising a sequence as set forth in SEQ ID NO: 10, 12, 14, 16, 27 or 28, or a sequence that is at least 80% identical to the sequence as set forth in SEQ ID NO: 10, 12, 14, 16, 27 or 28.

[0011] According to one embodiment of the invention, the method further comprises a step of converting the amplified double-stranded L-nucleic acid oligonucleotide into a single-stranded oligonucleotide after step (b) and before step (c).

[0012] According to one embodiment of the present invention, in order to enrich the target-binding L-nucleic acid aptamers, steps (a), (b) and the converting step are repeated at least three times before the isolating step.

[0013] According to one embodiment of the invention, the method further comprises monitoring the concentration of target-binding L-nucleic acid aptamers.

[0014] According to one embodiment of the present invention, the monitoring is performed by electrophoretic mobility shift assay (EMSA).

[0015] According to one embodiment of the invention, the electrophoresis-based method is selected from the group consisting of native PAGE, denaturing PAGE, denaturing gradient gel electrophoresis (DGGE), constant-state denaturing gel electrophoresis (CDGE), and time-temperature gradient gel electrophoresis (TTGE).

[0016] According to one embodiment of the present invention, the electrophoresis-based method comprises DGGE.

[0017] According to one embodiment of the present invention, the target molecule is selected from the group consisting of peptides, polypeptides, small molecules, carbohydrates and nucleic acid molecules.

[0018] According to one embodiment of the invention, the target molecule is contained in a cell or tissue.

[0019] According to one embodiment of the invention, the amplification utilises a D-amino acid polymerase.

[0020] According to one embodiment of the invention, the D-amino acid polymerase is selected from the group consisting of D-ASFV pol X, D-Taq polymerase, D-Pfu polymerase, Sulfolobus solfataricus P2 DNA polymerase IV (DPO4), a fusion protein containing DPO4, and a polymerase having an amino acid sequence at least 80% identical to DPO4.

[0021] According to one embodiment of the invention, the polymerase has the amino acid sequence set forth in SEQ ID NO:38 or SEQ ID NO:40.

[0022] According to one embodiment of the present invention, the method further comprises sequencing the isolated members following step (c) to obtain the sequence of the L-nucleic acid aptamer having binding affinity for the target molecule.

[0023] According to one embodiment of the present invention, sequencing is performed using a method selected from the group consisting of L-DNA chemical sequencing, L-DNA phosphorothioate sequencing, L-DNA dideoxy sequencing, L-DNA Ion Torrent sequencing, L-DNA Illumina sequencing, and L-DNA Nanopore sequencing.

[0024] According to one embodiment of the invention, the method is an L-DNA phosphorothioate sequencing method.

[0025] According to one embodiment of the invention, the method further comprises contacting the amplified double-stranded L-nucleic acid oligonucleotide with a phosphatase prior to sequencing.

[0026] According to one embodiment of the invention, the phosphatase comprises calf intestinal phosphatase (CIP).

[0027] According to one embodiment of the invention, each of the L-nucleic acid aptamers in the plurality of L-nucleic acid aptamers is of the same length.

[0028] According to one embodiment of the present invention, the plurality of L-nucleic acid aptamers is a library, each member of the library having identical 5' and 3' nucleic acid sequences and a non-identical core sequence.

[0029] According to one embodiment of the present invention, the method further comprises constructing an additional aptamer library, each member of the library having identical 5' and 3' nucleic acid sequences and being up to 60% randomized compared to the sequence of the isolated L-nucleic acid aptamer.

[0030] According to one embodiment of the present invention, the method further comprises the step of synthesizing a plurality of L-nucleic acid aptamers prior to step (a).

[0031] According to one embodiment of the invention, the synthesis comprises error-prone PCR.

[0032] According to one embodiment of the present invention, error-prone PCR involves the use of an error-prone polymerase.

[0033] According to one embodiment of the invention, the core sequence comprises a random or semi-random sequence.

[0034] According to embodiments of the invention, the polymerase comprises Sulfolobus solfataricus P2 DNA polymerase IV (DPO4), or a polymerase having an amino acid sequence at least 80% identical to DPO4.

[0035] According to an embodiment of the invention, the polymerase has the amino acid sequence set forth in SEQ ID NO:38 or SEQ ID NO:40.

[0036] According to an embodiment of the invention, the thrombin-binding L-DNA aptamer comprises a sequence as set forth in SEQ ID NO: 10, 14 or 28 or a sequence at least 80% identical to the sequence as set forth in SEQ ID NO: 10, 12, 14, 16, 27 or 28.

[0037] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice or test embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will prevail. Additionally, the materials, methods, and examples are illustrative only and are not necessarily intended to be limiting.

[0038] Certain embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings, in which: Reference will now be made specifically in detail to the drawings, it being stressed that the particulars shown are by way of example and are for the purposes of illustrative discussion of embodiments of the invention, and in which the description taken with reference to the drawings will make apparent to those skilled in the art how embodiments of the invention may be practiced. [Brief description of the drawings]

[0039] [Figure 1]Figure 1A-B. Design of the mirror-image selection scheme. Figure 1A. Schematic of mirror-image selection of L-DNA aptamers from a large randomized L-DNA library (color). It avoids the need for chemical synthesis of the mirror-image target molecule, as in the indirect selection-mirror scheme (gray). PDB source: 1PPB (native human thrombin). Figure 1B. Schematic of the steps in the mirror-image selection scheme. Selection begins with a large randomized L-DNA library (e.g., about 1 × 1014 different L-DNA sequences in this study) bound to an immobilized protein target, such as native human thrombin. The bound L-DNA is eluted and amplified by mirror-image PCR. The amplified L-DNA pool is separated into single-stranded L-DNA for the next round. After the final round of selection, the enriched L-DNA pool is analyzed by DGGE, isolated, and sequenced by L-DNA sequencing-by-synthesis using a phosphorothioate approach. [Diagram 2] Figure 2A-C. Mirror-selection of L-DNA aptamers targeting native human thrombin. Figure 2A. Monitoring the progress of mirror-selection by EMSA with 200 nM of the corresponding L-DNA pool and 1 μM of native human thrombin or 1 μM of streptavidin. Analyzed by 8% native PAGE and stained with SYBR Green II. Figure 2B. Gel quantification results of (A). The bound fraction was measured with ImageJ software using the band intensity of the bound L-DNA pool relative to the total lane intensity. ND indicates not detected (bound). Figure 2C. DGGE analysis of the corresponding L-DNA pool and isolated bands L-9-1 and L-9-2. Re-amplified by mirror-PCR with D-Dpo4-5m and L-DNA primers, analyzed by 10% denaturing PAGE in 2.1 M-4.2 M urea and 12%-24% formamide, and stained with SYBR-Green II. [Figure 3-1]Figure 3A-N. Characterization of selected L-DNA aptamers. Figure 3A. Secondary structure of the L-9-1 aptamer predicted by Mfold. Nucleotides from the randomized region are shown in blue (SEQ ID NO: 9). ITC analysis of the binding of the BL-9-1 aptamer from Figure 3 to native human thrombin. Kd was measured to be 29 nM. Figure 3C. Secondary structure of the L-9-1t (truncated version) aptamer predicted by Mfold. Nucleotides from the randomized region are shown in cyan (SEQ ID NO: 10). ITC analysis of the binding of the DL-9-1t aptamer from Figure 3 to native human thrombin. Kd was measured to be 39 nM. Figure 3E. EMSA of the binding of 200 nM Cy5-L-9-1t aptamer to 1 μM native human thrombin or 1 μM streptavidin with or without 50 units / ml DNase I. Analyzed by 8% native PAGE. Figure 3F. EMSA of the binding of 35 nM Cy5-L-9-1t aptamer to various concentrations of native human thrombin analyzed by 8% native PAGE. Figure 3G. Gel quantification results of (f). The band intensity of bound Cy5-L-9-1t aptamer relative to the total lane intensity was used to measure the bound fraction with ImageJ software. [Figure 3-2]H in FIG. 3. Secondary structure of the L-9-2 (SEQ ID NO: 13) aptamer predicted by Mfold. Nucleotides from the randomized region are shown in green. ITC analysis of the binding of the IL-9-2 aptamer of FIG. 3 to native human thrombin. Kd was measured to be 168 nM. J in FIG. 3. Secondary structure of the L-9-2t (truncated version) aptamer (SEQ ID NO: 14) predicted by Mfold. Nucleotides from the randomized region are shown in light green. ITC analysis of the binding of the KL-9-2t aptamer of FIG. 3 to native human thrombin. Kd was measured to be 251 nM. L in FIG. 3. EMSA of the binding of 200 nM Cy5-L-9-2t aptamer to 1 μM native human thrombin or 1 μM streptavidin with or without 50 units / ml DNase I. Analyzed by 8% native PAGE. Figure 3M. EMSA of the binding of 200 nM Cy5-L-9-2t aptamer to various concentrations of native human thrombin analyzed on 10% native PAGE containing 5% (v / v) glycerol. Figure 3N. (M) Gel quantification results. The band intensity of bound Cy5-L-9-2t aptamer relative to the total lane intensity was used to measure the bound fraction with ImageJ software. [Figure 4-1]Figure 4A-I. Detection and inhibition of native human thrombin by selected L-DNA aptamers. Schematic of detection of native human thrombin using L-DNA aptamer sensor based on AL-9-1t aptamer in Figure 4. Figure 4B. Relative fluorescence measurements of L-DNA aptamer sensor upon incubation with 1 μM native human thrombin in physiological buffer alone or in physiological buffer containing 10% human serum for up to 48 min. Measurements were taken every 4 min at excitation wavelength 494 nm and emission wavelength 518 nm. NC1 is the negative control in physiological buffer alone. NC2 is the negative control in physiological buffer containing 10% human serum. RFU is relative fluorescence unit. Data are shown as mean ± SD (n=3 independent measurements). Figure 4C. Thrombin concentration measurements upon incubation of D- and L-DNA aptamer sensors with 300 nM native human thrombin for 1 or 4 h in physiological buffer alone or in physiological buffer containing 10% human serum or 50 units / ml DNase I. Data are shown as mean ± SD (n=3 independent determinations). Schematic of detection of native human thrombin using DL-DNA aptamer Western blot in Figure 4E. Native human thrombin separated by 15% SDS-PAGE was transferred to a nitrocellulose membrane, incubated with 500 nM Cy5-L-13t aptamer, and scanned with an Amersham Typhoon Biomolecular Imager operated in Cy5 mode. Figure 4F. Streptavidin separated on 15% SDS-PAGE was incubated with 500 nM Cy5-L-13t aptamer and scanned with an Amersham Typhoon Biomolecular Imager operated in Cy5 mode. Figure 4G. Native human thrombin separated on 15% SDS-PAGE was transferred to a nitrocellulose membrane, incubated with a monoclonal primary antibody targeting native human thrombin and an Alexa Fluor 647-labeled polyclonal secondary antibody, and scanned with an Amersham Typhoon Biomolecular Imager operated in Cy5 mode. M indicates protein marker. [Figure 4-2] Schematic of inhibition of native human thrombin enzymatic activity using HL-DNA aptamers in Figure 4. Figure 4I. Relative thrombin enzymatic activity of L-9-2 and L-9-2t aptamers incubated with 10 nM native human thrombin and 100 μM fluorogenic substrate Benzoyl-Phe-Val-Arg-AMC in physiological buffer. IC50 measurements were 317 ± 128 nM and 479 ± 65 nM, respectively. Data are presented as mean ± SD (n = 3 independent measurements). [Diagram 5] Figure 5A-C. Sequencing of L-DNA aptamers isolated by DGGE using the phosphorothioate approach. Band L-9-1 amplified by D-Dpo4-5m using AL-dNTPαS and 5'-FAM-labeled L-DNA forward sequencing primer in Figure 5 was cleaved with 2-iodoethanol and analyzed by 10% denaturing PAGE. Band L-9-1 (SEQ ID NO: 9) amplified by D-Dpo4-5m using BL-dNTPαS and 5'-FAM-labeled L-DNA forward sequencing primer in Figure 5 was cleaved with 2-iodoethanol, treated with CIP, and analyzed by 10% denaturing PAGE. Band L-9-2 (SEQ ID NO: 13) amplified by D-Dpo4-5m using CL-dNTPαS and 5'-FAM-labeled L-DNA forward sequencing primer in Figure 5 was cleaved with 2-iodoethanol, treated with CIP, and analyzed by 10% denaturing PAGE. Incorrect nucleotide positions are marked with an asterisk to indicate the most likely alternative nucleotides (A and G) or deletions (-). [Figure 6]Figure 6A-B. Exclusion of incorrect sequences from band L-9-2 sequencing results by DGGE. Figure 6A. Schematic of exclusion of incorrect sequences by DGGE. Correct sequences should co-migrate with band L-9-2 if they have the same Tm. Figure 6B. The native versions of the eight most likely L-DNA aptamer sequences of band L-9-2 (DL-9-2-1 to DL-9-2-8) (Table 1A) (Tm calculations shown in brackets) amplified by native PCR with FastPfu Fly DNA polymerase and D-DNA primers, together with L-DNA pools from R0 and R9, were analyzed by 10% denaturing PAGE in 2.1M-4.2M urea and 12%-24% formamide and stained with SYBR-Green II. Co-migration of DL-9-2-7 with band L-9-2 is shown by a straight dashed blue line. [Figure 7-1] Figure 7A-D. Reselection and optimization of L-DNA aptamers from a partially randomized L-DNA library. Figure 7A. Schematic diagram of reselection and optimization of L-DNA aptamers from a partially randomized L-DNA library. Based on the L-9-2 aptamer, 34 nucleotides were partially randomized at a frequency of 10%. Figure 7B. Monitoring of the progress of mirror-image selection by EMSA with 200 nM of the corresponding L-DNA pool and 1 μM of native human thrombin or 1 μM of streptavidin. Analyzed on 8% native PAGE and stained with SYBR Green II. Figure 7C. Gel quantification results of (B). The band intensity of the bound L-DNA pool relative to the total lane intensity was used to measure the bound fraction with ImageJ software. ND indicates not detected (bound). Figure 7D. DGGE analysis of the corresponding L-DNA pool and isolated band L-13. It was reamplified by mirror-image PCR with D-Dpo4-5m and L-DNA primers, analyzed by 10% denaturing PAGE in 2.1 M–4.2 M urea and 12%–24% formamide, and stained with SYBR-Green II. Sequencing chromatogram after native CIP treatment of band L-13 by D-Dpo4-5m using EL-dNTPαS and 5′-FAM-labeled L-DNA sequencing primer in Figure 7 (two mutations are highlighted in yellow). [Figure 7-2] F of FIG. 7. Secondary structure of the L-13 aptamer (SEQ ID NO: 27) predicted by Mfold. Nucleotides from the reselection are shown in red and two mutations (adenosine to cytidine) are shown. ITC analysis of the binding of the GL-13 aptamer of FIG. 7 to native human thrombin. Kd was measured to be 22 nM. H of FIG. 7. Secondary structure of the L-13t (truncated version) aptamer (SEQ ID NO: 28) predicted by Mfold. Nucleotides from the reselection are shown in pink and two mutations (adenosine to cytidine) are shown. ITC analysis of the binding of the IL-13t aptamer of FIG. 7 to native human thrombin. Kd was measured to be 34 nM. [Figure 7-3] Figure 7J. EMSA of binding of 35 nM Cy5-L-13t aptamer to various concentrations of native human thrombin. Analyzed by 8% native PAGE. Figure 7K. Gel quantification results of (J). The band intensity of bound Cy5-L-13t aptamer relative to the total lane intensity was used to measure the bound fraction with ImageJ software. Figure 7L. Schematic of inhibition of native human thrombin enzymatic activity with reselected L-DNA aptamer. Figure 7M. Relative thrombin enzymatic activity of L-13 and L-13t aptamers incubated with 10 nM native human thrombin and 100 μM fluorogenic substrate Benzoyl-Phe-Val-Arg-AMC in physiological buffer. IC50 was measured to be 27 ± 3 nM and 46 ± 4 nM, respectively. Data are shown as mean ± SD (n = 3, independent measurements). [Figure 7-4] Schematic of anticoagulant effect using NL-DNA aptamers in Figure 7. Prothrombin time measured with 2.5 μM L-9-1t, L-13t, and native version L-9-1t (DL-9-1t) aptamers in the presence of 50% (v / v) human plasma. NC is a negative control with physiological buffer alone. Data are presented as mean ± SD (n = 3, independent measurements, two-tailed unpaired Student's t test). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] In some embodiments, the present invention relates to methods for selecting L-nucleotide aptamers and for sequencing L-nucleotide aptamers.

