Nucleic acid aptamer

Abstract

The present invention addresses the problem of providing a method for improving the affinity of a nucleic acid aptamer containing 7-(2-thienyl) imidazo [4, 5-b] pyridine (Ds base) to a target protein. Provided is a nucleic acid aptamer comprising a non-natural base represented by general formula (I) and / or general formula (II).

Description

Technical Field

[0001] The present invention relates to unnatural bases, nucleosides / nucleotides containing unnatural bases, nucleic acid aptamers containing unnatural bases, pharmaceutical compositions, and the like. Background Art

[0002] A nucleic acid aptamer is a nucleic acid strand that specifically binds to a target such as a low molecular weight substance, protein, or cell. Generally, a nucleic acid aptamer is generated by repeatedly selecting and amplifying a nucleic acid library having a random sequence by the SELEX method (systematic evolution of ligands by exponential enrichment).

[0003] After determining the base sequence of the nucleic acid aptamer generated by the SELEX method, it is mass-produced and modified by chemical synthesis. Since the nucleic acid aptamer can be provided in high purity, it can be an excellent alternative to antibodies in terms of quality control.

[0004] However, generally, nucleic acids are more hydrophilic than protein-based antibodies. Therefore, the hydrophobic interaction between a nucleic acid aptamer and a target protein is weaker than that of an antibody. Therefore, it is difficult for existing nucleic acid aptamers to provide a binding force exceeding that of antibodies, and practical application is insufficient.

[0005] To solve the above problems of nucleic acid aptamers, the present inventors developed the ExSELEX (genetic alphabet Expansion for SELEX) method (Non-Patent Document 1) as a new aptamer generation method capable of improving the hydrophobic interaction with a target protein by introducing the hydrophobic unnatural base 7-(2-thienyl)imidazo[4,5-b]pyridine (described as "Ds" or "Ds base" in this specification) as the fifth base into the nucleic acid aptamer. The Ds base pairs with 2-nitro-4-propynylpyrrole modified with diol (described as "Px" or "Px base" in this specification). Then, the Ds-Px base pair formed by base pairing between the Ds base and the Px base functions as the third base pair with high fidelity in PCR amplification (Non-Patent Documents 1 to 6). For the nucleic acid aptamer obtained by the ExSELEX method, the K D value based on the inclusion of the Ds base and showing affinity for the target protein is below the nanomolar concentration, and high affinity can be achieved.

[0006] Prior Art Documents

[0007] Non-Patent Documents

[0008] Non-Patent Document 1: Kimoto, M., et al., Nat. Biotechnol., 2013, 31: 453-457.

[0009] Non-Patent Document 2: Kimoto, M., et al., Nucleic Acids Res., 2009, 37: e14.

[0010] Non-Patent Document 3: Yamashige, R., et al., Nucleic Acids Res., 2012, 40: 2793-2806.

[0011] Non-Patent Document 4: Matsunaga, K., et al., J. Am. Chem. Soc., 2017, 139: 324-334.

[0012] Non-Patent Document 5: Matsunaga, K., et al., Nucleic Acids Res., 2021, 49: 11407-11424.

[0013] Non-Patent Document 6: Kimoto, M. and Hirao, I., Chem. Soc, Rev., 2020, 49: 7602-7626. Summary of the Invention

[0014] Problems to be Solved by the Invention

[0015] An object of the present invention is to provide a method for improving the affinity of a nucleic acid aptamer containing a Ds base for a target protein.

[0016] Means for Solving the Problems

[0017] In the ExSELEX method developed by the present inventors in the past, the Ds base pairs with the Px base, and the Ds-Px base pair functions with high fidelity as the third base pair in PCR amplification. Since the ExSELEX method is based on this Ds-Px base pair, modifications of the Ds base that can affect the base pair with the Px base have not been considered so far.

[0018] However, the Ds base may not be the best hydrophobic artificial base for bringing high affinity except for the steps in the ExSELEX method, and there may be hydrophobic artificial bases that further strengthen hydrophobic interactions in terms of, for example, van der Waals forces, base stacking, and polarity. Therefore, it may be possible to improve the affinity by replacing the Ds base with a new artificial base (hereinafter often referred to as "Ds substitute") in a nucleic acid aptamer containing the Ds base.

[0019] To solve the above problems, the inventors of the present invention created a new artificial base group (collectively referred to as "Ds substitutes" in this specification) by replacing the 1-deazapurine (imidazo[4,5-b]pyridine) moiety and the thiophene side chain in the Ds base with pyrrolo[2,3-b]pyridine, purine, indole, benzimidazole, or pyrrolo[2,3-d]pyrimidine in two modules, and replacing the thiophene side chain with a furyl, imidazolyl, thiazolyl, pyridazinyl, or their modified structures (e.g., methylthiophenyl, dithienyl moieties). The inventors of the present invention found that by replacing the Ds base with a Ds substitute in the nucleic acid aptamers developed in the past using the ExSELEX method, new nucleic acid aptamers with improved affinity were obtained, thus completing the present invention.

[0020] Based on the above new knowledge, the present invention provides the following solutions.

[0021] [1] A nucleic acid aptamer comprising an unnatural base represented by the following general formula (I) and / or general formula (II):

[0022]

[0023] [In the above formula, R 1 represents the sugar moiety in the nucleoside or an alkyl group of C 1 or C 2 and R 2 represents any one of the following formulas (III) to (IX):

[0024]

[0025] [2] The nucleic acid aptamer according to [1], comprising two of the unnatural bases.

[0026] [3] The nucleic acid aptamer according to [1] or [2], comprising the unnatural bases represented by the general formula (I) and general formula (II).

[0027] [4] The nucleic acid aptamer according to any one of [1] to [3], wherein the unnatural base is any one of the following formulas (X) to (XVII) and formulas (XXI) to (XXII):

[0028]

[0029] [5] The nucleic acid aptamer according to any one of [1] to [4], comprising the unnatural base represented by the following formula (XVIII):

[0030]

[0031] [6] The nucleic acid aptamer according to any one of [1] to [5] is a DNA aptamer.

[0032] [7] The nucleic acid aptamer according to [1], the nucleic acid aptamer

[0033] (1) contains the base sequence shown in SEQ ID NO: 1 or 2 and binds to von Willebrand Factor (vWF protein);

[0034] (2) contains the base sequence shown in SEQ ID NO: 15 or 16 and binds to interferon-γ (IFNγ);

[0035] (3) contains the base sequence shown in SEQ ID NO: 26 and binds to vascular endothelial growth factor (VEGF);

[0036] (4) contains the base sequence shown in SEQ ID NO: 27 or 28 and binds to Dengue Virus NS1 Protein Serotype 1 (DEN1 protein), or

[0037] (5) contains the base sequence shown in SEQ ID NO: 38 or 39 and binds to Dengue Virus NS1 Protein Serotype 3 (DEN3 protein), and

[0038] at least one of the bases represented by n in the base sequence is a non-natural base represented by the general formula (I) and / or general formula (II).

[0039] [8] The nucleic acid aptamer according to [7], as the base represented by n in the base sequence, includes two of the non-natural bases.

[0040] [9] The nucleic acid aptamer according to [7] or [8] contains the non-natural bases represented by the general formula (I) and general formula (II).

[0041]

[10] The nucleic acid aptamer according to [7], as the base represented by n in the base sequence, contains 7-(2-thienyl)-3H-imidazo[4,5-b]pyridin-3-yl.

[0042]

[11] The nucleic acid aptamer according to [7], the non-natural base is any one of the following formula (X) to formula (XVII) and formula (XXI) to formula (XXII):

[0043]

[0044]

[12] The nucleic acid aptamer according to [7] contains an unnatural base represented by the following formula (XVIII):

[0045]

[0046]

[13] The nucleic acid aptamer according to [7] is a DNA aptamer.

[0047]

[14] A pharmaceutical composition contains the nucleic acid aptamer described in [7].

[0048]

[15] The pharmaceutical composition according to

[14] , which is used for the treatment and / or prevention of diseases selected from thrombosis, thrombotic thrombocytopenic purpura, intracranial embolism, cerebral embolism, carotid artery stenosis, thrombotic microangiopathy, and acute myocardial infarction, contains the nucleic acid aptamer described in (1) as an active ingredient.

[0049]

[16] The pharmaceutical composition according to

[14] , which is used for inhibiting the function of interferon-γ, contains the nucleic acid aptamer described in (2) as an active ingredient.

[0050]

[17] The pharmaceutical composition according to

[14] , which is used for inhibiting the function of vascular endothelial growth factor, contains the nucleic acid aptamer described in (3) as an active ingredient.

[0051]

[18] The pharmaceutical composition according to

[14] , which is used for the treatment and / or prevention of dengue fever, contains the nucleic acid aptamer described in (4) and / or the nucleic acid aptamer described in (5) as an active ingredient.

[0052]

[19] An unnatural base represented by any one of the following formulas (XI) to (XIII) and formulas (XV) to (XVII):

[0053]

[0054]

[20] A nucleoside containing the unnatural base described in

[19] .

[0055]

[21] A nucleotide containing the unnatural base described in

[19] .

[0056] This specification incorporates the disclosures of Singapore Patent Application No. 10202260206Y, Singapore Patent Application No. 10202260204R, and Singapore Patent Application No. 10202300790X, which form the basis of the priority of this application. In addition, Sequence Nos. 1 to 54 in this application are the same as Sequence Nos. 1 to 54 in Singapore Patent Application No. 10202260206Y and Singapore Patent Application No. 10202260204R. Further, Sequence Nos. 55 to 141 in this application are the same as Sequence Nos. 1 to 87 in Singapore Patent Application No. 10202300790X.

[0057] Effects of the Invention

[0058] According to the present invention, there is provided a method for improving the affinity of a nucleic acid aptamer containing Ds bases for a target protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 It is a diagram showing Ds bases, which are hydrophobic unnatural bases, Px bases that base-pair with Ds bases, and four natural DNA bases.

[0060] Figure 2 It is a diagram showing examples of various Ds substitutes in which the 1-deazapurine moiety is replaced with pyrrolo[2,3-b]pyridine, purine, indole, benzimidazole, or pyrrolo[2,3-d]pyrimidine, and the thiophene side chain is replaced with a furyl group, an imidazolyl group, a thiazolyl group, a pyridazinyl group, and their modified structures (e.g., methylthienyl, dithienyl moieties).

[0061] Figure 3 It is a diagram showing examples of an anti-VEGF aptamer, an anti-IFNγ aptamer, and an anti-vWF aptamer.

[0062] Figure 4 It is a diagram showing examples of an anti-DEN1-NS1 aptamer, an anti-DEN2-NS1 aptamer, an anti-DEN3-NS1 aptamer, and an anti-DEN4-NS1 aptamer.

[0063] Figure 5 It is a diagram showing the results of analyzing the affinity of Ds aptamer (AptIFNγ-DsDs) and 11 UB aptamers (AptIFNγ-BsBs, AptIFNγ-IsIs, AptIFNγ-YsYs, AptIFNγ-YiYi, AptIFNγ-DoDo, AptIFNγ-DpDp, AptIFNγ-DssDss, AptIFNγ-DtDt, AptIFNγ-YoYo, AptIFNγ-PsPs, and AptIFNγ-EsEs) for IFNγ by EMSA method.Figure 5 A shows a gel stained with SYBR Gold. Figure 5 B shows the relative binding rate (%) of the nucleic acid aptamer bound to the target.

[0064] Figure 6 It is a graph showing the results of analyzing the affinity of the Ds aptamer (AptvWF-DsDs) and 11 UB aptamers (AptvWF-BsBs, AptvWF-IsIs, AptvWF-YsYs, AptvWF-YiYi, AptvWF-DoDo, AptvWF-DpDp, AptvWF-DssDss, AptvWF-DtDt, AptvWF-YoYo, AptvWF-PsPs, and AptvWF-EsEs) for vWF by EMSA. Figure 6 A shows a gel stained with SYBR Gold. Figure 6 B shows the relative binding rate (%) of the nucleic acid aptamer bound to the target.

[0065] Figure 7 It is a graph showing the results of analyzing the affinity of the Ds aptamer (AptvWF-DsDs) and 6 UB aptamers (AptvWF-YsYs, AptvWF-BsYs, AptvWF-BsYo, AptvWF-YsBs, AptvWF-BsBs, and AptvWF-YoYo) for vWF by EMSA. Figure 7 A shows a gel stained with SYBR Gold. Figure 7 B shows the relative binding rate (%) of the nucleic acid aptamer bound to the target.

[0066] Figure 8 It is a graph showing the results of analyzing the affinity of the Ds aptamer (AptD1-DsDs) and 11 UB aptamers (AptD1-BsBs, AptD1-IsIs, AptD1-YsYs, AptD1-YiYi, AptD1-DoDo, AptD1-DpDp, AptD1-DssDss, AptD1-DtDt, AptD1-YoYo, AptD1-PsPs, and AptD1-EsEs) for DEN1-NS1 by EMSA. Figure 8 A shows a gel stained with SYBR Gold. Figure 8 B shows the relative binding rate (%) of the nucleic acid aptamer bound to the target.

[0067] Figure 9This is a graph showing the results of analyzing the affinity of the Ds aptamer (AptD1-DsDs) and nine UB aptamers (AptD1-DsYs, AptD1-YsDs, AptD1-YsYs, AptD1-DsBs, AptD1-BsDs, AptD1-BsBs, AptD1-DsDt, AptD1-DtDs, and AptD1-DtDt) for DEN1-NS1 using the EMSA method. Figure 9 A shows the gel stained with SYBR Gold. Figure 9 B shows the relative binding rate (%) of the nucleic acid aptamer bound to the target.

[0068] Figure 10 This is a graph showing the results of analyzing the affinity of the Ds aptamer (AptD1-DsDs) and seven UB aptamers (AptD1-BsBs, AptD1-YsYs, AptD1-YoYo, AptD1-YsBs, AptD1-YoBs, AptD1-YsmBs, and AptD1-BsYs) for DEN1-NS1 using the EMSA method. Figure 10 A shows the gel stained with SYBR Gold. Figure 10 B shows the relative binding rate (%) of the nucleic acid aptamer bound to the target.

[0069] Figure 11 This is a graph showing the results of analyzing the affinity of the Ds aptamer (AptD3-DsDs) and eleven UB aptamers (AptD3-BsBs, AptD3-IsIs, AptD3-YsYs, AptD3-YiYi, AptD3-DoDo, AptD3-DpDp, AptD3-DssDss, AptD3-DtDt, AptD3-YoYo, AptD3-PsPs, and AptD3-EsEs) for DEN3-NS1 using the EMSA method. Figure 11 A shows the gel stained with SYBR Gold. Figure 11 B shows the relative binding rate (%) of the nucleic acid aptamer bound to the target.

[0070] Figure 12 This is a graph showing the results of analyzing the affinity of the Ds aptamer (AptD3-DsDs) and seven UB aptamers (AptD3-YsYs, AptD3-YsDss, AptD3-DssYs, AptD3-DssDss, AptD3-YoYo, AptD3-YssYss, and AptD3-YsmYsm) for DEN3-NS1 using the EMSA method. Figure 12 A shows the gel stained with SYBR Gold. Figure 12B shows the relative binding rate (%) of the nucleic acid aptamer bound to the target.

[0071] Figure 13 It is a graph showing the results of analyzing the affinity of Ds aptamer (AptD3-DsDs) and 8 UB aptamers (AptD3-YssYss, AptD3-YssYs, AptD3-YssYo, AptD3-YssYsm, AptD3-YsmYss, AptD3-YsmYsm, AptD3-YsYs, and AptD3-YoYo) for DEN3-NS1 by EMSA method. Figure 13 A shows the gel stained with SYBR Gold. Figure 13 B shows the relative binding rate (%) of the nucleic acid aptamer bound to the target.

[0072] Figure 14 It is a schematic diagram showing the method for measuring the detection sensitivity of anti-IFNγ antibody and IFNγ target nucleic acid aptamer for IFNγ by ELISA method. Figure 14 A shows the ELISA method for anti-IFNγ antibody. Figure 14 B shows the ELISA method for IFNγ target nucleic acid aptamer.

[0073] Figure 15 It is a graph showing the results of measuring the detection sensitivity of anti-IFNγ antibody and IFNγ target nucleic acid aptamer for IFNγ by ELISA method. Figure 15 A shows the results of measuring the detection sensitivity of anti-IFNγ antibody, AptIFNγ-DsDs, AptIFNγ-YsYs, and AptIFNγ-YoYo aptamers for IFNγ. The horizontal axis shows the concentration of the antibody used for immobilizing IFNγ. Figure 15 B shows the signal intensity when using various concentrations of antibody and nucleic acid aptamer in ELISA method.

[0074] Figure 16 It is a graph showing the results of analyzing the affinity of 7 nucleic acid aptamers (AptIFNγ-1, AptIFNγ-2, AptIFNγ-3, AptIFNγ-4, AptIFNγ-5, AptIFNγ-6, and Bio-AptIFNγ-3) for human IFNγ by EMSA method.

[0075] Figure 17This is a graph showing the results of analyzing the affinity of five nucleic acid aptamers (AptIFNγ-1, AptIFNγ-3, and Bio-AptIFNγ-3, as well as AptDs-IFNγ and Bio-AptDs-IFNγ as comparative controls) for non-glycosylated IFNγ and glycosylated IFNγ by EMSA method.

[0076] Figure 18 This is a graph showing the results of analyzing the affinity of ten nucleic acid aptamers (XL2-01a, XL2-01b, XL2-01c, XL2-01d, XL2-01e, XL2-01h, XL2-01i, XL2-01j, XL2-01k, and XL2-01l) for non-glycosylated IFNγ and glycosylated IFNγ by EMSA method.

[0077] Figure 19 This is a graph showing the results of analyzing the affinity of three nucleic acid aptamers (Bio-XL2-01a, Bio-XL2-01i, and Bio-AptDs-IFNγ) for glycosylated IFNγ by ELONA method.

[0078] Figure 20 This is a graph showing the results of analyzing the affinity of three nucleic acid aptamers (Bio-XL2-01a, Bio-XL2-01i, and Bio-AptDs-IFNγ) for glycosylated IFNγ by ELONA method.

[0079] Figure 21 This is a graph showing the results of analyzing the affinity of nine nucleic acid aptamers (Thr101, Thr102, Thr104, Thr106, Thr201, Thr202, Thr203, Thr204, and Thr205) for human thrombin by EMSA method.

[0080] Figure 22 This is a graph showing the results of analyzing the affinity of seven nucleic acid aptamers (Thr104, Bio-Thr104b, Thr204, Bio-Thr204, Thr205, Bio-Thr205, and BioMH-RE31) for human thrombin by EMSA method.

[0081] Figure 23 This is a graph showing the results of analyzing the affinity of four nucleic acid aptamers (Bio-Thr104b, Bio-Thr204, Bio-Thr205, and BioMH-RE31) for human thrombin by ELONA method.

[0082] Figure 24It is a graph showing the results of analyzing the affinity of 6 nucleic acid aptamers (Thr204-DD, Thr204-BB, Thr204-BY, Thr204-YB, Thr204-YY, and Thr204-AA) for human thrombin by EMSA method.

[0083] Figure 25 It is a graph showing the results of analyzing the affinity of 6 nucleic acid aptamers (Bio-HMGB1-301-DD, Bio-HMGB1-301-BB, Bio-HMGB1-301-BY, Bio-HMGB1-301-YB, Bio-HMGB1-301-YY, and Bio-HMGB1-301-AA) for human HMGB1 protein by EMSA method.

[0084] Figure 26 It is a graph showing the results of analyzing the affinity of 6 nucleic acid aptamers (IFNg-201AAD, IFNg-201AAY, IFNg-201AAB, IFNg-201ADD, IFNg-201AYD, and IFNg-201ABD) for human IFNγ by EMSA method.

[0085] Figure 27 It is a graph showing the results of analyzing the detection sensitivity for human IFNγ of 4 nucleic acid aptamers (B-I-Apt1-DD, B-I-Apt1-YY, B-IFNg-201AAD, B-IFNg-201AAB, and B-IFNg-201AYD) by ELISA method. Nucleic acid aptamers were used for IFNγ capture, and B133.5 antibody was used as the detection antibody. As a control, the results using 2G1 antibody as the capture antibody and Bio-B133.5 antibody as the detection antibody are shown in the right column.

[0086] Figure 28 It is a graph showing the results for Figure 19 the same 4 nucleic acid aptamers as above, analyzing the detection sensitivity for human IFNγ by ELISA method. B133.5 antibody was used as the IFNγ capture antibody, and nucleic acid aptamers were used for detection. As a control, the results using B133.5 antibody as the capture antibody and Bio-2G1 antibody as the detection antibody are shown in the right column.

[0087] Figure 29This is a graph showing the results of analyzing the detection sensitivity for human IFNγ by ELISA using two nucleic acid aptamers (B-IFNg-201AAD and B-IFNg-201AAB). The B133.5 antibody was used as the IFNγ capture antibody, and the nucleic acid aptamers shown in the figure were used for detection. As a control, the results using the 2G1 antibody as the capture antibody and the Bio-B133.5 antibody as the detection antibody are shown in the right column.

[0088] Figure 30 This is a graph showing the structure of the AptD1c(DsDs) aptamer that binds to the DEN1-NS1 protein as a target.

[0089] Figure 31 This is a graph showing the results of analyzing the affinity of eight nucleic acid aptamers (AptD1c(DsDs), AptD1c2(DsDs), AptD1c2(AA), AptD1c2a(YsYs), AptD1c2b(YsBs), AptD1c2c(BsYs), and AptD1c2d(BsBs)) for the DEN1-NS1 protein by EMSA.

[0090] Figure 32 This is a graph showing the results of analyzing the affinity of six nucleic acid aptamers (AptD2-b1, AptD2-b2, AptD2-b3, AptD2-b4, AptD2bb(Ys), and AptD2bb(Bs)) for DEN2-NS1 protein, DEN1-NS1 protein, Zika NS1 protein, etc. by EMSA.

[0091] Figure 33 This is a graph showing the results of analyzing the affinity of five nucleic acid aptamers (AptD4-1(DsDs), AptD4-2(YsYs), AptD4-3(YsBs), AptD4-4(BsYs), and AptD4-5(BsBs)) for the DENV4 NS1 protein by EMSA.

[0092] Figure 34 This is a graph showing the results of analyzing the affinity of seven nucleic acid aptamers (V-Apt1(DsDs), V-Apt4(AA), b58(YsYs), c58(YsBs), d58(BsYs), e58(BsBs), and f58(YoYo)) for human VEGF 165 protein by EMSA.

[0093] Figure 35This is a figure showing the results of analyzing the affinity of six nucleic acid aptamers (MB45-Ds, MB45-Ys, MB45-Yo, MB45-Bs, MB45-Bo, and MB45-A) for human transferrin receptor 1 (TrfR1) using EMSA for display. Detailed implementation mode

[0094] <Definition of terms>

[0095] The definitions of general terms used in this specification are described below.

[0096] In this specification, the so-called "nucleic acid" or "nucleic acid molecule" generally refers to a polymer composed of nucleosides as constituent units, with these nucleosides linked by internucleoside bonds.

[0097] In this specification, the so-called "natural nucleoside" refers to a nucleoside that exists in nature. Examples include ribonucleosides formed by ribose and bases such as adenine, cytosine, guanine, or uracil, and deoxyribonucleosides formed by deoxyribose and the aforementioned bases such as adenine, cytosine, guanine, or thymine.

[0098] In this specification, the so-called "unnatural nucleoside" refers to any nucleoside other than natural nucleosides, including modified nucleosides and nucleoside mimics.

[0099] In this specification, the so-called "modified nucleoside" refers to a nucleoside having a modified moiety such as a modified base and / or a modified sugar moiety.

[0100] In this specification, the so-called "nucleotide" refers to a molecule in which a phosphate group is covalently bonded to the sugar moiety of a nucleoside. In the case of nucleotides containing furanose pentose, generally, a phosphate group is attached to the hydroxyl group at the 2', 3', or 5' position of the sugar. Phosphoramidites used as raw materials for nucleic acid synthesis are included in nucleotides.

[0101] In this specification, the so-called "oligonucleotide" refers to a linear oligomer formed by covalently bonding the hydroxyl group of the sugar moiety and the phosphate group between adjacent nucleotides in several to dozens. Additionally, the so-called "polynucleotide" refers to a linear polymer formed by covalently bonding more nucleotides than oligonucleotides in dozens or more, preferably hundreds or more. Inside the structure of natural oligonucleotides or polynucleotides, phosphate groups generally form internucleoside bonds.

[0102] As used in this specification, the term "natural base" refers to naturally occurring adenine, cytosine, guanine, thymine, and uracil, as well as modified bases of any of them. In addition, in the base sequences in this specification, purine bases selected from adenine and guanine are expressed as "R", and pyrimidine bases selected from cytosine, thymine, and uracil are expressed as "Y".

[0103] As used in this specification, the term "unnatural base" or "artificial base" refers to any nucleic acid base other than natural bases. Unnatural bases or artificial bases can replace natural bases that make up natural nucleosides. In addition, the unnatural bases or artificial bases referred to in this specification can either form artificial base pairs or not form artificial base pairs.

[0104] As used in this specification, the term "artificial base pair" refers to a pair of artificial bases that can form base pairing, such as adenine and thymine, adenine and uracil, or guanine and cytosine of natural bases. The base pairing formed by artificial base pairs can include hydrogen bonds also visible in the base pairing between natural bases, stacking effects mediated by hydrophobic interactions, etc. Artificial base pairs that can base pair can sometimes be correctly replicated and transcribed through the complementarity between artificial bases, and nucleic acid chains containing artificial bases can also be amplified by nucleic acid amplification methods such as PCR.

[0105] As specific examples of the above artificial bases, unnatural bases represented by the following general formula (I) and / or general formula (II) can be cited, as well as Ds (7-(2-thienyl)-3H-imidazo[4,5-b]pyridin-3-yl; referred to as "Ds" in this specification), Pn (2-nitropyrrol-1-yl; referred to as "Pn" in this specification), Pa (2-formyl-1H-pyrrol-1-yl; referred to as "Pa" in this specification), P (2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one; referred to as "P" in this specification), Z (6-amino-5-nitro-2(1H)-pyridone; referred to as "Z" in this specification), 5SICS (6-methylisoquinoline-1(2H)-trione; referred to as "5SICS" in this specification), NaM (3-methoxynaphthalen-2-yl; referred to as "NaM" in this specification), and MMO2 (2-methoxy-4-methylphenyl; referred to as "MMO2" in this specification). Among these artificial bases, complementary artificial bases of Ds can include Pn and Pa. Complementary artificial bases of P can include Z. Complementary artificial bases of 5SICS can include NaM and MMO2.

[0106] When an unnatural nucleoside having a complementary artificial base is not included in the substrate during replication or transcription, the artificial base sometimes base-pairs with a natural base that is structurally and / or functionally similar to the complementary artificial base instead. In such a case, the unnatural nucleoside in the nucleic acid molecule serving as the template is replaced with a natural nucleoside after replication or transcription. For example, in the case of Ds described above, it is known to be replaced with A or T.

[0107] As used herein, the term "modified base" refers to a base that has been chemically modified. Examples of modified bases include modified pyrimidines (e.g., 5-hydroxycytosine, 5-fluorouracil, 4-thiouracil, 5-(3-indol-2-yl)uracil, 5-(4-hydroxyphenyl-2-yl)uracil), modified purines (e.g., 6-methyladenine, 6-thioguanosine), and other heterocyclic bases.

[0108] As used herein, the term "stem structure" or "stem region" refers to a double-stranded structure formed by complete or partial base pairing between a part of the bases, e.g., two or more consecutive bases. The length of the stem structure is, for example, 1 bp or more, 2 bp or more, 3 bp or more, 4 bp or more, or 5 bp or more, and / or 30 bp or less, 25 bp or less, 20 bp or less, 15 bp or less, 10 bp or less, or 8 bp or less. Examples of stem structures include those formed by base pairing of two base sequences represented by SEQ ID NO: 105 and 106, SEQ ID NO: 107 and 108, SEQ ID NO: 109 and 110, SEQ ID NO: 111 and 112, SEQ ID NO: 113 and 114, SEQ ID NO: 115 and 116, SEQ ID NO: 117 and 118, SEQ ID NO: 119 and 120, SEQ ID NO: 121 and 122, SEQ ID NO: 123 and 124, SEQ ID NO: 125 and 126, SEQ ID NO: 127 and 128, SEQ ID NO: 129 and 130, SEQ ID NO: 131 and 132, SEQ ID NO: 133 and 134, SEQ ID NO: 135 and 136, or SEQ ID NO: 138 and 139.

[0109] In this specification, the "miniature hairpin structure" or "miniature hairpin" has a structure in which three DNA nucleic acid regions, namely, a first nucleic acid region, a second nucleic acid region, and a third nucleic acid region described below, are sequentially connected from the 5'-terminal side to the 3'-terminal side. The so-called "first nucleic acid region" is a nucleic acid region composed of 2 to 5 arbitrary nucleotides. The bases of this nucleic acid region are not limited, but guanine or cytosine is preferred. The so-called "second nucleic acid region" is a nucleic acid region having a base sequence of 5'-gna-3' or 5'-gnna-3'. Each n in the sequence independently consists of any one of natural bases, the above-mentioned base analogs, or modified bases. The so-called "third nucleic acid region" is a nucleic acid region having a base sequence complementary to the first nucleic acid region. Therefore, the base sequence of the third nucleic acid region is determined by the base sequence of the first nucleic acid region, and the first nucleic acid region and the third nucleic acid region form base pairs within the molecule. As a result, the first nucleic acid region and the third nucleic acid region form a stem portion in which complete base pairing is achieved with each other, and the second nucleic acid region existing between the first nucleic acid region and the third nucleic acid region constitutes a loop portion. As an example of the miniature hairpin sequence, 5'-CGCGTAGCG-3' (SEQ ID NO: 214; the base T can be biotinylated) can be cited. The miniature hairpin structure can improve the degradation resistance to nucleic acid degrading enzymes and / or increase the Tm value of the DNA aptamer, thereby increasing the thermal stability of the DNA aptamer.

[0110] In this specification, the so-called "internal loop structure" refers to a loop structure within a stem structure generated when there are one or more bases that do not form base pairs at corresponding positions on both strands forming the stem structure.

[0111] In this specification, the so-called "bulge structure" refers to a protruding structure within a stem structure generated when there are one or more bases that do not form base pairs on only one strand at corresponding positions on the double strand forming the stem structure.

[0112] In this specification, the so-called "loop structure" refers to a circular structure within a nucleic acid that is located between the double strands constituting the stem structure and is generated by the formation of the stem structure and does not form base pairs.

[0113] In this specification, the "hairpin structure" or "stem-loop structure" refers to a structure composed of one stem structure and one loop structure (a set of stem structure and loop structure).

[0114] As used in this specification, the term "target molecule" refers to a substance that can be a binding target of an aptamer. The types of target molecules are not particularly limited as long as they are biological substances to which aptamers can bind. Examples include proteins such as peptides (oligopeptides or polypeptides), nucleic acids, lipids, sugars (including sugar chains), and low molecular weight compounds. In principle, the molecule to which the aptamer of the present invention binds as a target is a protein, and thus the target molecule is often referred to as "target protein" in this specification.

[0115] The term "complementary" as used in this specification refers to a relationship in which nucleic acid bases can form so-called Watson-Crick base pairs (natural base pairs) or non-Watson-Crick base pairs (Hoogsteen base pairs, etc.) via hydrogen bonds. In the present invention, a base sequence is acceptable as long as it has at least 80%, preferably at least 90% (for example, 95%, 96%, 97%, 98%, or 99% or more) complementarity. The complementarity of a base sequence can be determined by using a BLAST program or the like.

[0116] As used in this specification, the term "plurality" means, for example, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, or 2.

[0117] <Aptamer>

[0118] In one aspect of the present invention, an aptamer is provided.

[0119] As used in this specification, the term "aptamer" is an aptamer composed of nucleic acid molecules, and is a ligand molecule that binds firmly and specifically to a target molecule through a three-dimensional structure formed based on the secondary structure and then the tertiary structure of single-stranded nucleic acid molecules via hydrogen bonds, etc. When the aptamer of the present invention has the ability to specifically inhibit or suppress the physiological activity of a target molecule, etc., the aptamer can become a functional inhibitor of the target molecule. As used in this specification, the term "functional inhibition of a target molecule" refers to inhibiting or suppressing the function of a target molecule, such as a catalytic function or a gene expression control function. As used in this specification, the term "target molecule" refers to a substance that can be a binding target of an aptamer. The type of nucleic acid constituting the aptamer in this specification is not particularly limited, and it can be, for example, a DNA aptamer composed only of DNA, an RNA aptamer composed only of RNA, or a nucleic acid aptamer composed of a combination of DNA and RNA.

[0120] The aptamer of the present invention contains at least one unnatural base represented by the following general formula (I) and / or general formula (II).

[0121]

[0122] [In the above formula, R1 represents the sugar moiety in a nucleoside or C 1 or C 2 alkyl, and R 2 represents any one of the following formulas (III) to (IX):

[0123]

[0124] In the nucleic acid aptamer of the present invention, R in the above general formula (I) and / or general formula (II) 1 represents the sugar moiety in a nucleoside or C 1 or C 2 alkyl. When the nucleic acid aptamer is a DNA aptamer, the sugar moiety in the nucleoside is a deoxyribose moiety, and the unnatural base is bound to the 1'-position carbon atom in the deoxyribose. When the nucleic acid aptamer is an RNA aptamer, the sugar moiety in the nucleoside is a ribose moiety, and the unnatural base is bound to the 1'-position carbon atom in the ribose.