[0041] Before describing at least one embodiment of the present invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by way of examples. The invention is capable of other embodiments and of being practiced or carried out in various ways.

[0042] Mirror aptamers prepared with chiral inverted nucleic acids are nuclease-resistant and extremely biologically stable. Despite their diagnostic and therapeutic potential, only a few mirror aptamers have been selected by indirect selection schemes such as "mirror selection", because most biologically important target molecules such as large proteins cannot be chemically synthesized or folded properly. Here, we have developed a "mirror selection" scheme for discovering L-DNA aptamers by directly selecting from a large-scale randomized L-DNA library using mirror molecular tools (see A in Figure 1). By repeated rounds of enrichment and D-amino acid polymerase chain reaction (PCR) amplification of L-DNA sequences that bind to native human thrombin, coupled with isolation of enriched L-DNA aptamers by denaturing gradient gel electrophoresis (DGGE) and sequencing of enriched L-DNA aptamers by L-DNA sequencing-by-synthesis, we identified several high-affinity thrombin-binding L-DNA aptamers (as shown in Figure 1B).

[0043] While further implementing the present invention, the inventors designed sensors and inhibitors based on the selected L-DNA aptamers, which functioned in physiologically relevant nuclease-rich environments even in the presence of human serum, which rapidly degrades the D-DNA aptamers (as shown in Figures 4B-C, 4H-I, and 7N).

[0044] The realization of a direct mirror-image selection scheme for L-DNA aptamers significantly expands the applications of mirror-image biological systems toward fully unlocking the potential of mirror-aptamers as biostable biosensors, therapeutics, and fundamental research tools. Due to the biological stability of the L-DNA pool and mirror-image molecular tools, the system is also completely resistant to degradation by contaminating nucleases and proteases, especially for low purity target molecules and cell- and tissue-based selections.

[0045] Thus, according to a first aspect of the present invention, A method for screening a plurality of L-nucleic acid aptamers to obtain an L-nucleic acid aptamer having a binding affinity for a target molecule, comprising: (a) contacting a plurality of L-nucleic acid aptamers with a target molecule under conditions that selectively capture target-binding L-nucleic acid aptamers from among the plurality of L-nucleic acid aptamers; (b) amplifying the L-nucleic acid aptamer of the target-binding L-nucleic acid aptamer to produce an amplified double-stranded L-nucleic acid oligonucleotide; (c) isolating the amplified double-stranded L-nucleic acid oligonucleotides using an electrophoresis-based method, thereby screening a plurality of L-nucleic acid aptamers; A method is provided, comprising:

[0046] As used herein, the term "aptamer" refers to a nucleic acid molecule that exhibits specific binding affinity to a target molecule, such targets being primarily non-polynucleotide targets that bind to the aptamer sequence through a mechanism that is dependent on Watson / Crick base pairing.

[0047] The phrase "L-nucleic acid aptamer" refers to an aptamer that comprises at least one L-deoxyribonucleotide or at least one L-ribonucleotide. According to a particular embodiment, at least 50% of the nucleotides of the L-nucleic acid aptamer are L-nucleotides. In yet another embodiment, the nucleotides of the L-nucleic acid aptamer are all L-nucleotides. Here, it is also intended that instead of deoxyribose or ribose, other sugars form the sugar component of the nucleotide. In addition, it is also included to use nucleotides with further modifications at the 2' position, such as NH2, OMe, OBt, O-alkyl, NH-alkyl, and to use natural and unnatural nucleobases (such as isocytidine, isoguanosine).

[0048] L-nucleic acid aptamers can be double-stranded or single-stranded. Typically, L-nucleic acid aptamers are single-stranded L-nucleic acids, but due to their primary sequence, they can form defined secondary structures, and even tertiary structures. In these secondary structures, many of the L-nucleic acids have double-stranded portions.

[0049] 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 excludes a nucleotide or polynucleotide molecule.

[0050] According to certain embodiments, the target molecule is a protein (or a part thereof).

[0051] The binding affinity (Kd) of the aptamer for 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, or even less than 50 nM, as measured by EMSA (in the absence of serum).

[0052] As discussed above, an aptamer selected according to the methods described herein specifically (or selectively) binds to its target, i.e., the aptamer binds to the target molecule with at least 10, 20, or even 50 times greater affinity than the same type of non-target molecule. Thus, for example, if an aptamer selectively binds to a protein (e.g., human thrombin), the aptamer binds to human thrombin with at least 10 times greater affinity than a similarly sized protein (e.g., bovine thrombin).

[0053] The method for selecting aptamer candidates begins with contacting a plurality of L-nucleic acid aptamer candidates with a target molecule under conditions that selectively capture target-binding L-nucleic acid aptamers from the plurality of L-nucleic acid aptamer candidates.

[0054] Synthesis of L-nucleic acid aptamer candidates: The plurality of L-nucleic acid aptamer candidates includes any number of candidates, for example, at least 10, at least 100, at least 1000 candidates, each candidate having a non-identical sequence. The L-nucleic acid aptamer candidates may all be the same length or may all be different lengths. Exemplary lengths of the L-nucleic acid aptamer are 20-500 nucleotides, 20-400 nucleotides, 20-300 nucleotides, 20-200 nucleotides, and 20-100 nucleotides.

[0055] Chemical synthesis of L-nucleic acid aptamers can be carried out by solid-phase synthesis using L-DNA phosphoramidite chemistry known in the art. L-nucleic acid aptamer candidates can be purified after synthesis using methods known in the art, including but not limited to native polyacrylamide gel electrophoresis, to remove aggregation-prone L-nucleic acid aptamers.

[0056] In one embodiment, the plurality of L-nucleic acid aptamer candidates are members of a library, each member of the library having identical 5' and 3' nucleic acid sequences and a non-identical (e.g. random) core sequence, which 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, 10-30 nucleotides long.

[0057] The generation of such combinatorial libraries is described, for example, in Conrad, RC, Giver, L., Tian, ​​Y. and Ellington, AD, 1996, Methods Enzymol., Vol 267, 336-367.

[0058] To efficiently enrich for library members with identical 5' and 3' sequences, chemically synthesized aptamers may be amplified by error-prone PCR, where the 5' and 3' ends (which are the primer binding sites) are kept constant by the primers used during the PCR reaction, and the core is subjected to error-prone PCR. In one embodiment, 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 are selected to promote the insertion of errors (e.g., Mn 2+ (addition of).

[0059] It will be understood that the mutation content of the L-DNA candidate pool may be controlled by doping the wild type nucleotide with each of the other three L-DNA nucleotides during chemical synthesis.

[0060] Furthermore, it is in principle possible to generate an RNA library from double-stranded DNA with a suitable DNA-dependent RNA polymerase (e.g., T7 RNA polymerase) if a T7 promoter is already included. By employing the method described, it is possible to generate an RNA library from 10 15 It is possible to create a library of such DNA or RNA molecules, where every molecule from the library has a different sequence and therefore a different three-dimensional structure.

[0061] Capture of target-binding L-nucleic acid aptamers To separate the L-nucleic acid aptamers that bind to targets with high affinity from the L-nucleic acid aptamers that bind to targets with lower affinity, the target can be used as bait to capture the target-binding aptamers, thereby enriching the pool of target-binding L-nucleic acid aptamers.

[0062] To capture the target-binding aptamer, the target molecule may be immobilized on a solid support. Exemplary solid supports include, but are not limited to, stacked graphene, carbon nanotubes, fullerenes, and particles. Examples of materials that can be used to make particles include, but are not limited to, silica beads, polystyrene beads, latex beads, and may include metal colloids. According to certain embodiments, the particles are magnetic particles. The target molecule may be immobilized on the solid support surface by hydrophobic interaction, electrostatic interaction, covalent bond, coordinate bond, or non-covalent intermolecular action (such as biotin-streptavidin).

[0063] In another embodiment, the target molecule may be bound to a readable label, e.g., a fluorescent label, so that the signal from the aptamer-bound target molecule can be read and recorded, e.g., using FACS. In another embodiment, the target molecule may not include a readable label. In such a scenario, the aptamers of the library to be screened may have a specific scaffold (e.g., a hairpin scaffold and a displaced strand) that undergoes a structural change upon binding of the aptamer to the target molecule. The conformational change induced by the binding of the target molecule can generate a readable signal (e.g., due to a FRET interaction) to be recorded.

[0064] It will be understood that before capturing the L-nucleic acid aptamers that bind to the target molecule, the candidate pool may be pre-enriched by at least one round of negative selection (i.e. depletion of the candidate pool of sequences that bind non-specifically to non-targets). For example, selection may be performed against bead-immobilized human serum to reduce the number of aptamers that bind non-specifically to non-targets.

[0065] Amplification of target-binding L-nucleic acid aptamers After separating the target-binding aptamers from the non-target-binding aptamers, they may be amplified using a mirror-image PCR reaction.

[0066] As used herein, the term "mirror image" refers to an isomer that is a mirror image in chirality to the natural material.

[0067] The phrase "mirror-image PCR reaction" refers to a polymerase chain reaction that incorporates L-nucleotides into the sequence being amplified.

[0068] Mirror-image PCR reactions typically use a mirror-image polymerase, which is a D-amino acid polymerase that is the mirror image of the native polymerase (i.e., an L-type polymerase). The term "mirror-image polymerase" is used interchangeably with "D-type polymerase" or "D-amino acid polymerase." For example, "D-Dpo4" refers to a D-type Dpo4 polymerase that is the mirror image of the native L-type Dpo4 polymerase.

[0069] Polymerases particularly suitable for the present invention include D-ASFV pol X, D-Dpo4, D-Taq polymerase, D-Pfu polymerase, and functional variants thereof.

[0070] Dpo4 (Sulfolobus solfataricus P2 DNA polymerase IV) is a thermostable polymerase that can synthesize DNA even at 37°C. Its mismatch rate is 8×10 -3 ~3×10 -4 It is a polymerase that can replace Taq in multi-cycle PCR reactions. The length of its amino acid sequence is within the range of current chemical synthesis technology.

[0071] Taq polymerase is a thermostable polymerase that maintains activity even at DNA denaturing temperatures. The optimum temperature for Taq is 75°C to 80°C, and its half-life at 92.5°C is approximately 2 hours.

[0072] Pfu polymerase is contained in Pyrococcus furiosus, and its function in microorganisms is to replicate DNA during cell division. Pfu polymerase has 3'-5' exonuclease activity and is superior to Taq in that it can cleave mis-added nucleotides on the elongating strand during DNA synthesis. The mismatch rate of commercially available Pfu is approximately 1 in 1.3 million.

[0073] As used herein, the term "functional variant" refers to a variant that contains one or more (e.g. 1-5, 1-10 or 1-15, in particular, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15 or more) amino acid substitutions, deletions or additions in the amino acid sequence of a wild-type enzyme, which variant substantially retains the biological properties of the wild-type enzyme. For example, 50%, 60%, 70%, 80% or 90% or more of the biological activity of the wild-type enzyme is retained. A "functional variant" may be a naturally occurring variant or an artificial variant, such as a variant obtained by site-directed mutagenesis or a variant produced by recombinant gene technology.

[0074] In a preferred embodiment of the invention, the mirror image nucleic acid polymerase may contain an affinity tag that facilitates purification and recycling of the protein, such as a polyhistidine tag (His-Tag or His Tag), a polyarginine tag, a glutathione S-transferase tag, etc.

[0075] A particular functional variant of the Dpo4 protein is Dpo4-5m, which contains an amino acid mutation at position 5. In one embodiment, the Dpo4 protein contains at least one, two, three, four, or each of the mutations C31S, S86C, Nl23A, S207A, and S313A.

[0076] The amino acid sequence of the Dpo4-5m polymerase may comprise the sequence set forth in SEQ ID NO:38.

[0077] In another embodiment, the Dpo4-5m polymerase comprises an Sso7d domain fused to the C-terminus of Dpo4-5m (an exemplary sequence is set forth in SEQ ID NO:40).

[0078] In one embodiment, mirror image PCR is carried out in a buffer of 50 mM Tris-HCl (pH 7.5), 20 mM MgCl 2 , 1 mM DTT, and 50 mM KCl.

[0079] The present invention also provides D-ASFV pol X, the sequence of which is set forth in SEQ ID NO:39, in which, except for the non-chiral glycine, all other amino acids are D-amino acids.

[0080] In some embodiments, the mirror nucleic acid, mirror nucleic acid template, mirror nucleic acid primer, and mirror dNTPs / rNTPs are L-type and the mirror nucleic acid polymerase is D-type.

[0081] Here, the nucleic acid replication reaction may be carried out in one cycle or multiple cycles, which can be determined by those skilled in the art according to actual needs.

[0082] As used herein, the term "multiple" refers to at least two. For example, "multiple cycles" refers to two or more cycles, such as, for example, 3 cycles, 4 cycles, or 10 cycles.

[0083] As used herein, the term "replication" includes obtaining one or more copies of target DNA in the presence of DNA template and dNTPs, and also includes obtaining one or more copies of target RNA in the presence of DNA template and rNTPs (this process may be known as RNA "transcription"). In the process of nucleic acid replication, the template and primer are usually DNA. When the target nucleic acid is DNA, dNTPs need to be added to the reaction system, and when the target nucleic acid is RNA, rNTPs need to be added to the reaction system.

[0084] In a particularly preferred embodiment, the reaction is carried out in a buffer of 50 mM Tris-HCl (pH 7.5), 20 mM MgCl 2 , 1 mM DTT, and 50 mM KCl.

[0085] It will be understood that if the L-nucleic acid aptamer is an RNA aptamer, a reverse transcription reaction must be performed before the polymerase chain reaction amplification step.The library enriched after the first round of selection may be used for a new round of selection, so that the molecules enriched in the first round of selection have the opportunity to become dominant again through selection and amplification and proceed to further rounds of selection with even more daughter molecules.In this way, an enriched pool is generated, whose members are then separated using electrophoresis-based methods, as described in more detail below.

[0086] In multiple rounds of selection, the amplified aptamer sequences (which are double-stranded) are converted to single-stranded nucleic acid sequences prior to addition of the target.

[0087] Methods for obtaining single-stranded nucleic acid are known in the art, and the present invention contemplates the use of any of these methods.In certain embodiments, a spacer is used to block the reverse primer, so that the PCR product contains two strands of unequal length (e.g., Sp18 spacer).The two strands can then be separated using denaturing PAGE (see the "Examples" section below).

[0088] In another embodiment, one of the reverse primers is modified with a binding site (e.g., with biotin) and the double-stranded DNA is captured with an agent that specifically binds to the binding site (e.g., streptavidin-coated beads). The strand without the binding site is eluted with NaOH, while the strand with the binding site remains attached to the agent.

[0089] The present invention contemplates 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, at least 6 rounds of selection, amplification and conversion to single-stranded aptamers. In one embodiment, 10 or fewer rounds of selection, amplification and conversion to single-stranded aptamers are performed. In yet another embodiment, 15 or fewer rounds of selection, amplification and conversion to single-stranded aptamers are performed.

[0090] The enrichment of the L-nucleic acid aptamer pool towards target-binding L-nucleic acid aptamers may be monitored using methods known in the art, such as electromobility shift assays (EMSA).

[0091] As mentioned above, after sufficient enrichment of the L-nucleic acid aptamer pool, the resulting aptamers are further purified using electrophoresis-based methods as described in more detail below.

[0092] Isolation of amplified L-nucleic acid oligonucleotides Electrophoresis-based methods for isolating target-binding aptamers include, but are not limited to, native PAGE, denaturing PAGE, denaturing gradient gel electrophoresis (DGGE), constant-state denaturing gel electrophoresis (CDGE), capillary electrophoresis, and time-temperature gradient gel electrophoresis (TTGE).

[0093] According to certain embodiments, the electrophoresis-based method for separating target-binding aptamer candidates is DGGE.

[0094] Denaturing / Temperature Gradient Gel Electrophoresis (DGGE / TGGE): This is a method that relies on the detection of changes in electrophoretic mobility in response to small changes in sequence. One of these methods, called "denaturing gradient gel electrophoresis" (DGGE), is based on the observation that slightly different sequences show different local melting patterns when electrophoretically separated in a gradient gel. In this method, the difference in melting properties of homoduplexes and heteroduplexes that differ by a single nucleotide allows the presence of SNPs in the target sequence to be detected from the corresponding change in electrophoretic mobility, and therefore the identification of mutants. The fragment to be analyzed (usually a PCR product) is "clamped" at one end with a long stretch (30-80) of GC base pairs in order to completely denature the sequence of interest without completely dissociating the strands. The attachment of a GC "clamp" to the DNA fragment increases the proportion of mutations that can be recognized by DGGE (Abrams et al., Genomics 7:463-475, 1990). The attachment of a GC clamp to one of the primers is important to ensure that the amplified sequence has a low melting temperature (Sheffield et al., Proc. Natl. Acad. Sci., 86:232-236, 1989, and Lerman and Silverstein, Meth. Enzymol., 155:482-501, 1987). A variation of this technique using a temperature gradient has been developed (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).

[0095] Limitations to the usefulness of DGGE include the need to optimize denaturing conditions for each type of DNA to be tested. In addition, the method requires specialized equipment to prepare the gel and maintain the high temperatures required during electrophoresis. Another major problem is the expense associated with the synthesis of clamping tails for one of the oligonucleotides for each sequence to be tested. In addition, DGGE requires long run times. The long run times of DGGE are reduced by a variant of DGGE called constant denaturing gel electrophoresis (CDGE) (Borrensen et al., Proc. Natl. Acad. Sci. USA 88:8405, 1991). CDGE requires gel electrophoresis to be performed under a variety of denaturing conditions for efficient detection of SNPs.

[0096] Similar to DGGE is a technique called temperature gradient gel electrophoresis (TGGE), which uses a temperature gradient rather than a chemical denaturant gradient (Scholz, et al., Hum. Mol. Genet. 2:2155, 1993). TGGE requires the use of specialized equipment that can generate a temperature gradient perpendicular to the electric field. Because TGGE can detect mutations in relatively small DNA fragments, scanning large gene segments requires the use of multiple PCR products prior to gel electrophoresis.

[0097] After electrophoretic separation, the isolated L-DNA aptamers may be sequenced using methods known in the art.

[0098] Exemplary methods for sequencing L-DNA aptamers include, but are not limited to, L-DNA chemical sequencing, L-DNA phosphorothioate sequencing, L-DNA dideoxy sequencing, L-DNA Ion Torrent sequencing, L-DNA Illumina sequencing, and L-DNA Nanopore sequencing.

[0099] High throughput methods can include technologies for rapidly sequencing large numbers of nucleic acids, including next generation technologies such as massively parallel signature sequencing (MPSS), Polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing, SOLiD sequencing, Ion Torrent semiconductor sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, single molecule real-time (SMRT) sequencing, or other methods such as Nanopore DNA sequencing, tunneling current DNA sequencing, sequencing by hybridization, sequencing by mass spectrometry, microfluidic Sanger sequencing, microscope-based technologies, RNAP sequencing, in vitro viral high throughput sequencing, etc.

[0100] The isolated L-nucleotide aptamers may be subjected to automated dideoxy terminator sequencing reactions using dye terminator (unlabeled primer and labeled dideoxynucleotide) or dye primer (labeled primer and unlabeled dideoxynucleotide) cycle sequencing protocols. In dye terminator reactions, a PCR reaction is performed using unlabeled PCR primers, followed by a sequencing reaction in the presence of one of the primer, deoxynucleotide, and labeled dideoxynucleotide mixes. In dye primer reactions, a PCR reaction is performed using PCR primers (one in each direction) bound to a universal or reverse primer, followed by a sequencing reaction in the presence of four separate mixes (corresponding to A, G, C, T nucleotides), each of which contains a labeled primer specific to the universal or reverse sequence and the corresponding unlabeled dideoxynucleotide.

[0101] Pyrosequencing™ Analysis (Pyrosequencing, Inc., Westborough, Massachusetts, USA): This technology is based on hybridization of a sequencing primer to a PCR-amplified single-stranded DNA template in the presence of DNA polymerase, ATP sulfurylase, luciferase and apyrase enzymes, adenosine 5'-phosphosulfate (APS) and luciferin substrate. In a second step, the first of four deoxynucleotide triphosphates (dNTPs) is added to the reaction, and if it is complementary to a base in the template strand, the DNA polymerase catalyzes the incorporation of that deoxynucleotide triphosphate into the DNA strand. Each incorporation event is accompanied by the release of an amount of pyrophosphate (PPi) equimolar to the amount of nucleotide incorporated. In the final step, ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5'-phosphosulfate. This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin, producing an amount of visible light proportional to the amount of ATP. Light produced by the luciferase-catalyzed reaction is detected by a charge-coupled device (CCD) camera and displayed as peaks on a pyrogram™, with each light signal being proportional to the number of nucleotides incorporated.