[0125] As used in this specification, "alkyl" refers to a saturated hydrocarbon having one or more carbon atoms. Specific examples of the alkyl include, for example, methyl, ethyl, propyl, butyl, etc. "C 1 or C 2 alkyl" has 1 or 2 carbon atoms, and in this case, the alkyl is methyl or ethyl.

[0126] As an example of the substitution of the hydrogen group in the alkyl, substitution with a hydroxyl group, a carboxyl group, a cyano group, a nitro group, a halogen (for example, Cl, F, Br, or I), a thiol group, an alkoxy group, an ester group, a thioether group, a thioester group, a nitro group, or an amine group, etc. can be cited. The number of substitutions of the hydrogen group in the alkyl is not limited, and can be, for example, 1 or 2 or more substitutions.

[0127] In the nucleic acid aptamer of the present invention, the unnatural bases represented by the above general formula (I) and / or general formula (II) are not limited as long as there is one or more, and can be, for example, one or more, two or more, three or more, or four or more, and / or can be 20 or less, 10 or less, 8 or less, 7 or less, 6 or less, or 5 or less. For example, the nucleic acid aptamer of the present invention contains 1 to 9, 1 to 6, or 1 to 3, for example, 1, 2, 3, or 4, of the unnatural bases represented by the above general formula (I) and / or general formula (II).

[0128] In addition, in the nucleic acid aptamer of the present invention, the positions of the unnatural bases represented by the above general formula (I) and / or general formula (II) are not limited, and can be, for example, at the 5'-end and / or 3'-end of the nucleic acid chain constituting the nucleic acid aptamer, and / or inside the nucleic acid chain.

[0129] In one embodiment, the aptamer of the present invention comprises unnatural bases represented by the above general formula (I) and general formula (II). In this embodiment, the number of unnatural bases represented by the above general formula (I) and general formula (II) is not limited as long as it is 1 or more respectively. For example, it is 1 or more and 20 or less respectively, and more specifically, it can be 1, 2, 3, or 4 respectively.

[0130] In one embodiment, the unnatural bases represented by the above general formula (I) and general formula (II) in the aptamer of the present invention are any unnatural bases other than the unnatural base represented by the following formula (XVIII) (referred to as "Ds" in this specification).

[0131]

[0132] In one embodiment, the unnatural bases represented by the above general formula (I) and / or general formula (II) in the aptamer of the present invention can be any of the unnatural bases represented by the following formula (X) to formula (XVII) and formula (XXI) to formula (XXII). In addition, in this specification, the unnatural base represented by the following formula (X) is referred to as "Ys", the unnatural base represented by the following formula (XI) is referred to as "Yi", the unnatural base represented by the following formula (XII) is referred to as "Yo", the unnatural base represented by the following formula (XIII) is referred to as "Ysm", the unnatural base represented by the following formula (XIV) is referred to as "Yss", the unnatural base represented by the following formula (XV) is referred to as "Bs", the unnatural base represented by the following formula (XVI) is referred to as "Bss", the unnatural base represented by the following formula (XVII) is referred to as "Bo", the unnatural base represented by the following formula (XXI) is referred to as "Dss", and the unnatural base represented by the following formula (XXII) is referred to as "Dt".

[0133]

[0134] In one embodiment, in addition to the unnatural bases represented by the above general formula (I) and / or general formula (II), the aptamer of the present invention further comprises the unnatural base represented by the above formula (XVIII) (Ds). In this embodiment, the number of Ds is not limited as long as it is 1 or more, and can be, for example, 1 or more and 20 or less, and more specifically, it can be 1, 2, 3, or 4.

[0135] In one embodiment, the aptamer of the present invention is composed of a 5'-region, a central region, and a 3'-region. As used herein, the "5'-region" and "3'-region" are regions that are respectively disposed on the 5'-terminal side and 3'-terminal side of the aptamer and contain a pair of base sequences complementary to each other so as to form a stem structure. As used herein, the "central region" is a region that is disposed between the 5'-region and the 3'-region and mainly functions to bind to a target molecule. In principle, the aptamer of the present invention can contain the unnatural bases represented by the above general formula (I) and / or general formula (II) at least in the central region.

[0136] In one embodiment, the aptamer of the present invention contains a microhairpin sequence on its 5'-terminal side, 3'-terminal side, and / or within its sequence, for example, on the 3'-terminal side. A modification group such as biotin can be bound to the microhairpin sequence. For example, the aptamer of the present invention sequentially contains the above-mentioned 5'-region, central region, and 3'-region, and a microhairpin sequence from the 5'-side, or sequentially contains a microhairpin sequence, and the above-mentioned 5'-region, central region, and 3'-region from the 5'-side.

[0137] In addition, in one embodiment, the aptamer of the present invention contains an internal loop structure, a bulge structure, a loop structure, a hairpin structure, and / or a stem-loop structure at a position that does not affect its binding ability to the target protein.

[0138] The length of the aptamer of the present invention is, for example, 10 mer or more, 15 mer or more, 20 mer or more, 30 mer or more, 40 mer or more, 50 mer or more, 60 mer or more, 70 mer or more, 80 mer or more, 90 mer or more, or 100 mer or more, and / or 300 mer or less, 200 mer or less, 150 mer or less, 140 mer or less, 130 mer or less, 120 mer or less, 110 mer or less, 100 mer or less, 90 mer or less, 80 mer or less, 70 mer or less, 60 mer or less, or 50 mer or less.

[0139] In addition to the above-mentioned unnatural bases and / or Ds, the aptamer of the present invention can optionally contain base analogs, other artificial bases, or other modified bases, etc.

[0140] The aptamers of the present invention can be modified by adding other substances, such as polyethylene glycol (PEG) (e.g., a PEG polymer of about 20 to about 60 kDa), amino acids, peptides, inverted dT, lipids, pigments, fluorescent substances, enzymes, radioactive substances, biotin, etc. These substances can be linked via known linkers as needed. Examples of the linkers in this specification include nucleotide linkers, peptide linkers, and linkers containing disulfide bonds. It is known that by linking PEG, the half-life of DNA aptamers can generally be extended. In addition, the modification position of the aptamers of the present invention is not limited and can be, for example, the 5'-terminal portion, the 3'-terminal portion, or the inside of the aptamer sequence. For example, a microhairpin sequence can be modified.

[0141] The method for producing the aptamers of the present invention is not particularly limited. A method well known in the art can be used. For example, the aptamers of the present invention can be chemically synthesized based on the above sequences according to a known solid-phase synthesis method. Regarding the chemical synthesis method of nucleic acids, refer to, for example, Current Protocols in Nucleic Acid Chemistry, Volume 1, Section 3. In addition, for example, after synthesizing several fragments based on the sequence of the aptamer, the fragments can be ligated by intramolecular annealing, ligation using a ligase, etc. to produce the aptamer. The aptamers of the present invention after chemical synthesis are preferably purified by a method well known in the art before use. Examples of the purification method include gel purification, affinity column purification, HPLC method, etc.

[0142] Furthermore, according to the present invention, there are also provided a nucleoside or nucleotide containing an unnatural base represented by the above general formula (I) or general formula (II), and a nucleic acid (e.g., DNA or RNA) containing an unnatural base represented by the above general formula (I) and / or general formula (II). For example, there are also provided a nucleoside or nucleotide (e.g., amidite) containing an unnatural base represented by any one of the above formula (XI) to formula (XIII) and the above formula (XV) to formula (XVII), and a nucleic acid (e.g., an oligonucleotide or polynucleotide such as DNA or RNA) containing an unnatural base represented by the above formula (XI) to formula (XIII) and the above formula (XV) to formula (XVII).

[0143] The following (1) to (10) specifically describe the constitution of the aptamers of the present invention for each binding target.

[0144] <(1) Aptamer that binds to vWF>

[0145] In a further aspect of the aptamers of the present invention, there is provided an aptamer that binds to vWF protein.

[0146] In the present specification, "vWF" refers to von Willebrand Factor protein (also expressed as "vWF protein" in the present specification). It is known that vWF is one of the blood coagulation factors present in blood, and its gene mutations are associated with various diseases such as von Willebrand disease. Acquired thrombotic thrombocytopenic purpura, etc. are caused by the production of autoantibodies against vWF. In the present invention, the biological species that is the source of vWF is not limited, and examples include mammals such as primates like humans and chimpanzees, experimental animals such as rats and mice, domestic animals such as pigs, cows, horses, sheep, and goats, and pets such as dogs and cats. Preferably, it is human vWF.

[0147] The nucleic acid aptamer of the present scheme contains the base sequence shown in SEQ ID NO: 205, 206, or 187, or a sequence obtained by adding a pair of mutually complementary base sequences capable of forming a stem structure to the 5'-end side and 3'-end side of the base sequence shown in SEQ ID NO: 205, 206, or 187, and at least one of the bases represented by n in the base sequence is the unnatural base represented by the above general formula (I) and / or general formula (II). For example, at least one of the bases represented by n in the base sequence is any of the unnatural bases represented by formula (X) to formula (XVII) and formula (XXI) to formula (XXII). When two or more of the bases represented by n in the base sequence are the above-mentioned any unnatural bases, they can be independently selected from the unnatural bases represented by the above general formula (I) and / or general formula (II), such as the unnatural bases represented by the above general formula (X) to formula (XVII) and formula (XXI) to formula (XXII).

[0148] The base sequence shown in SEQ ID NO: 206 (5'-GAnGAGYACCTAACnGGTCTCRRYRnYGGA-3') is included in Figure 1 (b) the base sequence of "Anti-vWF" in the literature (Kimoto M,.et al., bioRxiv 2022.06.27.497860), and Figure 2(c) shows the sequences after removing the stem regions composed of 7 bases each at the 5'-end and 3'-end from the base sequences (SEQ ID NOs: 9 to 14) of substitutions (C14T, A29G, A30G, C31T, G32A, and T34C) that do not impair the binding activity. The base sequence shown in SEQ ID NO: 205 (5'-GAnGAGYACCGAAGGTCTCRRYRnY-3') is included in the base sequence of the nucleic acid aptamer vWF2-DsDsDs-2mhGC shown in SEQ ID NO: 21 in WO2017 / 073536, and in the sequence obtained by extending the positions corresponding to the above-mentioned allowable substitutions (C14T, A29G, A30G, C31T, G32A, and T34C) to allowable bases, the sequence after removing the stem regions composed of 7 bases each at the 5'-end and 3'-end. The base sequence shown in SEQ ID NO: 187 (5'-GAnGAGCACCGAAGGTCTCAACGnTGGA-3') corresponds to the central region after removing the stem region and the mini-hairpin sequence from the base sequence of nucleic acid aptamers such as AptvWF-BsBs in (2) of Example 3, is included in the base sequence shown in SEQ ID NO: 205, and is a sequence obtained by adding the base sequence GGA to the 3'-end side of the base sequence in which purine bases and pyrimidine bases are specified as specific bases.

[0149] The nucleic acid aptamer that binds to vWF protein contains at least 1 non-natural base shown in the above general formula (I) and / or general formula (II) as the base shown as n in the above base sequence, for example, it contains 2, 3, or 4.

[0150] In a further embodiment, the nucleic acid aptamer that binds to vWF protein may contain Ds as at least 1 of the bases shown as n in the base sequence.

[0151] In one embodiment of the present solution, the non-natural base shown in the above general formula (I) and / or general formula (II) is any one of those shown in the above formula (X) to formula (XVII) and formula (XXI) to formula (XXII).

[0152] In a further embodiment of the present solution, the nucleic acid aptamer that binds to vWF protein is selected from the group consisting of the following (1-a) to (1-d):

[0153] (1-a) A nucleic acid aptamer containing the base sequence shown in SEQ ID NO: 1 or 3, wherein the bases shown as n at positions 10 and 31 in the base sequence are respectively (I) base Bs and base Bs, (II) base Ys and base Ys, (III) base Yo and base Yo, (IV) base Bs and base Ys, (V) base Bs and base Yo, or (VI) base Ys and base Bs;

[0154] (1-b) A nucleic acid aptamer comprising a base sequence in SEQ ID NO: 1 or 3 that has one or more bases deleted, substituted, or added except at positions 10 and 31, wherein the positions corresponding to the bases shown as n at positions 10 and 31 of SEQ ID NO: 1 or 3 are respectively (I) base Bs and base Bs, (II) base Ys and base Ys, (III) base Yo and base Yo, (IV) base Bs and base Ys, (V) base Bs and base Yo, or (VI) base Ys and base Bs;

[0155] (1-c) A nucleic acid aptamer that sequentially includes a 5'-region, a central region, and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region include a pair of complementary base sequences capable of forming a stem structure, the central region includes the base sequence shown in SEQ ID NO: 205 or 187, and the bases shown as n at positions 3 and 24 in the base sequence shown in SEQ ID NO: 205 or 187 are respectively (I) base Bs and base Bs, (II) base Ys and base Ys, (III) base Yo and base Yo, (IV) base Bs and base Ys, (V) base Bs and base Yo, or (VI) base Ys and base Bs;

[0156] (1-d) A nucleic acid aptamer that sequentially includes a 5'-region, a central region, and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region include a pair of complementary base sequences capable of forming a stem structure, the central region includes a base sequence in SEQ ID NO: 205 or 187 that has one or more bases deleted, substituted, or added except at positions 3 and 24, and the positions corresponding to the bases shown as n at positions 3 and 24 in the base sequence in the central region are respectively (I) base Bs and base Bs, (II) base Ys and base Ys, (III) base Yo and base Yo, (IV) base Bs and base Ys, (V) base Bs and base Yo, or (VI) base Ys and base Bs.

[0157] In addition, the above SEQ ID NOs: 205 and 187 are sequences obtained by adding a pair of complementary base sequences capable of forming a stem structure to the 5'-terminal side and 3'-terminal side of SEQ ID NOs: 1 and 3, respectively. Additionally, the base sequence shown in SEQ ID NO: 3 is included in the base sequence shown in SEQ ID NO: 1, and purine bases and pyrimidine bases are specified as specific bases.

[0158] <(2) Nucleic acid aptamer that binds to IFNγ>

[0159] In a further aspect of the nucleic acid aptamer of the present invention, a nucleic acid aptamer that binds to IFNγ protein is provided.

[0160] In the present specification, "IFNγ (Interferon γ)" is a cytokine secreted by immune cells such as T cells and NK cells and produced in the immune response against infections by viruses, bacteria, etc. In the present invention, the biological species that can be the source of IFNγ is not limited, and examples include mammals, such as primates like humans and chimpanzees, experimental animals like rats and mice, domestic animals like pigs, cows, horses, sheep, and goats, and pets like dogs and cats, and preferably human IFNγ.

[0161] The nucleic acid aptamer of the present solution comprises the base sequence shown in SEQ ID NO: 207, 208, 188, 56, 59, 61, 65, 67, 189, or 213, or a sequence obtained by adding a pair of base sequences complementary to each other capable of forming a stem structure to the 5'-terminal side and 3'-terminal side of the base sequence shown in SEQ ID NO: 207, 208, 188, 56, 59, 61, 65, 67, 189, or 213, and at least one of the bases represented by n in the base sequence is the unnatural base shown in the above general formula (I) and / or general formula (II). For example, at least one of the bases represented by n in the base sequence is any of the unnatural bases shown in formula (X) to formula (XVII) and formula (XXI) to formula (XXII). When two or more of the bases represented by n in the base sequence are the above-mentioned any unnatural base, they can be independently selected from the unnatural bases shown in the above general formula (I) and / or general formula (II), such as the unnatural bases shown in the above general formula (X) to formula (XVII) and formula (XXI) to formula (XXII).

[0162] The base sequence shown in SEQ ID NO: 188 (5'-CGGGTCCGCGAAGCGGTAGGTnTGGGCTAGGCnGCT-3') corresponds to the central region excluding the stem region and the mini-hairpin sequence in the base sequence of nucleic acid aptamers such as AptIFNγ-YoYo in the result (1) of Example 3. The base sequence shown in SEQ ID NO: 207 (5'-CGGGTCCGCGAAGCGGTAGGTnYGGGCTAGGCnGYY-3') is included in the Figure 1 (b) base sequence of "Anti-vWF" in the literature (Kimoto M,.et al., bioRxiv 2022.06.27.497860), and includes the Figure 2(b) shows the base sequences of substitutions (T30C, C42T, T43C) that do not impair the binding activity (SEQ ID NOs: 23-25), excluding the stem regions each consisting of 6 bases at the 5'-end and 3'-end. The base sequence shown in SEQ ID NO: 208 (5'-CGGGTCATTTAnTAATGTAGGTnYGGGCTAGGCnGYY-3') contains the Figure 1 (b) the base sequence of "Anti-IFNγ", and the sequences in which the positions corresponding to the above-mentioned allowable substitutions are extended to allowable bases. The base sequence shown in SEQ ID NO: 56 (5'-TGTGGTGGGACnGGGCTnATGTAnGGGATCT-3'), the base sequence shown in SEQ ID NO: 59 (5'-TGTGGTGGGACTGGGCTnATGTAnGGGATCT-3'), and the base sequence shown in SEQ ID NO: 61 (5'-TGTGGTGGGACnGGGCTAATGTAnGGGATCT-3') correspond to the central regions of the base sequences of nucleic acid aptamers such as AptIFNγ-1 in (b) of Example 4, excluding the sequences containing the terminal portions of the stem regions. The base sequence shown in SEQ ID NO: 65 (5'-CCGTCATnATTGTTnTTTTGATCCTTGnAGTGGGGA-3') and the base sequence shown in SEQ ID NO: 67 (5'-CCGTCATAnTTGTTnTTTTGATCCTTGnAGTGGGGA-3') correspond to the central regions of the base sequences of nucleic acid aptamers such as XL2-01a in (c) of Example 4, excluding the sequences containing the terminal portions of the stem regions. The base sequence shown in SEQ ID NO: 189 (5'-CCGTCATAATTGTTnTTTTGATCCTTGnAGTGGGGA-3') and the base sequence shown in SEQ ID NO: 213 (5'-CCGTCATAATTGTTATTTTGATCCTTGnAGTGGGGA-3') correspond to the central regions of the stem regions removed from the base sequences of nucleic acid aptamers such as IFNg-201AAY in Example 7.

[0163] The nucleic acid aptamer that binds to IFNγ contains at least 1 unnatural base represented by the above general formula (I) and / or general formula (II) as the base represented by n in the above base sequence, for example, contains 2, 3, or 4.

[0164] In a further embodiment, the nucleic acid aptamer that binds to IFNγ contains Ds as at least 1 of the bases represented by n in the base sequence.

[0165] In one embodiment of the present solution, the unnatural base represented by the above general formula (I) and / or general formula (II) is any one of those represented by the above formula (X) to formula (XVII) and formula (XXI) to formula (XXII).

[0166] In a further embodiment of the present solution, the nucleic acid aptamer that binds to IFNγ is selected from the group consisting of the following (2-i-a) to (2-iii-d):

[0167] (2-i-a) A nucleic acid aptamer containing the base sequence shown in SEQ ID NO: 15 or 17, wherein the bases represented by n at positions 28 and 39 in the base sequence are (I) base Ys and base Ys, or (II) base Yo and base Yo;

[0168] (2-i-b) A nucleic acid aptamer containing a base sequence in which one or more bases are deleted, substituted, or added at positions other than positions 28 and 39 in SEQ ID NO: 15 or 17, and the positions corresponding to the bases represented by n at positions 28 and 39 in SEQ ID NO: 15 or 17 in the base sequence are (I) base Ys and base Ys, or (II) base Yo and base Yo;

[0169] (2-i-c) A nucleic acid aptamer that sequentially includes a 5'-region, a central region, and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region include a pair of complementary base sequences capable of forming a stem structure, the central region includes the base sequence shown in SEQ ID NO: 207 or 188, and the bases represented by n at positions 22 and 33 in the base sequence shown in SEQ ID NO: 207 or 188 are (I) base Ys and base Ys, or (II) base Yo and base Yo;

[0170] (2-i-d) A nucleic acid aptamer that sequentially includes a 5'-region, a central region, and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region include a pair of complementary base sequences capable of forming a stem structure, the central region includes a base sequence in which one or more bases are deleted, substituted, or added at positions other than positions 22 and 33 in SEQ ID NO: 207 or 188, and the positions corresponding to the bases represented by n at positions 22 and 33 in SEQ ID NO: 207 or 188 in the base sequence in the central region are (I) base Ys and base Ys, or (II) base Yo and base Yo;

[0171] (2-ii-a) A nucleic acid aptamer containing the base sequence shown in SEQ ID NO: 62, wherein the bases represented by n at positions 24 and 37 in the base sequence are (I) base Ys and base Ds, or (II) base Bs and base Ds;

[0172] (2-ii-b) A nucleic acid aptamer comprising a base sequence in SEQ ID NO: 62 in which one or more bases are deleted, substituted, or added except at positions 24 and 37, wherein the positions corresponding to the bases shown as n at positions 24 and 37 of SEQ ID NO: 62 are (I) base Ys and base Ds, or (II) base Bs and base Ds;

[0173] (2-ii-c) A nucleic acid aptamer comprising a 5'-region, a central region, and a 3'-region in that order from the 5'-side, wherein the 5'-region and the 3'-region each contain a pair of complementary base sequences capable of forming a stem structure, the central region contains the base sequence shown in SEQ ID NO: 189, and the bases shown as n at positions 15 and 28 in the base sequence shown in SEQ ID NO: 189 are (I) base Ys and base Ds, or (II) base Bs and base Ds;

[0174] (2-ii-d) A nucleic acid aptamer comprising a 5'-region, a central region, and a 3'-region in that order from the 5'-side, wherein the 5'-region and the 3'-region each contain a pair of complementary base sequences capable of forming a stem structure, the central region contains a base sequence in which one or more bases are deleted, substituted, or added except at positions 15 and 28 in SEQ ID NO: 189, and the positions corresponding to the bases shown as n at positions 15 and 28 of SEQ ID NO: 189 in the base sequence in the central region are (I) base Ys and base Ds, or (II) base Bs and base Ds;

[0175] (2-iii-a) A nucleic acid aptamer comprising the base sequence shown in SEQ ID NO: 212, wherein the base shown as n at position 37 in the base sequence is base Ys or base Bs;

[0176] (2-iii-b) A nucleic acid aptamer comprising a base sequence in which one or more bases are deleted, substituted, or added except at position 37 in SEQ ID NO: 212, and the position corresponding to the base shown as n at position 37 of SEQ ID NO: 212 in the base sequence is base Ys or base Bs;

[0177] (2-iii-c) A nucleic acid aptamer comprising a 5'-region, a central region, and a 3'-region in that order from the 5'-side, wherein the 5'-region and the 3'-region each contain a pair of complementary base sequences capable of forming a stem structure, the central region contains the base sequence shown in SEQ ID NO: 213, and the base shown as n at position 28 in the base sequence shown in SEQ ID NO: 213 is base Ys or base Bs;

[0178] (2-iii-d) A nucleic acid aptamer that sequentially includes a 5' region, a central region, and a 3' region starting from the 5' side. The 5' region and the 3' region contain a pair of base sequences that are complementary to each other and can form a stem structure. The central region contains a base sequence in which 1 or more bases are deleted, substituted, or added except at position 28 in SEQ ID NO: 213. At the position corresponding to the base shown as n at position 28 of SEQ ID NO: 213 in the base sequence in the central region, the base is base Ys or base Bs.

[0179] In addition, the above SEQ ID NOs: 15, 17, 62, and 212 are sequences obtained by adding a pair of complementary sequences that can form a stem structure to the 5' end side and the 3' end side of SEQ ID NOs: 207, 188, 189, and 213, respectively. Additionally, the base sequence shown in SEQ ID NO: 17 is included in the base sequence shown in SEQ ID NO: 15, and purine bases and pyrimidine bases are specified as specific bases.

[0180] <(3) Nucleic acid aptamer that binds to thrombin>

[0181] In a further aspect of the nucleic acid aptamer of the present invention, a nucleic acid aptamer that binds to thrombin is provided.

[0182] As used in this specification, "thrombin" is a serine protease that plays a central role in hemostasis through the conversion of fibrinogen to fibrin. In the present invention, the biological species that is the source of thrombin is not limited, and examples include mammals such as primates such as humans and chimpanzees, experimental animals such as rats and mice, domestic animals such as pigs, cows, horses, sheep, and goats, and pets such as dogs and cats. Preferably, it is human thrombin.

[0183] The nucleic acid aptamer of this aspect includes the base sequence shown in SEQ ID NO: 190, or a sequence obtained by adding a pair of complementary base sequences that can form a stem structure to the 5' end side and the 3' end side of the base sequence shown in SEQ ID NO: 190. At least 1 of the bases shown as n in the base sequence is a non-natural base represented by the above general formula (I) and / or general formula (II). For example, at least 1 of the bases shown as n in the base sequence is any non-natural base represented by formula (X) to formula (XVII) and formula (XXI) to formula (XXII). When 2 or more of the bases shown as n in the base sequence are the above non-natural bases, they can be independently selected from the non-natural bases represented by the above general formula (I) and / or general formula (II), such as the non-natural bases represented by the above formula (X) to formula (XVII) and formula (XXI) to formula (XXII).

[0184] The base sequence shown by serial number 190 (5'-TGTGGTnGTTTTnTTGGGGGGGTGGTTAAG-3') is for the central region other than the stem region in the base sequences of nucleic acid aptamers such as Thr204-BB in Example 5.

[0185] The nucleic acid aptamer that binds to thrombin contains at least 1 unnatural base shown by the above general formula (I) and / or general formula (II) as the base shown by n in the above base sequence, for example, it contains 2, 3, or 4.

[0186] In a further embodiment, the nucleic acid aptamer that binds to thrombin may contain Ds as at least 1 of the bases shown by n in the base sequence.

[0187] In one embodiment of this scheme, the unnatural base shown by the above general formula (I) and / or general formula (II) is any one shown by the above formula (X) to formula (XVII) and formula (XXI) to formula (XXII).

[0188] In a further embodiment of this scheme, the nucleic acid aptamer that binds to thrombin is selected from the group consisting of the following (3-a) to (3-d):

[0189] (3-a) A nucleic acid aptamer containing the base sequence shown by serial number 81, wherein the bases shown by n at positions 16 and 22 in the base sequence are respectively (I) base Bs and base Bs, (II) base Bs and base Ys, (III) base Ys and base Bs, or (IV) base Ys and base Ys;

[0190] (3-b) A nucleic acid aptamer containing a base sequence in which 1 or more bases are deleted, replaced, or added in serial number 81 except at positions 16 and 22, and the positions corresponding to the bases shown by n at positions 16 and 22 in serial number 81 in the base sequence are respectively (I) base Bs and base Bs, (II) base Bs and base Ys, (III) base Ys and base Bs, or (IV) base Ys and base Ys;

[0191] (3-c) A nucleic acid aptamer that sequentially includes a 5'-region, a central region, and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region contain a pair of complementary base sequences capable of forming a stem structure, the central region contains the base sequence shown by serial number 190, and the bases shown by n at positions 7 and 13 in the base sequence shown by serial number 190 are respectively (I) base Bs and base Bs, (II) base Bs and base Ys, (III) base Ys and base Bs, or (IV) base Ys and base Ys;

[0192] (3-d) An aptamer that sequentially includes a 5'-region, a central region, and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region include a pair of base sequences that are complementary to each other and can form a stem structure, and the central region includes a base sequence in which 1 or more bases are deleted, substituted, or added at positions other than the 7th and 13th positions of SEQ ID NO: 190. At the positions corresponding to the bases shown as n at the 7th and 13th positions of SEQ ID NO: 190 in the base sequence in the central region, they are respectively (I) base Bs and base Bs, (II) base Bs and base Ys, (III) base Ys and base Bs, or (IV) base Ys and base Ys.

[0193] In addition, the above SEQ ID NO: 81 is a sequence obtained by adding sequences that are complementary to each other and can form a stem structure to the 5'-terminal side and the 3'-terminal side of SEQ ID NO: 190, respectively.

[0194] <(4) Aptamer that binds to VEGF>

[0195] In a further aspect of the aptamer of the present invention, an aptamer that binds to VEGF is provided.

[0196] In the present specification, "VEGF (vascular endothelial growth factor)" is a growth factor that functions as an angiogenesis promoting factor and is known as one of the etiological factors of age-related macular degeneration (AMD). Age-related macular degeneration is a progressive retinal disease that causes serious symptoms such as reduced visual function and blindness in the middle age in adults, and the condition deteriorates and worsens as angiogenesis in the retina progresses.

[0197] In the present invention, the biological species that is the source of VEGF is not limited, and examples include mammals such as primates such as humans and chimpanzees, experimental animals such as rats and mice, livestock animals such as pigs, cows, horses, sheep, and goats, and pets such as dogs and cats. Preferably, it is human VEGF.

[0198] The aptamer of the present solution comprises the base sequence shown in SEQ ID NO: 209 or 192, or a sequence obtained by adding a pair of complementary base sequences capable of forming a stem structure to the 5'-end side and 3'-end side of the base sequence shown in SEQ ID NO: 209 or 192, and at least one of the bases represented by n in the base sequence is the unnatural base represented by the above general formula (I) and / or general formula (II). For example, at least one of the bases represented by n in the base sequence is any one of the unnatural bases represented by formula (X) to formula (XVII) and formula (XXI) to formula (XXII). When two or more of the bases represented by n in the base sequence are the above-mentioned any unnatural base, they can be independently selected from the unnatural bases represented by the above general formula (I) and / or general formula (II), for example, the unnatural bases represented by the above general formula (X) to formula (XVII) and formula (XXI) to formula (XXII).

[0199] The base sequence shown in SEQ ID NO: 192 (5'-TAAGCCGCGTCCGAAGGGGCnTGCGGCGAnCCCGAATGGGT-3') corresponds to the central region excluding the stem region and the minihairpin sequence in the base sequences of aptamers such as b58 (YsYs) in Example 11. The base sequence shown in SEQ ID NO: 209 (5'-TAAACTGCGTCCGAAGGGGCnTGCAGTGAnCCCGAATGGGT-3') corresponds to the sequence excluding the stem regions each consisting of 3 bases at the 5'-end and 3'-end in the base sequence of " Figure 1 (b) "Anti-VEGF 165 " in the literature (Kimoto M,.et al., bioRxiv 2022.06.27.497860).

[0200] The aptamer that binds to VEGF contains at least one unnatural base represented by the above general formula (I) and / or general formula (II) as the base represented by n in the above base sequence, for example, it contains 2, 3, or 4 such bases.

[0201] In a further embodiment, the aptamer that binds to VEGF contains Ds as at least one of the bases represented by n in the base sequence.

[0202] In one embodiment of the present solution, the unnatural base represented by the above general formula (I) and / or general formula (II) is any one of the above formula (X) to formula (XVII) and formula (XXI) to formula (XXII).

[0203] In a further embodiment of the present solution, the nucleic acid aptamer that binds to VEGF is selected from the group consisting of the following (4-a) to (4-d):

[0204] (4-a) A nucleic acid aptamer comprising the base sequence shown in SEQ ID NO: 191, wherein the bases represented by n at positions 25 and 34 in the base sequence are respectively (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, (IV) base Bs and base Bs, or (V) base Yo and base Yo;

[0205] (4-b) A nucleic acid aptamer comprising a base sequence in which one or more bases are deleted, substituted, or added in SEQ ID NO: 191 except at positions 25 and 34, and the positions corresponding to the bases represented by n at positions 25 and 34 in SEQ ID NO: 191 in the base sequence are respectively (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, (IV) base Bs and base Bs, or (V) base Yo and base Yo;

[0206] (4-c) A nucleic acid aptamer comprising a 5'-region, a central region, and a 3'-region in this order from the 5'-side, wherein the 5'-region and the 3'-region comprise a pair of complementary base sequences capable of forming a stem structure, the central region comprises the base sequence shown in SEQ ID NO: 192, and the bases represented by n at positions 21 and 30 in the base sequence shown in SEQ ID NO: 192 are respectively (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, (IV) base Bs and base Bs, or (V) base Yo and base Yo;

[0207] (4-d) A nucleic acid aptamer comprising a 5'-region, a central region, and a 3'-region in this order from the 5'-side, wherein the 5'-region and the 3'-region comprise a pair of complementary base sequences capable of forming a stem structure, the central region comprises a base sequence in which one or more bases are deleted, substituted, or added in SEQ ID NO: 192 except at positions 21 and 30, and the positions corresponding to the bases represented by n at positions 21 and 30 in SEQ ID NO: 192 in the base sequence in the central region are respectively (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, (IV) base Bs and base Bs, or (V) base Yo and base Yo.

[0208] In addition, the above SEQ ID NO: 191 is a sequence obtained by adding a pair of complementary sequences capable of forming a stem structure to the 5'-terminal side and the 3'-terminal side of SEQ ID NO: 192, respectively.

[0209] <(5) Nucleic acid aptamer that binds to HMGB1>

[0210] In a further aspect of the nucleic acid aptamer of the present invention, a nucleic acid aptamer that binds to HMGB1 is provided.

[0211] In the present specification, "HMGB1" (High Mobility Group Box 1) functions not only as a chromatin protein but also as a cytokine mediator of inflammation secreted by immune cells such as macrophages and monocytes. In the present invention, the biological species that is the source of HMGB1 is not limited, and examples include mammals such as primates such as humans and chimpanzees, experimental animals such as rats and mice, livestock animals such as pigs, cows, horses, sheep, and goats, and pets such as dogs and cats, and preferably HMGB1 of humans.

[0212] The nucleic acid aptamer of this aspect contains the base sequence shown in SEQ ID NO: 194, or a sequence obtained by adding a pair of complementary base sequences capable of forming a stem structure to the 5'-terminal side and 3'-terminal side of the base sequence shown in SEQ ID NO: 194, and at least one of the bases represented by n in the base sequence is the unnatural base represented by the above general formula (I) and / or general formula (II). For example, at least one of the bases represented by n in the base sequence is any of the unnatural bases represented by formula (X) to formula (XVII) and formula (XXI) to formula (XXII). When two or more of the bases represented by n in the base sequence are the above-mentioned any unnatural base, they can be independently selected from the unnatural bases represented by the above general formula (I) and / or general formula (II), such as the unnatural bases represented by the above general formula (X) to formula (XVII) and formula (XXI) to formula (XXII).

[0213] The base sequence shown in SEQ ID NO: 194 (5'-GCCACCGTATGnACGCTCACAGnAGTTGCCGA-3') corresponds to the central region excluding the stem region and the mini-hairpin sequence in the base sequences of nucleic acid aptamers such as Bio-HMGB1-301-BB in Example 6.

[0214] The nucleic acid aptamer that binds to HMGB1 contains at least one unnatural base represented by the above general formula (I) and / or general formula (II) as the base represented by n in the above base sequence, for example, 2, 3, or 4.

[0215] In a further embodiment, the nucleic acid aptamer that binds to HMGB1 contains Ds as at least one of the bases represented by n in the base sequence.

[0216] In one embodiment of this solution, the unnatural base represented by the above general formula (I) and / or general formula (II) is any one of those represented by the above formulas (X) to (XVII) and formulas (XXI) to (XXII).

[0217] In a further embodiment of this solution, the nucleic acid aptamer that binds to the HMGB1 protein is selected from the group consisting of the following (5-a) to (5-d):

[0218] (5-a) A nucleic acid aptamer comprising the base sequence shown in SEQ ID NO: 193, wherein the bases represented by n at positions 22 and 33 in the base sequence are respectively (I) base Bs and base Bs, (II) base Bs and base Ys, (III) base Ys and base Bs, (IV) base Ys and base Ys;

[0219] (5-b) A nucleic acid aptamer comprising a base sequence in which one or more bases are deleted, substituted or added at positions other than positions 22 and 33 in SEQ ID NO: 193, and the positions corresponding to the bases represented by n at positions 22 and 33 in SEQ ID NO: 193 in the base sequence are respectively (I) base Bs and base Bs, (II) base Bs and base Ys, (III) base Ys and base Bs, (IV) base Ys and base Ys;

[0220] (5-c) A nucleic acid aptamer sequentially comprising a 5' region, a central region and a 3' region from the 5' side, wherein the 5' region and the 3' region comprise a pair of complementary base sequences capable of forming a stem structure, the central region comprises the base sequence shown in SEQ ID NO: 194, and the bases represented by n at positions 12 and 23 in the base sequence shown in SEQ ID NO: 194 are respectively (I) base Bs and base Bs, (II) base Bs and base Ys, (III) base Ys and base Bs, (IV) base Ys and base Ys;

[0221] (5-d) A nucleic acid aptamer sequentially comprising a 5' region, a central region and a 3' region from the 5' side, wherein the 5' region and the 3' region comprise a pair of complementary base sequences capable of forming a stem structure, the central region comprises a base sequence in which one or more bases are deleted, substituted or added at positions other than positions 12 and 23 in SEQ ID NO: 194, and the positions corresponding to the bases represented by n at positions 12 and 23 in SEQ ID NO: 194 in the base sequence in the central region are respectively (I) base Bs and base Bs, (II) base Bs and base Ys, (III) base Ys and base Bs, (IV) base Ys and base Ys.

[0222] In addition, the above SEQ ID NO: 193 is a sequence obtained by adding complementary sequences capable of forming a stem structure to the 5' end side and the 3' end side of SEQ ID NO: 194, respectively.

[0223] <(6) Nucleic acid aptamer that binds to transferrin receptor 1>

[0224] In a further aspect of the nucleic acid aptamer of the present invention, a nucleic acid aptamer that binds to transferrin receptor 1 is provided.

[0225] In the present specification, "transferrin receptor 1" (TrfR1) is a type of transferrin receptor that functions as a receptor for transferrin, which is responsible for binding and transporting iron ions. Transferrin receptor 1 transports iron into cells by internalizing the transferrin / iron complex. In the present invention, the biological species from which transferrin receptor 1 is derived is not limited, and examples include mammals such as primates such as humans and chimpanzees, laboratory animals such as rats and mice, livestock animals such as pigs, cows, horses, sheep, and goats, and pets such as dogs and cats, and preferably human transferrin receptor 1.

[0226] The nucleic acid aptamer of this aspect contains the base sequence shown in SEQ ID NO: 196, or a sequence obtained by adding a pair of complementary base sequences capable of forming a stem structure to the 5'-terminal side and 3'-terminal side of the base sequence shown in SEQ ID NO: 196, and at least one of the bases represented by n in the base sequence is a non-natural base represented by the above general formula (I) and / or general formula (II). For example, at least one of the bases represented by n in the base sequence is any non-natural base represented by formula (X) to formula (XVII) and formula (XXI) to formula (XXII). When two or more of the bases represented by n in the base sequence are the above non-natural bases, they can be independently selected from the non-natural bases represented by the above general formula (I) and / or general formula (II), such as the non-natural bases represented by the above general formula (X) to formula (XVII) and formula (XXI) to formula (XXII).

[0227] The base sequence shown in SEQ ID NO: 196 (5'-GGGGTGTTTGTGCCGTGAGnTG-3') corresponds to the central region of the nucleic acid aptamers such as MB45-Bs in Example 12 excluding the stem region and the mini-hairpin sequence.

[0228] The nucleic acid aptamer that binds to transferrin receptor 1 contains at least one non-natural base represented by the above general formula (I) and / or general formula (II) as the base represented by n in the above base sequence, for example, contains 2, 3, or 4.

[0229] In a further embodiment, the nucleic acid aptamer that binds to transferrin receptor 1 contains at least one Ds as the base represented by n in the base sequence.

[0230] In one embodiment of the present solution, the unnatural base shown in the above general formula (I) and / or general formula (II) is any one of those shown in the above formulas (X) to (XVII) and formulas (XXI) to (XXII).

[0231] In a further embodiment of the present solution, the nucleic acid aptamer that binds to the transferrin receptor 1 protein is selected from the group consisting of the following (6-a) to (6-d):

[0232] (6-a) A nucleic acid aptamer comprising the base sequence shown in SEQ ID NO: 195, wherein the base represented by n at position 27 in the base sequence is base Bs;

[0233] (6-b) A nucleic acid aptamer comprising a base sequence in which one or more bases are deleted, substituted or added in SEQ ID NO: 195 except at position 27, and the position corresponding to the base represented by n at position 27 in SEQ ID NO: 195 in the base sequence is base Bs;

[0234] (6-c) A nucleic acid aptamer sequentially comprising a 5'-region, a central region and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region comprise a pair of complementary base sequences capable of forming a stem structure, the central region comprises the base sequence shown in SEQ ID NO: 196, and the base represented by n at position 20 in the base sequence shown in SEQ ID NO: 196 is base Bs;

[0235] (6-d) A nucleic acid aptamer sequentially comprising a 5'-region, a central region and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region comprise a pair of complementary base sequences capable of forming a stem structure, the central region comprises a base sequence in which one or more bases are deleted, substituted or added in SEQ ID NO: 196 except at position 20, and the position corresponding to the base represented by n at position 20 in SEQ ID NO: 196 in the base sequence in the central region is base Bs.

[0236] In addition, the above SEQ ID NO: 195 is a sequence obtained by adding complementary sequences capable of forming a stem structure to the 5'-terminal side and the 3'-terminal side of SEQ ID NO: 196, respectively.

[0237] <(7) Nucleic acid aptamer that binds to DEN1 protein>

[0238] In a further aspect of the nucleic acid aptamer of the present invention, a nucleic acid aptamer that binds to dengue virus serotype 1 NS1 protein (Dengue Virus NS1 Protein Serotype 1; DEN1 protein) is provided.

[0239] In this specification, the "NS1 protein" refers to non-structural protein 1 (Nonstructural protein 1) derived from dengue virus. Among NS1 proteins, the dengue virus type 1 NS1 protein is referred to as "DEN1 protein", the dengue virus type 2 NS1 protein is referred to as "DEN2 protein", the dengue virus type 3 NS1 protein is referred to as "DEN3 protein", and the dengue virus type 4 NS1 protein is referred to as "DEN4 protein".

[0240] The nucleic acid aptamer of this scheme comprises the base sequence shown in SEQ ID NO: 210, 197 or 199, or a sequence obtained by adding a pair of complementary base sequences capable of forming a stem structure to the 5'-terminal side and 3'-terminal side of the base sequence shown in SEQ ID NO: 210, 197 or 199, and at least one of the bases represented by n in the base sequence is the unnatural base shown in the above general formula (I) and / or general formula (II). For example, at least one of the bases represented by n in the base sequence is any of the unnatural bases shown in formula (X) to formula (XVII) and formula (XXI) to formula (XXII). When two or more of the bases represented by n in the base sequence are the above-mentioned any unnatural bases, they can be independently selected from the unnatural bases shown in the above general formula (I) and / or general formula (II), such as the unnatural bases shown in the above general formula (X) to formula (XVII) and formula (XXI) to formula (XXII).

[0241] The base sequence shown in SEQ ID NO: 197 (5'-ACTGGTGTnCTCGGnATGG-3') corresponds to the central region of the base sequence of nucleic acid aptamers such as AptD1-YsDs in the result (3) of Example 3 after removing the stem region and the mini-hairpin sequence. The base sequence shown in SEQ ID NO: 210 (5'-RCTGGTGYnCTCGGnATGR-3') is included in the Figure 2 (d) The sequence after removing the stem regions each consisting of 4 bases at the 5'-terminal and 3'-terminal of the base sequences (SEQ ID NOs: 35 to 37) showing substitutions (A7G, T14C, G25A) that do not impair the binding activity. In addition, the base sequence shown in SEQ ID NO: 199 (5'-GTAnTCAGACGTATACnCATCA-3') corresponds to the central region of the base sequence of nucleic acid aptamers such as AptD1c2a (YsYs) in Example 8 after removing the stem region and the mini-hairpin sequence.

[0242] The nucleic acid aptamer that binds to the DEN1 protein contains at least 1 unnatural base represented by the general formula (I) and / or general formula (II) as the base represented by n in the above base sequence, for example, 2, 3, or 4.

[0243] In a further embodiment, at least 1 of the bases represented by n in the base sequence of the nucleic acid aptamer that binds to the DEN1 protein may contain Ds.

[0244] In one embodiment of this solution, the unnatural base represented by the general formula (I) and / or general formula (II) is any one of the formulas (X) to (XVII) and formulas (XXI) to (XXII).

[0245] In a further embodiment of this solution, the nucleic acid aptamer that binds to the DEN1 protein is selected from the group consisting of the following (7-i-a) to (7-ii-d):

[0246] (7-i-a) A nucleic acid aptamer containing the base sequence shown in SEQ ID NO: 27 or 29, wherein the bases represented by n at positions 19 and 25 in the base sequence are respectively (I) base Yss and base Ds, (II) base Ds and base Dt, (III) base Ys and base Bs, (IV) base Yo and base Bs, or (V) base Ysm and base Bs;

[0247] (7-i-b) A nucleic acid aptamer containing a base sequence in which 1 or more bases are deleted, substituted, or added in SEQ ID NO: 27 or 29 except at positions 19 and 25, and the positions corresponding to the bases represented by n at positions 19 and 25 in SEQ ID NO: 27 or 29 are respectively (I) base Yss and base Ds, (II) base Ds and base Dt, (III) base Ys and base Bs, (IV) base Yo and base Bs, or (V) base Ysm and base Bs;

[0248] (7-i-c) A nucleic acid aptamer that sequentially includes a 5' region, a central region, and a 3' region from the 5' side, wherein the 5' region and the 3' region contain a pair of complementary base sequences capable of forming a stem structure, and the central region contains the base sequence shown in SEQ ID NO: 210 or 197, and the bases represented by n at positions 9 and 15 in the base sequence shown in SEQ ID NO: 210 or 197 are respectively (I) base Yss and base Ds, (II) base Ds and base Dt, (III) base Ys and base Bs, (IV) base Yo and base Bs, or (V) base Ysm and base Bs;

[0249] (7-i-d) A nucleic acid aptamer comprising a 5' region, a central region, and a 3' region in sequence from the 5' side, wherein the 5' region and the 3' region each contain a pair of base sequences complementary to each other capable of forming a stem structure, the central region contains a base sequence in which one or more bases are deleted, substituted, or added at positions other than positions 9 and 15 in SEQ ID NO: 210 or 197, and the positions corresponding to the bases represented by n at positions 9 and 15 in SEQ ID NO: 210 or 197 in the base sequence in the central region are respectively (I) base Yss and base Ds, (II) base Ds and base Dt, (III) base Ys and base Bs, (IV) base Yo and base Bs, or (V) base Ysm and base Bs;

[0250] (7-ii-a) A nucleic acid aptamer comprising the base sequence shown in SEQ ID NO: 198, wherein the bases represented by n at positions 14 and 27 in the base sequence are respectively (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, or (IV) base Bs and base Bs;

[0251] (7-ii-b) A base sequence in which one or more bases are deleted, substituted, or added at positions other than positions 14 and 27 in SEQ ID NO: 198, wherein the positions corresponding to the bases represented by n at positions 14 and 27 in SEQ ID NO: 198 in the base sequence are respectively (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, or (IV) base Bs and base Bs;

[0252] (7-ii-c) A nucleic acid aptamer comprising a 5' region, a central region, and a 3' region in sequence from the 5' side, wherein the 5' region and the 3' region each contain a pair of base sequences complementary to each other capable of forming a stem structure, the central region contains the base sequence shown in SEQ ID NO: 199, and the bases represented by n at positions 4 and 17 in the base sequence shown in SEQ ID NO: 199 are respectively (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, or (IV) base Bs and base Bs;

[0253] (7-ii-d) A nucleic acid aptamer that sequentially includes a 5' region, a central region, and a 3' region starting from the 5' side. The 5' region and the 3' region contain a pair of base sequences that are complementary to each other and can form a stem structure. The central region contains a base sequence in which one or more bases are deleted, substituted, or added except at positions 4 and 17 in SEQ ID NO: 199. The positions corresponding to the bases represented by n at positions 4 and 17 in SEQ ID NO: 199 in the base sequence in the central region are respectively (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, or (IV) base Bs and base Bs.

[0254] In addition, the above SEQ ID NOs: 27, 29, and 198 are sequences obtained by adding a pair of complementary sequences that can form a stem structure to the 5' end side and the 3' end side of SEQ ID NOs: 210, 197, and 199, respectively. Additionally, the base sequence shown in SEQ ID NO: 29 is included in the base sequence shown in SEQ ID NO: 27, and purine bases and pyrimidine bases are specified as specific bases.

[0255] <(8) Nucleic acid aptamer that binds to DEN2 protein>

[0256] In a further aspect of the nucleic acid aptamer of the present invention, a nucleic acid aptamer that binds to Dengue Virus NS1 Protein Serotype 2 (DEN2 protein) is provided.

[0257] The nucleic acid aptamer of this aspect includes the base sequence shown in SEQ ID NO: 201, or a sequence obtained by adding a pair of complementary base sequences that can form a stem structure to the 5' end side and the 3' end side of the base sequence shown in SEQ ID NO: 201. At least one of the bases represented by n in the base sequence is a non-natural base represented by the above general formula (I) and / or general formula (II). For example, at least one of the bases represented by n in the base sequence is any non-natural base represented by formula (X) to formula (XVII) and formula (XXI) to formula (XXII). When two or more of the bases represented by n in the base sequence are the above non-natural bases, they can be independently selected from the non-natural bases represented by the above general formula (I) and / or general formula (II), such as the non-natural bases represented by the above formula (X) to formula (XVII) and formula (XXI) to formula (XXII).

[0258] The base sequence shown in SEQ ID NO: 201 (5'-CGnCCAACCTCCACCAATCAAAGC-3') corresponds to the central region excluding the stem region and the mini-hairpin sequence in the base sequences of nucleic acid aptamers such as AptD2bb(Ys) in Example 9.

[0259] The nucleic acid aptamer that binds to the DEN2 protein contains at least 1 unnatural base represented by the general formula (I) and / or general formula (II) as the base represented by n in the above base sequence, for example, contains 1 to 3, such as 1, 2, 3, or 4.

[0260] In a further embodiment, at least 1 of the bases represented by n in the base sequence of the nucleic acid aptamer that binds to the DEN2 protein may contain Ds.

[0261] In one embodiment of this scheme, the unnatural base represented by the general formula (I) and / or general formula (II) is any one of those represented by the above formula (X) to formula (XVII) and formula (XXI) to formula (XXII).

[0262] In a further embodiment of this scheme, the nucleic acid aptamer that binds to the DEN2 protein is selected from the group consisting of the following (8-a) to (8-d):

[0263] (8-a) A nucleic acid aptamer containing the base sequence shown in SEQ ID NO: 200, wherein the base represented by n at position 13 in the base sequence is base Bs;

[0264] (8-b) A nucleic acid aptamer containing a base sequence in which 1 or more bases are deleted, substituted, or added at positions other than position 13 in SEQ ID NO: 200, and the position corresponding to the base represented by n at position 13 in SEQ ID NO: 200 in the base sequence is base Ys or base Bs;

[0265] (8-c) A nucleic acid aptamer that sequentially includes a 5'-region, a central region, and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region contain a pair of complementary base sequences capable of forming a stem structure, the central region contains the base sequence shown in SEQ ID NO: 201, and the base represented by n at position 3 in the base sequence shown in SEQ ID NO: 201 is base Ys or base Bs;

[0266] (8-d) A nucleic acid aptamer that sequentially includes a 5'-region, a central region, and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region contain a pair of complementary base sequences capable of forming a stem structure, the central region contains a base sequence in which 1 or more bases are deleted, substituted, or added at positions other than position 3 in SEQ ID NO: 201, and the position corresponding to the base represented by n at position 3 in SEQ ID NO: 201 in the base sequence in the central region is base Ys or base Bs.

[0267] In addition, the above SEQ ID NO: 200 is a sequence obtained by adding complementary sequences capable of forming a stem structure to the 5'-terminal side and 3'-terminal side of SEQ ID NO: 201, respectively.

[0268] <Nucleic acid aptamer that binds to DEN3 protein>

[0269] In a further aspect of the nucleic acid aptamer of the present invention, there is provided a nucleic acid aptamer that binds to Dengue Virus NS1 Protein Serotype 3 (DEN3 protein).

[0270] The nucleic acid aptamer of this aspect comprises the base sequence shown in SEQ ID NO: 39 or 202, or a sequence obtained by adding a pair of complementary base sequences capable of forming a stem structure to the 5'-end side and 3'-end side of the base sequence shown in SEQ ID NO: 39 or 202, and at least one of the bases represented by n in the base sequence is a non-natural base represented by the above general formula (I) and / or general formula (II). For example, at least one of the bases represented by n in the base sequence is any non-natural base represented by formula (X) to formula (XVII) and formula (XXI) to formula (XXII). When two or more of the bases represented by n in the base sequence are the above-mentioned non-natural bases, they can be independently selected from the non-natural bases represented by the above general formula (I) and / or general formula (II), for example, the non-natural bases represented by the above general formula (X) to formula (XVII) and formula (XXI) to formula (XXII).

[0271] The base sequence shown in SEQ ID NO: 202 (5'-TCTAnCCTGGCCnTGTGGTACTGTAACGGC-3') corresponds to the central region of the stem region removed from the base sequences of nucleic acid aptamers such as AptD3-YsDss in the result (3) of Example 3. The base sequence shown in SEQ ID NO: 39 (5'-TCYAnCCYGGCCnYGTGGYRYYGTARCGGY-3') is a sequence extended in such a way as to further include substitutions corresponding to substitutions (T9C, T14C, T20C, T25C, A26G, C27T, T28C, A32G, and C36T) shown not to impair the binding activity in (e) of the literature (Kimoto M,.et al., bioRxiv 2022.06.27.497860). Figure 2 (e) The sequences (SEQ ID NOs: 46 to 54) of the substitutions corresponding to the substitutions that do not impair the binding activity shown in

[0272] The nucleic acid aptamer that binds to DEN3 protein contains at least one non-natural base represented by the above general formula (I) and / or general formula (II) as the base represented by n in the above base sequence, for example, contains 1 to 3, for example, 1, 2, 3, or 4.

[0273] In a further embodiment, the nucleic acid aptamer that binds to the DEN3 protein may contain at least one Ds as the base represented by n in the base sequence.

[0274] In one embodiment of the present solution, the unnatural base represented by the above general formula (I) and / or general formula (II) is any one of the bases represented by the above formula (X) to formula (XVII) and formula (XXI) to formula (XXII).

[0275] In a further embodiment of the present solution, the nucleic acid aptamer that binds to the DEN3 protein is selected from the group consisting of the following (9-a) to (9-d):

[0276] (9-a) A nucleic acid aptamer containing the base sequence shown in SEQ ID NO: 38 or 40, wherein the bases represented by n at positions 15 and 23 in the base sequence are respectively (I) base Dss and base Dss, (II) base Ys and base Dss, (III) base Dss and base Ys, (IV) base Ysm and base Ysm, (V) base Yss and base Ys, (VI) base Yss and base Yo, or (VII) base Yss and base Ysm;

[0277] (9-b) A nucleic acid aptamer containing a base sequence in which one or more bases are deleted, substituted, or added in SEQ ID NO: 38 or 40 except at positions 15 and 23, and the positions corresponding to the bases represented by n at positions 15 and 23 in SEQ ID NO: 38 or 40 are respectively (I) base Dss and base Dss, (II) base Ys and base Dss, (III) base Dss and base Ys, (IV) base Ysm and base Ysm, (V) base Yss and base Ys, (VI) base Yss and base Yo, or (VII) base Yss and base Ysm;

[0278] (9-c) A nucleic acid aptamer that sequentially includes a 5' region, a central region, and a 3' region from the 5' side, wherein the 5' region and the 3' region contain a pair of complementary base sequences capable of forming a stem structure, the central region contains the base sequence shown in SEQ ID NO: 39 or 202, and the bases represented by n at positions 5 and 13 in the base sequence shown in SEQ ID NO: 39 or 202 are respectively (I) base Dss and base Dss, (II) base Ys and base Dss, (III) base Dss and base Ys, (IV) base Ysm and base Ysm, (V) base Yss and base Ys, (VI) base Yss and base Yo, or (VII) base Yss and base Ysm;

[0279] (9-d) A nucleic acid aptamer that sequentially includes a 5' region, a central region, and a 3' region from the 5' side, wherein the 5' region and the 3' region include a pair of base sequences that are complementary to each other and can form a stem structure, the central region includes a base sequence in which one or more bases are deleted, substituted, or added except at positions 5 and 13 in SEQ ID NO: 39 or 202, and the positions corresponding to the bases shown as n at positions 5 and 13 in SEQ ID NO: 39 or 202 in the base sequence in the central region are respectively (I) base Dss and base Dss, (II) base Ys and base Dss, (III) base Dss and base Ys, (IV) base Ysm and base Ysm, (V) base Yss and base Ys, (VI) base Yss and base Yo, or (VII) base Yss and base Ysm.

[0280] In addition, SEQ ID NOs: 38 and 40 above are sequences obtained by adding a pair of complementary sequences that can form a stem structure to the 5' end side and the 3' end side of SEQ ID NOs: 39 and 202, respectively. Further, the base sequence shown in SEQ ID NO: 40 is included in the base sequence shown in SEQ ID NO: 38, and purine bases and pyrimidine bases are specified as specific bases.

[0281] <(10) Nucleic acid aptamer that binds to DEN4 protein>

[0282] In a further aspect of the nucleic acid aptamer of the present invention, a nucleic acid aptamer that binds to Dengue Virus NS1 Protein Serotype 4 (DEN4 protein) is provided.

[0283] The nucleic acid aptamer of this aspect includes the base sequence shown in SEQ ID NO: 204, or a sequence obtained by adding a pair of complementary base sequences that can form a stem structure to the 5' end side and the 3' end side of the base sequence shown in SEQ ID NO: 204, and at least one of the bases shown as n in the base sequence is a non-natural base shown in the above general formula (I) and / or general formula (II). For example, at least one of the bases shown as n in the base sequence is any of the non-natural bases shown in formulas (X) to (XVII) and formulas (XXI) to (XXII). When two or more of the bases shown as n in the base sequence are the above non-natural bases, they can be independently selected from the non-natural bases shown in the above general formula (I) and / or general formula (II), such as the non-natural bases shown in the above formulas (X) to (XVII) and formulas (XXI) to (XXII).

[0284] The base sequence shown by SEQ ID NO: 204 (5'-GACGTAACGCnTATCAAATCnAAACAGCTTAGGG-3') corresponds to the central region from which the stem region and the microhairpin sequence have been removed in the base sequences of nucleic acid aptamers such as AptD4-2 (YsYs) in Example 10.

[0285] The nucleic acid aptamer that binds to the DEN4 protein contains at least 1 unnatural base shown by the above general formula (I) and / or general formula (II) as the base shown by n in the above base sequence, for example, it contains 2, 3, or 4.

[0286] In a further embodiment, at least 1 of the bases shown by n in the base sequence of the nucleic acid aptamer that binds to the DEN4 protein may contain Ds.

[0287] In one embodiment of this scheme, the unnatural base shown by the above general formula (I) and / or general formula (II) is any one shown by the above formula (X) to formula (XVII) and formula (XXI) to formula (XXII).

[0288] In a further embodiment of this scheme, the nucleic acid aptamer that binds to the DEN4 protein is selected from the group consisting of the following (10-a) to (10-d):

[0289] (10-a) A nucleic acid aptamer containing the base sequence shown by SEQ ID NO: 203, wherein the bases shown by n at positions 18 and 28 in the base sequence are respectively (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, or (IV) base Bs and base Bs;

[0290] (10-b) A nucleic acid aptamer containing a base sequence in which 1 or more bases are deleted, substituted, or added in SEQ ID NO: 203 except at positions 18 and 28, and the positions corresponding to the bases shown by n at positions 18 and 28 in SEQ ID NO: 203 in the base sequence are respectively (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, or (IV) base Bs and base Bs;

[0291] (10-c) A nucleic acid aptamer that sequentially includes a 5' region, a central region, and a 3' region from the 5' side, wherein the 5' region and the 3' region contain a pair of complementary base sequences capable of forming a stem structure, the central region contains the base sequence shown by SEQ ID NO: 204, and the bases shown by n at positions 11 and 21 in the base sequence shown by SEQ ID NO: 204 are respectively (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, or (IV) base Bs and base Bs;

[0292] (10-d) A nucleic acid aptamer comprising a 5' region, a central region, and a 3' region in this order from the 5' side, wherein the 5' region and the 3' region each contain a pair of base sequences complementary to each other capable of forming a stem structure, the central region contains a base sequence in which one or more bases are deleted, substituted, or added at positions other than positions 11 and 21 in SEQ ID NO: 204, and the positions corresponding to the bases represented by n at positions 11 and 21 in SEQ ID NO: 204 in the base sequence in the central region are (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, or (IV) base Bs and base Bs.

[0293] In addition, SEQ ID NO: 203 above is a sequence obtained by adding sequences complementary to each other capable of forming a stem structure to the 5'-terminal side and the 3'-terminal side of SEQ ID NO: 204, respectively.

[0294] <Drug composition>

[0295] In one aspect of the present invention, there is provided a drug composition comprising any of the above nucleic acid aptamers. The drug composition of the invention comprises any one or more of the above nucleic acid aptamers. The drug composition of the present invention may also comprise any two or more of the above nucleic acid aptamers in combination. Additionally, the drug composition may contain one or more other agents within a range not losing the binding ability of the nucleic acid aptamer of the present invention.

[0296] The diseases that are the subject of prevention and / or treatment with the drug composition comprising the above (1) nucleic acid aptamer that binds to vWF are diseases such as gene mutations or overexpressions of vWF, and the generation of autoantibodies against vWF, etc., which can be the cause thereof (hereinafter also referred to as "vWF-related diseases" in this specification). Examples of vWF-related diseases include, for example, thrombosis, thrombotic thrombocytopenic purpura, intracranial embolism, cerebral embolism, carotid artery stenosis, thrombotic microangiopathy, and acute myocardial infarction.

[0297] The diseases that are the subject of prevention and / or treatment with the pharmaceutical composition containing the above-mentioned (2) nucleic acid aptamer that binds to IFNγ are diseases that require inhibition of the function of IFNγ (hereinafter also referred to as "IFNγ-related diseases" in this specification). As examples of IFNγ-related diseases, for example, autoimmune diseases or inflammatory lesions can be cited. Autoimmune diseases or inflammatory lesions can be, for example, Crohn's disease, systemic lupus erythematosus, psoriasis, rheumatoid arthritis, vasculitis, uveitis, atopic dermatitis, type I diabetes, multiple sclerosis, schizophrenia, corneal transplant rejection, alopecia areata, psoriasis vulgaris, vitiligo, pemphigus vulgaris, epidermolysis bullosa, acne vulgaris, herpes simplex virus type 1, or Hunner's interstitial cystitis.

[0298] The diseases that are the subject of prevention and / or treatment with the pharmaceutical composition containing the above-mentioned (3) nucleic acid aptamer that binds to thrombin are diseases that require inhibition of the function of thrombin (hereinafter also referred to as "thrombin-related diseases" in this specification). As examples of thrombin-related diseases, for example, thrombotic diseases can be cited. As examples of thrombotic diseases, for example, venous thrombosis (e.g., deep vein thrombosis), pulmonary embolism, atrial fibrillation, myocardial infarction, arterial thrombosis, stroke (e.g., thrombosis-induced stroke), atherosclerosis, and disseminated intravascular coagulation can be cited.

[0299] The diseases that are the subject of prevention and / or treatment with the pharmaceutical composition containing the above-mentioned (4) nucleic acid aptamer that binds to VEGF are diseases that require inhibition of the function of VEGF (hereinafter also referred to as "VEGF-related diseases" in this specification). As examples of VEGF-related diseases, for example, age-related macular degeneration, diabetic retinopathy, rheumatoid arthritis, heart disease after myocardial infarction, wound healing, or cancer can be cited.

[0300] The diseases that are the subject of prevention and / or treatment with the pharmaceutical composition containing the above-mentioned (5) nucleic acid aptamer that binds to HMGB1 are diseases that require inhibition of the function of HMGB1 (hereinafter also referred to as "HMGB1-related diseases" in this specification). HMGB1-related diseases can include autoimmune diseases and cardiovascular diseases, etc. As further specific examples, rheumatoid arthritis, inflammatory bowel disease, sepsis, cancer, lupus, Sjogren's syndrome, myocardial infarction, arteriosclerosis, stroke, cerebral infarction, cerebral edema, cerebral vasospasm, traumatic brain injury, atherosclerosis, neuropathic pain, arthritis, acute lung injury, cerebral ischemia, renal ischemia, or hepatic ischemia, etc. can be cited.

[0301] A disease that is a subject for prevention and / or treatment with a pharmaceutical composition containing the above-mentioned nucleic acid aptamer that binds to transferrin receptor 1 is a disease that requires inhibition of the function of transferrin receptor 1 (hereinafter also referred to as "transferrin receptor 1-related disease" in this specification). Examples of transferrin receptor 1-related diseases include neurological disorders, iron deficiency anemia, iron overload, cancer, neurodegenerative diseases, and infectious diseases. As further specific examples, there may be mentioned eye diseases, seizure disorders, lysosomal storage diseases, amyloidosis, viral infections, microbial infections, ischemia, behavioral abnormalities, and CNS inflammation, etc.

[0302] A disease that is a subject for prevention and / or treatment with a pharmaceutical composition containing the above-mentioned (7) nucleic acid aptamer that binds to DEN1 protein, (8) nucleic acid aptamer that binds to DEN2 protein, (9) nucleic acid aptamer that binds to DEN3 protein, and / or (10) nucleic acid aptamer that binds to DEN4 protein is dengue virus infection. Dengue virus infection includes dengue fever and severe dengue (e.g., dengue hemorrhagic fever and dengue shock syndrome).

[0303] The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. The so-called "pharmaceutically acceptable carrier" refers to a substance that is commonly used in the field of pharmaceutical technology to facilitate the formulation of a pharmaceutical composition, its application to an organism, and is added within a range that does not hinder or inhibit its action. Examples of carriers include, for example, excipients, binders, disintegrants, fillers, emulsifiers, flow additive regulators, lubricants, or stabilizers.

[0304] Examples of "excipients" include sugars such as monosaccharides, disaccharides, cyclodextrins, and polysaccharides (not specifically limited, but including glucose, sucrose, lactose, raffinose, mannitol, sorbitol, inositol, dextrin, maltodextrin, starch, and cellulose), metal salts (e.g., sodium phosphate or calcium phosphate, calcium sulfate, magnesium sulfate), citric acid, tartaric acid, glycine, low, medium, and high molecular weight polyethylene glycols (PEG), pluronics, or combinations thereof.

[0305] Examples of "binders" include, for example, starch paste made from corn, wheat, rice, or potato starch, gelatin, tragacanth, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone, etc.

[0306] Examples of "disintegrants" include, for example, the aforementioned starch, carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate or their salts.

[0307] Examples of "fillers" include, for example, the aforementioned sugars and / or calcium phosphate (e.g., tricalcium phosphate, or calcium hydrogen phosphate).

[0308] Examples of the "emulsifier" include, for example, sorbitan fatty acid esters, glycerol fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

[0309] Examples of the "flow additive regulator" and "lubricant" include, for example, silicates, talc, stearates, or polyethylene glycols.