[0102] Phosphorothioate sequencing: Phosphorothioate sequencing may be performed by performing a mirror image PCR reaction (e.g., using D-Dpo4-5m) in which one of the L-dNTPs is replaced with 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 DNA fragments cleaved with 2-iodoethanol using a phosphatase (e.g., calf intestinal phosphatase (CIP)) before running on a denaturing sequencing gel. Details regarding phosphorothioate sequencing are described in Fan, C., et al Nat. Biotechnol. 39: 1548-1555 (2021), the contents of which are incorporated herein by reference.

[0103] After obtaining the sequences, L-DNA aptamers may be chemically synthesized and their binding activity against the corresponding targets verified.

[0104] Once a lead candidate sequence is obtained, it may be used as a starting point to generate new libraries, thereby identifying further candidates with improved affinity / specificity. The lead sequence may be partially randomized (e.g., 1-60% randomized). In one embodiment, the lead candidate sequence is mutated with 10% randomization (about 3.3% for each base other than the original base). Thus, the doping rate may be 1%-60%.

[0105] The agents used to isolate the L-DNA aptamers of the present invention may be provided in a kit, if desired, which may be accompanied by instructions for use.

[0106] According to certain embodiments, the kit comprises: (i) calf intestinal phosphatase (CIP), (ii) L-deoxyribonucleotide triphosphates (L-dNTPs) or modified L-dNTPs, and / or (iii) a polymerase capable of adding one or more L-nucleotides to the 3' end of a first L-nucleic acid, Includes.

[0107] Modified L-dNTPs include deoxynucleoside α-thiotriphosphates.

[0108] The aptamers of the present invention can be used in various methods to evaluate the presence or level of a biomarker in a biological sample (e.g., a biological entity of interest, such as a protein, sugar, cell, or microvesicle). The aptamer functions as a binder to evaluate the presence or level of a cognate target molecule. Thus, in various embodiments of the present invention directed to diagnosis, prognostics, or theranostics, one or more aptamers of the present invention are configured in a ligand-target-based assay, one or more aptamers of the present invention are contacted with a selected biological sample, and one or more aptamers of the present invention associate with or bind to its target molecule. The aptamers of the present invention are used to identify biosignature candidates based on the biological sample evaluated and the biomarkers detected.

[0109] The L-nucleic acid aptamers discovered by the method of the invention include those having a nucleic acid sequence as set forth in SEQ ID NO: 10, 12, 14, 16, 27 or 28. In one embodiment, the L-nucleic acid aptamer is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 10, 14 or 28. In another embodiment, the L-nucleic acid aptamer has a sequence as set forth in SEQ ID NO: 10, 12, 14, 16, 27 or 28, in which up to 10 nucleotides of the sequence are mutated, and the site of the mutation is a single-stranded region of the aptamer (predicted by computational analysis such as Mfold). In one embodiment, the single-stranded region is as shown in A, C, H, J of FIG. 3, F or H of FIG. 7.

[0110] The aptamers described herein may be conjugated to a detectable moiety or label.

[0111] Suitable labels include, but are not limited to, magnetic labels, fluorescent moieties, enzymes, chemiluminescent probes, metal particles, non-metallic colloidal particles, polymeric dye particles, pigment molecules, pigment particles, electrochemically active species, semiconductor nanocrystals, or other nanoparticles, including quantum dots or gold particles, fluorophores, quantum dots, or radioactive labels. Protein labels include luminescent proteins such as green fluorescent protein (GFP) and its variants (e.g., cyan fluorescent protein and yellow fluorescent protein), as well as luciferase, as described below. Radioactive labels include, but are not limited to, radioisotopes (radionuclides), 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, fluorescein types including but not limited to FITC, 5-carboxyfluorescein, 6-carboxyfluorescein, rhodamine types including but not limited to TAMRA, dansyl, lissamine, cyanine, phycoerythrin, Texas Red, Cy3, Cy5, dapoxyl, NBD, Cascade Yellow, dansyl, PyMPO, pyrene, 7-diethylaminocoumarin-3-carboxylic acid and other coumarin derivatives, Marina Blue™, Pacific Blue™, Cascade Blue™, 2-anthracenesulfonyl, PyMPO, 3,4,9,10-perylene-tetracarboxylic acid, 2,7-difluorofluorescein (Oregon Green™ 488-X), 5-carboxyfluorescein, Texas Red™-X, Alexa Fluor, among others. 430, 5-carboxytetramethylrhodamine (5-TAMRA), 6-carboxytetramethylrhodamine (6-TAMRA), BODIPY FL, bimane, Alexa Fluor 350, 405, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700 and 750, and derivatives thereof.See, e.g., "The Handbook - A Guide to Fluorescent Probes and Labeling Technologies" (10th Edition), available online at probes.invitrogen.com / handbook. The fluorescent label can be one or more of FAM, dRHO, 5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET, dTAMRA, TAMRA, NED, dROX, PET, BHQ, Gold540 and LIZ.

[0112] Using conventional techniques, the L-nucleic acid aptamer can be directly or indirectly labeled. For example, the label is attached to the aptamer by biotin-streptavidin (for example, a biotinylated aptamer is synthesized to which a streptavidin molecule (itself attached to a detectable label) can be attached. A non-limiting example is phycoerythrin-conjugated streptavidin (SAPE)). Methods of chemical coupling involving a multi-step procedure include biotinylation, i.e. coupling of trinitrophenol (TNP) or digoxigenin, for example, with succinimide esters of these compounds. Biotinylation can be achieved, for example, by using D-biotinyl-N-hydroxysuccinimide. The succinimide group reacts efficiently with amino groups at pH values ​​above 7, preferentially between about pH 8.0 and about pH 8.5. Alternatively, the aptamer is not labeled and is contacted with a labeled secondary antibody after the antigen of interest has bound to the primary antibody.

[0113] Various enzyme-substrate labels may also be used in combination with the L-nucleic acid aptamers. Such enzyme-substrate labels are commercially available (e.g., U.S. Pat. No. 4,275,149). These enzymes generally catalyze a chemical change in a chromogenic substrate, which can be measured using various techniques. For example, these enzymes may catalyze a color change in the substrate, which can be measured spectrophotometrically. Alternatively, these enzymes may change the fluorescence or chemiluminescence of the substrate. Examples of enzyme labels include luciferases (e.g., firefly luciferase and bacterial luciferase, U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Examples of enzyme-substrate combinations include, but are not limited to, horseradish peroxidase (HRP) with hydrogen peroxidase as a substrate (hydrogen peroxidase oxidizes a dye precursor (e.g., orthophenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB))), alkaline phosphatase (AP) with paranitrophenyl phosphate as a chromogenic substrate, β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate 4-methylumbelliferyl-pD-galactosidase.

[0114] The L-nucleic acid aptamer can be bound to a substrate, such as a planar substrate. A planar array generally comprises addressable sites (e.g., pads, addresses, or microsites) of biomolecules in an array format. The size of the array depends on the composition and end use of the array. An array can be manufactured to comprise from two different molecules to thousands of molecules. In general, an array comprises from two molecules to as many as 100,000 or more molecules, based on the end use of the array and the method of its manufacture. The microarray used in the present invention comprises at least one biomolecule for identifying or capturing a biosignature of interest, for example, a microRNA or other biomolecule that constitutes a biosignature or a biomarker present in a vesicle. In some arrays, multiple substrates of different or identical composition are used. Thus, a planar array may comprise multiple small substrates.

[0115] The present inventors have shown that the use of calf intestinal phosphatase (CIP) followed by iodoethanol cleavage of DNA fragments is useful for sequencing oligonucleotides containing L-deoxynucleoside α-thiotriphosphates.

[0116] Therefore, according to yet another aspect of the invention, there is provided a method for sequencing purified L-DNA molecules, comprising the steps of: (a) treating a sample containing purified L-DNA molecules with a phosphatase (e.g. CIP) under conditions that remove the 3'-monophosphate from the L-DNA molecules; (b) subjecting the sample to phosphorothioate sequencing, thereby sequencing the purified L-DNA molecules; A method is provided, comprising:

[0117] As used herein, the term "about" refers to ±10%.

[0118] The terms "comprises," "comprising," "includes," "including," "having" and their cognates mean "including but not limited to."

[0119] The term "consisting of" means "including and limited to."

[0120] The term "consisting essentially of" means that a composition, method, or structure may include additional components, steps, and / or moieties, but only if the additional components, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0121] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0122] Throughout this application, various embodiments of the present invention may be described in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as indefinitely limiting the scope of the present invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as the individual numbers within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed the individual numbers within that range, e.g., 1, 2, 3, 4, 5, and 6, as well as the subranges 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc. This applies regardless of the breadth of the range.

[0123] Whenever a numerical range is given herein, it is meant to include any cited numbers (decimals or integers) within the stated range. The phrases "range / between" a first and second numerical value and "to" and "range / from" a first numerical value to a second numerical value are used interchangeably herein and are meant to include the first and second numerical values ​​and all decimals and integers therebetween.

[0124] As used herein, the term "method" refers to methods, means, techniques and procedures for accomplishing a given task, including but not limited to those methods, means, techniques and procedures known to practitioners in the chemical, pharmacological, biological, biochemical and medical fields, or readily developed from methods, means, techniques and procedures known to practitioners in the chemical, pharmacological, biological, biochemical and medical fields.

[0125] Where a particular sequence listing is referred to, the reference is understood to also include sequences substantially corresponding to its complementary sequences, including minor sequence variations due to, for example, sequencing errors, cloning errors, or other modifications resulting in base substitutions, deletions or additions, provided that the frequency of such mutations is less than 1 in 50 nucleotides, or less than 1 in 100 nucleotides, or less than 1 in 200 nucleotides, or less than 1 in 500 nucleotides, or less than 1 in 1000 nucleotides, or less than 1 in 5,000 nucleotides, or less than 1 in 10,000 nucleotides.

[0126] It will be understood that any sequence identification number (SEQ ID NO) disclosed in this application, even if the SEQ ID NO is presented only in DNA or RNA sequence format, can refer to either a DNA or RNA sequence depending on the context in which the SEQ ID NO is referred to. Similarly, depending on the actual type of molecule being described, some sequences are presented in RNA sequence format (e.g., uracil is represented as U), but the sequence can refer to either the sequence of an RNA molecule, including dsRNA, or the sequence of a DNA molecule that corresponds to the RNA sequence shown. In any event, both DNA and RNA molecules having the disclosed sequences and with any substitutions are envisioned.

[0127] It will be understood that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or in any other described embodiment of the invention, as appropriate. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperable without those elements.

[0128] Various embodiments and aspects of the present invention as described in detail herein and claimed below are provided with experimental support in the following examples. EXAMPLES

[0129] Reference will now be made to the following examples which, together with the above descriptions, illustrate some embodiments of the invention in a non-limiting manner.

[0130] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbial, and recombinant DNA techniques. Such techniques are explained thoroughly in the literature. General references are provided throughout the specification.

[0131] material All L-DNA oligos (Tables 1A and 1B below) were synthesized on an H-8 oligo synthesizer (K&A Laborgeraete, Germany). All D-DNA oligos (Tables 1A-1B below) were ordered from Genewiz (Jiangsu, China). L-deoxynucleoside phosphoramidites were purchased from ChemGenes (MA, USA). Hexaethylene glycol spacer (Sp18) phosphoramide was purchased from Glen Research (VA, USA). Fluorescein (FAM) cyanine 5 (Cy5) phosphoramide, as well as 4-(4-dimethyl-aminophenylazo)benzoic acid (DABCYL) and monophosphate controlled pore glass (CPG) were purchased from Ruibiotech (Beijing, China). All D- and L-DNA oligos were purified by HPLC or denaturing PAGE before use. L-deoxynucleoside triphosphates (L-dNTPs) and L-deoxynucleoside α-thiotriphosphates (L-dNTPαS) were synthesized from L-deoxynucleosides (ChemGenes, Massachusetts, USA). 1 D-dNTPαS was purchased from TriLink Biotechnologies Inc. (CA, USA). L-Dpo4-5m with an N-terminal His6 tag was expressed in Escherichia coli BL21 strain and synthesized as described in the literature. 2 The DNA polymerase was purified as described in. FastPfu Fly DNA polymerase was purchased from TransGen Biotech (Beijing, China). D-Dpo4-5m was synthesized and folded using an automated peptide synthesizer according to a previously published method, except that norleucine (Nle) was replaced with methionine (Met). 2,32-Iodoethanol was purchased from Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). Plasma-derived native human α-thrombin and native bovine α-thrombin were purchased from Haematologic Technologies (VT, USA). Streptavidin, calf intestinal alkaline phosphatase (CIP), and DNase I were purchased from New England Biolabs (MA, USA). Human serum was purchased from Zhong Ke Chen Yu Biotech (Beijing, China). Monoclonal primary antibodies targeting native human thrombin and Alexa Fluor 647-labeled polyclonal secondary antibodies 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, USA). Benzoyl-Phe-Val-Arg-AMC (AMC, 7-amino-4-methylcoumarin) was purchased from Sigma-Aldrich (MO, USA).