[0310] Examples of the "stabilizer" include, for example, antioxidants such as ascorbic acid and sulfites, and sugars such as trehalose and glucose.

[0311] Such carriers can be appropriately used as needed. In addition to the above additives, the pharmaceutical composition of the present invention may contain, as needed, flavoring and deodorizing agents, solubilizers (cosolvents), suspending agents, diluents, surfactants, absorption promoters (e.g., quaternary ammonium salts, sodium lauryl sulfate, etc.), bulking agents, humectants, moisturizing agents (e.g., glycerol, starch, etc.), adsorbents (e.g., starch, lactose, kaolin, bentonite, colloidal silica, etc.), disintegration inhibitors (e.g., white sugar, glyceryl tristearate, cocoa butter, hydrogenated oil, etc.), coating agents, coloring agents, preservatives, fragrances, flavoring agents, sweeteners, buffering agents, isotonic agents, analgesics, solubilizers, etc.

[0312] Examples of the "surfactant" include, for example, alkali metal salts, alkaline earth metal salts, and ammonium salts of lignin sulfonic acid, naphthalene sulfonic acid, hydroxybenzenesulfonic acid, and dibutylnaphthalenesulfonic acid, alkylaryl sulfonates, alkyl sulfates, alkyl sulfonates, fatty alcohol sulfates, fatty acids and sulfated fatty alcohol ethylene glycol ethers, and condensates of sulfonated naphthalene and naphthalene derivatives with formaldehyde, condensates of naphthalene or naphthalene sulfonic acid with phenol and formaldehyde, polyoxyethylene octylphenyl ether, ethoxylated isooctylphenol, octylphenol, nonylphenol, alkylphenyl polyethylene glycol ether, tributylphenyl polyethylene glycol ether, tristearylphenyl polyethylene glycol ether, alkylaryl polyether alcohol, condensates of alcohols and fatty alcohols / ethylene oxide, ethoxylated castor oil, polyoxyethylene alkyl ether, ethoxylated polyoxypropylene, dodecyl alcohol polyethylene glycol ether acetal, sorbitan esters, lignosulfite waste liquor, and methylcellulose.

[0313] The pharmaceutical composition of the present embodiment may contain one or more of the above carriers in one pharmaceutical composition.

[0314] The dosage form of the pharmaceutical composition of the present invention is not particularly limited as long as it is a form that does not inactivate the active ingredient and can exert its pharmacological effect in a living body after administration. It usually varies depending on the administration method and / or prescription conditions.

[0315] For example, as dosage forms suitable for oral administration, solid dosage forms (including tablets, pills, sublingual tablets, capsules, drops, lozenges), granules, powders, powders, liquid preparations, etc. can be mentioned. Furthermore, solid dosage forms can be made into dosage forms with a dosage form coating known in the art as needed. For example, sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, bilayer tablets, and multilayer tablets.

[0316] Parenteral administration can be subdivided into systemic administration and local administration, and local administration can be further subdivided into intratissue administration, transdermal administration, transmucosal administration, and rectal administration. The pharmaceutical composition can also be made into a dosage form suitable for the administration method. As dosage forms suitable for systemic or intratissue administration, for example, injections as liquid preparations can be mentioned. As dosage forms suitable for transdermal administration or transmucosal administration, for example, liquid preparations (including coating agents, eye drops, nasal drops, inhalants), suspensions (including emulsions, creams), powders (including nasal drops, inhalants), pastes, gels, ointments, plasters, etc. can be mentioned. As dosage forms suitable for rectal administration, for example, suppositories, etc. can be mentioned.

[0317] In addition, regarding the specific shape and size of each of the above dosage forms, for each dosage form, it is only necessary to be within the range of dosage forms known in the art, and it is not particularly limited.

[0318] In principle, the pharmaceutical composition of the present invention can be prepared by using a pharmaceutical formulation method known in the art. For example, the methods described in Remington’s Pharmaceutical Sciences (Merck Publishing Co., Easton, Pa.) can be referred to.

[0319] For example, in the case of an injection, the aptamer of the present invention can be dissolved in a pharmaceutically acceptable solvent, and a pharmaceutically acceptable carrier can be added as needed, and it can be manufactured by a method commonly used in the art.

[0320] As the "pharmaceutically acceptable solvent", for example, water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. can be mentioned. These solvents are preferably adjusted to be isotonic with blood as needed.

[0321] The pharmaceutical composition of the present invention can be administered to an organism in a pharmaceutically effective amount for the treatment or prevention of the target disease. The organism to be administered is a vertebrate, preferably a mammal, and more preferably a human.

[0322] The pharmaceutical composition of the present invention can be administered by any means of systemic administration or local administration, which can be appropriately selected according to the type of disease, the location of onset, or the degree of progression, etc. In the case of a disease with a local onset location, local administration directly to the onset location and its surrounding area by injection or the like is preferred. This is because it is possible to administer a sufficient amount of the aptamer of the present invention to the location (tissue or organ) to be treated, and it is not easy to affect other tissues. On the other hand, in the case where the treatment location cannot be specified or the onset is a systemic disease, although not limited, systemic administration such as intravenous injection is preferred. This is because by allowing the aptamer of the present invention to spread throughout the body via the bloodstream, it is possible to administer even to lesion sites that cannot be detected in the diagnosis.

[0323] The pharmaceutical composition of the present invention can be administered by any suitable method that does not inactivate the active ingredient. For example, it can be parenteral (e.g., injection, aerosol, coating, eye drop, nose drop) or oral. Injection is preferred.

[0324] In the case of administration by injection, the injection site is not particularly limited. As long as the DNA aptamer as the active ingredient can bind to the target substance, it can be any site. Examples include intravenous, intra-arterial, intrahepatic, intramuscular, intra-articular, intramedullary, intramedullary cavity, intraventricular, transpulmonary, transdermal, subcutaneous, intradermal, and intraperitoneal, etc.

[0325] <Treatment and / or prevention method>

[0326] In one aspect, there is provided a method for treating and / or preventing a disease, comprising administering the aptamer of the present invention or the above-mentioned pharmaceutical composition to a subject.

[0327] In the present specification, species that can be "subjects" are, for example, mammals, such as primates like humans and chimpanzees, experimental animals like rats and mice, domestic animals like pigs, cows, horses, sheep, and goats, and pets like dogs and cats, preferably humans.

[0328] <Detection agent>

[0329] In one aspect of the present invention, a detection agent is also provided. The detection agent of the present invention utilizes the binding ability of the aptamer of the present invention to a target protein and is a reagent for detecting the target protein in vivo or in vitro. The target protein is vWF, IFNγ, thrombin, VEGF, HMGB1, transferrin receptor 1, DEN1 protein, DEN2 protein, DEN3 protein, or DEN4 protein. For example, by pre-labeling the aptamer with a fluorescent reagent or the like and administering it, the expression intensity of any of the above target proteins can be determined in a living organism, and its localization can also be investigated. Thus, it is possible to assist in the diagnosis of the above diseases. The aptamer of the present invention is also useful in imaging and tissue staining, etc.

[0330] In one embodiment, the present invention relates to a composition for detecting a target protein containing the nucleic acid aptamer of the present invention. The constitution of the composition is the same as that of the above-mentioned pharmaceutical composition, and the description thereof is omitted here.

[0331] In one aspect, the present invention relates to a kit for detecting a target protein containing the nucleic acid aptamer of the present invention. The kit of the present invention may contain a buffer, a labeling reagent, and / or an instruction manual, etc., in addition to the nucleic acid aptamer of the present invention.

[0332] <Detection method>

[0333] In one aspect, the present invention relates to a method for detecting any of the above-mentioned target proteins. This method includes: a step of bringing a sample obtained from a subject into contact with the nucleic acid aptamer of the present invention, and a step of detecting any of the above-mentioned target proteins based on the binding of the sample to the nucleic acid aptamer. By this method, the diagnosis of diseases related to the above-mentioned target protein can be assisted.

[0334] Examples of the sample used in this method include tissues and biological specimens. Examples of tissues include lesion sites, such as the brain, heart, liver, pancreas, lung, bone marrow, lymph nodes, and spleen, etc. For example, biopsy samples of these tissues can be used. Examples of biological specimens include, for example, body fluids (e.g., blood, plasma, lymph, tissue fluid, or urine), and cells, such as peripheral blood cells, hair matrix cells, oral cells, nasal cells, intestinal cells, vaginal cells, mucosal cells, and sputum (which may contain alveolar cells or tracheal cells, etc.).

[0335] The detection step of the detection method of the present invention is not particularly limited as long as it utilizes the binding of the sample to the nucleic acid aptamer, and a known method can be used. For example, SPR method, quartz crystal microbalance method, turbidimetry, colorimetry, or fluorescence method.

[0336] SPR (surface plasmon resonance) refers to the phenomenon that when a metal thin film is irradiated with laser light, the intensity of the reflected light significantly attenuates at a specific incident angle (resonance angle). The SPR method is a measurement method that utilizes this phenomenon and can measure the adsorbate on the surface of the metal thin film serving as the sensor part with high sensitivity. In the present invention, for example, the nucleic acid aptamer of the present invention is immobilized on the surface of the metal thin film in advance, the sample is passed on the surface of the metal thin film, and the difference in the adsorbate on the metal surface before and after the passage of the sample due to the binding of the nucleic acid molecule to the target substance is detected, thereby enabling the detection of the target substance in the sample. The SPR method is known as the replacement method, indirect competition method, etc., and can be used arbitrarily.

[0337] The QCM (Quartz Crystal Microbalance) method is a method that utilizes the phenomenon that if a substance is adsorbed on the electrode surface installed on the crystal oscillator, the resonant frequency of the crystal oscillator decreases corresponding to its mass. The QCM sensor using this method can quantitatively capture a very small amount of adsorbate by the change in the water resonant frequency. In the present invention, the DNA aptamer is pre-immobilized on the electrode surface in the same manner as the above-mentioned SPR method, and the sample is contacted with the electrode surface, so that the change in the water resonant frequency generated by the combination of the DNA aptamer and the target substance can be quantitatively detected in the sample. This technology is well known in the field. For example, reference can be made to Christopher J., et al. (2005) Self-Assembled Monolayers of a Form of Nanotechnology, Chemical Review, 105: 1103-1169.

[0338] Turbidimetry is a method of measuring the amount of substance in a solution by irradiating a solution with light and optically measuring the attenuation of scattered light scattered by a substance suspended in the solution or the transmitted light passing through the solution using a colorimeter or the like. In the present invention, the target substance in the sample can be quantitatively detected by measuring the absorbance before and after the nucleic acid aptamer of the present invention is added to the sample.

[0339] In addition, the target substance can also be detected by combining with an antibody for the target substance. For example, a sandwich method using the ELISA method can be used. In this method, first, the nucleic acid aptamer of the present invention is fixed in advance on a solid phase carrier, and then a sample is added to bind the target substance present in the sample to the nucleic acid aptamer. Next, the sample is rinsed, and then an anti-target substance antibody is added to bind it to the target substance. After cleaning, the anti-target substance antibody is detected by using a properly labeled secondary antibody, so that the target substance in the sample can be detected. As a solid phase carrier, an insoluble carrier in the shape of beads, microplates, test tubes, strips or test pieces made of materials such as polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, latex, gelatin, agarose, cellulose, Sepharose, glass, metal, ceramic or magnetic body can be used.

[0340] In addition, the target substance can also be detected by combining with an antibody against the target substance. For example, a sandwich method using the ELISA method can be used.

[0341] When a target protein is detected by the detection process of this solution, it indicates that the subject from whom the sample is derived has a disease related to the target protein. For example, when DEN1 - DEN4 proteins are detected by the nucleic acid aptamers of the present invention that bind to DEN1 - DEN4 proteins, it indicates that the subject under test has dengue virus type 1 - type 4 respectively.

[0342] In a further aspect of the present invention, there is also provided any of the above nucleic acid aptamers of the present invention for use in the treatment and / or prevention of diseases in a subject such as a human. Furthermore, according to the present invention, there is also provided any of the above nucleic acid aptamers of the present invention for use in the manufacture of a drug for the treatment and / or prevention of diseases.

[0343] In a further aspect, the present invention relates to a method for assisting in the diagnosis of whether a subject has a disease. This method includes the step of administering the nucleic acid aptamer of the present invention to the subject and the step of detecting the nucleic acid aptamer. For example, when the nucleic acid aptamer is detected at a high concentration in a specific site in the living body, it can be determined that a disease has occurred at that site. The detection process can use a known method, for example, the above fluorescence method can be used.

[0344] Examples

[0345] <Example 1: Chemical synthesis of Ds substitute>

[0346] (Purpose)

[0347] The structure of the highly hydrophobic artificial base 7-(2-thienyl)imidazo[4,5-b]pyridine (hereinafter referred to as “Ds”) was modified to create a new artificial base (hereinafter referred to as “Ds substitute”). The highly hydrophobic Ds base consists of two modules, a 1-deazapurine (imidazo[4,5-b]pyridine) moiety and a thiophene side chain. Thus, by replacing the 1-deazapurine moiety with pyrrolo[2,3-b]pyridine, purine, indole, benzimidazole, or pyrrolo[2,3-d]pyrimidine, and replacing the thiophene side chain with furyl, imidazolyl, thiazolyl, pyridazinyl, and their modified structures (such as methylthienyl, dithienyl moieties), various Ds substitutes (Bs, Is, Ys, Yi, Do, Dp, Dt, Yo, Ps, Es, Ysm, Dss, Yss, Bss, and Bo) as shown were synthesized. Figure 2 shown.

[0348] (Method and results)

[0349] Figure 2Among the Ds and Ds substitutes shown, the unnatural phosphoramidites of dDs, dDss, and dPs (also expressed as "s'") were prepared by the methods described in the past literature (Hirao, I., et al., Nat. Methods, 2006, 3: 729-735.; Kimoto, M., et al., J. Am. Chem. Soc., 2010, 132: 4988-4989.; Fujiwara, T., et al., Bioorg. Med. Chem. Lett., 2001, 11: 2221-2223). The unnatural phosphoramidites of dDt, dYi, dDp, and dDo were also synthesized by the same method. The chemical synthesis methods of other unnatural phosphoramidites are described in the item of chemical synthesis. The Ds substitutes other than the above were synthesized by the following methods.

[0350] (1) Chemical synthesis of dBs phosphoramidite

[0351] Compounds 2 to 6 corresponding to (2) to (6) in the reaction pathway diagram shown below were synthesized by the following methods.

[0352]

[0353] Compound 2: 4-(Thiophen-2-yl)-1H-benzo[d]imidazole

[0354] A mixture of 4-bromo-1h-benzoimidazole (1.00 g, 5.08 mmol) and Pd(PPh3)2Cl2 (0.357 g, 0.508 mmol) in DMF (25 mL) was degassed under vacuum, and the gas phase was replaced with argon. After 10 minutes, 2-(tributylstannyl)thiophene (1.97 mL, 6.10 mmol) was added, and the mixture was further degassed under argon for 10 minutes and stirred at 100 °C for 6 hours. The reaction mixture was liquid-extracted with brine and ethyl acetate. The organic layer was concentrated in vacuo to obtain a crude product. After two stages of silica gel column chromatography using the solvent hexane / EtOAc (20-50% EtOAc in hexane), compound 2 was obtained as a mixture with the unreacted starting material as a beige solid (0.39 g, 1.94 mmol, 38.2%).

[0355] Compound 3: ((2R,3S,5R)-3-((4-Methylbenzoyl)oxy)-5-(4-(thiophen-2-yl)-1H-benzo[d]imidazol-1-yl)tetrahydrofuran-2-yl) methyl 4-methylbenzoate

[0356] Compound 2 (0.38 g, 1.94 mmol) was dissolved in ACN (25 mL), sodium hydride (60%) (0.093 g, 2.33 mmol) was added, and the mixture was stirred at ambient temperature for 20 minutes. Then, 3,5-di-o-(p-tolyl)-2-deoxy-ribofuranosyl chloride (0.91 g, 2.33 mmol) was added, and the solution was further stirred at ambient temperature for 3 hours. After removing the solvent, the reaction mixture was subjected to liquid extraction with brine and ethyl acetate. The combined organic layers were dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain a crude product. The desired product, compound 3 (0.70 g, 1.27 mmol, 65.4%), was obtained as a colorless oil by silica gel column chromatography with the solvent being hexane / EtOAc (20% - 30% EtOAc in hexane). 1 1H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.58 (s, 1H), 8.07 (dd, 1H, J = 1.2, 3.6 Hz), 8.00 (d, 2H, J = 8.2 Hz), 7.84 (d, 2H, J = 8.2 Hz), 7.66 (dd, 1H, J = 0.8, 8.1 Hz), 7.60 - 7.58 (m, 2H), 7.39 (d, 2H, J = 8.0 Hz), 7.31 (d, 2H, J = 8.0 Hz), 7.19 - 7.15 (m, 2H), 6.64 - 6.60 (m, 1H), 5.76 - 5.74 (m, 1H), 4.66 - 4.54 (m, 3H), 2.84 - 2.78 (m, 1H), 2.42, 2.36 (2s, 6H), 2.33 - 2.32 (m, 1H).

[0357] Compound 4: (2R,3S,5R)-2-(hydroxymethyl)-5-(4-(thiophen-2-yl)-1H-benzo[d]imidazol-1-yl)tetrahydrofuran-3-ol

[0358] To a solution of compound 3 (0.70 g, 1.27 mmol) in DCM / MeOH (1:1) (9.6 mL) was added sodium methoxide (5 M in MeOH) (0.660 mL, 3.30 mmol), and the solution was stirred at ambient temperature for 30 minutes. The solvent was removed in vacuo to obtain a crude product. After silica gel column chromatography (0 - 6% MeOH in DCM), followed by C18 RP-HPLC (isocratic elution with 35% CH3CN in H2O), compound 4 was isolated as a white solid (0.31 g, 0.97 mmol, 76.9%). 11H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.57 (s, 1H), 8.09 (dd, 1H, J = 1.2, 3.6 Hz), 7.65 - 7.58 (m, 3H), 7.29 (t, 1H, J = 7.8 Hz), 7.18 (dd, 1H, J = 3.6, 5.1 Hz), 6.41 - 6.37 (m, 1H), 5.36 (d, 1H, J = 4.2 Hz), 4.98 (t, 1H, J = 5.3 Hz), 4.43 - 4.39 (m, 1H), 3.90 - 3.87 (m, 1H), 3.61 - 3.52 (m, 2H), 2.66 - 2.59 (m, 1H), 2.37 - 2.31 (m, 1H).

[0359] Compound 5: (2R,3S,5R)-2-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(4-(thiophen-2-yl)-1H-benzo[d]imidazol-1-yl)tetrahydrofuran-3-ol

[0360] Before adding pyridine (1.0 mL) and DMTrCl (0.27 g, 0.81 mmol), Compound 4 (0.25 g, 0.81 mmol) was co-evaporated with pyridine (3 times). The reaction mixture was stirred at ambient temperature for 1 hour. Work-up was carried out using liquid extraction with NaHCO3 solution and ethyl acetate. The combined organic layers were dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. Using the solvent hexane / EtOAc (0 - 100% EtOAc in hexane) (1% TEA) in silica gel column chromatography, the target product, Compound 5 (0.35 g, 0.57 mmol, 70.2%), was obtained as a colorless oil. 1 1H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.50 (s, 1H), 8.10 (dd, 1H, J = 1.1, 3.6 Hz), 7.66 - 7.58 (m, 3H), 7.31 - 7.13 (m, 11H), 6.77 - 6.71 (m, 4H), 6.44 (t, 1H, J = 6.2 Hz), 5.43 (d, 1H, J = 4.8 Hz), 4.48 - 4.43 (m, 1H), 4.00 - 3.99 (m, 1H), 3.67, 3.65 (2s, 6H), 3.17 - 3.08 (m, 2H), 2.84 - 2.77 (m, 1H), 2.45 - 2.39 (m, 1H).

[0361] Compound 6: (2R,3S,5R)-2-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(4-(thiophen-2-yl)-1H-benzo[d]imidazol-1-yl)tetrahydrofuran-3-yl (2-cyanoethyl)diisopropylphosphoramidite

[0362] Before adding ACN (14.5 mL), DIPA (0.037 mL, 0.26 mmol), and 2-cyanoethyl N,N,N'-tetraisopropylphosphorodiamidite (0.288 mL, 0.91 mmol), compound 5 (0.35 g, 0.57 mmol) and 1H-tetrazole in ACN (0.4 M) (0.653 mL, 0.26 mmol) were co-evaporated with ACN (3 times), and the reaction mixture was stirred at ambient temperature for 3 hours. The combined organic layers were washed with water, dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. After silica gel column chromatography with the solvent hexane / EtOAc (0 - 40% EtOAc in hexane) (1% TEA), compound 6 was obtained as a white foamy solid (0.35 g, 0.43 mmol, 75.5%). 1 1H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.49 (d, 1H, J = 2.6 Hz), 8.10 - 8.09 (m, 1H), 7.67 - 7.58 (m, 3H), 7.33 - 7.15 (m, 11H), 6.80 - 6.74 (m, 4H), 6.49 - 6.45 (m, 1H), 4.74 - 4.68 (m, 1H), 4.18 - 4.10 (m, 1H), 3.79 - 3.50 (m, 10H), 3.28 - 3.14 (m, 2H), 2.95 - 2.89 (m, 1H), 2.78 (t, 1H, J = 5.9 Hz), 2.66 (t, 1H, J = 5.9 Hz), 1.15 - 1.11 (m, 10H), 1.01 - 1.00 (m, 2H). 31 31P NMR (DMSO-d6, 162 MHz) δ, ppm: 148.02, 147.32 (diastereomers).

[0363] (2) Chemical synthesis of dIs phosphoramidite

[0364] Compounds 2 - 6 corresponding to (2) - (6) in the reaction pathway diagram shown below were synthesized by the following methods.

[0365]

[0366] Compound 2: 4-(Thiophen-2-yl)-1H-indole

[0367] A mixture of 4-bromoindole (0.50 g, 2.55 mmol) and Pd(PPh3)2Cl2 (0.179 g, 0.255 mmol) in DMF (13 mL) was degassed under vacuum, and the gas phase was replaced with argon for 10 minutes. After adding 2-(tributylstannyl)thiophene (0.989 mL, 3.06 mmol), the mixture was further degassed under argon for 10 minutes and stirred at 100 °C for 3 hours. The reaction mixture was subjected to liquid extraction with brine and ethyl acetate. The organic layer was dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. After silica gel column chromatography using the solvent hexane / EtOAc (5 - 100% EtOAc in hexane), compound 2 (0.36 g, 1.84 mmol, 72.4%) was obtained as a brown oil. 1 H NMR (DMSO-d6, 400 MHz) δ, ppm: 11.47 (bs, 1H), 7.57 (dd, 1H, J = 1.2, 5.1 Hz), 7.52 (dd, 1H, J = 1.2, 3.6 Hz), 7.46 (t, 1H, J = 2.8 Hz), 7.39 (dt, 1H, J = 0.9, 8.1 Hz), 7.26 (dd, 1H, J = 0.9, 7.3 Hz), 7.19 (dd, 1H, J = 3.6, 5.1 Hz), 7.16 - 7.12 (m, 1H), 6.80 - 6.79 (m, 1H).

[0368] Compound 3: Methyl ((2R,3S,5R)-3-((4-methylbenzoyl)oxy)-5-(4-(thiophen-2-yl)-1H-indol-1-yl)tetrahydrofuran-2-yl) 4-methylbenzoate

[0369] Sodium hydride (60%) (0.088 g, 2.21 mmol) was added to a mixture obtained by dissolving compound 2 (0.36 g, 1.84 mmol) in ACN (24 mL), and the mixture was stirred at ambient temperature for 20 minutes. Then, 3,5-di-O-(p-tolyl)-2-deoxyribosyl chloride (0.86 g, 2.21 mmol) was added, and the solution was further stirred at ambient temperature for 3 hours. After removing the solvent, the reaction mixture was subjected to liquid extraction with brine and ethyl acetate. The combined organic layers were dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. Using the solvent hexane / EtOAc (5 - 10% EtOAc in hexane) in silica gel column chromatography, the target product, compound 3 (0.89 g, 1.60 mmol, 87.4%), was obtained as a pale yellow oil. 11H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.02 - 7.98 (m, 2H), 7.89 - 7.86 (m, 3H), 7.73 - 7.68 (m, 2H), 7.60 (dd, 1H, J = 1.1, 5.1 Hz), 7.51 (dd, 1H, J = 1.1, 3.6 Hz), 7.40 - 7.31 (m, 6H), 7.20 (dd, 1H, J = 3.6, 5.1 Hz), 7.15 (t, 1H, J = 7.8 Hz), 6.88 (d, 1H, J = 3.4 Hz), 6.67 - 6.59 (m, 1H), 5.73 - 5.70 (m, 1H), 4.63 - 4.50 (m, 3H), 3.04 - 2.97 (m, 1H), 2.74 - 2.69 (m, 1H), 2.42, 2.38 (2s, 6H).

[0370] Compound 4: (2R,3S,5R)-2-(Hydroxymethyl)-5-(4-(thiophen-2-yl)-1H-indol-1-yl)tetrahydrofuran-3-ol

[0371] To a stirred solution of compound 3 (0.88 g, 1.60 mmol) in DCM / MeOH (1:1) (12.2 mL) was added sodium methoxide (5 M in MeOH) (0.832 mL, 4.16 mmol), and the solution was stirred at ambient temperature for 30 minutes. The solvent was removed in vacuo to afford the crude product. After repurification by silica gel column chromatography (0 - 5% MeOH in DCM), followed by C18 RP-HPLC (isocratic elution with 40% CH3CN in H2O), and C18 RP-HPLC (isocratic elution with 45% CH3CN in H2O), compound 4 was isolated as a white solid (0.22 g, 0.682 mmol, 47.7%). 1 1H NMR (DMSO-d6, 400 MHz) δ, ppm: 7.71 (d, 1H, J = 3.5 Hz), 7.60 - 7.58 (m, 2H), 7.51 (dd, 1H, J = 1.1, 3.6 Hz), 7.31 (dd, 1H, J = 0.8, 7.4 Hz), 7.22 - 7.18 (m, 2H), 6.86 (d, 1H, J = 3.4 Hz), 6.44 - 6.40 (m, 1H), 5.30 (d, 1H, J = 4.3 Hz), 4.90 (t, 1H, J = 5.4 Hz), 4.38 - 4.34 (m, 1H), 3.85 - 3.82 (m, 1H), 3.58 - 3.47 (m, 2H), 2.55 - 2.52 (m, 1H), 2.28 - 2.23 (m, 1H).

[0372] Compound 5: (2R,3S,5R)-2-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(4-(thiophen-2-yl)-1H-indol-1-yl)tetrahydrofuran-3-ol

[0373] Compound 4 (0.21 g, 0.68 mmol) was co-evaporated with pyridine (3 times) before adding pyridine (1.0 mL) and DMTrCl (0.23 g, 0.68 mmol). The reaction mixture was stirred at ambient temperature for 1 hour. Work-up was carried out by liquid extraction with NaHCO3 solution and ethyl acetate. The combined organic layers were dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. Using the solvent hexane / EtOAc (0 - 50% EtOAc in hexane) (1% TEA) in silica gel column chromatography, the desired product, Compound 5 (0.28 g, 0.45 mmol, 67.0%), was obtained as a pale yellow oil. 1 1H NMR (DMSO-d6, 400 MHz) δ, ppm: 7.64 (d, 1H, J = 8.3 Hz), 7.60 - 7.58 (m, 2H), 7.51 (dd, 1H, J = 1.1, 3.6 Hz), 7.35 - 7.32 (m, 3H), 7.25 - 7.16 (m, 9H), 6.83 (d, 1H, J = 3.1 Hz), 6.80 - 6.76 (m, 4H), 6.46 (t, 1H, J = 6.4 Hz), 5.38 (d, 1H, J = 4.7 Hz), 4.43 - 4.38 (m, 1H), 3.96 - 3.95 (m, 1H), 3.69, 3.68 (2s, 6H), 3.13 - 3.11 (m, 2H), 2.63 - 2.59 (m, 1H), 2.37 - 2.30 (m, 1H).

[0374] Compound 6: (2R,3S,5R)-2-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(4-(thiophen-2-yl)-1H-indol-1-yl)tetrahydrofuran-3-yl (2-cyanoethyl)diisopropylphosphoramidite

[0375] Before adding ACN (11.5 mL), DIPA (0.029 mL, 0.21 mmol), and 2-cyanoethyl N,N,N'-tetraisopropylphosphorodiamidite (0.228 mL, 0.72 mmol), compound 5 (0.28 g, 0.45 mmol) and 1H-tetrazole in acetonitrile (ACN) (0.4 M) (0.518 mL, 0.21 mmol) were co-evaporated with ACN (3 times). The reaction mixture was stirred at ambient temperature for 3 hours. The combined organic layers were washed with water, dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. Using the solvent hexane / EtOAc (0 - 20% EtOAc in hexane) (1% TEA) in silica gel column chromatography, compound 6 (0.21 g, 0.26 mmol, 57.4%) was obtained as a white foamy solid. 1 H NMR (DMSO-d6, 400 MHz) δ, ppm: 7.65 (dd, 1H, J = 3.1, 8.3 Hz), 7.60 - 7.59 (m, 2H), 7.51 (dd, 1H, J = 1.0, 3.6 Hz), 7.37 - 7.32 (m, 3H), 7.26 - 7.16 (m, 9H), 6.86 - 6.77 (m, 5H), 6.51 - 6.46 (m, 1H), 4.71 - 4.65 (m, 1H), 4.13 - 4.05 (m, 1H), 3.72 - 3.52 (m, 10H), 3.28 - 3.13 (m, 2H), 2.79 - 2.75 (m, 2H), 2.67 (t, 1H, J = 5.9 Hz), 1.16 - 1.11 (m, 10H), 1.02 - 1.01 (m, 2H). 31 P NMR (DMSO-d6, 162 MHz) δ, ppm: 147.81, 147.16 (diastereomers).

[0376] (3) Chemical synthesis of dYs phosphoramidite

[0377] Compounds 2 - 6 corresponding to (2) - (6) in the reaction pathway diagram shown below were synthesized by the following methods.

[0378]

[0379] Compound 2: 4-(thiophen-2-yl)-1H-pyrrolo[2,3-b]pyridine

[0380] A mixture of 4-bromo-1H-pyrrolo[2,3-b]pyridine (1.00 g, 5.07 mmol) in DMF (25 mL) and Pd(PPh3)2Cl2 (0.180 g, 0.253 mmol) was degassed under vacuum and the gas phase was replaced with argon for 10 minutes. After adding 2-(tributylstannyl)thiophene (2.46 mL, 7.61 mmol), the mixture was further degassed under argon for 10 minutes and stirred at 100 °C for 4 hours. The reaction mixture was subjected to liquid extraction with brine and ethyl acetate. The organic layer was dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. Compound 2 (0.82 g, 4.11 mmol, 81.1%) was obtained as a pale yellow solid by two precipitation operations using a mixed solvent of ethyl acetate and dichloromethane. 1 H NMR (DMSO-d6, 400 MHz) δ, ppm: 11.85 (s, 1H), 8.23 (d, 1H, J = 5.0 Hz), 7.81 (dd, 1H, J = 1.2, 3.6 Hz), 7.76 (dd, 1H, J = 1.1, 5.0 Hz), 7.58 (dd, 1H, J = 2.6, 3.4 Hz), 7.34 (d, 1H, J = 5.0 Hz), 7.26 (dd, 1H, J = 3.7, 5.1 Hz), 6.83 (dd, 1H, J = 1.9, 3.6 Hz).

[0381] Compound 3: Methyl ((2R,3S,5R)-3-((4-methylbenzoyl)oxy)-5-(4-(thiophen-2-yl)-1H-pyrrolo[2,3-b]pyridin-1-yl)tetrahydrofuran-2-yl) 4-methylbenzoate

[0382] Sodium hydride (60%) (0.20 g, 4.93 mmol) was added to a mixture obtained by dissolving compound 2 (0.82 g, 4.11 mmol) in ACN (52 mL), and the mixture was stirred at ambient temperature for 20 minutes. Then, 3,5-di-O-(p-tolyl)-2-deoxy-ribofuranosyl chloride (1.92 g, 4.93 mmol) was added, and the solution was further stirred at ambient temperature for 3 hours. After removing the solvent, the reaction mixture was subjected to liquid extraction with brine and ethyl acetate. The combined organic layers were dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. Using the solvent hexane / EtOAc (0 - 30% EtOAc in hexane) in silica gel column chromatography, the target product, compound 3 (1.83 g, 3.32 mmol, 81.0%), was obtained as a yellow oil. 11H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.29 (d, 1H, J = 5.0 Hz), 7.99 - 7.97 (m, 2H), 7.90 - 7.88 (m, 2H), 7.86 (d, 1H, J = 3.9 Hz), 7.83 (dd, 1H, J = 1.1, 3.7 Hz), 7.80 (dd, 1H, J = 1.1, 5.1 Hz), 7.44 (d, 1H, J = 5.0 Hz), 7.40 - 7.38 (m, 2H), 7.34 - 7.32 (m, 2H), 7.28 (dd, 1H, J = 3.7, 5.1 Hz), 6.96 (d, 1H, J = 3.8 Hz), 6.91 - 6.88 (m, 1H), 5.76 - 5.74 (m, 1H), 4.64 - 4.53 (m, 3H), 3.18 - 3.11 (m, 1H), 2.75 - 2.69 (m, 1H), 2.42, 2.38 (2s, 6H).

[0383] Compound 4: (2R,3S,5R)-2-(hydroxymethyl)-5-(4-(thiophen-2-yl)-1H-pyrrolo[2,3-b]pyridin-1-yl)tetrahydrofuran-3-ol

[0384] To a solution of compound 3 (1.83 g, 3.32 mmol) in DCM / MeOH (1:1) (25.2 mL) was added sodium methoxide (5 M in MeOH) (1.726 mL, 8.63 mmol), and the solution was stirred at ambient temperature for 30 minutes. The solvent was removed under vacuum to obtain the crude product. After silica gel column chromatography (0 - 5% MeOH in DCM) and lyophilization, compound 4 (0.85 g, 2.71 mmol, 81.6%) was isolated as a white foamy solid. 1 1H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.27 (d, 1H, J = 5.0 Hz), 7.87 (d, 1H, J = 3.8 Hz), 7.83 (dd, 1H, J = 1.1, 3.6 Hz), 7.79 (dd, 1H, J = 1.1, 5.1 Hz), 7.41 (d, 1H, J = 5.0 Hz), 7.28 (dd, 1H, J = 3.7, 5.1 Hz), 6.93 (d, 1H, J = 3.8 Hz), 6.75 (dd, 1H, J = 6.0, 8.2 Hz), 5.29 (d, 1H, J = 4.1 Hz), 5.04 (t, 1H, J = 5.6 Hz), 4.40 - 4.37 (m, 1H), 3.86 - 3.84 (m, 1H), 3.62 - 3.50 (m, 2H), 2.60 - 2.53 (m, 1H), 2.26 - 2.21 (m, 1H).

[0385] Compound 5: (2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(4-(thiophen-2-yl)-1H-pyrrolo[2,3-b]pyridin-1-yl)tetrahydrofuran-3-ol

[0386] Compound 4 (0.85 g, 2.71 mmol) was co-evaporated with pyridine (3 times) before adding pyridine (2.7 mL) and DMTrCl (0.92 g, 2.71 mmol). The reaction mixture was stirred at ambient temperature for 1 h. Work-up was carried out by liquid extraction with NaHCO3 solution and ethyl acetate. The combined organic layers were dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to give the crude product. The desired product, compound 5 (1.44 g, 2.33 mmol, 86.2 %), was obtained as a pale green foamy solid by silica gel column chromatography using solvent DCM / MeOH (0 - 1 % MeOH in DCM) (1 % TEA). 1 H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.27 (d, 1H, J = 5.0 Hz), 7.83 (dd, 1H, J = 1.1, 3.7 Hz), 7.79 (dd, 1H, J = 1.1, 5.1 Hz), 7.70 (d, 1H, J = 3.8 Hz), 7.43 (d, 1H, J = 5.0 Hz), 7.38 - 7.36 (m, 2H), 7.29 - 7.19 (m, 8H), 6.90 (d, 1H, J = 3.8 Hz), 6.85 - 6.81 (m, 4H), 6.77 (t, 1H, J = 6.7 Hz), 5.36 (d, 1H, J = 4.5 Hz), 4.42 - 4.38 (m, 1H), 3.98 - 3.95 (m, 1H), 3.71, 3.70 (2s, 6H), 3.17 - 3.16 (m, 2H), 2.65 - 2.59 (m, 1H), 2.34 - 2.28 (m, 1H).

[0387] Compound 6: (2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(4-(thiophen-2-yl)-1H-pyrrolo[2,3-b]pyridin-1-yl)tetrahydrofuran-3-yl (2-cyanoethyl)diisopropylphosphoramidite

[0388] Before adding ACN (49 mL), DIPA (0.124 mL, 0.88 mmol), and 2-cyanoethyl N,N,N'-tetraisopropylphosphorodiamidite (0.98 mL, 3.09 mmol), compound 5 (1.19 g, 1.93 mmol) and 1H-tetrazole in ACN (0.4 M) (2.22 mL, 0.88 mmol) were co-evaporated with ACN (3 times). The reaction mixture was stirred at ambient temperature for 3.5 h. The combined organic layers were washed with water, dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. Using the solvent hexane / EtOAc (0 - 40% EtOAc in hexane) (1% TEA) in silica gel column chromatography, compound 6 (1.20 g, 1.47 mmol, 76.3%) was obtained as a white foamy solid. 1 H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.25 (dd, 1H, J = 0.9, 5.1 Hz), 7.83 (dd, 1H, J = 1.1, 3.7 Hz), 7.79 (dd, 1H, J = 1.1, 5.1 Hz), 7.73 (d, 1H, J = 3.8 Hz), 7.43 (dd, 1H, J = 0.6, 5.0 Hz), 7.39 - 7.35 (m, 2H), 7.28 - 7.19 (m, 8H), 6.93 - 6.92 (m, 1H), 6.85 - 6.79 (m, 4H), 6.75 (t, 1H, J = 6.8 Hz), 4.72 - 4.66 (m, 1H), 4.13 - 4.06 (m, 1H), 3.78 - 3.52 (m, 10H), 3.30 - 3.14 (m, 2H), 2.86 - 2.81 (m, 1H), 2.78 (t, 1H, J = 5.9 Hz), 2.68 (t, 1H, J = 5.9 Hz), 2.55 - 2.41 (m, 1H), 1.16 - 1.12 (m, 9H), 1.06 - 1.04 (m, 3H). 31 P NMR (DMSO-d6, 162 MHz) δ, ppm: 147.57, 146.96 (diastereomers).

[0389] (4) Chemical synthesis of dYo phosphoramidite

[0390] Compounds 2 - 6 corresponding to (2) - (6) in the reaction pathway diagram shown below were synthesized by the following methods.

[0391]

[0392] Compound 2: 4-(furan-2-yl)-1H-pyrrolo[2,3-b]pyridine

[0393] A mixture of 4-bromo-1H-pyrrolo[2,3-b]pyridine (0.50 g, 2.54 mmol) in DMF (12.5 mL) and Pd(PPh3)2Cl2 (0.089 g, 0.1273 mmol) was degassed under vacuum, and the gas phase was replaced with argon for 10 minutes. After adding 2-(tributylstannyl)furan (1.20 mL, 3.81 mmol), the mixture was further degassed under argon for 10 minutes and stirred at 100 °C for 3 hours. The reaction mixture was subjected to liquid extraction with brine and ethyl acetate. The organic layer was dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. Compound 2 (0.39 g, 2.12 mmol, 83.5%) was obtained as a pale yellow solid by washing twice with dichloromethane. 1 H NMR (DMSO-d6, 400 MHz) δ, ppm: 11.78 (bs, 1H), 8.23 (d, 1H, J = 5.1 Hz), 7.92 (dd, 1H, J = 0.6, 1.8 Hz), 7.55 (dd, 1H, J = 2.6, 3.3 Hz), 7.38 (d, 1H, J = 5.0 Hz), 7.29 (dd, 1H, J = 0.6, 3.5 Hz), 6.87 (dd, 1H, J = 1.9, 3.5 Hz), 6.72 (dd, 1H, J = 1.8, 3.4 Hz).

[0394] Compound 3: (2R,3S,5R)-5-(4-(Furan-2-yl)-1H-pyrrolo[2,3-b]pyridin-1-yl)-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl 4-methylbenzoate

[0395] Sodium hydride (60%) (0.10 g, 2.54 mmol) was added to a mixture obtained by dissolving compound 2 (0.39 g, 2.12 mmol) in ACN (27 mL), and the mixture was stirred at ambient temperature for 20 minutes. Then, 3,5-di-O-(p-tolyl)-2-deoxyribosyl chloride (0.99 g, 2.54 mmol) was added, and the solution was further stirred at ambient temperature for 3 hours. After removing the solvent, the reaction mixture was subjected to liquid extraction with brine and ethyl acetate. The combined organic layers were dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. The desired product, compound 3 (0.63 g, 1.17 mmol, 55.3%), was obtained as a colorless oil by silica gel column chromatography using a solvent of hexane / EtOAc (0 - 30% EtOAc in hexane). 11H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.29 (d, 1H, J = 5.1 Hz), 7.99 - 7.96 (m, 2H), 7.95 (dd, 1H, J = 0.5, 1.7 Hz), 7.90 - 7.88 (m, 2H), 7.83 (d, 1H, J = 3.8 Hz), 7.49 (d, 1H, J = 5.0 Hz), 7.40 - 7.32 (m, 5H), 7.02 (d, 1H, J = 3.8 Hz), 6.89 (dd, 1H, J = 5.9, 8.7 Hz), 6.73 (dd, 1H, J = 1.8, 3.4 Hz), 5.76 - 5.74 (m, 1H), 4.65 - 4.60 (m, 1H), 4.56 - 4.51 (m, 2H), 3.18 - 3.10 (m, 1H), 2.74 - 2.68 (m, 1H), 2.41, 2.38 (2s, 6H).

[0396] Compound 4: (2R,3S,5R)-5-(4-(Furan-2-yl)-1H-pyrrolo[2,3-b]pyridin-1-yl)-2-(hydroxymethyl)tetrahydrofuran-3-ol

[0397] To a solution of compound 3 (0.63 g, 1.17 mmol) in DCM / MeOH (1:1) (9.0 mL) was added sodium methoxide (5 M in MeOH) (0.608 mL, 3.04 mmol), and the solution was stirred at ambient temperature for 30 minutes. The solvent was removed in vacuo to afford the crude product. Compound 4 (0.29 g, 0.96 mmol, 82.3%) was isolated as a white solid by silica gel column chromatography (0 - 5% MeOH in DCM). 1 1H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.27 (d, 1H, J = 5.1 Hz), 7.95 (dd, 1H, J = 0.6, 1.8 Hz), 7.84 (d, 1H, J = 3.8 Hz), 7.45 (d, 1H, J = 5.1 Hz), 7.34 (dd, 1H, J = 0.6, 3.5 Hz), 6.98 (d, 1H, J = 3.7 Hz), 6.76 - 6.72 (m, 2H), 5.28 (d, 1H, J = 4.1 Hz), 5.04 (t, 1H, J = 5.6 Hz), 4.40 - 4.36 (m, 1H), 3.86 - 3.83 (m, 1H), 3.62 - 3.49 (m, 2H), 2.60 - 2.49 (m, 1H), 2.25 - 2.19 (m, 1H).

[0398] Compound 5: (2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(4-(furan-2-yl)-1H-pyrrolo[2,3-b]pyridin-1-yl)tetrahydrofuran-3-ol

[0399] Compound 4 (0.29 g, 0.96 mmol) was co-evaporated with pyridine (3 times) before adding pyridine (1.0 mL) and DMTrCl (0.33 g, 0.96 mmol). The reaction mixture was stirred at ambient temperature for 1 h. Workup was carried out by liquid extraction with NaHCO3 solution and ethyl acetate. The combined organic layers were dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to give the crude product. The desired product, Compound 5 (0.40 g, 0.67 mmol, 69.7%), was obtained as a white solid by silica gel column chromatography using solvent DCM / MeOH (0 - 1% MeOH in DCM) (1% TEA). 1 H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.27 (d, 1H, J = 5.1 Hz), 7.94 (dd, 1H, J = 0.6, 1.7 Hz), 7.67 (d, 1H, J = 3.8 Hz), 7.47 (d, 1H, J = 5.0 Hz), 7.38 - 7.34 (m, 3H), 7.28 - 7.19 (m, 7H), 6.96 (d, 1H, J = 3.8 Hz), 6.84 - 6.80 (m, 5H), 6.74 (dd, 1H, J = 1.8, 3.5 Hz), 5.35 (d, 1H, J = 4.5 Hz), 4.42 - 4.37 (m, 1H), 3.97 - 3.94 (m, 1H), 3.71, 3.70 (2s, 6H), 3.16 - 3.15 (m, 2H), 2.66 - 2.59 (m, 1H), 2.33 - 2.27 (m, 1H).

[0400] Compound 6: (2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(4-(furan-2-yl)-1H-pyrrolo[2,3-b]pyridin-1-yl)tetrahydrofuran-3-yl (2-cyanoethyl)diisopropylphosphoramidite

[0401] Before adding ACN (17 mL), DIPA (0.043 mL, 0.31 mmol) and 2-cyanoethyl N,N,N'-tetraisopropylphosphorodiamidite (0.339 mL, 1.07 mmol), compound 5 (0.40 g, 0.66 mmol) and 1H-tetrazole in ACN (0.4 M) (0.77 mL, 0.31 mmol) were co-evaporated with ACN (3 times). The reaction mixture was stirred at ambient temperature for 3 hours. The reaction was quenched by adding aqueous NaHCO3 solution, followed by liquid extraction with aqueous NaHCO3 solution and ethyl acetate. The combined organic layers were washed with water, dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. Using solvent hexane / EtOAc (0 - 30% EtOAc in hexane) (1% TEA) in silica gel column chromatography, compound 6 (0.32 g, 0.39 mmol, 60.2%) was obtained as a white foamy solid. 1H NMR (DMSO-d6, 400 MHz) δ, ppm: 88.26 (dd, 1H, J = 1.0, 5.1 Hz), 7.94 (dd, 1H, J = 0.6, 1.7 Hz), 7.71 (d, 1H, J = 3.8 Hz), 7.47 (d, 1H, J = 5.1 Hz), 7.39 - 7.35 (m, 3H), 7.27 - 7.21 (m, 7H), 6.98 (dd, 1H, J = 1.4, 3.8 Hz), 6.84 - 6.73 (m, 6H), 4.71 - 4.65 (m, 1H), 4.13 - 4.04 (m, 1H), 3.71 - 3.51 (m, 10H), 3.28 - 3.14 (m, 2H), 2.87 - 2.81 (m, 1H), 2.78 (t, 1H, J = 5.9 Hz), 2.68 (t, 1H, J = 5.9 Hz), 2.51 - 2.40 (m, 1H with DMSO peak), 1.16 - 1.12 (m, 10H), 1.05 - 1.04 (m, 2H). 31P NMR (DMSO-d6, 162 MHz) δ, ppm: 147.57, 146.94 (diastereomers).

[0402] (5) Chemical synthesis of dEs phosphoramidite

[0403] Compounds 2 - 6 corresponding to (2) - (6) in the reaction pathway diagram shown below were synthesized by the following methods.

[0404]

[0405] Compound 2: 4-(thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidine

[0406] A mixture of 4-bromo-7H-pyrrolo[2,3-d]pyrimidine (0.594 g, 3.0 mmol) and Pd(PPh3)2Cl2 (0.105 g, 0.15 mmol) in DMF (14.8 mL) was degassed under vacuum, and the gas phase was replaced with argon for 10 minutes. After adding 2-(tributylstannyl)thiophene (1.455 mL, 4.5 mmol), the mixture was further degassed under argon for 10 minutes and stirred at 100 °C for 3 hours. The reaction mixture was subjected to liquid extraction with brine and ethyl acetate. The organic layer was dried over anhydrous MgSO4, filtered, and the filtrate was concentrated under vacuum to obtain the crude product. After washing twice with dichloromethane, compound 2 (0.44 g, 2.22 mmol, 74.2%) was obtained as a pale yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ, ppm: 12.26 (bs, 1H), 8.70 (s, 1H), 8.14 (dd, 1H, J = 1.0, 3.8 Hz), 7.83 (dd, 1H, J = 1.0, 5.0 Hz), 7.66 (dd, 1H, J = 2.4, 3.5 Hz), 7.29 (dd, 1H, J = 3.8, 5.1 Hz), 7.04 (dd, 1H, J = 1.6, 3.6 Hz).

[0407] Compound 3: Methyl ((2R,3S,5R)-3-((4-methylbenzoyl)oxy)-5-(4-(thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-2-yl) 4-methylbenzoate

[0408] Sodium hydride (60%) (0.106 g, 2.664 mmol) was added to a mixture obtained by dissolving compound 2 (0.44 g, 2.22 mmol) in ACN (28.4 mL), and the mixture was stirred at ambient temperature for 20 minutes. Then, 3,5-di-O-(p-tolyl)-2-deoxy-ribonucleosyl chloride (1.035 g, 2.664 mmol) was added, and the solution was further stirred at ambient temperature for 3 hours. After removing the solvent, the reaction mixture was subjected to liquid extraction with brine and ethyl acetate. The combined organic layers were dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. Using the solvent hexane / EtOAc (0 - 30% EtOAc in hexane) in silica gel column chromatography, the target product, compound 3 (1.11 g, 2.00 mmol, 90.4%), was obtained as a pale yellow solid. 11H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.75 (s, 1H), 8.17 (dd, 1H, J = 1.0, 3.8 Hz), 7.97 (d, 2H, J = 8.2 Hz), 7.93 (d, 1H, J = 3.9 Hz), 7.88 - 7.86 (m, 3H), 7.38 (d, 2H, J = 8.0 Hz), 7.32 - 7.29 (m, 3H), 7.20 (d, 1H, J = 3.9 Hz), 6.83 (dd, 1H, J = 6.0, 8.4 Hz), 5.78 - 5.76 (m, 1H), 4.66 - 4.61 (m, 1H), 4.57 - 4.53 (m, 2H), 3.21 - 3.14 (m, 1H), 2.79 - 2.73 (m, 1H), 2.41, 2.37 (2s, 6H).

[0409] Compound 4: (2R,3S,5R)-2-(hydroxymethyl)-5-(4-(thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-3-ol

[0410] To a solution of compound 3 (1.11 g, 2.00 mmol) in DCM / MeOH (1:1) (15.2 mL) was added sodium methoxide (5 M in MeOH) (1.04 mL, 5.20 mmol), and the solution was stirred at ambient temperature for 30 minutes. The solvent was removed in vacuo to afford the crude product. Compound 4 (0.48 g, 1.5 mmol, 76.2%) was isolated as a white solid by silica gel column chromatography (0 - 4% MeOH in DCM). 1 1H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.75 (s, 1H), 8.17 (dd, 1H, J = 1.1, 3.8 Hz), 7.95 (d, 1H, J = 3.8 Hz), 7.86 (dd, 1H, J = 1.0, 5.0 Hz), 7.31 (dd, 1H, J = 3.8, 5.0 Hz), 7.16 (d, 1H, J = 3.8 Hz), 6.70 (dd, 1H, J = 6.1, 7.9 Hz), 5.32 (d, 1H, J = 4.2 Hz), 4.99 (t, 1H, J = 5.5 Hz), 4.41 - 4.38 (m, 1H), 3.88 - 3.85 (m, 1H), 3.63 - 3.51 (m, 2H), 2.60 - 2.53 (m, 1H), 2.29 - 2.24 (m, 1H).

[0411] Compound 5: (2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(4-(thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-3-ol

[0412] Before adding pyridine (1.5 mL) and DMTrCl (0.514 g, 1.52 mmol), compound 4 (0.48 g, 1.52 mmol) was co-evaporated with pyridine (3 times). The reaction mixture was stirred at ambient temperature for 1 hour. Work-up was carried out by liquid extraction using NaHCO3 solution and ethyl acetate. The combined organic layers were dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to afford the crude product. The desired product, compound 5 (0.42 g, 0.69 mmol, 45.4%), was obtained as a white foamy solid by silica gel column chromatography using solvent DCM / MeOH (0 - 1% MeOH in DCM) (1% TEA). 1 H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.74 (s, 1H), 8.17 (dd, 1H, J = 1.0, 3.8 Hz), 7.86 (dd, 1H, J = 1.0, 5.0 Hz), 7.77 (d, 1H, J = 3.8 Hz), 7.37 - 7.35 (m, 2H), 7.30 (dd, 1H, J = 3.8, 5.0 Hz), 7.27 - 7.19 (m, 7H), 7.15 (d, 1H, J = 3.8 Hz), 6.84 - 6.79 (m, 4H), 6.70 (t, 1H, J = 6.6 Hz), 5.38 (d, 1H, J = 4.6 Hz), 4.46 - 4.41 (m, 1H), 3.99 - 3.96 (m, 1H), 3.70, 3.69 (2s, 6H), 3.18 - 3.17 (m, 2H), 2.72 - 2.65 (m, 1H), 2.37 - 2.31 (m, 1H).

[0413] Compound 6: (2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(4-(thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-3-yl (2-cyanoethyl)diisopropylphosphoramidite

[0414] Before adding ACN (18 mL), DIPA (0.044 mL, 0.317 mmol), and 2-cyanoethyl N,N,N'-tetraisopropylphosphorodiamidite (0.350 mL, 1.10 mmol), compound 5 (0.42 g, 0.69 mmol) in ACN (0.4 M) (0.794 mL, 0.317 mmol) and 1H-tetrazole were co-evaporated (3 times). The reaction mixture was stirred at ambient temperature for 3.5 h. The reaction was quenched by the addition of NaHCO3 solution, followed by liquid extraction with NaHCO3 solution and ethyl acetate. The combined organic layers were washed with water, dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. Using the solvent hexane / EtOAc (0 - 30% EtOAc in hexane) (1% TEA) in silica gel column chromatography, compound 6 (0.43 g, 1.52 mmol, 75.7%) was obtained as a white foamy solid. 1 H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.72 (d, 1H, J = 2.2 Hz), 8.18 (dd, 1H, J = 1.0, 3.8 Hz), 7.86 (dd, 1H, J = 1.0, 5.0 Hz), 7.81 (t, 1H, J = 3.4 Hz), 7.37 - 7.16 (m, 11H), 6.84 - 6.78 (m, 4H), 6.70 - 6.66 (m, 1H), 4.75 - 4.69 (m, 1H), 4.15 - 4.06 (m, 1H), 3.78 - 3.52 (m, 10H), 3.29 - 3.15 (m, 2H), 2.92 - 2.85 (m, 1H), 2.78 (t, 1H, J = 5.9 Hz), 2.67 (t, 1H, J = 5.9 Hz), 2.59 - 2.44 (m, 1H, with DMSO peak), 1.16 - 1.12 (m, 10H), 1.05 - 1.03 (m, 2H). 31 P NMR (DMSO-d6, 162 MHz) δ, ppm: 147.68, 147.05 (diastereomers).

[0415] (6) Chemical synthesis of dYsm phosphoramidite

[0416] Compounds 2 - 6 corresponding to (2) - (6) in the following reaction pathway diagram were synthesized by the following methods.

[0417]

[0418] Tributyl(5-methylthiophen-2-yl)stannane

[0419] A stirred solution of 2-methylthiophene (0.405 mL, 4.21 mmol) in THF (21 mL) at -78 °C was added n-BuLi (2 M in hexanes) (2.11 mL, 4.21 mmol), and the mixture was stirred for 30 minutes. Then, tributyl(chloromethyl)stannane (1.14 mL, 4.21 mmol) was added, and the solution was warmed to ambient temperature and further stirred for 30 minutes. The reaction mixture was quenched by the addition of ice-cold H2O and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to afford the Sn reagent.

[0420] Compound 2: 4-(5-methylthiophen-2-yl)-1H-pyrrolo[2,3-b]pyridine

[0421] A mixture of 4-bromo-1H-pyrrolo[2,3-b]pyridine (0.50 g, 2.54 mmol) and Pd(PPh3)2Cl2 (0.089 g, 0.127 mmol) in DMF (12.5 mL) was degassed under vacuum and the gas phase was replaced with argon for 10 minutes. After addition of tributyl(5-methylthiophen-2-yl)stannane (4.21 mmol), the mixture was further degassed under argon for 10 minutes and stirred at 100 °C for 3 hours. The mixture was cooled to room temperature and concentrated in vacuo. The reaction mixture was subjected to liquid-liquid extraction with brine and ethyl acetate. The organic layer was dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to afford the crude product. After washing twice with dichloromethane, Compound 2 (0.33 g, 1.54 mmol, 60.7%) was obtained as a yellow solid. The washings were concentrated in vacuo and further purified by silica gel column chromatography using a solvent hexanes / EtOAc (0 - 50% EtOAc in hexanes) to recover Compound 2 (0.18 g, 0.85 mmol, 33.4%) as a yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ, ppm: 11.80 (bs, 1H), 8.19 (d, 1H, J = 5.0 Hz), 7.61 (d, 1H, J = 3.6 Hz), 7.55 (dd, 1H, J = 2.7, 3.4 Hz), 7.25 (d, 1H, J = 5.0 Hz), 6.95 (dd, 1H, J = 1.1, 3.6 Hz), 6.80 (dd, 1H, J = 1.9, 3.5 Hz), 2.53 (dd, 3H, J = 0.8 Hz).

[0422] Compound 3: Methyl ((2R,3S,5R)-3-((4-methylbenzoyl)oxy)-5-(4-(5-methylthiophen-2-yl)-1H-pyrrolo[2,3-b]pyridin-1-yl)tetrahydrofuran-2-yl) 4-methylbenzoate

[0423] To a stirred mixture of Compound 2 (0.33 g, 1.54 mmol) in ACN (20 mL) was added sodium hydride (60%) (0.074 g, 1.848 mmol), and the mixture was stirred at ambient temperature for 20 minutes. Then, 3,5-di-O-(p-tolyl)-2-deoxy-ribofuranosyl chloride (0.718 g, 1.848 mmol) was added, and the mixture was stirred further at ambient temperature for 3.5 hours. After removing the solvent, the reaction mixture was subjected to liquid extraction with brine and ethyl acetate. The combined organic layers were dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. The crude product was first purified by silica gel column chromatography using solvent hexane / EtOAc (0 - 20% EtOAc in hexane) to obtain Compound 3 (0.37 g, 0.66 mmol, 42.9%). The reaction was repeated with the recovered Compound 2 (0.18 g, 0.85 mmol) to further obtain Compound 3 (0.24 g, 0.42 mmol, 49.8%). The two batches were combined and the purification by silica gel column chromatography using solvent hexane / EtOAc (0 - 20% EtOAc in hexane) was repeated to obtain the desired product, Compound 3 (0.53 g, 0.94 mmol, 39.3%) as a pale yellow oil. 1 1H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.25 (d, 1H, J = 5.1 Hz), 7.98 - 7.96 (m, 2H), 7.90 - 7.88 (m, 3H), 7.83 (d, 1H, J = 3.9 Hz), 7.64 (d, 1H, J = 3.6 Hz), 7.40 - 7.32 (m, 6H), 6.98 - 6.96 (m, 1H), 6.93 (d, 1H, J = 3.8 Hz), 6.88 (dd, 1H, J = 5.9, 8.6 Hz), 5.76 - 7.74 (m, 1H), 4.65 - 4.60 (m, 1H), 4.56 - 4.51 (m, 2H), 3.17 - 3.09 (m, 1H), 2.74 - 2.68 (m, 1H), 2.54 (m, 3H), 2.41 - 2.38 (m, 13H).

[0424] Compound 4: (2R,3S,5R)-2-(Hydroxymethyl)-5-(4-(5-methylthiophen-2-yl)-1H-pyrrolo[2,3-b]pyridin-1-yl)tetrahydrofuran-3-ol

[0425] To a solution of compound 3 (0.53 g, 0.94 mmol) in DCM / MeOH (1:1) (7.2 mL) was added sodium methoxide (5 M in MeOH) (0.491 mL, 2.45 mmol), and the solution was stirred at ambient temperature for 30 minutes. The solvent was removed in vacuo to afford the crude product. Compound 4 (0.20 g, 0.62 mmol, 66.7%) was isolated as a white solid by silica gel column chromatography (0 - 5% MeOH in DCM). 1 H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.23 (d, 1H, J = 5.1 Hz), 7.83 (d, 1H, J = 3.8 Hz), 7.63 (d, 1H, J = 3.6 Hz), 7.33 (d, 1H, J = 5.1 Hz), 6.96 (dd, 1H, J = 1.1, 3.6 Hz), 6.89 (d, 1H, J = 3.8 Hz), 6.73 (dd, 1H, J = 6.0, 8.2 Hz), 5.29 (d, 1H, J = 4.1 Hz), 5.05 (t, 1H, 5.6 Hz), 4.40 - 4.36 (m, 1H), 3.86 - 3.83 (m, 1H), 3.61 - 3.49 (m, 2H), 2.59 - 2.52 (m, 4H), 2.25 - 2.19 (m, 1H).

[0426] Compound 5: (2R,3S,5R)-2-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(4-(5-methylthiophen-2-yl)-1H-pyrrolo[2,3-b]pyridin-1-yl)tetrahydrofuran-3-ol

[0427] Compound 4 (0.20 g, 0.62 mmol) was co-evaporated with pyridine (3 times) before the addition of pyridine (1.0 mL) and DMTrCl (0.21 g, 0.62 mmol), and the reaction mixture was stirred at ambient temperature for 1 hour. The reaction was quenched by the addition of water, and the solvent was removed in vacuo. The work-up was carried out by liquid extraction with NaHCO3 solution and ethyl acetate. The combined organic layers were dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to afford the crude product. The desired product, compound 5 (0.29 g, 0.46 mmol, 74.4%), was obtained as a white foamy solid by silica gel column chromatography using solvent DCM / MeOH (0 - 0.5% MeOH) (1% TEA). 11H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.23 (d, 1H, J = 5.1 Hz), 7.67 (d, 1H, J = 3.8 Hz), 7.63 (d, 1H, J = 3.6 Hz), 7.38 - 7.34 (m, 3H), 7.28 - 7.18 (m, 7H), 6.96 (dd, 1H, J = 1.1, 3.6 Hz), 6.87 (d, 1H, J = 3.8 Hz), 6.85 - 6.81 (m, 4H), 6.76 (t, 1H, J = 6.7 Hz), 5.35 (d, 1H, J = 4.5 Hz), 4.41 - 4.37 (m, 1H), 3.97 - 3.94 (m, 1H), 3.71 (2s, 6H), 3.17 - 3.15 (m, 2H), 2.64 - 2.58 (m, 1H), 2.54 (m, 3H), 2.32 - 2.27 (m, 1H).

[0428] Compound 6: (2R,3S,5R)-2-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(4-(5-methylthiophen-2-yl)-1H-pyrrolo[2,3-b]pyridin-1-yl)tetrahydrofuran-3-yl (2-cyanoethyl)diisopropylphosphoramidite

[0429] Before adding ACN (12 mL), DIPA (0.030 mL, 0.211 mmol), and 2-cyanoethyl N,N,N'-tetraisopropylphosphorodiamidite (0.234 mL, 0.736 mmol), compound 5 (0.29 g, 0.46 mmol) in ACN (0.4 M) (0.529 ml, 0.211 mmol) and 1H-tetrazole were co-evaporated with ACN (3 times), and the reaction mixture was stirred at ambient temperature for 3 hours. The reaction was quenched by adding an aqueous NaHCO3 solution, followed by liquid extraction with an aqueous NaHCO3 solution and ethyl acetate. The combined organic layers were washed with water, dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. Using the solvent hexane / EtOAc (0 - 30% EtOAc in hexane) (1% TEA) in silica gel column chromatography, compound 6 (0.22 g, 0.26 mmol, 58.2%) was obtained as a white foamy solid. 11H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.22 (d, 1H, J = 5.1 Hz), 7.70 (d, 1H, J = 3.8 Hz), 7.64 (d, 1H, J = 3.6 Hz), 7.39 - 7.34 (m, 3H), 7.28 - 7.19 (m, 7H), 6.96 (dd, 1H, J = 1.1, 3.6 Hz), 6.89 (d, 1H, J = 1.7, 3.8 Hz), 6.85 - 6.80 (m, 4H), 6.74 (t, 1H, J = 6.8 Hz), 4.71 - 7.65 (m, 1H), 4.13 - 4.04 (m, 1H), 3.78 - 3.54 (m, 10H), 3.28 - 3.15 (m, 2H), 2.85 - 2.76 (m, 2H), 2.68 (t, 1H, J = 5.9 Hz), 2.54 (m, 3H), 2.45 - 2.39 (m, 1H), 1.16 - 1.12 (m, 9H), 1.05 - 1.04 (m, 3H). 31 31P NMR (DMSO-d6, 162 MHz) δ, ppm: 147.57, 146.95 (diastereomers).

[0430] (7) Chemical synthesis of dBss phosphoramidite

[0431] Compounds 2 - 6 corresponding to (2) - (6) in the following reaction pathway diagram were synthesized by the following method.

[0432]

[0433] 5'-Tributylstannyl-2,2'-dithiophene

[0434] To a stirred solution of 2,2'-dithiophene (1.40 g, 8.42 mmol) in THF (42 mL) at -78 °C was added n-BuLi (2 M in hexanes) (4.21 mL, 8.42 mmol), and the mixture was stirred for 30 minutes. Then, tributyl(chloromethyl)stannane (2.28 mL, 8.42 mmol) was added, and the solution was warmed to ambient temperature and stirred for a further 30 minutes. The reaction mixture was quenched by addition to ice-cold H2O and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to afford the Sn reagent.

[0435] Compound 2: 4-([2,2'-dithiophen]-5-yl)-1H-benzo[d]imidazole

[0436] A mixture of 4-bromo-1H-benzoimidazole (1.00 g, 5.07 mmol) and Pd(PPh3)2Cl2 (0.178 g, 0.253 mmol) in DMF (25 mL) was degassed under vacuum and the gas phase was replaced with argon for 10 minutes. Then 5’-tributylstannyl-2,2’-dithiophene (8.42 mmol) was added and the mixture was further degassed under argon for 10 minutes and then stirred at 100 °C for 17 hours. The reaction mixture was subjected to liquid extraction with brine and ethyl acetate. The organic layer was dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. The crude product was precipitated with DCM and, without further purification, the desired product, compound 2 (1.00 g, 3.56 mmol, 70.4 %), was obtained as a brown solid. 1 1H NMR (DMSO-d6, 400 MHz) δ, ppm: 12.68 (bs, 1H), 8.34 (s, 1H), 8.04 (d, 1H, J = 3.8 Hz), 7.60 - 7.47 (m, 4H), 7.38 - 7.24 (m, 4H), 7.13 - 7.07 (m, 2H).