[0132] [Table 1A]

[0133] [Table 1B]

[0134] Construction of L-DNA library The 30 nt randomized regions of the 65 nt full-length D- or L-DNA libraries were synthesized using molar ratios of D- or L-dA, dC, dG, and dT phosphoramidites of 1.5:1.25:1.15:1 to ensure approximately equal coupling efficiency. 4 . literature 5Native polyacrylamide gel electrophoresis (PAGE) purification was performed to remove aggregated DNA as described in. Briefly, 5 nmol of synthetic D- or L-DNA libraries were loaded onto a 1 mm × 200 mm × 550 mm slab and separated by PAGE consisting of a denaturing upper section (1 mm × 200 mm × 50 mm) containing 7 M urea, 8% acrylamide in 0.5 × Tris-borate-EDTA (TBE) and a non-denaturing lower section (1 mm × 200 mm × 500 mm) containing 10% acrylamide, 10 mM Mg(OAc)2 in 0.5 × TBE. Gel electrophoresis was performed for 6 h at 10 W (constant power) and stained with SYBR-Green II. One-third of the fastest migrating band was isolated and separated by the "crush and soak" method. 6 Approximately 165 pMol of native PAGE-purified library (approximately 1 × 10 14 The different sequences of the 5'-terminal end ... 20 PCR products were generated that contained tails and had strands of different lengths for strand separation by denaturing PAGE. 7 The program settings for native and mirror PCR were 86°C for 3 min (initial denaturation); 15 cycles of 86°C for 30 s, 50°C for 1 min, and 65°C for 2 min; 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 7 M urea and used as the starting D- or L-DNA library for aptamer selection.

[0135] Selection of D- or L-DNA aptamers targeting native human thrombin Magnetic beads coupled with native human thrombin were prepared from N-hydroxy-succinimide (NHS)-activated magnetic beads according to the manufacturer's instructions (Thermo Fisher Scientific, MA, USA). Briefly, 300 μl of native human thrombin at a concentration of 0.1 mg / ml was mixed with 3 mg of NHS-activated magnetic beads in 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 and then quenched with 3 M ethanolamine, pH 9.0. After coupling, the beads were resuspended in 300 μl of 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). In round 1 (R1), a 250 μl volume of D- or L-DNA library of approximately 600 pMol (approximately 1 × 10 14 Approximately 3.6 x 10 14The mixture was heated to 85°C for 5 min in selection buffer and slowly cooled to 25°C over 10 min, after which 50 μl of protein-free NHS-activated magnetic beads were added and the mixture was incubated at room temperature for 1 h with gentle rotation. In each selection round, a negative selection step was performed on 50 μl of protein-free NHS-activated magnetic beads. The supernatant was mixed with 100 μl of native human thrombin-coupled magnetic beads in a total volume of 400 μl and incubated at room temperature for 1 h with gentle rotation, after which the beads were separated from the supernatant with a DynaMag-2 magnet (Thermo Fisher Scientific, MA, USA) and briefly washed three times (10 s per wash) with 400 μl of selection buffer. Bound DNA was eluted from the beads with 25 mM NaOH and 5 mM EDTA and precipitated with ethanol. The recovered D- or L-DNA was used as a template to perform native or mirror PCR amplification with L- or D-Dpo4-5m to generate D- or L-DNA pools for the next round. The number of native or mirror PCR cycles for each selection round was determined based on the results of 10 μl-scale PCR. As shown in Tables 2 and 3, the amount of DNA pool gradually decreased from about 600 pMol in R1 to about 50 pMol in R6 (for D-DNA pool), and from about 600 pMol in R1 to about 30 pMol in R9 (for L-DNA pool), respectively. The volume of magnetic beads coupled with native human thrombin gradually decreased from 100 μl in R1 to 10 μl in R6 (for D-DNA pool), and from 100 μl in R1 to 3 μl in R9 (for L-DNA pool), respectively. The washing steps were gradually increased from three washes for 10 s in R1 to six washes for 10 min in R6 (for the D-DNA pool) and from three washes for 10 s in R1 to eight washes for 10 min in R9 (for the L-DNA pool), respectively.

[0136] [Table 2]

[0137] [Table 3]

[0138] Electrophoretic mobility shift assay (EMSA) D- or L-DNA pools and D- or L-DNA aptamers were heated to 85°C for 5 min in selection buffer, slowly cooled to 25°C over 10 min, and then mixed with native human thrombin or streptavidin in selection buffer containing 10% (v / v) glycerol. The mixtures were incubated at room temperature for 30 min and analyzed by 8% native PAGE in 1× running buffer (20 mM HEPES-NaOH, 50 mM NaOAc, 5 mM KOAc, 2 mM Mg(OAc)2, 1 mM CaCl2, pH 7.4) (for D- or L-DNA pools and D-6, Cy5-L-9-1t, and Cy5-L-13t aptamers) or 10% native PAGE in 1× running buffer supplemented with 5% (v / v) glycerol in both the gel and running buffer (for Cy5-L-9-2t aptamer). Gel electrophoresis was performed at 150 V (constant voltage) for 1–2 h, stained with SYBR-Green II, and scanned with an Amersham Typhoon Biomolecular Imager (Cytiva, USA) operated in Cy2 mode (for D- or L-DNA pools and D-6 aptamers) or Cy5 mode (for Cy5-labeled L-DNA aptamers). Gel quantification was performed by ImageJ software, and dissociation constants (K d ) was calculated by fitting the bound fraction to a sigmoidal model using KaleidaGraph software (Synergy Software, PA, USA).

[0139] Denaturing gradient gel electrophoresis (DGGE) D- or L-DNA pools, and D- or L-DNA aptamers were amplified by native or mirror-image PCR using L- or D-Dpo4-5m with D- or L-DNA primers listed in Table 1A. The forward primer contains a GC-rich region (GC clamp) to prevent complete melting of double-stranded PCR products during DGGE. 8 Native or mirror-image PCR products were purified by 3% sieving agarose gel electrophoresis, mixed with 2× loading buffer (100 mM Tris-HCl, 10 mM EDTA, 30% glycerol, pH 7.0), and separated on a 7.5% polyacrylamide gel (for D-DNA pool) or 10% polyacrylamide gel (for L-DNA pool) composed of a linear denaturing gradient from 2.1 M urea, 12% (v / v) formamide (top) to 4.2 M urea, 24% (v / v) formamide (bottom) in 1× Tris-acetate-EDTA (TAE). Gel electrophoresis was performed at 100 V and 60 °C (constant temperature) for 6 h (for D-DNA pool) or at 75 V and 60 °C for 13 h (for L-DNA pool). For DGGE isolation of D- or L-DNA aptamer sequences, 500 ng of native or mirror-image PCR products were separated by DGGE, stained with SYBR-Green II, and isolated by cutting the gel on a 254 nm ultraviolet transilluminator using the "crush-and-soak" method. 6 The L-9-2 bands were purified by HPLC and re-amplified by native or mirror-image PCR using L- or D-Dpo4-5m and D- or L-DNA primers listed in Table 1A. To exclude incorrect sequences from the sequencing results of the L-9-2 bands, the native versions of the eight most likely L-DNA aptamer sequences in band L-9-2 (from DL-9-2-1 to DL-9-2-8, Figure 6B and Table 4 below) were amplified by native PCR using D-DNA primers and FastPfu Fly DNA polymerase, separated by DGGE, stained by SYBR-Green II, and scanned by an Amersham Typhoon Biomolecular Imager operating in Cy2 mode. Melting temperatures (T m) were calculated with OligoCalc using the default parameters of the nearest-neighbor thermodynamic model. 9 .

[0140] [Table 4]

[0141] High-throughput sequencing of selected D-DNA aptamers The R6 D-DNA pool and the D-6 bands isolated by DGGE were amplified by native PCR using the D-DNA primers listed in Table 1A and L-Dpo4-5m. PCR products were purified with 2.5% agarose and sequenced on an Illumina HiSeq system (Illumina, CA, USA). Raw Illumina reads were processed and sorted by abundance using the Galaxy server (www.usegalaxy.org).

[0142] Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) Dephosphorylation of L-DNA by CIP was analyzed using MALDI-TOF MS. Approximately 100 ng of 3'-monophosphate-labeled L-DNA oligos (Table 1A) were treated with 20 units of CIP, incubated in 1x CutSmart buffer (New England Biolabs, MA, USA) at 37°C for 1 h, desalted on a C18 spin column (Thermo Fisher Scientific, MA, USA) and analyzed by MALDI-TOF MS (Applied Biosystems 4800 plus, CA, USA) in positive linear mode.

[0143] Sequencing of L-DNA aptamers The L-DNA aptamers isolated by DGGE were subjected to four separate PCR reactions in which one of the L-dNTPs was replaced by the corresponding L-dNTPαS using the 5'-FAM-labeled forward sequencing primer and unlabeled reverse primer described in Table 1A. 10 The 5'-FAM-labeled L-DNA was amplified by mirror-image PCR using D-Dpo4-5m. The 5'-FAM-labeled PCR products were purified by 10% denaturing PAGE in 7 M urea and dissolved in 10 mM Tris-HCl, 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 cleavage solution containing 2% (v / v) 2-iodoethanol in ddH2O, followed by heating to 95 °C for 3 min and immediately placing on ice. To remove the 3'-monophosphate from the DNA fragments cleaved with 2-iodoethanol, each sequencing reaction was treated with 5 units of CIP and incubated in 1 × CutSmart buffer at 37 °C for 1 h before mixing with 10 μl of 2 × loading buffer containing 95% formamide and 10 mM EDTA. The sample was loaded into a slab of 0.4 mm × 340 mm × 300 mm and measured by the previously reported method. 10 The results were analyzed by 10% denaturing PAGE in 7 M urea according to the method described above.

[0144] Isothermal titration calorimetry (ITC) Native human thrombin, native bovine thrombin, and streptavidin in 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) for 16 h at 4 °C. D- and L-DNA aptamers were equilibrated with physiological buffer by ultrafiltration, then heated to 85 °C for 5 min and slowly cooled to 25 °C over 10 min. ITC was performed using a MicroCal iTC 200A Microcalorimeter (GE Healthcare, UK) was used with 7 μM-20 μM of native human thrombin, native bovine thrombin, or streptavidin in the reaction cell and 70 μM-200 μM of D- or L-DNA aptamer in the injection syringe, and stirring at 750 rpm at 25 °C. To measure the heat of dilution, 70 μM-200 μM of D- or L-DNA aptamer was injected into physiological buffer in the absence of protein. Data fitting was performed using MicroCal Origin software (GE Healthcare, UK).

[0145] L-DNA aptamer sensor D- or L-DNA aptamer sensors containing 250 nM 5'-FAM-labeled fluorescent strand, 750 nM 3'-DABCYL-labeled quencher strand, and 500 nM aptamer strand based on D-6 or L-9-1t aptamer (Table 1A) were incubated with 300 nM native human thrombin in physiological buffer alone or in physiological buffer containing 10% (v / v) human serum for 1 or 4 h at 37°C. Relative fluorescence was measured with a Varioskan Flash system (Thermo Fisher Scientific, MA, USA) at an excitation wavelength of 494 nm and an emission wavelength of 518 nm. Standard curves were plotted using 0, 125, 250, 500, or 1000 nM native human thrombin, and relative fluorescence was measured after 1 h of incubation at 37°C in physiological buffer. The change in relative fluorescence units (ΔRFU) over background (RFU measured with D- or L-DNA aptamer sensors in physiological buffer alone) was used for data fitting. For measurements in physiological buffer containing 10% (v / v) human serum, standard curves were plotted using 0, 250, 500, 1000, or 2000 nM native human thrombin, and relative fluorescence was measured after 1 h of incubation at 37°C in physiological buffer containing 10% human serum. To evaluate the biological stability of the D- and L-DNA aptamer sensors, the sensors were incubated in physiological buffer containing 10% human serum at 37°C for up to 24 h (for D-DNA aptamer sensors) or in physiological buffer containing 83% (v / v) human serum at 37°C for up to 24 h (for D-DNA aptamer sensors) or up to 30 days (720 h) (for L-DNA aptamer sensors). Samples were mixed with 2x loading buffer containing 95% formamide and 10 mM EDTA and immediately placed at -20°C before analysis by 10% denaturing PAGE in 7 M urea. Gel quantification was performed with ImageJ software and half-life (t 1 / 2 ) were calculated by fitting relative band intensities to an exponential decay model using KaleidaGraph software (Synergy Software, PA, USA).