[0437] Compound 3: (2R,3S,5R)-5-(4-([2,2’-dithiophen]-5-yl)-1H-benzo[d]imidazol-1-yl)-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl 4-methylbenzoate

[0438] Sodium hydride (60 %) (0.171 g, 4.27 mmol) was added to a mixture obtained by dissolving compound 2 (1.00 g, 3.56 mmol) in ACN (45 mL) and the mixture was stirred at ambient temperature for 30 minutes. Then 3,5-di-O-(p-tolyl)-2-deoxy-ribofuranosyl chloride (1.66 g, 4.27 mmol) was added and the mixture was further stirred at ambient temperature for 3 hours. After removing the solvent, the reaction mixture was subjected to liquid extraction with brine and ethyl acetate. The combined organic layers were dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. Using the solvent hexane / EtOAc (0 - 40 % EtOAc in hexane) in silica gel column chromatography, the desired product, compound 3 (1.69 g, 2.66 mmol, 74.7 %), was obtained as a yellow solid. 11H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.61 (s, 1H), 8.01 - 7.99 (m, 2H), 7.85 - 7.83 (m, 2H), 7.69 - 7.63 (m, 2H), 7.53 (dd, 1H, J = 1.1, 5.1 Hz), 7.40 - 7.30 (m, 7H), 7.18 (t, 1H, J = 7.9 Hz), 7.12 (dd, 1H, J = 3.6, 5.1 Hz), 6.63 (dd, 1H, J = 6.1, 8.6 Hz), 5.77 - 5.74 (m, 1H), 4.66 - 4.55 (m, 3H), 3.14 - 3.08 (m, 1H), 2.85 - 2.79 (m, 1H), 2.42, 2.36 (2s, 6H).

[0439] Compound 4: (2R,3S,5R)-5-(4-([2,2'-bithiophen]-5-yl)-1H-benzo[d]imidazol-1-yl)-2-(hydroxymethyl)tetrahydrofuran-3-ol

[0440] To a solution of compound 3 (1.69 g, 2.66 mmol) in DCM / MeOH (1:1) (20 mL) was added sodium methoxide (5 M in MeOH) (1.38 mL, 6.92 mmol), and the solution was stirred at ambient temperature for 30 minutes. The solvent was removed in vacuo to afford the crude product. Compound 4 (0.73 g, 1.84 mmol, 69.4%) was isolated as a yellow solid by silica gel column chromatography (0 - 5% MeOH in DCM). 1 1H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.60 (s, 1H), 8.02 (d, 1H, J = 3.9 Hz), 7.67 - 7.64 (m, 2H), 7.53 (dd, 1H, J = 1.1, 5.1 Hz), 7.38 (dd, 1H, J = 1.1, 3.6 Hz), 7.35 (d, 1H, J = 3.8 Hz), 7.31 (t, 1H, J = 7.9 Hz), 7.12 (dd, 1H, J = 3.6, 5.1 Hz), 6.40 (t, 1H, J = 6.7 Hz), 5.36 (d, 1H, J = 4.2 Hz), 4.98 (t, 1H, J = 5.3 Hz), 4.44 - 4.40 (m, 1H), 3.90 - 3.87 (m, 1H), 3.62 - 3.52 (m, 2H), 2.67 - 2.60 (m, 1H), 2.38 - 2.32 (m, 1H).

[0441] Compound 5: (2R,3S,5R)-5-(4-([2,2'-dithiophen]-5-yl)-1H-benzo[d]imidazol-1-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-ol

[0442] Before adding pyridine (2.0 mL) and DMTrCl (0.623 g, 1.84 mmol), compound 4 (0.73 g, 1.84 mmol) was co-evaporated with pyridine (3 times). The reaction mixture was stirred at ambient temperature for 1 hour. Workup was carried out by liquid extraction using NaHCO3 solution and ethyl acetate. The combined organic layers were dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. Using the solvent hexane / EtOAc (0 - 100% EtOAc) (1% TEA) in silica gel column chromatography, the desired product, compound 5 (0.97 g, 1.38 mmol, 75.3%), was obtained as a pale yellow solid. 1 1H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.53 (s, 1H), 8.02 (d, 1H, J = 3.9 Hz), 7.66 (dd, 2H, J = 2.7, 8.0 Hz), 7.53 (dd, 1H, J = 1.1, 5.1 Hz), 7.38 (dd, 1H, J = 1.1, 3.5 Hz), 7.36 (d, 1H, J = 3.8 Hz), 7.31 - 7.11 (m, 11H), 6.78 - 6.72 (m, 4H), 6.45 (t, 1H, J = 6.1 Hz), 5.43 (d, 1H, J = 4.8 Hz), 4.49 - 4.44 (m, 1H), 4.02 - 3.99 (m, 1H), 3.68, 3.66 (2s, 6H), 3.17 - 3.08 (m, 2H), 2.85 - 2.78 (m, 1H), 2.46 - 2.39 (m, 1H).

[0443] Compound 6: (2R,3S,5R)-5-(4-([2,2'-dithiophen]-5-yl)-1H-benzo[d]imidazol-1-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl (2-cyanoethyl)diisopropylphosphoramidite

[0444] Before adding ACN (35 mL), DIPA (0.089 mL, 0.634 mmol), and 2-cyanoethyl N,N,N'-tetraisopropylphosphoramidite (0.701 mL, 2.20 mmol), compound 5 (0.97 g, 1.38 mmol) and 1H-tetrazole in ACN (0.4 M) (1.587 mL, 0.634 mmol) were co-evaporated with ACN (3 times). The reaction mixture was stirred at ambient temperature for 3 hours. The reaction was quenched by adding aqueous NaHCO3, and extracted with aqueous NaHCO3 and ethyl acetate. The combined organic layers were washed with water, dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. Using the solvent hexane / EtOAc (0 - 50% EtOAc in hexane) (1% TEA) in silica gel column chromatography, compound 6 (0.83 g, 0.925 mmol, 67.0%) was obtained as a yellow foamy solid. 1 H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.52 (d, 1H, J = 2.7 Hz), 8.02 (dd, 1H, J = 1.3, 3.8 Hz), 7.69 - 7.65 (m, 2H), 7.53 (d, 1H, J = 1.0, 5.1 Hz), 7.38 (dd, 1H, J = 1.0, 3.6 Hz), 7.36 (d, 1H, J = 3.8 Hz), 7.33 - 7.11 (m, 11H), 6.80 - 6.74 (m, 4H), 6.48 (dd, 1H, J = 6.2, 12.4 Hz), 4.75 - 4.68 (m, 1H), 4.19 - 4.10 (m, 1H), 3.81 - 3.50 (m, 10H), 3.30 - 3.13 (m, 2H), 2.97 - 2.90 (m, 1H), 2.79 (t, 1H, J = 5.9 Hz), 2.66 (t, 1H, J = 6.0 Hz), 2.62 - 2.57 (m, 1H), 1.15 - 1.11 (m, 10H), 1.01 - 0.99 (m, 2H). 31 P NMR (DMSO-d6, 162 MHz) δ, ppm: 147.99, 147.28 (diastereomers).

[0445] (8) Chemical synthesis of dYss phosphoramidite

[0446] Compounds 2 - 6 corresponding to (2) - (6) in the following reaction pathway diagram were synthesized by the following methods.

[0447]

[0448] 5'-tributylstannyl-2,2'-dithiophene

[0449] To a stirred solution of 2,2'-dithiophene (1.40 g, 8.42 mmol) in THF (42 mL) at -78 °C was added n-BuLi (2 M in hexanes) (4.21 mL, 8.42 mmol), and the mixture was stirred for 30 minutes. Then, tributyl(chloromethyl)stannane (2.28 mL, 8.42 mmol) was added, and the solution was kept at ambient temperature and stirred for a further 30 minutes. The reaction mixture was quenched by addition to ice-cold H2O and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to afford the Sn reagent.

[0450] Compound 2: 4-([2,2'-dithiophen]-5-yl)-1H-pyrrolo[2,3-b]pyridine

[0451] A mixture of 4-bromo-1H-pyrrolo[2,3-b]pyridine (1.0 g, 5.07 mmol) and Pd(PPh3)2Cl2 (0.178 g, 0.253 mmol) in DMF (25 mL) was degassed under vacuum, and the gas phase was replaced with argon for 10 minutes. Then, 5'-tributylstannyl-2,2'-dithiophene (8.42 mmol) was added, and the mixture was further degassed with argon for 10 minutes and then stirred at 100 °C for 3 hours. After cooling the mixture to room temperature, the mixture was concentrated in vacuo. The reaction mixture was subjected to liquid-liquid extraction with brine and ethyl acetate. The organic layer was dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to afford the crude product. The desired product, Compound 2 (0.46 g, 1.65 mmol, 32.6%), was obtained as a brown solid using solvent hexane / EtOAc (20 - 50 - 80% EtOAc in hexane) in silica gel column chromatography. 1 H NMR (DMSO-d6, 400 MHz) δ, ppm: 11.88 (s, 1H), 8.24 (d, 1H, J = 5.1 Hz), 7.79 (d, 1H, J = 3.9 Hz), 7.64 - 7.54 (m, 4H), 7.45 (dd, 1H, J = 1.2, 3.6 Hz), 7.44 (d, 1H, J = 5.0 Hz), 7.36 (d, 1H, J = 5.0 Hz), 7.15 (dd, 1H, J = 3.6, 5.1 Hz), 6.87 (dd, 1H, J = 1.9, 3.5 Hz).

[0452] Compound 3: (2R,3S,5R)-5-(4-([2,2'-dithiophen]-5-yl)-1H-pyrrolo[2,3-b]pyridin-1-yl)-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl 4-methylbenzoate

[0453] Compound 2 (0.44 g, 0.78 mmol) was dissolved in ACN (10 mL), and sodium hydride (60%) (0.037 g, 0.936 mmol) was added. The mixture was stirred at ambient temperature for 30 minutes and further heated at 40 °C for 30 minutes. After cooling to ambient temperature, 3,5-di-O-(p-toluoyl)-2-deoxy-α-D-ribofuranosyl chloride (0.363 g, 0.936 mmol) was added, and the mixture was further stirred at ambient temperature for 13 hours. After removing the solvent, the reaction mixture was subjected to liquid extraction with brine and ethyl acetate. The combined organic layers were dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. The desired product, compound 3 (0.25 g, 0.40 mmol, 51.4%), was obtained as a yellow solid by silica gel column chromatography using hexane / EtOAc (0 - 20% EtOAc in hexane) as the solvent. 1 1H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.29 (d, 1H, J = 5.1 Hz), 7.98 (d, 2H, J = 8.2 Hz), 7.90 - 7.88 (m, 3H), 7.82 (d, 1H, J = 3.9 Hz), 7.60 (dd, 1H, J = 1.1, 5.1 Hz), 7.47 - 7.45 (m, 3H), 7.39 (d, 2H, J = 8.0 Hz), 7.33 (d, 2H, J = 8.0 Hz), 7.15 (dd, 1H, J = 3.6, 5.1 Hz), 7.00 (d, 1H, J = 3.8 Hz), 6.90 (dd, 1H, J = 5.9, 8.6 Hz), 5.77 - 5.75 (m, 1H), 4.66 - 4.61 (m, 1H) 4.57 - 4.52 (m, 2H), 3.19 - 3.11 (m, 1H), 2.76 - 2.70 (m, 1H), 2.42, 2.38 (2s, 6H).

[0454] Compound 4: (2R,3S,5R)-5-(4-([2,2'-bithiophen]-5-yl)-1H-pyrrolo[2,3-b]pyridin-1-yl)-2-(hydroxymethyl)tetrahydrofuran-3-ol

[0455] To a solution of compound 3 (0.53 g, 0.84 mmol) in DCM / MeOH (1:1) (6.4 mL) was added sodium methoxide (5 M in MeOH) (0.436 mL, 2.18 mmol), and the solution was stirred at ambient temperature for 30 minutes. The solvent was removed in vacuo to obtain the crude product. Compound 4 (0.25 g, 0.64 mmol, 76.9%) was isolated as a yellow solid by silica gel column chromatography (0 - 5% MeOH in DCM).1 1H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.27 (d, 1H, J = 5.1 Hz), 7.89 (d, 1H, J = 3.8 Hz), 7.81 (d, 1H, J = 3.9 Hz), 7.59 (dd, 1H, J = 1.2, 5.1 Hz), 7.47 - 7.43 (m, 3H), 7.15 (dd, 1H, J = 3.6, 5.1 Hz), 6.97 (d, 1H, J = 3.8 Hz), 6.75 (dd, 1H, J = 6.0, 8.2 Hz), 5.29 (d, 1H, J = 4.1 Hz), 5.03 (t, 1H, J = 5.6 Hz), 4.41 - 4.37 (m, 1H), 3.87 - 3.84 (m, 1H), 3.62 - 3.50 (m, 2H), 2.61 - 2.54 (m, 1H), 2.26 - 2.21 (m, 1H).

[0456] Compound 5: (2R,3S,5R)-5-(4-([2,2'-Bithiophen]-5-yl)-1H-pyrrolo[2,3-b]pyridin-1-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-ol

[0457] Before adding pyridine (1.0 mL) and DMTrCl (0.201 g, 0.59 mmol), compound 4 (0.24 g, 0.59 mmol) was co-evaporated with pyridine (3 times). The reaction mixture was stirred at ambient temperature for 1 hour. Work-up was carried out by liquid extraction using NaHCO3 solution and ethyl acetate. The combined organic layers were dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. Using solvent hexane / EtOAc (0 - 80% EtOAc) (1% TEA) in silica gel column chromatography, the desired product, compound 5 (0.28 g, 0.40 mmol, 69.0%), was obtained as a yellow foamy solid. 11H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.28 (d, 1H, J = 5.1 Hz), 7.81 (d, 1H, J = 3.9 Hz), 7.72 (d, 1H, J = 3.8 Hz), 7.60 (dd, 1H, J = 1.2, 5.1 Hz), 7.47 - 7.44 (m, 3H), 7.38 - 7.36 (m, 2H), 7.28 - 7.20 (m, 7H), 7.15 (dd, 1H, J = 3.6, 5.1 Hz), 6.94 (d, 1H, J = 3.8 Hz), 6.85 - 6.81 (m, 4H), 6.77 (t, 1H, J = 6.7 Hz), 5.36 (d, 1H, J = 4.5 Hz), 4.43 - 4.38 (m, 1H), 4.05 - 3.95 (m, 1H), 3.71 (2s, 6H), 3.18 - 3.16 (m, 2H), 2.67 - 2.60 (m, 1H), 2.34 - 2.29 (m, 1H).

[0458] Compound 6: (2R, 3S, 5R)-5-(4-([2,2'-Bithiophen]-5-yl)-1H-pyrrolo[2,3-b]pyridin-1-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl (2-cyanoethyl)diisopropylphosphoramidite

[0459] Before adding ACN (10 mL), DIPA (0.026 mL, 0.184 mmol) and 2-cyanoethyl N,N,N'-tetraisopropylphosphorodiamidite (0.203 mL, 0.64 mmol), Compound 5 (0.28 g, 0.40 mmol) and 1H-tetrazole in ACN (0.4 M) (0.46 mL, 0.184 mmol) were co-evaporated with pyridine (3 times). The reaction mixture was stirred at ambient temperature for 3.5 h. The reaction was quenched by adding NaHCO3 solution, followed by liquid extraction with NaHCO3 solution and ethyl acetate. The combined organic layers were washed with water, dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. Using the solvent hexane / EtOAc (0 - 30% EtOAc in hexane) (1% TEA) in silica gel column chromatography, Compound 6 (0.21 g, 0.24 mmol, 60.5%) was obtained as a yellow foamy solid. 11H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.26 (dd, 1H, J = 0.8, 5.1 Hz), 7.82 (d, 1H, J = 3.9 Hz), 7.75 (d, 1H, J = 3.8 Hz), 7.59 (dd, 1H, J = 1.1, 5.1 Hz), 7.47 - 7.44 (m, 3H), 7.39 - 7.35 (m, 2H), 7.29 - 7.19 (m, 7H), 7.15 (dd, 1H, J = 3.6, 5.1 Hz), 6.97 (dd, 1H, J = 2.1, 3.8 Hz), 6.85 - 6.80 (m, 4H), 6.75 (t, 1H, J = 6.9 Hz), 4.72 - 4.66 (m, 1H), 4.14 - 4.05 (m, 1H), 3.81 - 3.52 (m, 10H), 3.24 - 3.17 (m, 2H), 2.87 - 2.82 (m, 1H), 2.78 (t, 1H, J = 5.9 Hz), 2.68 (t, 1H, J = 5.9 Hz), 2.47 - 2.41 (m, 1H), 1.16 - 1.12 (m, 10H), 1.06 - 1.04 (m, 2H). 31 31P NMR (DMSO-d6, 162 MHz) δ, ppm: 147.57, 146.96 (diastereomers).

[0460] (9) Chemical synthesis of dBo phosphoramidite

[0461] Compounds 2 - 6 corresponding to (2) - (6) in the following reaction pathway diagram were synthesized by the following methods.

[0462]

[0463] Compound 2: 4-(furan-2-yl)-1H-benzo[d]imidazole

[0464] A mixture of 4-bromo-1H-benzoimidazole (2.00 g, 10.1 mmol) and Pd(PPh3)2Cl2 (0.354 g, 0.505 mmol) in DMF (50 mL) was degassed under vacuum and the gas phase was replaced with argon. After 10 minutes, 2-(tributylstannyl)furan (4.77 mL, 15.15 mmol) was added and the mixture was further degassed under argon for 10 minutes and stirred at 100 °C for 24 hours. The reaction mixture was subjected to liquid extraction with brine and ethyl acetate. The combined organic layers were dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. Compound 2 was obtained as a pink solid (1.36 g, 7.36 mmol, 72.8%) as a mixture with the unreacted starting material by silica gel column chromatography using the solvent hexane / EtOAc (20 - 90% EtOAc in hexane).

[0465] Compound 3: (2R,3S,5R)-5-(4-(furan-2-yl)-1H-benzo[d]imidazol-1-yl)-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl 4-methylbenzoate

[0466] To a stirred mixture of compound 2 (1.35 g, 7.36 mmol) in ACN (94 mL) was added sodium hydride (60%) (0.353 g, 8.83 mmol) and the mixture was stirred at ambient temperature for 20 minutes. Then, 3,5-di-O-(p-toluoyl)-2-deoxy-α-D-ribofuranosyl chloride (3.43 g, 8.83 mmol) was added and the solution was stirred at ambient temperature for a further 3 hours. After removal of the solvent, the reaction mixture was subjected to liquid extraction with brine and ethyl acetate. The combined organic layers were dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to obtain the crude product. The desired product, compound 3, was obtained as a white foamy solid (2.99 g, 5.57 mmol, 75.7%) by silica gel column chromatography using the solvent hexane / EtOAc (0 - 40% EtOAc in hexane). 11H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.59 (s, 1H), 8.00 - 7.98 (m, 2H), 7.85 - 7.83 (m, 2H), 7.79 (dd, 1H, J = 0.8, 1.8 Hz), 7.67 (dd, 1H, J = 0.8, 8.2 Hz), 7.60 (dd, 1H, J = 0.7, 8.2 Hz), 7.55 (dd, 1H, J = 0.6, 4.0 Hz), 7.40 - 7.38 (m, 2H), 7.32 - 7.30 (m, 2H), 7.21 (t, 1H, J = 7.9 Hz), 6.66 (dd, 1H, J = 1.8, 3.3 Hz), 6.62 (dd, 1H, J = 5.8, 8.4 Hz), 5.76 - 5.74 (m, 1H), 4.66 - 4.54 (m, 3H), 3.15 - 3.07 (m, 1H), 2.84 - 2.78 (m, 1H), 2.41, 2.36 (2s, 6H).

[0467] Compound 4: (2R,3S,5R)-5-(4-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)-2-(hydroxymethyl)tetrahydrofuran-3-ol

[0468] To a solution of Compound 3 (2.99 g, 5.57 mmol) in DCM / MeOH (1:1) (42 mL) was added sodium methoxide (5 M in MeOH) (2.90 mL, 14.5 mmol), and the solution was stirred at ambient temperature for 30 minutes. The solvent was removed under vacuum to obtain the crude product. After silica gel column chromatography (0 - 10% MeOH in DCM), followed by C18 RP-HPLC (isocratic elution with 25% CH3CN in H2O), Compound 4 (1.08 g, 3.61 mmol, 64.8%) was isolated as a white solid. 1 1H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.57 (s, 1H), 7.79 (dd, 1H, J = 0.8, 1.8 Hz), 7.66 - 7.60 (m, 2H), 7.55 (dd, 1H, J = 0.7, 3.3 Hz), 7.32 (t, 1H, J = 7.9 Hz), 6.66 (dd, 1H, J = 1.8, 3.3 Hz), 6.39 (dd, 1H, J = 6.3, 7.2 Hz), 5.35 (d, 1H, J = 4.2 Hz), 4.97 (t, 1H, J = 5.3 Hz), 4.43 - 4.39 (m, 1H), 3.90 - 3.87 (m, 1H), 3.62 - 3.52 (m, 2H), 2.66 - 2.59 (m, 1H), 2.36 - 2.30 (m, 1H).

[0469] Compound 5: (2R,3S,5R)-2-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(4-(furan-2-yl)-1H-benzo[d]imidazol-1-yl)tetrahydrofuran-3-ol

[0470] Compound 4 (0.80 g, 2.66 mmol) was co-evaporated with pyridine (3 times) before the addition of pyridine (2.7 mL) and DMTrCl (0.90 g, 2.66 mmol). The reaction mixture was stirred at ambient temperature for 1 hour. Work-up was carried out using liquid extraction with NaHCO3 solution and ethyl acetate. The combined organic layers were dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo to give the crude product. The desired product, compound 5 (1.18 g, 1.97 mmol, 74.1%), was obtained as a white foamy solid by silica gel column chromatography using the solvent hexane / EtOAc (20 - 100% EtOAc in hexane) (1% TEA). 1 1H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.49 (s, 1H), 7.79 (d, 1H, J = 0.8, 1.8 Hz), 7.67 - 7.60 (m, 2H), 7.57 (dd, 1H, J = 0.7, 3.3 Hz), 7.30 - 7.13 (m, 10H), 6.78 - 6.71 (m, 4H), 6.66 (dd, 1H, J = 1.8, 3.3 Hz), 6.44 (t, 1H, J = 6.2 Hz), 5.42 (d, 1H, J = 4.8 Hz), 4.48 - 4.43 (m, 1H), 4.02 - 3.98 (m, 1H), 3.68, 3.66 (2s, 6H), 3.16 - 3.08 (m, 2H), 2.83 - 2.77 (m, 1H), 2.45 - 2.38 (m, 1H).

[0471] Compound 6: (2R,3S,5R)-2-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(4-(furan-2-yl)-1H-benzo[d]imidazol-1-yl)tetrahydrofuran-3-yl (2-cyanoethyl)diisopropylphosphoramidite

[0472] Before adding ACN (50 mL), DIPA (0.127 mL, 0.906 mmol), and 2-cyanoethyl N,N,N'-tetraisopropylphosphorodiamidite (1.00 mL, 3.152 mmol), co-evaporate compound 5 (1.18 g, 1.97 mmol) and 1H-tetrazole with pyridine in ACN (0.4 M) (2.27 mL, 0.906 mmol) (3 times). Stir the reaction mixture at ambient temperature for 3 hours. Wash the combined organic layers with water, dry over anhydrous MgSO4, filter, and concentrate the filtrate in vacuo to obtain the crude product. After silica gel column chromatography, solvent hexane / EtOAc (0 - 50% EtOAc in hexane) (1% TEA), obtain compound 6 (1.06 g, 1.33 mmol, 68.5%) as a white foamy solid. 1 H NMR (DMSO-d6, 400 MHz) δ, ppm: 8.49 (d, 1H, J = 2.6 Hz), 7.79 (m, 1H), 7.68 - 7.56 (m, 3H), 7.33 - 7.13 (m, 10H), 6.79 - 6.73 (m, 4H), 6.67 - 6.65 (m, 1H), 6.47 (dd, 1H, J = 6.3, 12.4 Hz), 4.74 - 4.67 (m, 1H), 4.18 - 4.09 (m, 1H), 3.79 - 3.50 (m, 10H), 3.27 - 3.12 (m, 2H), 2.96 - 2.89 (m, 1H), 2.78 (t, 1H, J = 5.9 Hz), 2.67 - 2.54 (m, 2H), 1.16 - 1.11 (m, 10H), 1.00 - 0.99 (m, 2H). 31 P NMR (DMSO-d6, 162 MHz) δ, ppm: 147.98, 147.27 (diastereomers).

[0473] <Example 2: Preparation of Nucleic Acid Aptamer>

[0474] (Objective)

[0475] Synthesize a nucleic acid aptamer containing the Ds and / or Ds substitutes synthesized in Example 1.

[0476] (Method and Result)

[0477] The nucleic acid aptamers shown in Table 1 below are nucleic acid aptamers that bind to interferon-γ (IFNγ), von Willebrand factor A1-domain (vWF), NS1 proteins of dengue virus serotype 1 and serotype 3 (DEN1-NS1 and DEN3-NS1) respectively, contain 2 Ds (hereinafter referred to as "IFNγ-DsDs", "vWF-DsDs", "AptD1-DsDs", and "AptD3-DsDs" respectively in the following table; hereinafter often referred to as "Ds aptamers"), and nucleic acid aptamers in which at least 1 D is replaced with various D substitutes or combinations thereof in each nucleic acid aptamer (hereinafter often referred to as "UB aptamers"). The Ds aptamers are shown as Anti-IFNγ and Anti-VWF in Figure 3 and shown as Anti-DEN1-NS1 and Anti-DEN3-NS1 in Figure 4 , based on sequences isolated by the ExSELEX (genetic alphabet Expansion for SELEX) method using a DNA library containing Ds base as the 5th base in previous literatures (Kimoto, M., et al., Nat. Biotechnol., 2013, 31:453-457.; Matsunaga, K., et al., J. Am. Chem. Soc., 2017, 139:324-334.; Matsunaga, K., et al., Nucleic Acids Res., 2021, 49:11407-11424.). All Ds aptamers contain 2 Ds bases, are linked with a biotin-binding minihairpin sequence GGCG(biotin-T)AGCC, and the internal stem-loop structure is replaced with a minihairpin sequence GCGAAGC in IFNγ-DsDs and vWF-DsDs. The nucleic acid aptamers shown in Table 1 below were chemically synthesized using commercially available natural base phosphoramidites, biotin-dT phosphoramidite, and the above-mentioned synthetic unnatural base phosphoramidites by a DNA / RNA synthesizer using phosphoramidite chemistry, deprotected with concentrated ammonia solution, and the full-length DNA fragments were purified by denaturing polyacrylamide gel electrophoresis.

[0478] Table 1: List of nucleic acid aptamers

[0479]

[0480]

[0481]

[0482]

[0483]

[0484] * The underlines indicate sequences that are complementary to each other and form the stem region. The black boxes indicate the microhairpin sequences. "L" in the sequences indicates biotin-dT.

[0485] <Example 3: Binding Assay of Nucleic Acid Aptamers>

[0486] (Purpose)

[0487] For the nucleic acid aptamers prepared in Example 2, the affinity for the target protein was evaluated.

[0488] (Method)

[0489] The affinity of various nucleic acid aptamers for the target protein was evaluated by the EMSA method, SPR method, and ELISA method described below.

[0490] (1) EMSA method (Gel Electrophoresis Mobility Shift Assay)

[0491] The EMSA method was performed as follows: The gel after electrophoresis was stained with SYBR Gold, and the shifted band pattern on the gel was detected with a BIOIMAGER. The band densities of the nucleic acid aptamer bound to the target and the free nucleic acid aptamer were quantified using the software attached to the BIOIMAGER and normalized by the shifted band ratio of the Ds aptamer in the same gel, thereby calculating the relative binding (%). The experimental conditions of the EMSA method are shown in Table 2 below. In addition, the Ds aptamers of IFNγ-DsDs, vWF-DsDs, AptD1-DsDs, and AptD3-DsDs, and each UB aptamer derived from each Ds aptamer are hereinafter referred to as "IFNγ target nucleic acid aptamer", "vWF target nucleic acid aptamer", "DEN1-NS1 target nucleic acid aptamer", and "DEN3-NS1 target nucleic acid aptamer", respectively.

[0492] Table 2: Experimental Conditions of the EMSA Method

[0493]

[0494] (2) SPR method (Surface Plasmon Resonance Method)

[0495] To compare the binding affinity profiles of the DS aptamer and the UB aptamer, SPR analysis was performed using a Biacore T200 (GE Healthcare) platform. Each nucleic acid aptamer (ligand) was immobilized on the flow cell of a streptavidin-coated sensor chip by injecting a 0.5 nM ligand solution into the running buffer. Regarding the running buffer, 1×PBS containing 0.05% Nonidet P-40 was used for the vWF target nucleic acid aptamer, 1×PBS containing 50 mM NaCl and 0.05% Nonidet P-40 was used for the IFNγ target nucleic acid aptamer, and 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 2.7 mM KCl, 0.05% Tween20 was used for the DEN1-NS1 target nucleic acid aptamer and the DEN3-NS1 target nucleic acid aptamer. The conditions were 150 - 960 seconds at 25°C and a flow rate of 5 μL / min. A target protein solution with a concentration ranging from 0.15625 nM to 40 nM was prepared in the running buffer and injected at a flow rate of 100 μL / min. The binding time was 150 seconds, and the dissociation time was 450 seconds (for the IFNγ target nucleic acid aptamer and the vWF target nucleic acid aptamer) or 600 seconds (for the DEN1-NS1 target nucleic acid aptamer and the DEN3-NS1 target nucleic acid aptamer). The sensor chip was regenerated by injecting 50 mM NaOH for 5 seconds and then equilibrated with the running buffer for 10 minutes. Using BIAevaluation T200 software version 1.0 (GE Healthcare), the kinetic data after subtracting the blank were fitted with a 1:1 binding model to determine the K D value. The K D values of each Ds aptamer (IFNγ-DsDs, vWF-DsDs, AptD1-DsDs, and AptD3-DsDs) are shown in Figures 3 - 4 .

[0496] (3) ELISA method

[0497] The experimental protocol for installing the marker used a commercially available ELISA kit (Thermo Fisher Scientific, #88-7316-88). The sensitivity of the IFNγ antibody was measured by replacing the biotinylated detection antibody in the ELISA kit with various nucleic acid aptamers ( Figure 14)。The detection concentration of the antibody used in the experimental protocol was deduced to be 120 nM from the absorbance at 280 nm of the stock solution. Therefore, 120 nM of biotinylated aptamer was used as the standard operating concentration. As the target protein, two kinds of recombinant human IFNγ proteins were used: the product of Peprotech (#300-02, the amino acid residue at position 160 is Q) and the product of Abcam (#ab51240, the amino acid residue at position 160 is R).

[0498] (Results)

[0499] (1) IFNγ target nucleic acid aptamer

[0500] The results of analyzing the affinity of Ds aptamer (AptIFNγ-DsDs) and 11 UB aptamers (AptIFNγ-BsBs, AptIFNγ-IsIs, AptIFNγ-YsYs, AptIFNγ-YiYi, AptIFNγ-DoDo, AptIFNγ-DpDp, AptIFNγ-DssDss, AptIFNγ-DtDt, AptIFNγ-YoYo, AptIFNγ-PsPs, and AptIFNγ-EsEs) for IFNγ by EMSA method are shown in Figure 5 . The AptIFNγ-YsYs and AptIFNγ-YoYo aptamers showed stronger band shifts compared with the AptIFNγ-DsDs aptamer. The K D values determined by SPR method were 42.4 pM for AptIFNγ-DsDs, 19.5 pM for AptIFNγ-YsYs, and 24.4 pM for AptIFNγ-YoYo. The binding ability of these two UB aptamers was enhanced compared with the Ds aptamer.

[0501] (2) vWF target nucleic acid aptamer

[0502] The results of analyzing the affinity of Ds aptamer (AptvWF-DsDs) and 11 UB aptamers (AptvWF-BsBs, AptvWF-IsIs, AptvWF-YsYs, AptvWF-YiYi, AptvWF-DoDo, AptvWF-DpDp, AptvWF-DssDss, AptvWF-DtDt, AptvWF-YoYo, AptvWF-PsPs, and AptvWF-EsEs) for vWF by EMSA method are shown in Figure 6。The AptvWF-YsYs, AptvWF-YoYo aptamers, and AptvWF-BsBs aptamer showed a band shift equal to or greater than that of the AptvWF-DsDs aptamer. Next, the results of analyzing the affinity of three UB aptamers (AptvWF-BsYs, AptvWF-BsYo, and AptvWF-YsBs) obtained by replacing two Ds in AptvWF-DsDs with a combination of different Ds substitutes by EMSA are shown in Figure 7 。AptvWF-BsYs, AptvWF-BsYo, and AptvWF-YsBs showed a stronger band shift compared to the AptvWF-DsDs aptamer. The K D value of AptvWF-DsDs was 54.7 pM, AptvWF-BsBs was 24.0 pM, ptvWF-BsYs was 24.2 pM, AptvWF-BsYo was 22.3 pM, and AptvWF-YsBs was 27.4 pM. It can be seen that the binding ability of these UB aptamers was enhanced compared to the Ds aptamer, and AptvWF-BsYo had the highest affinity.

[0503] (3) DEN1-NS1 target nucleic acid aptamer

[0504] The results of analyzing the affinity of the Ds aptamer (AptD1-DsDs) and 11 UB aptamers (AptD1-BsBs, AptD1-IsIs, AptD1-YsYs, AptD1-YiYi, AptD1-DoDo, AptD1-DpDp, AptD1-DssDss, AptD1-DtDt, AptD1-YoYo, AptD1-PsPs, and AptD1-EsEs) for DEN1-NS1 by EMSA are shown in Figure 8 。Any UB aptamer showed a weaker binding compared to the AptIFNγ-DsDs aptamer, but AptD1-BsBs, AptD1-YsYs, and AptD1-DtDt showed a clear band shift. Therefore, aptamers containing Bs, Ys, and / or Dt in combination in addition to Ds (AptD1-DsYs, AptD1-YsDs, AptD1-DsBs, AptD1-BsDs, AptD1-DsDt, and AptD1-DtDs) were prepared and analyzed by EMSA ( Figures 9 - 10 ). The results showed that AptD1-YsDs, AptD1-BsDs, and AptD1-DsDt ( Figure 9 ), as well as AptD1-YsBs, AptD1-YoBs, and AptD1-YsmBs ( Figure 10) showed a stronger band shift compared to the AptIFNγ-DsDs aptamer. The K D values determined by SPR were 254 - 439 pM for AptD1-DsDs, 48.6 pM for AptD1-DsBs, 35.9 - 44.2 pM for AptD1-YsBs, 56.2 pM for AptD1-YoBs, 63.1 pM for AptD1-YsmBs, 8.55 pM for AptD1-BsYs. AptD1-YsBs and AptD1-BsYs showed the strongest affinity.

[0505] (4) DEN3-NS1 target nucleic acid aptamer

[0506] The results of analyzing the affinity of Ds aptamer (AptD3-DsDs) and 11 UB aptamers (AptD3-BsBs, AptD3-IsIs, AptD3-YsYs, AptD3-YiYi, AptD3-DoDo, AptD3-DpDp, AptD3-DssDss, AptD3-DtDt, AptD3-YoYo, AptD3-PsPs, and AptD3-EsEs) for DEN3-NS1 by EMSA are shown in Figure 11 . AptD3-YsYs, AptD3-DssDss, and AptD3-YoYo aptamers showed relatively strong band shifts. Based on this result, aptamers containing Ds, Ys, Dss, Yss, Yo, and / or Ysm (AptD3-YsDss, AptD3-DssYs, AptD3-YssYs, AptD3-YssYo, AptD3-YssYsm, and AptD3-YsmYss) were prepared and analyzed by EMSA ( Figures 12 - 13 ). The results showed that AptD3-YsDss, AptD3-DssYs, AptD3-DssDss, and AptD3-YsmYsm ( Figure 12 ), and AptD3-YssYs, AptD3-YssYo, AptD3-YssYsm, and AptD3-YsmYsm ( Figure 13 ) showed a stronger band shift compared to the AptIFNγ-DsDs aptamer. The K D values determined by SPR were 71.4 pM for AptD3-DsDs, 41.9 pM for AptD3-DssYs, 41.9 pM for AptD3-YssYs, 55.7 pM for AptD3-YssYo, 91.1 pM for AptD3-YssYsm. AptD1-DssYs and AptD1-YssYs showed the strongest affinity.

[0507] (5) Comparison of IFNγ-targeting nucleic acid aptamers with anti-IFNγ antibodies (ELISA method)

[0508] For the AptIFNγ-DsDs, AptIFNγ-YsYs, and AptIFNγ-YoYo aptamers, the detection sensitivity of IFNγ was studied by ELISA. The results of ELISA using 120 nM nucleic acid aptamers and anti-IFNγ antibodies in the detection showed that the AptIFNγ-DsDs, AptIFNγ-YsYs, and AptIFNγ-YoYo aptamers all showed higher signal intensities compared to the anti-IFNγ antibody attached to the ELISA kit (ThermoFisher Scientific, #88-7316-88). Figure 15 A). In addition, the AptIFNγ-YsYs and AptIFNγ-YoYo aptamers showed significantly higher signal intensities compared to AptIFNγ-DsDs.

[0509] Next, the dependence of the detection reagent concentration was studied. In the experimental protocol of the ELISA kit, 120 nM is recommended as the use concentration of the detection antibody. If the antibody concentration is decreased, the signal intensity is significantly decreased. On the other hand, it was shown that even if the use concentrations of AptIFNγ-DsDs, AptIFNγ-YsYs, and AptIFNγ-YoYo were decreased, there was no great influence on the signal intensity, and AptIFNγ-YsYs and AptIFNγ-YoYo could detect with sufficient sensitivity even at 10 nM. Figure 15 B).

[0510] <Example 4: Isolation of new IFNγ-targeting nucleic acid aptamers, thrombin-targeting nucleic acid aptamers, and HMGB1-targeting nucleic acid aptamers based on the 6-base ExSELEX method>

[0511] (Objective)

[0512] Isolate new nucleic acid aptamers targeting IFNγ, nucleic acid aptamers targeting thrombin, and nucleic acid aptamers targeting HMGB1 by the ExSELEX method.

[0513] (Methods and Results)

[0514] (1) Isolation and analysis of new IFNγ-targeting nucleic acid aptamers

[0515] (a) 6-base ExSELEX method

[0516] In order to discover novel nucleic acid aptamers that bind to human IFNγ, the ExSELEX method (hereinafter referred to as the "6-base ExSELEX method" to distinguish it from the existing 5-base ExSELEX method) was implemented, which uses two non-natural bases out of the following three types represented by the general formulas (XVIII) to (XXX) added to the natural 4 bases. In addition, the non-natural base (7-(2-thienyl)-3H-imidazo[4,5-b]pyridin-3-yl) represented by the following general formula (XVIII) is referred to as "Ds" or "Ds base" in this specification. Further, the non-natural base (4-propynylpyrrole-2-carboxaldehyde) represented by the following general formula (XIX) is referred to as "Pa'" or "Pa' base" in this specification. The non-natural base (2-nitro-4-propynylpyrrole) represented by the following general formula (XX) is referred to as "Px" or "Px base" in this specification.

[0517]

[0518] (In the formula, R 1 represents methyl)

[0519] As two libraries in which Ds bases of two bases are arranged at different positions in a 29-base-long random region composed of five bases in which the non-natural 4-propynylpyrrole-2-carboxaldehyde base (hereinafter referred to as "Pa'" or "Pa' base") is added to A base, G base, C base, and T base, the XL1 library and the XL2 library (including 46 sub-libraries) were prepared. The XL1 library contains a 7-base region arranged on the 5' side of the random region and a 6-base region arranged on the 3' side of the random region, and contains primer sequences for PCR on their further 5' side and 3' side, and contains 40 sub-libraries. The XL2 library contains a 4-base region arranged on the 5' side of the random region and a 3-base region arranged on the 3' side of the random region, and contains a 5-base complementary sequence and a primer sequence for PCR on their further 5' side and 3' side, and contains 46 sub-libraries.

[0520] Each sub-library was chemically synthesized using natural 4 bases, Ds, and Pa' phosphoramidites by the standard phosphoramidite method and purified by denaturing polyacrylamide gel electrophoresis (PAGE). In ExSELEX, each sub-library was mixed in equal amounts.

[0521] In the initial screening, 3.6 nmol of the library (containing 2.2×10 15 different sequences composed of 6 bases) was used. The 50 nM or 100 nM library was rapidly cooled from 90 °C to 4 °C and then mixed with the target protein (human IFNγ).

[0522] (b) XL1 library

[0523] Using commercially available glycosylated human IFNγ expressed in HEK293 cells as the target protein, 6-base ExSELEX was performed using the above XL1 library. The library at each stage of 6-base ExSELEX was incubated with the target protein in binding buffer, and the target protein was biotinylated with NHS-activated biotin reagent. The biotinylated protein bound to the DNA fragment was captured with streptavidin-conjugated magnetic beads, and the DNA fragment in the complex on the beads was eluted with 150 mM NaOH. The isolated DNA fragment was amplified by PCR using dNTP, dDsTP (deoxynucleoside triphosphate containing Ds base), and dPxTP (deoxynucleoside triphosphate containing 2-nitro-4-propynylpyrrole base (hereinafter referred to as "Px" or "Px base")). The Pa' base in the original library was replaced with Px during PCR amplification and used in the subsequent screening. After the 5th to 7th screening, the DNA-protein complex on the beads was washed with 2 - 3 M urea. After the 7th screening, the binding of the enriched library to the target protein was confirmed by EMSA. As the final 8th screening, the bound DNA fragment was isolated from the complex of the band shifted in the gel.

[0524] The enriched library after 7 screenings (R7) and the enriched library after 8 screenings (R8) obtained from the XL1 library were amplified by replacement PCR method that replaced the unnatural bases in the library with natural bases, and 87,013 (R7) and 78,581 (R8) extracted sequences READ were obtained in deep sequencing using the Ion-PGM system. They were classified into families with similar sequences, the top families with more READ numbers were analyzed, and some of them were further modified to obtain the IFNγ target nucleic acid aptamers shown in Table 3 below.

[0525] Table 3: IFNγ target nucleic acid aptamers obtained from XL1 library

[0526]

[0527] * Underlines indicate sequences complementary to each other forming the stem region, black boxes indicate microhairpin sequences, "L" in the sequence represents biotin-dT, "Ds" represents Ds base, and "Pa'" represents Pa' base.

[0528] The affinity of the IFNγ target nucleic acid aptamers shown in Table 3 above for human glycosylated IFNγ was analyzed by EMSA. The nucleic acid aptamers were incubated with human glycosylated IFNγ in binding buffer supplemented with 0.05% Nonidet P-40 or Tween 20 at 25 °C for 30 minutes. The sample solution was mixed with glycerol (final concentration 5%), and the complexes were analyzed by non-denaturing 8% PAGE at 26 - 28 °C. Other conditions were the same as in Example 3.

[0529] The results are shown in Figure 16 . Nucleic acid aptamers containing 2 Ds and 1 Pa’ (AptIFNγ-1, AptIFNγ-3, and Bio-AptIFNγ-3) strongly bound to glycosylated IFNγ, but no band shift was observed for nucleic acid bases that did not contain these artificial bases.

[0530] Next, the above-mentioned new nucleic acid aptamers were compared with the previously reported IFNγ target nucleic acid aptamer AptDs-IFNγ and its biotinylated minihairpin DNA conjugate (Bio-AptDs-IFNγ). AptDs-IFNγ and Bio-AptDs-IFNγ are aptamers isolated by 5-base ExSELEX using non-glycosylated human IFNγ expressed in Escherichia coli as the target protein, and their sequences are shown in Table 4 below.

[0531] Table 4: IFNγ target nucleic acid aptamers as comparative controls

[0532]

[0533] * Underlines indicate sequences complementary to each other forming the stem region, black boxes indicate minihairpin sequences, "L" in the sequence indicates biotin-dT, and "Ds" indicates Ds base.

[0534] For AptIFNγ-1, AptIFNγ-3, and Bio-AptIFNγ-3 derived from the XL1 library, and AptDs-IFNγ and Bio-AptDs-IFNγ as comparative controls, EMSA was performed in the same manner as the above method, and the results are shown in Figure 17 . It was shown that the binding of AptDs-IFNγ and Bio-AptDs-IFNγ to glycosylated IFNγ was significantly reduced, while AptIFNγ-1, AptIFNγ-3, and Bio-AptIFNγ-3 effectively bound to both glycosylated and non-glycosylated IFNγ.

[0535] (c) XL2 library

[0536] Using glycosylated IFNγ as the target protein, the 6-base ExSELEX method was implemented using the XL2 library. The concentrated library after 7 rounds of screening was sequenced using the Ion-PGM sequencing system, and the top families with a large number of READs were analyzed. A part of them was further modified to obtain the IFNγ-targeting nucleic acid aptamers shown in Table 5 below.

[0537] Table 5: IFNγ-targeting nucleic acid aptamers obtained from the XL2 library

[0538]

[0539] * The underlines indicate sequences complementary to each other that form the stem region. "Ds" in the sequence represents the Ds base, and "Pa'" represents the Pa' base.

[0540] The affinity of the IFNγ-targeting nucleic acid aptamers shown in Table 5 above for glycosylated IFNγ and non-glycosylated IFNγ was analyzed by EMSA. The nucleic acid aptamers were incubated with IFNγ in a binding buffer containing 0.05% Nonidet P-40 or Tween 20 at 25 °C for 30 minutes. The sample solution was mixed with glycerol (final concentration 5%), and the complexes were analyzed by non-denaturing 8% PAGE at 26 - 28 °C. Other conditions were the same as in Example 3.

[0541] The results are shown in Figure 18 . XL2-01a, XL2-01h, and XL2-01i also showed high affinity for glycosylated IFNγ and non-glycosylated IFNγ in a 3M urea gel.

[0542] Next, by conjugating a biotin-binding minihairpin to XL2-01a and XL2-01i, the IFNγ-targeting nucleic acid aptamers shown in Table 6 below were prepared.

[0543] Table 6: IFNγ-targeting nucleic acid aptamers conjugated with a biotin-binding minihairpin

[0544]

[0545] * The underlines indicate sequences complementary to each other that form the stem region. The black boxes indicate the minihairpin sequences. "L" in the sequence represents biotin-dT.

[0546] The affinity of the IFNγ-targeting nucleic acid aptamer shown in Table 6 above for glycosylated IFNγ was analyzed by the ELONA (enzyme-linked oligonucleotides assay) method. Specifically, human IFNγ (0 - 25 nM) was immobilized on a 96-well maxisorp plate and blocked with 1% BSA. After adding a biotinylated aptamer (100 nM / 100 μL per well) as the first detector and allowing it to bind for 1 hour, SA-HRP was added as the second detector and allowed to bind for 30 minutes for colorimetric detection. After reacting HRP with TMB for 30 minutes, the reaction was stopped with 1 N HCl, and the absorbance at 450 nm was measured using a NanoDrop.

[0547] The results are shown in Figure 19 and Figure 20 . For Bio-XL2-01a and Bio-XL2-01i, significantly higher signal intensities were observed compared to Bio-AptDs-IFNγ obtained by the 5-base ExSELEX method using non-glycosylated IFNγ as the target.

[0548] (2) Isolation of thrombin-targeting nucleic acid aptamer

[0549] By the same method as in (1) above, using thrombin isolated from human plasma (Enzyme Research Lab) as the target protein, 6-base ExSELEX was performed using the XL1 library and the XL2 library. The library concentrated after 5 rounds of screening was sequenced using the Ion PGM system, and the top families with a larger number of READs were analyzed to obtain the aptamers shown in Table 7 below. Any sequence contains 4 or more G tracts, suggesting the formation of a guanine quadruplex. In addition, as the consensus sequence in the sequences obtained from the XL1 and XL2 libraries, the TTADsTGG sequence and the AAGGADsGGTGGG sequence were found.

[0550] Table 7: Thrombin-targeting nucleic acid aptamer

[0551]

[0552] * Underlines indicate sequences complementary to each other that form a stem region.

[0553] The affinity of the thrombin-targeting nucleic acid aptamer shown in Table 7 above for thrombin was analyzed by EMSA. The nucleic acid aptamer (25 nM) was incubated with human (α) thrombin (50 nM) in binding buffer (1×D-PBS supplemented with 0.005% Nonidet P-40 and 1 mM MgCl2) at 25 °C for 30 minutes. The sample solution was mixed with glycerol (final concentration 5%), and the complex was analyzed using a non-denaturing or denaturing 8% acrylamide gel. Other conditions were the same as in Example 3.

[0554] Thr104, Thr204, and Thr205 showed high affinity for thrombin ( Figure 21 ). Thus, the following aptamers shown in Table 8, which had a biotinylated mini-hairpin sequence linked to their 3'-terminal side, were prepared. BioMH-RE31 in the following table was derived from the DNA aptamer (RE31) described in the literature (Russo, K. I. et al., Nucleic Acids Res., 2016, 44: 983-991).

[0555] Table 8: Thrombin-targeting nucleic acid aptamers linked with biotinylated mini-hairpin sequences

[0556]

[0557] * Underlines indicate sequences complementary to each other forming the stem region, black boxes indicate the mini-hairpin sequence, and "L" in the sequence indicates biotin-dT.

[0558] The affinity of the thrombin-targeting nucleic acid aptamer shown in Table 8 above for thrombin was analyzed by EMSA, ELONA, and SPR methods. The results are shown in Figure 22 and Figure 23 . In the EMSA and ELONA methods, Bio-Thr104, Bio-Thr204, and Bio-Thr205 showed significantly higher affinity compared to BioMH-RE31. The K D values determined by SPR method were 9.0 pM for Bio-Thr204 and 7.3 pM for Bio-Thr205.

[0559] (3) Isolation of HMGB1-targeting nucleic acid aptamer

[0560] By the same method as in (1) above, using HMGB1 protein as the target protein, 6-base ExSELEX was carried out, and as a result, the following Bio-HMGB1-301-DD aptamer (SEQ ID NO: 143) was isolated.

[0561] <Example 5: Thrombin-targeting nucleic acid aptamer>

[0562] (Objective)

[0563] To prepare a thrombin - targeted nucleic acid aptamer and evaluate its affinity for human thrombin.

[0564] (Methods and Results)

[0565] (1) Synthesis of nucleic acid aptamers

[0566] By the same method as in Example 2, the following nucleic acid aptamers shown in Table 9 were synthesized. The nucleic acid aptamers shown in Table 9 are nucleic acid aptamers that bind thrombin as a target and contain 2 Ds (denoted as "Thr204 - DD" in the following table), and nucleic acid aptamers in which at least 1 Ds in the Thr204 - DD aptamer was replaced with a Ds substitute or an adenine base. The Thr204 - DD aptamer is the same as Thr204 prepared in Example 4.

[0567] Table 9: Thrombin - targeted nucleic acid aptamers

[0568]

[0569] * Underlines indicate sequences complementary to each other forming the stem region, black boxes indicate mini - hairpin sequences, "L" in the sequence represents biotin - dT, "Ds" represents Ds base, and "Bs" and "Ys" represent Ds substitutes.

[0570] (2) Binding assay

[0571] The affinity of the nucleic acid aptamers shown in Table 9 above for human thrombin was analyzed by EMSA. The nucleic acid aptamer was incubated with human thrombin in a binding buffer (1×D - PBS supplemented with 0.005% Nonidet P - 40 and 1 mM MgCl2) at 25 °C for 30 minutes. The sample solution was mixed with glycerol (final concentration 5%), and complex formation was analyzed by non - denaturing 8% polyacrylamide gel electrophoresis (PAGE) at 25 °C or 37 °C or 8% PAGE in the presence of 1 M urea at 37 °C. Other conditions were the same as in Example 3.

[0572] The results are shown in Figure 24 . Nucleic acid aptamers in which Ds was replaced with a Ds substitute in the Thr204 - DD aptamer all showed binding activity equal to or higher than that of the Thr204 - DD aptamer prepared in Example 4. On the other hand, in Thr204 - AA in which the Ds base was replaced with a natural adenine base, the binding activity was significantly reduced.

[0573] Next, the K was determined by SPR method DValue. The SPR method was performed using a running buffer (20 mM Tris HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 2.7 mM KCl, 0.05% Tween 20) at a flow rate of 100 μL / min at 25°C. 50 mM NaOH was injected for 5 seconds and equilibrated with the running buffer for regeneration. As for the K D value, Bio-Thr204-DD was 9.1 pM and Bio-Thr204-BB was 7.95 pM. The affinity of the Bio-Thr204-BB aptamer was enhanced compared to the Ds aptamer.

[0574] <Example 6: HMGB1 Target Nucleic Acid Aptamer>

[0575] (Objective)

[0576] To produce a nucleic acid aptamer targeting HMGB1 (High Mobility Group Box 1) protein and evaluate its affinity for human HMGB1 protein.

[0577] (Method and Result)

[0578] (1) Synthesis of Nucleic Acid Aptamer

[0579] By the same method as in Example 2, the nucleic acid aptamers shown in Table 10 below were synthesized. The nucleic acid aptamers shown in Table 10 are nucleic acid aptamers that bind to HMGB1 protein as a target and contain 2 Ds (denoted as "Bio-HMGB1-301-DD" in the following table), and nucleic acid aptamers in which at least 1 Ds in the Bio-HMGB1-301-DD aptamer is replaced with a Ds substitute or an adenine base. The Bio-HMGB1-301-DD aptamer is the nucleic acid aptamer isolated by the above Example 4(3).

[0580] Table 10: HMGB1 Target Nucleic Acid Aptamer

[0581]

[0582] * Underlines indicate sequences complementary to each other forming the stem region, black boxes indicate microhairpin sequences, "L" in the sequence represents biotin-dT, "Ds" represents Ds base, and "Bs" and "Ys" represent Ds substitutes.

[0583] (2) Binding Assay

[0584] The affinity of the nucleic acid aptamers shown in Table 10 above for human HMGB1 protein was analyzed by EMSA. The nucleic acid aptamer was incubated with human thrombin in binding buffer (1×D-PBS supplemented with 0.005% Nonidet P-40 and 1 mM MgCl2) at 25 °C for 30 minutes. The sample solution was mixed with glycerol (final concentration 5%), and complex formation was analyzed by non-denaturing 6% polyacrylamide gel electrophoresis (PAGE) at 25 °C. Other conditions were the same as in Example 3.

[0585] The results are shown in Figure 25 . Nucleic acid aptamers in which Ds was replaced with a Ds substitute in the Bio-HMGB1-301-DD aptamer all showed binding activity equal to or higher than that of the Bio-HMGB1-301-DD aptamer. On the other hand, in Bio-HMGB1-301-AA in which the Ds base was replaced with the natural adenine base, the binding activity was significantly reduced.

[0586] Next, the K D value was determined by SPR method in the same manner as in Example 5. D As the K

[0587] <Example 7: IFNγ Target Nucleic Acid Aptamer>

[0588] (Purpose)

[0589] Further nucleic acid aptamers targeting IFNγ were prepared, and their affinity for IFNγ was evaluated.

[0590] (Method and Results)

[0591] (1) Synthesis of Nucleic Acid Aptamers

[0592] The following nucleic acid aptamers shown in Table 11 were synthesized by the same method as in Example 2. The nucleic acid aptamers shown in Table 11 are Ds nucleic acid aptamers and nucleic acid aptamers in which at least one Ds is replaced with a Ds substitute or an adenine base. IFNg-201ADD is the same as XL2-01a prepared in Example 4.

[0593] Table 11: IFNγ Target Nucleic Acid Aptamers

[0594]

[0595] * The underlines indicate sequences that are complementary to each other forming the stem region, the black boxes indicate the microhairpin sequences, "L" in the sequences represents biotin-dT, "Ds" represents the Ds base, and "Bs" and "Ys" represent Ds substitutes.

[0596] (2) Binding assay

[0597] The affinity of the nucleic acid aptamers from IFNγ-201AAD to B-I-Apt1-YY for human IFNγ in Table 11 above was analyzed by EMSA. The nucleic acid aptamers were incubated with non-glycosylated human IFNγ expressed in E. coli or glycosylated human IFNγ expressed in HEK293 cells in a binding buffer (1×D-PBS supplemented with 0.05% Nonidet P-40 and 1 mM MgCl2) at 25 °C for 30 minutes. The incubated complexes were analyzed by non-denaturing 8% PAGE or non-denaturing 8% PAGE containing 3 M urea at 25 °C. Other conditions were the same as in Example 3.

[0598] The results are shown in Figure 26 . Except for IFNγ-201AAY, IFNγ-201AAD, IFNγ-201AAB, IFNγ-201ADD, IFNγ-201AYD, and IFNγ-201ABD showed equivalent binding activities.

[0599] Next, for each of the nucleic acid aptamers B-I-Apt1-DD, B-I-Apt1-YY, B-IFNγ-201AAD, B-IFNγ-201AAB, and B-IFNγ-201AYD, the detection sensitivity for IFNγ was studied by ELISA. The 2G1 antibody and B133.5 antibody (Thermo Fisher Scientific), which are commercially available monoclonal antibodies, were used in the ELISA. The nucleic acid aptamer was used as the capture antibody for IFNγ, the B133.5 antibody was used as the detection antibody, and anti-mouse IgG Ab-HRP conjugated with HRP was used as the secondary antibody. The results are shown in Figure 27 (As a control, the 2G1 antibody was used as the capture antibody and the Bio-B133.5 antibody was used as the detection antibody, and the results are shown in Figure 27 in the right column). The B133.5 antibody was used as the capture antibody for IFNγ and the nucleic acid aptamer was used as the detection antibody. The results detected by binding HRP to streptavidin are shown in Figure 28 (As a control, the B133.5 antibody was used as the capture antibody and the Bio-2G1 antibody was used as the detection antibody, and the results are shown in Figure 28the right column). Further, the B133.5 antibody was used as the IFNγ capture antibody, and B-IFNg-201AAD or B-IFNg-201AAB was used as the detection antibody, and the results are shown in Figure 29 (As a control, the 2G1 antibody was used as the capture antibody, and the Bio-B133.5 antibody was used as the detection antibody, and the results are shown in Figure 29 the right column). B-I-Apt1-DD and B-I-Apt1-YY showed low detection sensitivity for glycosylated human IFNγ expressed by HEK293 cells. In contrast, B-IFNg-201AAD, B-IFNg-201AAB, and B-IFNg-201AYD detected both non-glycosylated human IFNγ expressed by Escherichia coli and glycosylated human IFNγ expressed by HEK293 cells with high sensitivity.

[0600] <Example 8: DEN1-NS1 Target Nucleic Acid Aptamer>

[0601] (Objective)

[0602] To produce further nucleic acid aptamers targeting the DEN1-NS1 protein and evaluate the affinity for the DEN1-NS1 protein.

[0603] (Method and Results)

[0604] (1) Synthesis of Nucleic Acid Aptamers

[0605] By the same method as in Example 2, the nucleic acid aptamers shown in Table 12 below were synthesized. The nucleic acid aptamers shown in Table 12 are nucleic acid aptamers containing 2 Ds that bind to the DEN1-NS1 protein as a target (shown as "AptD1c(DsDs)" in the following table), AptD2c(DsDs) in which the bulged loop region is deleted and the stem region is shortened in AptD1c(DsDs), and nucleic acid aptamers in which at least 1 D in the AptD2c(DsDs) aptamer is replaced with a D substitute or a combination of multiple D substitutes or a natural adenine base. The AptD1c aptamer is based on the sequence isolated by the ExSELEX method in a past literature (Matsunaga, K., et al., Nucleic Acids Res., 2021, 49: 11407-11424.). Figure 30 It is shown as "AptD1c(DsDs)" in the following table, AptD2c(DsDs) in which the bulged loop region is deleted and the stem region is shortened in AptD1c(DsDs), and nucleic acid aptamers in which at least 1 D in the AptD2c(DsDs) aptamer is replaced with a D substitute or a combination of multiple D substitutes or a natural adenine base. The AptD1c aptamer is based on the sequence isolated by the ExSELEX method in a past literature (Matsunaga, K., et al., Nucleic Acids Res., 2021, 49: 11407-11424.).

[0606] Table 12: DEN1-NS1 Target Nucleic Acid Aptamers

[0607]

[0608] * The underlines indicate sequences that are complementary to each other to form the stem region. The black boxes indicate the mini-hairpin sequences. "L" in the sequence represents biotin-dT, "Ds" represents the Ds base, and "Bs" and "Ys" represent Ds substitutes.

[0609] (2) Binding assay

[0610] The affinity of the aptamers shown in Table 12 above for the DEN1-NS1 protein was analyzed by EMSA. The aptamers were incubated with DENV1 NS1 mutant 1 purchased from Native Antigen Company or DENV1 NS1 mutant 2 expressed in CHO cells in the binding buffer described in the above literature. The sample solution was separated by non-denaturing 4% acrylamide gel. Other conditions were the same as in Example 3.

[0611] The results are shown in Figure 31 . All the aptamers in which Ds was replaced with Ds substitutes showed binding activity equal to or higher than that of AptD1c1 or AptD1c2. On the other hand, the binding activity of AptD1c2(AA) in which the Ds base was replaced with the natural adenine base was significantly reduced.

[0612] Next, the K D value was determined by SPR. As the K D value, Bio-AptD1C2(DD) was 143 pM, Bio-AptD1C2a(YsYs) was 102 pM, and Bio-AptD1C2c(BsYs) was 136 pM.

[0613] <Example 9: DEN1 / 2-NS1 target aptamer>

[0614] (Objective)

[0615] To prepare a DEN1 / 2-NS1 target aptamer targeting DENV1 NS1 (DEN2-NS1) protein and / or DENV2 NS1 (DEN1-NS1) protein, and evaluate its affinity for the target protein.

[0616] (Method and result)

[0617] (1) Synthesis of aptamer

[0618] By the same method as in Example 2, the following nucleic acid aptamers shown in Table 13 were synthesized. The nucleic acid aptamers shown in Table 13 are nucleic acid aptamers that target and bind to DENV1 NS1 (DEN2-NS1) protein and / or DENV2 NS1 (DEN1-NS1) protein, and nucleic acid aptamers in which at least one Ds has been replaced with a Ds substitute. Each of the aptamers AptD2-b1, AptD2-b2, AptD2-b3, and AptD2-b4 is based on a sequence isolated by the ExSELEX method in previous literature (Matsunaga, K., et al., Nucleic Acids Res., 2021, 49:11407-11424.). In addition, the consensus sequence motifs CCAACC and CCAATC in the nucleic acid aptamers shown in Table 13 below were frequently found in the library enriched by ExSELEX targeting DENV1 NS1 and DENV2 NS1 in the above literature, suggesting that such sequences interact with DENV NS1 protein with broad specificity.

[0619] Table 13: DEN1 / 2-NS1 Target Nucleic Acid Aptamers

[0620]

[0621] * Underlines indicate sequences complementary to each other forming the stem region, black boxes indicate microhairpin sequences, "L" in the sequence indicates biotin-dT, "Ds" indicates Ds base, and "Bs" and "Ys" indicate Ds substitutes.

[0622] (2) Binding Assay

[0623] The affinities of the nucleic acid aptamers shown in Table 13 above for DENV NS1 protein and ZIKA NS1 protein were analyzed by EMSA. The nucleic acid aptamers were incubated with DENV1 NS1 mutant 1 purchased from Native Antigen Company, DENV1 NS1 mutant 2 expressed in CHO cells, DENV2 NS1 mutant 1 purchased from Native Antigen Company, or DENV2 NS1 mutant 2 (with 178F, not 178S) expressed in CHO cells in the binding buffer described in the above literature. The incubated sample solution was separated by non-denaturing 4% acrylamide gel. Other conditions were the same as in Example 3.

[0624] The results are shown in Figure 32 . AptD2-b4 showed the strongest binding to DEN1-NS1 v1 and DEN2-NS1 v1 and v2, and the nucleic acid aptamers in which it was replaced with a Ds substitute all showed binding activity equal to or higher than that.

[0625] <Example 10: DEN4-NS1 Target Nucleic Acid Aptamer>

[0626] (Objective)

[0627] To produce a nucleic acid aptamer targeting DENV4 NS1 (DEN4-NS1) protein and evaluate its affinity for DEN4-NS1 protein.

[0628] (Methods and Results)

[0629] (1) Synthesis of Nucleic Acid Aptamer

[0630] By the same method as in Example 2, the nucleic acid aptamers shown in Table 14 below were synthesized. The nucleic acid aptamers shown in Table 14 are nucleic acid aptamers that bind to DEN4-NS1 protein as a target, and nucleic acid aptamers in which at least one Ds has been replaced with a Ds substitute or a combination of multiple Ds substitutes. The AptD4-1 aptamer is based on a sequence isolated by the ExSELEX method in previous literature (Matsunaga, K., et al., Nucleic Acids Res., 2021, 49:11407-11424.).

[0631] Table 14: DEN4-NS1 Target Nucleic Acid Aptamer

[0632]

[0633] * The underlines indicate sequences complementary to each other forming the stem region, the black boxes indicate the microhairpin sequences, "L" in the sequence indicates biotin-dT, "Ds" indicates the Ds base, and "Bs" and "Ys" indicate Ds substitutes.

[0634] (2) Binding Assay

[0635] The affinity of the nucleic acid aptamers shown in Table 14 above for DEN4-NS1 protein was analyzed by EMSA. The nucleic acid aptamers were incubated with DENV4 NS1 protein purchased from Native Antigen Company in the binding buffer described in the above literature. The incubated sample solution was separated by non-denaturing 4% acrylamide gel. Other conditions were the same as in Example 3.

[0636] The results are shown in Figure 33 . All nucleic acid aptamers in which Ds was replaced with a Ds substitute in AptD4-1 (DsDs) showed binding activity equal to or higher than that of AptD4-1 (DsDs).

[0637] <Example 11: VEGF 165 Target Nucleic Acid Aptamer>

[0638] (Objective)

[0639] To produce nucleic acid aptamers targeting VEGF165 protein and evaluate the affinity for human VEGF 165 protein.

[0640] (Methods and Results)

[0641] (1) Synthesis of nucleic acid aptamers

[0642] By the same method as in Example 2, the following nucleic acid aptamers shown in Table 15 were synthesized. The nucleic acid aptamers shown in Table 15 are nucleic acid aptamers that bind VEGF 165 protein as a target ("V-Apt1(DD)" in the following table), and nucleic acid aptamers in which at least one Ds is replaced with a Ds substitute or a combination of multiple Ds substitutes or a natural adenine base. The V-Apt1(DD) aptamer is based on the sequence isolated by the ExSELEX method in previous literature (Kimoto et al., Nature Biotechnology. 2013; 31(5):453-7.).