[0146] L-DNA aptamer Western blot The Cy5-L-13t aptamer was heated to 85°C for 5 min in physiological buffer and slowly cooled to 25°C over 10 min. Native human thrombin was separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane in 1x transfer buffer (25 mM Tris, 192 mM glycine, 20% (v / v) methanol, pH 8.3). The membrane was incubated in 1x 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) for 1 h at room temperature and incubated with 500 nM Cy5-L-13t aptamer in selection buffer for 1 h at room temperature. After incubation, the membrane was washed five times (5 min per wash) with selection buffer and scanned with an Amersham Typhoon Biomolecular Imager operating in Cy5 mode. Conventional Western blots with antibodies were performed according to the manufacturer's instructions (Abcam, UK).

[0147] L-DNA aptamer enzyme inhibitor The L-DNA aptamer was heated to 85°C for 5 min in physiological buffer, slowly cooled to 25°C over 10 min, and native human thrombin was added to a final concentration of 10 nM. The mixture was incubated at room temperature in physiological buffer for 30 min before the addition of 100 μM of the fluorescent substrate Benzoyl-Phe-Val-Arg-AMC. Relative fluorescence was measured with a Varioskan Flash system at an excitation wavelength of 350 nm and an emission wavelength of 450 nm. Relative thrombin enzyme activity was measured by setting the ΔRFU at 0 min as 0 and the ΔRFU of the negative control in physiological buffer alone as 100%, and the ΔRFU at 16 min was used to calculate the relative thrombin enzyme activity. The half-maximal inhibitory concentration (IC 50 ) was calculated by fitting relative thrombin enzyme activity to a sigmoidal model using KaleidaGraph software.

[0148] L-DNA aptamer clotting assay Human plasma was obtained from healthy volunteers. D- and L-DNA aptamers were heated in 180 μl physiological buffer at 85° C. for 5 min, slowly cooled to 25° C. over 10 min for annealing, and 180 μl human plasma was incubated at room temperature for up to 10 min to a final concentration of D- and L-DNA aptamers of 2.5 μM. Prothrombin time was measured using a Stago STA R Max automated coagulation analyzer (Stago, France) according to the manufacturer's instructions.

[0149] result Validation and optimization of a selection scheme to directly identify L-DNA aptamers from large randomized L-DNA libraries Dpo4-5m has been shown to efficiently amplify short DNA sequences, 7,8 However, it has not been tested for the amplification of large randomized DNA libraries. Here, we have a 30-nucleotide randomization vector with 30 randomized nucleotides flanked by two constant regions for primer binding, approximately 1 × 10 14 A large-scale randomized D-DNA library consisting of different sequences was generated by solid-phase oligo synthesis. The ability of L-Dpo4-5m to amplify a large-scale randomized D-DNA library was confirmed, and high-affinity D-DNA aptamers were previously selected. 30,31 Iterative rounds of selection were performed on D-DNA aptamers targeting commercially available native human thrombin purified from plasma (Materials and Methods). Selection progress was monitored by electrophoretic mobility shift assays (EMSA) that access the overall binding fraction of the sequence pool during each selection round. 32 After six rounds of selection, approximately 70% of the D-DNA pool bound to 1 μM native human thrombin but not to 1 μM streptavidin. The round 6 (R6) D-DNA pool was then sequenced by high-throughput sequencing, revealing enrichment of multiple DNA sequences, although the most abundant sequence represented only approximately 1.1% of the total reads.

[0150] To determine whether D-DNA sequences of similar length could be separated by differences in melting temperature, native PCR products from R4 to R6 and PCR products from unselected R0 were analyzed by DGGE. No clear bands were observed in R0 and R4, but a single band began to appear in R5, and the number and intensity of bands increased in R6. A single band (D-6) was then isolated from R6, which accounted for approximately 1.7% of the total lane fluorescence intensity of R6. This 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 dominant band that accounted for approximately 35% of the total lane fluorescence intensity. This band was recovered from DGGE and its composition was analyzed by high-throughput sequencing, revealing a single sequence that accounted for approximately 45% of the total reads (249272 reads out of 554081 reads). In fact, the same sequence (D-6) was also found in the R6 pool before DGGE separation, but only accounted for about 0.8% (fourth place) of the R6 reads. Thus, the D-6 sequence was rather rare in the R6 pool (about 1.7% by DGGE and about 0.8% by high-throughput sequencing, respectively), but became predominant after DGGE separation and PCR amplification with L-Dpo4-5m (about 35% by DGGE and about 45% by high-throughput sequencing).

[0151] Band D-6 was then sequenced using a phosphorothioate approach with D-deoxynucleoside α-thiotriphosphate (D-dNTPαS) and cleavage with 2-iodoethanol. 33 This sequencing method has recently been adopted for sequencing-by-synthesis of L-DNA. 13 The sequencing results were rather unclear, mainly due to the double band phenomenon caused by the presence of 3'-hydroxyl and 3'-monophosphate groups in the cleaved DNA fragments. 34To address this issue, the DNA fragments cleaved with 2-iodoethanol were treated with calf intestinal alkaline phosphatase (CIP). After CIP treatment, most of the doublet bands disappeared, presumably due to removal of 3'-monophosphate from the cleaved DNA fragments, and thus the sequence of band D-6 was easily determined. Prediction of the secondary structure of the D-6 aptamer by Mfold 35 The D-6 aptamer is the consensus sequence of the D-DNA aptamers targeting native human thrombin identified so far. 30 Finally, a dissociation constant (K) of 27 nM was 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). d The D-DNA aptamer D-6, which binds to native human thrombin at 1000 ng / ml, was prepared by solid-phase oligosynthesis. Furthermore, the D-6 aptamer formed a stable complex with native human thrombin as revealed by EMSA, which was digestible by DNase I, as expected.

[0152] Mirror-image selection of L-DNA aptamers targeting native human thrombin Similar to the D-DNA library, the library has 30 randomized nucleotides flanked by two constant regions for primer binding, approximately 1 × 10 14A large-scale randomized L-DNA library consisting of different sequences was generated by solid-phase oligo synthesis. The L-DNA library was amplified by mirror-image PCR using D-Dpo4-5m and L-DNA primers. As in the native system, the progress of mirror-image selection was monitored by EMSA (Figure 2A). After nine rounds of selection, about 70% of the L-DNA pool bound to 1 μM native human thrombin but not to 1 μM streptavidin (Figure 2A, Figure 2B). The mirror-image PCR products from R5 to R9 and the mirror-image PCR product from R0 before selection were analyzed by DGGE (Figure 2C). No clear bands were observed in R0 and R5, but bands began to appear in R6, and the number and intensity of bands increased from R7 to R9 (Figure 2C). Two bands (L-9-1 and L-9-2) were isolated from R9, accounting for approximately 1.7% and 1.6% of the total lane fluorescence intensity of R9, respectively (Fig. 2C). These bands were amplified by mirror-image PCR using D-Dpo4-5m and L-DNA primers in two separate reactions, and the mirror-image PCR products were analyzed by separate DGGE, revealing a predominant band in each case, accounting for approximately 18% and 12% of the corresponding total lane fluorescence intensity, respectively (Fig. 2C).

[0153] To determine the sequence of enriched L-DNA aptamers, band L-9-1 was purified using a phosphorothioate approach with L-deoxynucleoside α-thiotriphosphate (L-dNTPαS) and cleavage with 2-iodoethanol for sequencing-by-synthesis of L-DNA. 13 The L-DNA fragments were isolated in a 10-well plate. The sequencing results were still unclear due to double bands, similar to the phosphorothioate sequencing results in the native system (Fig. 5A). When the L-DNA fragments cleaved with 2-iodoethanol were treated with CIP, it was unexpectedly found that the CIP treatment substantially improved the L-DNA sequencing results (Fig. 5B). This is probably due to the removal of the 3'-monophosphate in the L-DNA by the cross-chiral dephosphorylation activity of CIP, which has not been reported before. Thus, the sequence of band L-9-1 was easily determined.

[0154] Furthermore, band L-9-2 was also sequenced using the phosphorothioate approach and it was observed that even after treatment with CIP, three nucleotide positions in the central region of the sequenced aptamer caused inaccurate reads (probably due to contaminating sequences) resulting in the eight most likely L-DNA aptamer sequences (Figure 5C and Table 4). Since the correct sequences should co-migrate with band L-9-2 at the same melting temperature (Figure 6A), it was thought that the incorrect sequences could be excluded by comparing the migration of the candidate aptamer sequences using DGGE. Therefore, the native versions (to save costs and mirror enzymes) of the eight most likely L-DNA aptamer sequences in band L-9-2 (from DL-9-2-1 to DL-9-2-8, Table 4) were screened by DGGE to exclude the incorrect sequences. Only the DL-9-2-7 sequence was observed to comigrate with band L-9-2 (Figure 6B), suggesting that DL-9-2-7 and band L-9-2 likely share the same sequence. Therefore, the sequence of band L-9-2 was determined by a combination of an initial DGGE to isolate it (Figure 2C), sequencing-by-synthesis of L-DNA using a phosphorothioate approach, and a second DGGE to exclude incorrect sequences (Figure 6B).

[0155] Characterization of selected L-DNA aptamers To evaluate the binding affinity of the sequenced L-DNA aptamers to native human thrombin, the L-DNA aptamer L-9-1 was prepared by solid-phase oligosynthesis (Figure 3A). This L-DNA aptamer L-9-1 had a K of 29 nM as measured by ITC in physiological buffer. d The D-DNA aptamer D-6 binds to native human thrombin at 1 kD (Fig. 3B), which is consistent with the K dThe L-9-1 aptamer was truncated from 65 nt to 36 nt based on the secondary structure predicted by Mfold (Figure 3C), and the truncated aptamer (L-9-1t) was observed to bind native human thrombin with only a slightly reduced affinity (K d = 39 nM, Figure 3D). On the other hand, no binding was detected between the L-9-1t aptamer and streptavidin, and no binding was detected between the natural version of the L-9-1t (DL-9-1t) aptamer and native human thrombin, suggesting that the binding between the L-9-1t aptamer and native human thrombin was target-specific and chiral-specific. Further shortening of the L-9-1t aptamer from 36 nt to 32 nt by truncating a portion of the stem region reduced the affinity by about three-fold (K d = 111 nM, possibly due to destabilization of the aptamer secondary structure). Furthermore, the 5'-cyanine 5 (Cy5)-labeled L-9-1t (Cy5-L-9-1t) aptamer had a K d had a kinetic activity of 21 nM, formed a stable complex with native human thrombin, and, as expected, was resistant to DNase I digestion (FIGS. 3E to 3G).

[0156] The L-DNA aptamer L-9-2 was also prepared by solid-phase oligosynthesis (Figure 3H). The L-DNA aptamer L-9-2 had a K of 168 nM as measured by ITC in physiological buffer. d The L-9-2 aptamer bound to native human thrombin at 65 nt (Fig. 3I). The L-9-2 aptamer was then truncated from 65 nt to 38 nt based on the secondary structure predicted by Mfold (Fig. 3J). The truncated aptamer (L-9-2t) was observed to bind to native human thrombin with only a slight decrease in affinity (K d= 251 nM, K in Figure 3). On the other hand, no binding was detected between the L-9-2t aptamer and streptavidin, and no binding was detected between the natural version of the L-9-2t (DL-9-2t) aptamer and native human thrombin, suggesting that the binding of the L-9-2t aptamer to native human thrombin is target- and chiral-specific. Furthermore, the 5'-Cy5-labeled L-9-2t (Cy5-L-9-2t) aptamer showed a K of 355 nM as measured by EMSA. d The L-9-2-1 aptamer bound to native human thrombin at 100 kb (Fig. 3L-N) and, as expected, was resistant to DNase I digestion (Fig. 3L). Furthermore, based on the contaminant sequence (DL-9-2-1) predicted by DGGE from R9, a truncated version of the L-9-2-1 (L-9-2-1t) aptamer was prepared by solid-phase oligosynthesis, and the binding affinity of the L-9-2-1t aptamer to native human thrombin was found to be similar to that of the L-9-2t aptamer (K d = 1337 nM), which was about 5-fold lower.

[0157] To further evaluate the target specificity of the L-DNA aptamers, we measured the binding affinity of the L-9-1t and L-9-2t aptamers to native bovine thrombin, which shows approximately 85% sequence identity with native human thrombin. 38 The L-9-1t and L-9-2t aptamers had K values ​​of 1027 nM and 426 nM, respectively. d We observed that the L-9-1t aptamer bound to native bovine thrombin at 100 nM and 1.7-fold reduced binding affinity compared to native human thrombin (39 nM and 251 nM, respectively). These results suggest that the L-9-1t aptamer binds to native human thrombin much more strongly than native bovine thrombin, while the L-9-2t aptamer binds to both with similar affinity.

[0158] L-DNA aptamer sensor To demonstrate the feasibility of practical application of the thrombin-binding L-DNA aptamer, the high-affinity thrombin-binding L-DNA aptamer L-9-1t was hybridized with an L-DNA fluorescent strand having a 5'-labeled fluorescein (FAM) and an L-DNA quencher strand having a 3'-labeled 4-(4-dimethyl-aminophenylazo)benzoic acid (DABCYL), both of which hybridize with the L-9-1t aptamer to form a stable L-DNA duplex. 39 By combining the L-9-1t aptamer with the 1-amino-3-pyridine aptamer (A in Figure 4), we synthesized a structure-switching L-DNA aptamer sensor. Upon binding to native human thrombin, the L-9-1t aptamer undergoes structure switching, releasing the quencher strand and causing an increase in relative fluorescence with a linear response in the range of approximately 125–1000 nM (B in Figure 4). In contrast, the L-DNA aptamer sensor did not respond to the addition of 1 μM streptavidin or 1 μM native bovine thrombin, consistent with the ITC results.

[0159] To evaluate the influence of serum enzymes on the biological stability and thrombin sensing ability of the L-DNA aptamer sensor, the L-DNA aptamer sensor was incubated in a physiological buffer containing 10% (v / v) human serum, which provides a physiologically relevant nuclease-rich environment. The L-DNA aptamer sensor showed a linear response in the range of about 250–2000 nM in a physiological buffer containing 10% human serum, responding to the addition of native human thrombin (Figure 4B). In parallel, a native structure-switching sensor was constructed based on the D-DNA aptamer D-6 (D-DNA aptamer sensor). Next, 300 nM (final concentration) of native human thrombin was incubated in a physiological buffer containing 10% human serum, or 50 units / ml of DNase I (one of the major nucleases in serum) at 20°C for 1 h. 41) was added to physiological buffer containing D- or L-DNA aptamer sensor. After 1 h incubation in physiological buffer containing 10% human serum, the thrombin concentrations measured with the D- and L-DNA aptamer sensors were 416 ± 62 nM and 457 ± 72 nM, respectively, which were similar to the concentrations measured in physiological buffer alone (334 ± 59 nM and 299 ± 12 nM, respectively, Figure 4C). However, after 4 h incubation in physiological buffer containing 10% human serum, the thrombin concentration measured with the D-DNA aptamer sensor was 784 ± 91 nM, and the thrombin concentration measured with the L-DNA aptamer sensor was 375 ± 54 nM (Figure 4C). Furthermore, after incubation in physiological buffer containing 50 units / ml DNase I for 1 and 4 h, the thrombin concentrations measured with the D-DNA aptamer sensor were 1219 ± 57 nM and 984 ± 52 nM, respectively, whereas those measured with the L-DNA aptamer sensor were 334 ± 58 nM and 251 ± 34 nM, respectively (Figure 4C).

[0160] The error-prone measurements with the D-DNA aptamer sensor but not with the L-DNA aptamer sensor may be due to the increased relative fluorescence and premature release of the FAM fluorophore and DABCYL quencher upon degradation of the D-DNA aptamer sensor by serum enzymes or DNase I, which is consistent with the estimated half-life (t 1 / 2 ) is approximately 1.7 hours. To further verify the biological stability of the L-DNA aptamer sensor, the sensor was incubated in a physiological buffer containing 83% human serum. No significant degradation of the L-DNA aptamer sensor was observed in denaturing PAGE after incubation for up to 30 days (720 hours). On the other hand, the D-DNA aptamer sensor showed an estimated t 1 / 2was rapidly degraded in approximately 2.1 h, which was in line with previous studies using other D-DNA aptamers in human serum. 3,42 .

[0161] L-DNA aptamer Western blot To further explore the practical application potential of the thrombin-binding L-DNA aptamer, the L-13t aptamer (selected and optimized in the "Reselection and optimization of L-DNA aptamers from a partially randomized L-DNA library" section below) was applied in a proof-of-concept Western blot experiment based on the L-DNA aptamer to detect native human thrombin immobilized on a nitrocellulose membrane (Figure 4D). 6 ng to 180 ng of native human thrombin was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then 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 consistent with the molecular weight of native human thrombin (approximately 36 kDa) was detected with a detection limit of less than 6 ng (Figure 4F). In a control experiment, when 6 ng to 180 ng of streptavidin was analyzed by SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with 500 nM Cy5-L-13t aptamer, no distinct band was identified at the expected molecular weight of streptavidin (~18 kDa) (Figure 4F). In contrast, when 6 ng to 180 ng of native human thrombin was analyzed by conventional Western blot using a mouse monoclonal primary antibody targeting native human thrombin and an Alexa Fluor 647-labeled (with excitation and emission wavelengths similar to Cy5) goat anti-mouse IgG polyclonal secondary antibody, a fluorescent band consistent with the molecular weight of native human thrombin (~36 kDa) was detected (Figure 4G).

[0162] L-DNA aptamer enzyme inhibitor Next, the fluorescent substrate for thrombin, benzoyl-Phe-Val-Arg-7-amino-4-methylcoumarin (AMC), 44(FIG. 4H) Inhibition of thrombin enzyme activity by the thrombin-binding L-DNA aptamers L-9-1 and L-9-2 was tested in physiological buffer containing 100 μM. The L-9-2 aptamer exhibited a half-maximal inhibitory concentration (IC 50 ) inhibited thrombin enzyme activity with a measured value of 317 ± 128 nM (Figure 4I), and the K d (168 nM, Figure 3I). In contrast, the pre-selection R0 L-DNA pool 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. It had a slightly higher IC of 479 ± 65 nM. 50 (I in Fig. 4) is shown, and its K measured by ITC. d (251 nM, K in Figure 3). However, the L-9-1 and L-9-1t aptamers had higher binding affinities (K d = 29 nM and 39 nM), did not inhibit thrombin enzymatic activity at concentrations up to 8 μM, suggesting that the binding sites of native human thrombin targeted by the L-9-1 and L-9-2 aptamers are different. Furthermore, inhibition of thrombin enzymatic activity by the L-9-2t aptamer was shown to be chiral specific, 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.

[0163] Reselection and optimization of L-DNA aptamers from partially randomized L-DNA libraries Binding of the L-9-2 aptamer to native human thrombin (K d was measured to be 168 nM) and inhibition of thrombin enzyme activity (IC 50 was measured to be 317±128 nM) was not optimal, calling for further improvement and optimization of the L-DNA aptamer, both in terms of binding and inhibition properties.

[0164] For reselection and optimization of the L-9-2 aptamer, approximately 1 × 10 11A partially randomized L-DNA library (R10) with different sequences was synthesized by solid-phase oligo synthesis. 34 nucleotides flanked by two constant regions for primer binding were partially randomized at a frequency of 10% based on the L-9-2 aptamer. Next, mirror selection of the partially randomized L-DNA library targeting native human thrombin was performed (Figure 7A). After three rounds of enrichment and mirror PCR amplification (Figure 7B, C), DGGE was applied to isolate a single band (L-13) from R13. This band accounted for approximately 0.2% of the total lane fluorescence intensity of R13 (Figure 7D). Band L-13 was amplified by mirror PCR using D-Dpo4-5m and L-DNA primers, and the mirror PCR product was analyzed by another DGGE, revealing a dominant band that accounted for approximately 13% of the corresponding total lane fluorescence intensity (Figure 7D). To determine the enriched L-DNA aptamer sequences, we performed sequencing-by-synthesis of L-DNA using a phosphorothioate approach, and identified a mutant sequence of the L-9-2 aptamer in which two adenosines were mutated to cytosines in the partially randomized region (Figure 7E). This reselected L-DNA aptamer (L-13) had a K of 22 nM in physiological buffer as measured by ITC. d The L-13 aptamer bound to native human thrombin at 68 nt (Fig. 7F, G) and showed approximately 8-fold improved binding affinity with native human thrombin compared to its parent aptamer L-9-2. The L-13 aptamer was truncated from 68 nt to 38 nt based on the secondary structure predicted by Mfold (Fig. 7H), and the truncated aptamer (L-13t) showed only a slight decrease in affinity (K d = 34 nM, Figure 7I), bound native human thrombin. Furthermore, the 5'-Cy5-labeled L-13t (Cy5-L-13t) aptamer had a K of 28 nM as measured by EMSA. d and was found to form a stable complex with native human thrombin (Fig. 7J and K).

[0165] Inhibition of thrombin enzyme 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). The L-13 aptamer had an IC measured at 27 ± 3 nM. 50 (Fig. 7M) and the K d The L-DNA pool showed inhibition of thrombin enzyme activity that was nearly consistent with the IC50 (22 nM, Figure 7G) and approximately 12-fold improved compared to its parent aptamer L-9-2. In contrast, the R10 partially randomized L-DNA pool before reselection did not inhibit thrombin enzyme activity at concentrations up to 1.4 μM. Inhibition of thrombin enzyme activity by the truncated aptamer (L-13t) was also measured, with a slightly higher IC50 of 46 ± 4 nM. 50 (M in Fig. 7) was observed, which is consistent with the K measured by ITC. d This was almost identical to (34 nM, Figure 7I).

[0166] As a final test of the selected L-DNA aptamers and a demonstration of their clinical potential, an in vitro clotting assay on human plasma was performed. Upon addition of 2.5 μM of the L-9-1t and L-13t aptamers, approximately 4-fold and 2-fold longer prothrombin times were measured, respectively, compared to the control without added L-DNA aptamer or the native version of L-9-1t (DL-9-1t) aptamer (Figure 7N).

[0167] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the present invention is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0168] It is the intention of the applicants that all publications, patents, and patent applications referenced herein are incorporated herein by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference when referenced. Furthermore, citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

[0169] Additionally, the priority documents of this application are incorporated herein by reference in their entireties.