[0643] Table 15: Nucleic acid aptamers targeting VEGF 165 Target nucleic acid aptamers

[0644]

[0645] * Underlines indicate sequences complementary to each other forming the stem region, black boxes indicate microhairpin sequences, "L" in the sequence indicates biotin-dT, "Ds" indicates Ds base, and "Bs", "Ys", and "Yo" indicate Ds substitutes.

[0646] (2) Binding assay

[0647] The affinity of the nucleic acid aptamers shown in Table 15 above for human VEGF 165 was analyzed by EMSA. The nucleic acid aptamers were incubated with human VEGF manufactured by Peprotech in binding buffer (1×D-PBS supplemented with 0.005% Nonidet P-40) at 25°C for 30 minutes. The incubated sample solution was mixed with glycerol (final concentration 5%) and analyzed by non-denaturing 8% PAGE at 25°C. Other conditions were the same as in Example 3. 165 The results are shown in

[0648] The results are shown in Figure 34The nucleic acid aptamers in which Ds was replaced with a Ds substitute all showed binding activity equal to or higher than that of V-Apt1 (DsDs). On the other hand, the binding activity of V-Apt4 (AA), in which the Ds base was replaced with a natural adenine base, was significantly reduced.

[0649] <Example 12: TrfR1 Target Nucleic Acid Aptamer>

[0650] (Purpose)

[0651] To prepare a nucleic acid aptamer targeting transferrin receptor 1 (TrfR1) and evaluate its affinity for human TrfR1.

[0652] (Method and Result)

[0653] (1) Synthesis of Nucleic Acid Aptamer

[0654] By the same method as in Example 2, the nucleic acid aptamers shown in Table 16 below were synthesized. The nucleic acid aptamers shown in Table 16 are nucleic acid aptamers that bind TrfR1 as a target ("MB45-Ds" in the following table), and nucleic acid aptamers in which Ds was replaced with a Ds substitute or a natural adenine base. The MB45-Ds aptamer is based on a sequence isolated by the ExSELEX method in a previous literature (Futami et al., Molecular Therapy Nucleic Acids. 2019 Mar 1:14:158-170.).

[0655] Table 16: TrfR1 Target Nucleic Acid Aptamer

[0656]

[0657] * Underlines indicate sequences complementary to each other that form a stem region, black boxes indicate microhairpin sequences, "L" in the sequence indicates biotin-dT, "Ds" indicates the Ds base, and "Bs", "Bo", "Ys", and "Yo" indicate Ds substitutes.

[0658] (2) Binding Assay

[0659] The affinity of the nucleic acid aptamers shown in Table 16 above for human TrfR1 was analyzed by EMSA. The nucleic acid aptamer was incubated with human TrfR1 manufactured by AcroBiosystems in a binding buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 0.05% Tween20) at 25 °C for 30 minutes. The incubated sample solution was mixed with glycerol (final concentration 5%), and analyzed by non-denaturing 6% PAGE at 25 °C. Other conditions were the same as in Example 3.

[0660] The results are shown in Figure 35 . Only MB45-Bs and MB45-Ds showed binding activities equal to or higher than that of the control. On the other hand, no band shift was observed in other nucleic acid aptamers. The binding activity of MB45-A in which the Ds base was replaced with a natural adenine base was significantly reduced.

[0661] Next, the K D value was determined by SPR method. The SPR method was carried out at 25 °C with a flow rate of 30 μL / min using a running buffer (1×D-PBS supplemented with 0.05% Nonidet P-40 and 1 mM MgCl2). Regeneration was performed by injecting 50 mM NaOH for 5 s and then equilibrating with the running buffer. As the K D value for human TrfR1 expressed in HEK293 cells, MB45-Ds was 42 pM and MB45-Bs was 51 pM. As the K D value for mouse TrfR1 expressed in HEK293 cells, MB45-Ds was 1 nM and MB45-Bs was 363 pM. The affinity of MB45-Bs for TrfR1 was enhanced compared to that of MB45-Ds.

[0662] All publications, patents, and patent applications cited in this specification are hereby incorporated by reference in their entirety.

Claims

1. A nucleic acid aptamer comprising at least one unnatural base represented by the following general formula (I) and / or general formula (II): In the above formula, R 1 represents the sugar moiety in the nucleoside, and R 2 represents any one of the following formulas (III) to (IX):

2. The nucleic acid aptamer according to claim 1, comprising 1 to 3 of the unnatural bases.

3. The nucleic acid aptamer according to claim 1 or 2, comprising the unnatural bases represented by the general formula (I) and general formula (II).

4. The nucleic acid aptamer according to any one of claims 1 to 3, wherein the unnatural base is any one of the following formulas (X) to (XVII) and formulas (XXI) to (XXII):

5. The nucleic acid aptamer according to any one of claims 1 to 4, comprising the unnatural base represented by the following formula (XVIII):

6. The nucleic acid aptamer according to any one of claims 1 to 5, which is a DNA aptamer.

7. The nucleic acid aptamer according to claim 1, wherein the nucleic acid aptamer (1) comprises the base sequence shown in SEQ ID NO: 205, 206 or 187, or a sequence obtained by adding a pair of complementary base sequences capable of forming a stem structure to the 5'-terminal side and 3'-terminal side of the base sequence shown in SEQ ID NO: 205, 206 or 187, and binds to von Willebrand factor, i.e., vWF protein, (2) comprises the base sequence shown in SEQ ID NO: 207, 208, 188, 56, 59, 61, 65, 67, 189 or 213, or a sequence obtained by adding a pair of complementary base sequences capable of forming a stem structure to the 5'-terminal side and 3'-terminal side of the base sequence shown in SEQ ID NO: 207, 208, 188, 56, 59, 61, 65, 67, 189 or 213, and binds to interferon-gamma, i.e., IFNγ, (3) comprises the base sequence shown in SEQ ID NO: 190, or a sequence obtained by adding a pair of complementary base sequences capable of forming a stem structure to the 5'-terminal side and 3'-terminal side of the base sequence shown in SEQ ID NO: 190, and binds to thrombin, (4) comprises the base sequence shown in SEQ ID NO: 209 or 192, or a sequence obtained by adding a pair of complementary base sequences capable of forming a stem structure to the 5'-terminal side and 3'-terminal side of the base sequence shown in SEQ ID NO: 209 or 192, and binds to vascular endothelial growth factor, i.e., VEGF, (5) comprises the base sequence shown in SEQ ID NO: 194, or a sequence obtained by adding a pair of complementary base sequences capable of forming a stem structure to the 5'-terminal side and 3'-terminal side of the base sequence shown in SEQ ID NO: 194, and binds to HMGB1 protein, (6) comprises the base sequence shown in SEQ ID NO: 196, or a sequence obtained by adding a pair of complementary base sequences capable of forming a stem structure to the 5'-terminal side and 3'-terminal side of the base sequence shown in SEQ ID NO: 196, and binds to transferrin receptor 1 (7) comprising the base sequence shown in SEQ ID NO: 210, 197 or 199, or a sequence obtained by adding a pair of base sequences complementary to each other capable of forming a stem structure to the 5'-end side and 3'-end side of the base sequence shown in SEQ ID NO: 210, 197 or 199, and binding to dengue virus type 1 NS1 protein, i.e., DEN1 protein, (8) comprising the base sequence shown in SEQ ID NO: 201, or a sequence obtained by adding a pair of base sequences complementary to each other capable of forming a stem structure to the 5'-end side and 3'-end side of the base sequence shown in SEQ ID NO: 201, and binding to dengue virus type 2 NS1 protein, i.e., DEN2 protein, (9) comprising the base sequence shown in SEQ ID NO: 39 or 202, or a sequence obtained by adding a pair of base sequences complementary to each other capable of forming a stem structure to the 5'-end side and 3'-end side of the base sequence shown in SEQ ID NO: 39 or 202, and binding to dengue virus type 3 NS1 protein, i.e., DEN3 protein, or (10) comprising the base sequence shown in SEQ ID NO: 204, or a sequence obtained by adding a pair of base sequences complementary to each other capable of forming a stem structure to the 5'-end side and 3'-end side of the base sequence shown in SEQ ID NO: 204, and binding to dengue virus type 4 NS1 protein, i.e., DEN4 protein, and at least one of the bases represented by n in the base sequence is an unnatural base represented by the general formula (I) and / or general formula (II).

8. The nucleic acid aptamer according to claim 7, wherein 1 to 3 of the unnatural bases are included as the bases represented by n in the base sequence.

9. The nucleic acid aptamer according to claim 7 or 8, comprising the unnatural bases represented by the general formula (I) and general formula (II).

10. The nucleic acid aptamer according to claim 7, wherein 7-(2-thienyl)-3H-imidazo[4,5-b]pyridin-3-yl is included as the base represented by n in the base sequence.

11. The nucleic acid aptamer according to claim 7, wherein the unnatural base is any one of the following formulas (X) to (XVII) and formulas (XXI) to (XXII):

12. The nucleic acid aptamer according to claim 7, comprising the unnatural base represented by the following formula (XVIII):

13. The nucleic acid aptamer according to claim 7, selected from the group consisting of the following (1) to (10), (1) a nucleic acid aptamer binding to vWF protein selected from the following (1-a) to (1-d): (1-a) a nucleic acid aptamer comprising the base sequence shown in SEQ ID NO: 1 or 3, wherein the bases represented by n at positions 10 and 31 in the base sequence are respectively (I) base Bs and base Bs, (II) base Ys and base Ys, (III) base Yo and base Yo, (IV) base Bs and base Ys, (V) base Bs and base Yo, or (VI) base Ys and base Bs; (1-b) A nucleic acid aptamer comprising a base sequence in SEQ ID NO: 1 or 3 that has 1 or more bases deleted, substituted, or added except at positions 10 and 31, wherein the positions corresponding to the bases represented by n at positions 10 and 31 of SEQ ID NO: 1 or 3 are respectively (I) base Bs and base Bs, (II) base Ys and base Ys, (III) base Yo and base Yo, (IV) base Bs and base Ys, (V) base Bs and base Yo, or (VI) base Ys and base Bs; (1-c) A nucleic acid aptamer comprising a 5'-region, a central region, and a 3'-region in sequence from the 5'-side, wherein the 5'-region and the 3'-region each comprise a pair of complementary base sequences capable of forming a stem structure, and the central region comprises the base sequence shown in SEQ ID NO: 205 or 187, and the bases represented by n at positions 3 and 24 in the base sequence shown in SEQ ID NO: 205 or 187 are respectively (I) base Bs and base Bs, (II) base Ys and base Ys, (III) base Yo and base Yo, (IV) base Bs and base Ys, (V) base Bs and base Yo, or (VI) base Ys and base Bs; (1-d) A nucleic acid aptamer comprising a 5'-region, a central region, and a 3'-region in sequence from the 5'-side, wherein the 5'-region and the 3'-region each comprise a pair of complementary base sequences capable of forming a stem structure, and the central region comprises a base sequence in SEQ ID NO: 205 or 187 that has 1 or more bases deleted, substituted, or added except at positions 3 and 24, and the positions corresponding to the bases represented by n at positions 3 and 24 of SEQ ID NO: 205 or 187 in the base sequence in the central region are respectively (I) base Bs and base Bs, (II) base Ys and base Ys, (III) base Yo and base Yo, (IV) base Bs and base Ys, (V) base Bs and base Yo, or (VI) base Ys and base Bs; (2) A nucleic acid aptamer that binds to IFNγ and is selected from (2-i-a) to (2-iii-d) below: (2-i-a) A nucleic acid aptamer comprising the base sequence shown in SEQ ID NO: 15 or 17, wherein the bases represented by n at positions 28 and 39 in the base sequence are respectively (I) base Ys and base Ys, or (II) base Yo and base Yo; (2-i-b) A nucleic acid aptamer comprising a base sequence in SEQ ID NO: 15 or 17 that has 1 or more bases deleted, substituted, or added except at positions 28 and 39, wherein the positions corresponding to the bases represented by n at positions 28 and 39 of SEQ ID NO: 15 or 17 in the base sequence are respectively (I) base Ys and base Ys, or (II) base Yo and base Yo; (2-i-c) A nucleic acid aptamer that sequentially includes a 5'-region, a central region, and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region include a pair of base sequences that are complementary to each other and can form a stem structure, the central region includes the base sequence shown in SEQ ID NO: 207 or 188, and the bases represented by n at positions 22 and 33 in the base sequence shown in SEQ ID NO: 207 or 188 are respectively (I) base Ys and base Ys, or (II) base Yo and base Yo; (2-i-d) A nucleic acid aptamer that sequentially includes a 5'-region, a central region, and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region include a pair of base sequences that are complementary to each other and can form a stem structure, the central region includes a base sequence in which one or more bases are deleted, substituted, or added except at positions 22 and 33 in SEQ ID NO: 207 or 188, and the positions corresponding to the bases represented by n at positions 22 and 33 in SEQ ID NO: 207 or 188 in the base sequence in the central region are respectively (I) base Ys and base Ys, or (II) base Yo and base Yo; (2-ii-a) A nucleic acid aptamer that includes the base sequence shown in SEQ ID NO: 62, and the bases represented by n at positions 24 and 37 in the base sequence are respectively (I) base Ys and base Ds, or (II) base Bs and base Ds; (2-ii-b) A nucleic acid aptamer that includes a base sequence in which one or more bases are deleted, substituted, or added except at positions 24 and 37 in SEQ ID NO: 62, and the positions corresponding to the bases represented by n at positions 24 and 37 in SEQ ID NO: 62 in the base sequence are respectively (I) base Ys and base Ds, or (II) base Bs and base Ds; (2-ii-c) A nucleic acid aptamer that sequentially includes a 5'-region, a central region, and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region include a pair of base sequences that are complementary to each other and can form a stem structure, the central region includes the base sequence shown in SEQ ID NO: 189, and the bases represented by n at positions 15 and 28 in the base sequence shown in SEQ ID NO: 189 are respectively (I) base Ys and base Ds, or (II) base Bs and base Ds; (2-ii-d) A nucleic acid aptamer that sequentially includes a 5'-region, a central region, and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region include a pair of base sequences that are complementary to each other and can form a stem structure, the central region includes a base sequence in which one or more bases are deleted, substituted, or added except at positions 15 and 28 in SEQ ID NO: 189, and the positions corresponding to the bases represented by n at positions 15 and 28 in SEQ ID NO: 189 in the base sequence in the central region are respectively (I) base Ys and base Ds, or (II) base Bs and base Ds; (2-iii-a) A nucleic acid aptamer that includes the base sequence shown in SEQ ID NO: 212, and the base represented by n at position 37 in the base sequence is base Ys or base Bs; (2-iii-b) An aptamer of a nucleic acid having a base sequence in SEQ ID NO: 212 in which one or more bases are deleted, substituted, or added except for the 37th position, where the base corresponding to the base shown as n at the 37th position of SEQ ID NO: 212 is base Ys or base Bs; (2-iii-c) An aptamer of a nucleic acid that sequentially includes a 5'-region, a central region, and a 3'-region from the 5'-side, where the 5'-region and the 3'-region include a pair of complementary base sequences capable of forming a stem structure, the central region includes the base sequence shown in SEQ ID NO: 213, and the base shown as n at the 28th position in the base sequence shown in SEQ ID NO: 213 is base Ys or base Bs; (2-iii-d) An aptamer of a nucleic acid that sequentially includes a 5'-region, a central region, and a 3'-region from the 5'-side, where the 5'-region and the 3'-region include a pair of complementary base sequences capable of forming a stem structure, the central region includes a base sequence in which one or more bases are deleted, substituted, or added except for the 28th position in SEQ ID NO: 213, and the base corresponding to the base shown as n at the 28th position of SEQ ID NO: 213 in the base sequence in the central region is base Ys or base Bs; (3) An aptamer of a nucleic acid that binds to thrombin, selected from (3-a) to (3-d) below: (3-a) An aptamer of a nucleic acid that includes the base sequence shown in SEQ ID NO: 81, where the bases shown as n at the 16th and 22nd positions in the base sequence are respectively (I) base Bs and base Bs, (II) base Bs and base Ys, (III) base Ys and base Bs, or (IV) base Ys and base Ys; (3-b) An aptamer of a nucleic acid that includes a base sequence in which one or more bases are deleted, substituted, or added except for the 16th and 22nd positions in SEQ ID NO: 81, where the bases corresponding to the bases shown as n at the 16th and 22nd positions of SEQ ID NO: 81 in the base sequence are respectively (I) base Bs and base Bs, (II) base Bs and base Ys, (III) base Ys and base Bs, or (IV) base Ys and base Ys; (3-c) An aptamer of a nucleic acid that sequentially includes a 5'-region, a central region, and a 3'-region from the 5'-side, where the 5'-region and the 3'-region include a pair of complementary base sequences capable of forming a stem structure, the central region includes the base sequence shown in SEQ ID NO: 190, and the bases shown as n at the 7th and 13th positions in the base sequence shown in SEQ ID NO: 190 are respectively (I) base Bs and base Bs, (II) base Bs and base Ys, (III) base Ys and base Bs, or (IV) base Ys and base Ys; (3-d) A nucleic acid aptamer that sequentially includes a 5' region, a central region, and a 3' region from the 5' side, wherein the 5' region and the 3' region include a pair of base sequences that are complementary to each other and can form a stem structure, the central region includes a base sequence in which one or more bases are deleted, substituted, or added in SEQ ID NO: 190 except at positions 7 and 13, and the positions corresponding to the bases shown as n at positions 7 and 13 of SEQ ID NO: 190 in the base sequence in the central region are respectively (I) base Bs and base Bs, (II) base Bs and base Ys, (III) base Ys and base Bs, or (IV) base Ys and base Ys; (4) A nucleic acid aptamer that binds to VEGF and is selected from (4-a) to (4-d) below: (4-a) A nucleic acid aptamer that includes the base sequence shown in SEQ ID NO: 191, wherein the bases shown as n at positions 25 and 34 in the base sequence are respectively (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, (IV) base Bs and base Bs, or (V) base Yo and base Yo; (4-b) A nucleic acid aptamer that includes a base sequence in which one or more bases are deleted, substituted, or added in SEQ ID NO: 191 except at positions 25 and 34, and the positions corresponding to the bases shown as n at positions 25 and 34 of SEQ ID NO: 191 in the base sequence are respectively (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, (IV) base Bs and base Bs, or (V) base Yo and base Yo; (4-c) A nucleic acid aptamer that sequentially includes a 5' region, a central region, and a 3' region from the 5' side, wherein the 5' region and the 3' region include a pair of base sequences that are complementary to each other and can form a stem structure, the central region includes the base sequence shown in SEQ ID NO: 192, and the bases shown as n at positions 21 and 30 in the base sequence shown in SEQ ID NO: 192 are respectively (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, (IV) base Bs and base Bs, or (V) base Yo and base Yo; (4-d) A nucleic acid aptamer that sequentially includes a 5' region, a central region, and a 3' region from the 5' side, wherein the 5' region and the 3' region include a pair of base sequences that are complementary to each other and can form a stem structure, the central region includes a base sequence in which one or more bases are deleted, substituted, or added in SEQ ID NO: 192 except at positions 21 and 30, and the positions corresponding to the bases shown as n at positions 21 and 30 of SEQ ID NO: 192 in the base sequence in the central region are respectively (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, (IV) base Bs and base Bs, or (V) base Yo and base Yo; (5) A nucleic acid aptamer that binds to HMGB1 protein, selected from the following (5-a) to (5-d): (5-a) A nucleic acid aptamer comprising the base sequence shown in SEQ ID NO: 193, wherein the bases represented by n at positions 22 and 33 in the base sequence are respectively (I) base Bs and base Bs, (II) base Bs and base Ys, (III) base Ys and base Bs, (IV) base Ys and base Ys; (5-b) A nucleic acid aptamer comprising a base sequence that has a deletion, substitution, or addition of one or more bases in SEQ ID NO: 193 except at positions 22 and 33, wherein the positions corresponding to the bases represented by n at positions 22 and 33 in SEQ ID NO: 193 are respectively (I) base Bs and base Bs, (II) base Bs and base Ys, (III) base Ys and base Bs, (IV) base Ys and base Ys; (5-c) A nucleic acid aptamer that sequentially comprises a 5'-region, a central region, and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region comprise a pair of complementary base sequences capable of forming a stem structure, the central region comprises the base sequence shown in SEQ ID NO: 194, and the bases represented by n at positions 12 and 23 in the base sequence shown in SEQ ID NO: 194 are respectively (I) base Bs and base Bs, (II) base Bs and base Ys, (III) base Ys and base Bs, (IV) base Ys and base Ys; (5-d) A nucleic acid aptamer that sequentially comprises a 5'-region, a central region, and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region comprise a pair of complementary base sequences capable of forming a stem structure, the central region comprises a base sequence that has a deletion, substitution, or addition of one or more bases in SEQ ID NO: 194 except at positions 12 and 23, and the positions corresponding to the bases represented by n at positions 12 and 23 in the base sequence in the central region are respectively (I) base Bs and base Bs, (II) base Bs and base Ys, (III) base Ys and base Bs, (IV) base Ys and base Ys; (6) A nucleic acid aptamer that binds to transferrin receptor 1, selected from the following (6-a) to (6-d): (6-a) A nucleic acid aptamer comprising the base sequence shown in SEQ ID NO: 195, wherein the base represented by n at position 27 in the base sequence is base Bs; (6-b) A nucleic acid aptamer comprising a base sequence that has a deletion, substitution, or addition of one or more bases in SEQ ID NO: 195 except at position 27, and the position corresponding to the base represented by n at position 27 in SEQ ID NO: 195 is base Bs; (6-c) A nucleic acid aptamer that sequentially comprises a 5'-region, a central region, and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region comprise a pair of complementary base sequences capable of forming a stem structure, the central region comprises the base sequence shown in SEQ ID NO: 196, and the base represented by n at position 20 in the base sequence shown in SEQ ID NO: 196 is base Bs; (6-d) A nucleic acid aptamer that sequentially includes a 5'-region, a central region, and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region include a pair of base sequences complementary to each other that can form a stem structure, the central region includes a base sequence in which one or more bases are deleted, substituted, or added at positions other than position 20 in SEQ ID NO: 196, and the position corresponding to the base shown as n at position 20 in SEQ ID NO: 196 in the base sequence in the central region is base Bs; (7) A nucleic acid aptamer that binds to DEN1 protein and is selected from (7-i-a) to (7-ii-d) below: (7-i-a) A nucleic acid aptamer that includes the base sequence shown in SEQ ID NO: 27 or 29, wherein the bases shown as n at positions 19 and 25 in the base sequence are respectively (I) base Yss and base Ds, (II) base Ds and base Dt, (III) base Ys and base Bs, (IV) base Yo and base Bs, or (V) base Ysm and base Bs; (7-i-b) A nucleic acid aptamer that includes a base sequence in which one or more bases are deleted, substituted, or added at positions other than positions 19 and 25 in SEQ ID NO: 27 or 29, and the positions corresponding to the bases shown as n at positions 19 and 25 in SEQ ID NO: 27 or 29 in the base sequence are respectively (I) base Yss and base Ds, (II) base Ds and base Dt, (III) base Ys and base Bs, (IV) base Yo and base Bs, or (V) base Ysm and base Bs; (7-i-c) A nucleic acid aptamer that sequentially includes a 5'-region, a central region, and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region include a pair of base sequences complementary to each other that can form a stem structure, the central region includes the base sequence shown in SEQ ID NO: 210 or 197, and the bases shown as n at positions 9 and 15 in the base sequence shown in SEQ ID NO: 210 or 197 are respectively (I) base Yss and base Ds, (II) base Ds and base Dt, (III) base Ys and base Bs, (IV) base Yo and base Bs, or (V) base Ysm and base Bs; (7-i-d) A nucleic acid aptamer that sequentially includes a 5'-region, a central region, and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region include a pair of base sequences complementary to each other that can form a stem structure, the central region includes a base sequence in which one or more bases are deleted, substituted, or added at positions other than positions 9 and 15 in SEQ ID NO: 210 or 197, and the positions corresponding to the bases shown as n at positions 9 and 15 in SEQ ID NO: 210 or 197 in the base sequence in the central region are respectively (I) base Yss and base Ds, (II) base Ds and base Dt, (III) base Ys and base Bs, (IV) base Yo and base Bs, or (V) base Ysm and base Bs; (7-ii-a) An aptamer comprising the nucleotide sequence shown in SEQ ID NO: 198, wherein the nucleotides represented by n at positions 14 and 27 in the nucleotide sequence are respectively (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, or (IV) base Bs and base Bs; (7-ii-b) An aptamer comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted or added in SEQ ID NO: 198 except at positions 14 and 27, and the positions corresponding to the nucleotides represented by n at positions 14 and 27 in SEQ ID NO: 198 are respectively (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, or (IV) base Bs and base Bs; (7-ii-c) An aptamer comprising a 5'-region, a central region and a 3'-region in sequence from the 5'-side, wherein the 5'-region and the 3'-region comprise a pair of complementary nucleotide sequences capable of forming a stem structure, the central region comprises the nucleotide sequence shown in SEQ ID NO: 199, and the nucleotides represented by n at positions 4 and 17 in the nucleotide sequence shown in SEQ ID NO: 199 are respectively (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, or (IV) base Bs and base Bs; (7-ii-d) An aptamer comprising a 5'-region, a central region and a 3'-region in sequence from the 5'-side, wherein the 5'-region and the 3'-region comprise a pair of complementary nucleotide sequences capable of forming a stem structure, the central region comprises a nucleotide sequence in which one or more nucleotides are deleted, substituted or added in SEQ ID NO: 199 except at positions 4 and 17, and the positions corresponding to the nucleotides represented by n at positions 4 and 17 in SEQ ID NO: 199 in the nucleotide sequence in the central region are respectively (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, or (IV) base Bs and base Bs; (8) An aptamer that binds to DEN2 protein and is selected from (8-a) to (8-d) below: (8-a) An aptamer comprising the nucleotide sequence shown in SEQ ID NO: 200, wherein the nucleotide represented by n at position 13 in the nucleotide sequence is base Bs; (8-b) An aptamer comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted or added in SEQ ID NO: 200 except at position 13, and the position corresponding to the nucleotide represented by n at position 13 in SEQ ID NO: 200 is base Ys or base Bs; (8-c) An aptamer comprising a 5'-region, a central region and a 3'-region in sequence from the 5'-side, wherein the 5'-region and the 3'-region comprise a pair of complementary nucleotide sequences capable of forming a stem structure, the central region comprises the nucleotide sequence shown in SEQ ID NO: 201, and the nucleotide represented by n at position 3 in the nucleotide sequence shown in SEQ ID NO: 201 is base Ys or base Bs; (8-d) An aptamer that sequentially includes a 5'-region, a central region, and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region include a pair of base sequences that are complementary to each other and can form a stem structure, the central region includes a base sequence in which one or more bases are deleted, substituted, or added at positions other than position 3 in SEQ ID NO: 201, and the position corresponding to the base shown as n at position 3 in SEQ ID NO: 201 in the base sequence in the central region is base Ys or base Bs; (9) An aptamer that binds to DEN3 protein and is selected from (9-a) to (9-d) below: (9-a) An aptamer that includes the base sequence shown in SEQ ID NO: 38 or 40, wherein the bases shown as n at positions 15 and 23 in the base sequence are respectively (I) base Dss and base Dss, (II) base Ys and base Dss, (III) base Dss and base Ys, (IV) base Ysm and base Ysm, (V) base Yss and base Ys, (VI) base Yss and base Yo, or (VII) base Yss and base Ysm; (9-b) An aptamer that includes a base sequence in which one or more bases are deleted, substituted, or added at positions other than positions 15 and 23 in SEQ ID NO: 38 or 40, and the positions corresponding to the bases shown as n at positions 15 and 23 in SEQ ID NO: 38 or 40 in the base sequence are respectively (I) base Dss and base Dss, (II) base Ys and base Dss, (III) base Dss and base Ys, (IV) base Ysm and base Ysm, (V) base Yss and base Ys, (VI) base Yss and base Yo, or (VII) base Yss and base Ysm; (9-c) An aptamer that sequentially includes a 5'-region, a central region, and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region include a pair of base sequences that are complementary to each other and can form a stem structure, the central region includes the base sequence shown in SEQ ID NO: 39 or 202, and the bases shown as n at positions 5 and 13 in the base sequence shown in SEQ ID NO: 39 or 202 are respectively (I) base Dss and base Dss, (II) base Ys and base Dss, (III) base Dss and base Ys, (IV) base Ysm and base Ysm, (V) base Yss and base Ys, (VI) base Yss and base Yo, or (VII) base Yss and base Ysm; (9-d) A nucleic acid aptamer that sequentially includes a 5'-region, a central region, and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region include a pair of base sequences that are complementary to each other and can form a stem structure, the central region includes a base sequence in which one or more bases are deleted, substituted, or added except at positions 5 and 13 in SEQ ID NO: 39 or 202, and the positions corresponding to the bases shown as n at positions 5 and 13 in SEQ ID NO: 39 or 202 in the base sequence in the central region are respectively (I) base Dss and base Dss, (II) base Ys and base Dss, (III) base Dss and base Ys, (IV) base Ysm and base Ysm, (V) base Yss and base Ys, (VI) base Yss and base Yo, or (VII) base Yss and base Ysm; (10) A nucleic acid aptamer that binds to DEN4 protein and is selected from (10-a) to (10-d) below: (10-a) A nucleic acid aptamer that includes the base sequence shown in SEQ ID NO: 203, wherein the bases shown as n at positions 18 and 28 in the base sequence are respectively (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, or (IV) base Bs and base Bs; (10-b) A nucleic acid aptamer that includes a base sequence in which one or more bases are deleted, substituted, or added except at positions 18 and 28 in SEQ ID NO: 203, and the positions corresponding to the bases shown as n at positions 18 and 28 in SEQ ID NO: 203 in the base sequence are respectively (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, or (IV) base Bs and base Bs; (10-c) A nucleic acid aptamer that sequentially includes a 5'-region, a central region, and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region include a pair of base sequences that are complementary to each other and can form a stem structure, the central region includes the base sequence shown in SEQ ID NO: 204, and the bases shown as n at positions 11 and 21 in the base sequence shown in SEQ ID NO: 204 are respectively (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, or (IV) base Bs and base Bs; (10-d) A nucleic acid aptamer that sequentially includes a 5'-region, a central region, and a 3'-region from the 5'-side, wherein the 5'-region and the 3'-region include a pair of base sequences that are complementary to each other and can form a stem structure, the central region includes a base sequence in which one or more bases are deleted, substituted, or added except at positions 11 and 21 in SEQ ID NO: 204, and the positions corresponding to the bases shown as n at positions 11 and 21 in SEQ ID NO: 204 in the base sequence in the central region are respectively (I) base Ys and base Ys, (II) base Ys and base Bs, (III) base Bs and base Ys, or (IV) base Bs and base Bs, The base Ys is an unnatural base represented by the following formula (X), the base Yo is an unnatural base represented by the following formula (XII), the base Ysm is an unnatural base represented by the following formula (XIII), the base Yss is an unnatural base represented by the following formula (XIV), the base Bs is an unnatural base represented by the following formula (XV), the base Ds is an unnatural base represented by the following formula (XVIII), the base Dss is an unnatural base represented by the following formula (XXI), and the base Dt is an unnatural base represented by the following formula (XXII).

14. The nucleic acid aptamer according to claim 7 is a DNA aptamer.

15. A pharmaceutical composition comprising the nucleic acid aptamer according to claim 7.

16. The pharmaceutical composition according to claim 15 is used for the treatment and / or prevention of diseases selected from thrombosis, thrombotic thrombocytopenic purpura, intracranial embolism, cerebral embolism, carotid artery stenosis, thrombotic microangiopathy, and acute myocardial infarction, and comprises the nucleic acid aptamer of (1) as an active ingredient.

17. The pharmaceutical composition according to claim 15 is used for the treatment and / or prevention of autoimmune diseases or inflammatory lesions, and comprises the nucleic acid aptamer of (2) as an active ingredient.

18. The pharmaceutical composition according to claim 15 is used for the treatment and / or prevention of thrombotic diseases, and comprises the nucleic acid aptamer of (3) as an active ingredient.

19. The pharmaceutical composition according to claim 15 is used for the treatment and / or prevention of age-related macular degeneration, diabetic retinopathy, rheumatoid arthritis, or cancer, and comprises the nucleic acid aptamer of (4) as an active ingredient.

20. The pharmaceutical composition according to claim 15 is used for the treatment and / or prevention of diseases selected from autoimmune diseases, cardiovascular diseases, rheumatoid arthritis, inflammatory bowel disease, sepsis, cancer, lupus, Sjogren's syndrome, myocardial infarction, arteriosclerosis, stroke, cerebral infarction, cerebral edema, cerebral vasospasm, traumatic brain injury, atherosclerosis, neuropathic pain, arthritis, acute lung injury, cerebral ischemia, renal ischemia, and hepatic ischemia, and comprises the nucleic acid aptamer of (5) as an active ingredient.

21. The pharmaceutical composition according to claim 15 is used for the treatment and / or prevention of iron deficiency anemia, iron overload, cancer, neurodegenerative diseases, or infectious diseases, and comprises the nucleic acid aptamer of (6) as an active ingredient.

22. The pharmaceutical composition according to claim 15 is used for the treatment and / or prevention of dengue virus infection, and comprises any one or more of the nucleic acid aptamers of (7)-(10) as an active ingredient.

23. An unnatural base represented by any one of the following formulas (XI)-(XIII) and formulas (XV)-(XVII):

24. A nucleoside comprising the unnatural base according to claim 23.

25. A nucleotide comprising the unnatural base according to claim 23.

26. The nucleotide according to claim 25 is a phosphoramidite.

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