[0170] References 1 Nolte, A., Klussmann, S., Bald, R., Erdmann, VA & Furste, JP Mirror-design of L-oligonucleotide ligands binding to L-arginine. Nat. Biotechnol. 14, 1116-1119 (1996). 2 Klussmann, S., Nolte, A., Bald, R., Erdmann, VA & Furste, JP Mirror-image RNA that binds D-adenosine. Nat. Biotechnol. 14, 1112-1115 (1996). 3 Williams, KP et al. Bioactive and nuclease-resistant L-DNA ligand of vasopressin. Proc. Natl Acad. Sci. USA 94, 11285-11290 (1997). 4 Dunn, M. R., Jimenez, R. M. & Chaput, J. C. Analysis of aptamer discovery and technology. Nat. Rev. Chem. 1, 0076 (2017). 5 Zhou, J. & Rossi, J. Aptamers as targeted therapeutics: current potential and challenges. Nat. Rev. Drug Discov. 16, 181-202 (2017). 6 Wang, Z., Xu, W., Liu, L. & Zhu, T. F. A synthetic molecular system capable of mirror-image genetic replication and transcription. Nat. Chem. 8, 698-704 (2016). 7 Xu, W. et al. Total chemical synthesis of a thermostable enzyme capable of polymerase chain reaction. Cell Discov. 3, 17008 (2017). 8 Jiang, W. et al. Mirror-image polymerase chain reaction. Cell Discov. 3, 17037 (2017). 9 Liu, X. & Zhu, T. F. Sequencing mirror-image DNA chemically. Cell Chem. Biol. 25, 1151-1156 (2018). 10 Wang, M. et al. Mirror-image gene transcription and reverse transcription. Chem 5, 848-857 (2019). 11 Ling, J.-J. et al. Mirror-image 5S ribonucleoprotein complexes. Angew. Chem. Int. Ed. 59, 3724-3731 (2020) 12 Chen, J., Chen, M. & Zhu, T. F. Translating protein enzymes without aminoacyl-tRNA synthetases. Chem 7, 786-798 (2021). 13 Fan, C., Deng, Q. & Zhu, T. F. Bioorthogonal information storage in L-DNA with a high-fidelity mirror-image Pfu DNA polymerase. Nat. Biotechnol. 39: 1548-1555 (2021). 14 Tuerk, C. & Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505-510 (1990). 15 Ellington, A. D. & Szostak, J. W. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818-822 (1990). 16 Wilson, D. S. & Szostak, J. W. In vitro selection of functional nucleic acids. Annu. Rev. Biochem. 68, 611-647 (1999). 17 Gawande, B. N. et al. Selection of DNA aptamers with two modified bases. Proc. Natl Acad. Sci. USA 114, 2898-2903 (2017). 18 Liu, Z. X., Chen, T. J. & Romesberg, F. E. Evolved polymerases facilitate selection of fully 2'-OMe-modified aptamers. Chem. Sci. 8, 8179-8182 (2017). 19 Dunn, M. R., McCloskey, C. M., Buckley, P., Rhea, K. & Chaput, J. C. Generating biologically stable TNA aptamers that function with high affinity and thermal stability. J. Am. Chem. Soc. 142, 7721-7724 (2020). 20 Pinheiro, V. B. et al. Synthetic genetic polymers capable of heredity and evolution. Science 336, 341-344 (2012). 21 Kimoto, M., Yamashige, R., Matsunaga, K.-i., Yokoyama, S. & Hirao, I. Generation of high-affinity DNA aptamers using an expanded genetic alphabet. Nat. Biotechnol. 31 (2013). 22 Hoshika, S. et al. Hachimoji DNA and RNA: A genetic system with eight building blocks. Science 363, 884-887 (2019). 23 Purschke, W. G., Eulberg, D., Buchner, K., Vonhoff, S. & Klussmann, S. An L-RNA-based aquaretic agent that inhibits vasopressin in vivo. Proc. Natl Acad. Sci. USA 103, 5173-5178 (2006). 24 Sczepanski, J. T. & Joyce, G. F. Binding of a structured D-RNA molecule by an L-RNA aptamer. J. Am. Chem. Soc. 135, 13290-13293 (2013). 25 Umar, M. I. & Kwok, C. K. Specific suppression of D-RNA G-quadruplex-protein interaction with an L-RNA aptamer. Nucleic Acids Res. 48, 10125-10141 (2020). 26 Olea Jr, C., Weidmann, J., Dawson, Philip E. & Joyce, Gerald F. An L-RNA aptamer that binds and inhibits RNase. Chem. Biol. 22, 1437-1441 (2015). 27 Yatime, L. et al. Structural basis for the targeting of complement anaphylatoxin C5a using a mixed L-RNA / L-DNA aptamer. Nat. Commun. 6, 6481 (2015). 28 Pech, A. et al. A thermostable D-polymerase for mirror-image PCR. Nucleic Acids Res. 45, 3997-4005 (2017). 29 Muyzer, G. & Smalla, K. Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie van Leeuwenhoek 73, 127-141 (1998). 30 Bock, L. C., Griffin, L. C., Latham, J. A., Vermaas, E. H. & Toole, J. J. Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature 355, 564-566 (1992). 31 Tasset, D. M., Kubik, M. F. & Steiner, W. Oligonucleotide inhibitors of human thrombin that bind distinct epitopes. J. Mol. Biol. 272, 688-698 (1997). 32 Dobbelstein, M. & Shenk, T. In vitro selection of RNA ligands for the ribosomal L22 protein associated with Epstein-Barr virus-expressed RNA by using randomized and cDNA-derived RNA libraries. J. Virol. 69, 8027-8034 (1995). 33 Gish, G. & Eckstein, F. DNA and RNA sequence determination based on phosphorothioate chemistry. Science 240, 1520-1522 (1988). 34 Nakamaye, K. L., Gish, G., Eckstein, F. & Vosberg, H. P. Direct sequencing of polymerase chain-reaction amplified DNA fragments through the incorporation of deoxynucleoside alpha-thiotriphosphates. Nucleic Acids Res. 16, 9947-9959 (1988). 35 Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406-3415 (2003). 36 DeAnda, A., Jr. et al. Pilot study of the efficacy of a thrombin inhibitor for use during cardiopulmonary bypass. Ann. Thorac. 58, 344-350 (1994). 37 Bode, W. et al. The refined 1.9 A crystal structure of human alpha-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J. 8, 3467-3475 (1989). 38 Liu, X. et al. RNA aptamers specific for bovine thrombin. J. Mol. Recognit. 16, 23-27 (2003). 39 Nutiu, R. & Li, Y. F. Structure-switching signaling aptamers. J. Am. Chem. Soc. 125, 4771-4778 (2003). 40 Li, L.-L., Ge, P., Selvin, P. R. & Lu, Y. Direct detection of adenosine in undiluted serum using a luminescent aptamer sensor attached to a terbium complex. Anal. Chem. 84, 7852-7856 (2012). 41 Barra, G. B. et al. EDTA-mediated inhibition of DNases protects circulating cell-free DNA from ex vivo degradation in blood samples. Clin. Biochem. 48, 976-981 (2015). 42 Kratschmer, C. & Levy, M. Effect of chemical modifications on aptamer stability in serum. Nucleic Acid Ther. 27, 335-344 (2017). 43 Wang, Y., Li, Z. & Yu, H. Aptamer-based Western blot for selective protein recognition. Front. Chem. 8, 870528 (2020) 44 Li, M.-L., Ren, Y.-J., Dong, M.-H. & Ren, W.-X. Design, synthesis and structural exploration of novel fluorinated dabigatran derivatives as direct thrombin inhibitors. Eur. J. Med. Chem. 96, 122-138 (2015). 45 Fang, X. H. & Tan, W. H. Aptamers generated from cell-SELEX for molecular medicine: a chemical biology approach. Acc. Chem. Res. 43, 48-57 (2010). 46 Li, S. et al. Identification of an aptamer targeting hnRNP A1 by tissue slide-based SELEX. J. Pathol. 218, 327-336 (2009). 47 Sczepanski, J. T. & Joyce, G. F. A cross-chiral RNA polymerase ribozyme. Nature 515, 440-442 (2014). 48 Scheitl, C. P. M., Ghaem Maghami, M., Lenz, A.-K. & Hobartner, C. Site-specific RNA methylation by a methyltransferase ribozyme. Nature 587, 663-667 (2020). 49 Chandrasekar, J. & Silverman, S. K. Catalytic DNA with phosphatase activity. Proc. Natl Acad. Sci. USA 110, 5315-5320 (2013). 50 Peplow, M. Mirror-image enzyme copies looking-glass DNA. Nature 533, 303-304 (2016). 51 Peplow, M. A Conversation with Ting Zhu. ACS Cent. Sci. 4, 783-784 (2018). 52 Mattheakis, L. C., Bhatt, R. R. & Dower, W. J. An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc. Natl Acad. Sci. USA 91, 9022-9026 (1994). 53 Roberts, R. W. & Szostak, J. W. RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc. Natl Acad. Sci. USA 94, 12297-12302 (1997). 54 Reuter, Jason A., Spacek, D. V. & Snyder, Michael P. High-throughput sequencing technologies. Mol. Cell 58, 586-597 (2015). 55 Iliuk, A. B., Hu, L. & Tao, W. A. Aptamer in bioanalytical applications. Anal. Chem. 83, 4440-4452 (2011). 56 Brumbt, A. et al. Chiral stationary phase based on a biostable L-RNA aptamer. Anal. Chem. 77, 1993-1998 (2005). 57 Idili, A., Parolo, C., Alvarez-Diduk, R. & Merkoci, A. Rapid and efficient detection of the SARS-CoV-2 spike protein using an electrochemical aptamer-based sensor. ACS sens. 6, 3093-3101 (2021).

Claims

1. A method for screening a plurality of L-nucleic acid aptamers to obtain an L-nucleic acid aptamer having binding affinity for a target molecule, comprising: (a) contacting the 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 the target-binding L-nucleic acid aptamer to generate an amplified double-stranded L-nucleic acid oligonucleotide; (c) isolating the amplified double-stranded L-nucleic acid oligonucleotide using an electrophoresis-based method; and screening the plurality of L-nucleic acid aptamers by these steps.

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

3. The method of claim 2, wherein steps (a), (b) and the converting step are repeated at least three times before the isolating step to enrich the target-binding L-nucleic acid aptamer.

4. The method of claim 3, further comprising the step of monitoring the enrichment of said target-binding L-nucleic acid aptamers.

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

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

7. The method of any one of claims 1 to 3, wherein the electrophoresis-based method comprises DGGE.

8. The method of any one of claims 1 to 5, wherein the target molecule is selected from the group consisting of peptides, polypeptides, small molecules, carbohydrates and nucleic acid molecules.

9. The method of any one of claims 1 to 5, wherein the target molecule is contained in a cell or tissue.

10. The method of any one of claims 1 to 5, wherein the amplification utilizes a D-amino acid polymerase.

11. The method of claim 10, wherein the D-amino acid polymerase is selected from the group consisting of D-ASFV pol X, D-Taq polymerase, D-Pfu polymerase, Sulfolobus solfataricus P2 DNA polymerase IV (DPO4), a fusion protein containing DPO4, and a polymerase having an amino acid sequence at least 80% identical to DPO4.

12. 12. The method of claim 11, wherein the polymerase has the amino acid sequence set forth in SEQ ID NO:38 or SEQ ID NO:

40.

13. The method of any one of claims 1 to 5, further comprising sequencing the isolated members following step (c) to obtain the sequence of the L-nucleic acid aptamer having binding affinity for the target molecule.

14. The method of claim 13, wherein the sequencing is performed using a method selected from the group consisting of L-DNA chemical sequencing, L-DNA phosphorothioate sequencing, L-DNA dideoxy sequencing, L-DNA Ion Torrent sequencing, L-DNA Illumina sequencing, and L-DNA Nanopore sequencing.

15. The method according to claim 14, wherein the method is an L-DNA phosphorothioate sequencing method.

16. The method of claim 15, further comprising contacting said amplified double-stranded L-nucleic acid oligonucleotide with a phosphatase prior to said sequencing.

17. 17. The method of claim 16, wherein the phosphatase comprises calf intestinal phosphatase (CIP).

18. The method according to any one of claims 1 to 5, wherein each of the L-nucleic acid aptamers in the plurality of L-nucleic acid aptamers has the same length.

19. The method according to any one of claims 1 to 5, wherein said plurality of L-nucleic acid aptamers is a library, and each member of said library has identical 5' and 3' nucleic acid sequences and non-identical core sequences.

20. The method of claim 13, further comprising constructing an additional aptamer library, wherein each member of the library has identical 5' and 3' nucleic acid sequences and is up to 60% randomized compared to the sequence of the isolated L-nucleic acid aptamer.

21. The method according to any one of claims 1 to 5, further comprising the step of synthesizing the plurality of L-nucleic acid aptamers before step (a).

22. 22. The method of claim 21, wherein the synthesis comprises error-prone PCR.

23. 23. The method of claim 22, wherein the error-prone PCR comprises the use of an error-prone polymerase.

24. 20. The method of claim 19, wherein the core sequence comprises a random or semi-random sequence.

25. A kit for identifying L-nucleic acid aptamers, comprising: (i) calf intestinal phosphatase (CIP); (ii) L-deoxyribonucleotide triphosphates (L-dNTPs) or modified L-dNTPs, and / or (iii) a polymerase capable of adding one or more L-nucleotides to the 3′ end of a first L-nucleic acid, Includes a kit.

26. 26. The kit of claim 25, wherein the polymerase comprises Sulfolobus solfataricus P2 DNA polymerase IV (DPO4), or a polymerase having an amino acid sequence at least 80% identical to DPO4.

27. 27. The kit of claim 26, wherein the polymerase has the amino acid sequence set forth in SEQ ID NO:38 or SEQ ID NO:

40.

28. 1. A method for sequencing purified L-DNA molecules, comprising: (a) treating a sample containing the purified L-DNA molecules with a phosphatase under conditions that remove the 3'-monophosphate from the L-DNA molecules; (b) subjecting said sample to phosphorothioate sequencing, thereby sequencing the purified L-DNA molecules; A method comprising:

29. An isolated thrombin-binding L-DNA aptamer comprising a sequence set forth in SEQ ID NO: 10, 12, 14, 16, 27 or 28, or a sequence that is at least 80% identical to said SEQ ID NO: 10, 12, 14, 16, 27 or 28.

30. An isolated thrombin-binding L-DNA aptamer comprising a sequence set forth in SEQ ID NO: 10, 14 or 28, or a sequence that is at least 80% identical to the sequence set forth in SEQ ID NO: 10, 12, 14, 16, 27 or 28.

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