Scaffold polynucleotide, conjugate, and preparation method thereof

Abstract

The present disclosure provides a scaffold polynucleotide including a linear polynucleotide or a branched polynucleotide; and a conjugator bound to the linear polynucleotide or the branched polynucleotide, wherein the branched polynucleotide has an arm linker emanating therefrom via the conjugator. The present disclosure also provides a conjugate including a biomolecule or an agent conjugated to the scaffold polynucleotide via the arm linker or the conjugator. The present disclosure also provides a method of preparing the scaffold polynucleotide and the conjugate.

Description

SCAFFOLD POLYNUCLEOTIDE, CONJUGATE, AND PREPARATION METHODTHEREOFCROSS REFERENCE

[0001] This application claims priority to, and the benefit of, U.S. Provisional Application No. US 63 / 626,068, filed on January 29, 2024, the content thereof is incorporated by reference herein.TECHNICAL FIELD

[0002] The present disclosure relates to a scaffold polynucleotide, more particularly to the scaffold polynucleotide with a plurality of arm linkers.SEQUENCE LISTING

[0003] The present disclosure is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled YDBPOOllPUSO-Sequence Listing, created on January 21, 2025, which is 8.40 kb in size. The information in the electronic format of Sequence Listing is incorporated herein by reference in its entirety.BACKGROUND OF THE DISCLOSURE

[0004] Description of the Prior Art

[0005] Antibody-drug conjugates (ADCs), which combine the advantages of monoclonal antibodies with precise targeting and payloads, show great clinical value in cancer therapy and allow for specific delivery of payloads to cancer cells. Challenges in the preparation of ADCs include heterogeneity, hydrophobicity of the drug linker, aggregation and stability of the linker. ADCs with hydrophobic linkers will aggregate and result in hepatotoxicity as well as immunogenicity in the bloodstream. In addition, ADCs suffer from systemic toxicity due to non-selective payloads. However, the selectivity of oligonucleotides for antibody-oligonucleotide conjugates (AOCs) could enhance the ability of conjugates to only affect target disease cells. The field of AOCs started as powerful diagnostic tools but has evolved recently as a therapeutic approach for many diseases. Binding one antibody to two or even more different cytotoxic payloads provides an attractive development for next-generation AOCs.

[0006] Therefore, there is a need to provide a simple and straight-forward approach toefficiently produce antibody-oligonucleotide conjugates with multiple cytotoxic payloads.SUMMARY OF THE DISCLOSURE

[0007] The present disclosure is based, at least in part, on the development of a scaffold polynucleotide with multiple arm linkers. The present disclosure accordingly provides polynucleotide conjugates and preparation and applications thereof.

[0008] In one aspect, the present disclosure provides a scaffold polynucleotide, including: a linear polynucleotide or a branched polynucleotide; and a conjugator bound to the linear polynucleotide or the branched polynucleotide, wherein the branched polynucleotide has an arm linker emanating therefrom via the conjugator.

[0009] In some embodiments, the linear polynucleotide or the branched polynucleotide of the present disclosure is a single-stranded form or a double-stranded form.

[0010] In some embodiments, the scaffold polynucleotide of the present disclosure has a plurality of the arm linkers.

[0011] In some embodiments, the arm linker of the present disclosure emanates from a nucleobase, a nucleosugar or a backbone of the branched polynucleotide.

[0012] In certain embodiments, the conjugator of the present disclosure is selected from the group consisting of a biotin moiety, a thiol group, a succinimide moiety, a maleimide moiety, an amine moiety, an acylhydrazide moiety, a hydrazine moiety, a tyrosine moiety, a hydrazone moiety, an azide moiety, a dibenzocyclooctyne group (DBCO), a bicyclononyne (BCN) group, an aminooxy moiety, a hydroxylamine moiety, a terminal alkyne moiety, a strained terminal alkyne moiety, a phosphine moiety, polyethylene glycol (PEG) chains, and any combinations thereof.

[0013] In some embodiments, the scaffold polynucleotide of the present disclosure further includes a biomolecule conjugated to the scaffold polynucleotide via the arm linker or the conjugator.

[0014] In certain embodiments, the biomolecule of the present disclosure is selected from the group consisting of a humanized antibody or antigen binding fragment thereof, a chimeric antibody or antigen binding fragment thereof, a monoclonal antibody or antigen binding fragment thereof, a monovalent Fab’, a divalent Fab2, a single-chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), a camelid antibody, antigen binding fragment thereof, a peptide, and any combinations thereof.

[0015] In some embodiments, the scaffold polynucleotide of the present disclosure further includes an agent conjugated to the scaffold polynucleotide via the arm linker or the conjugator.

[0016] In some embodiments, the agent of the present disclosure is selected from the groupconsisting of a therapeutic agent, a diagnostic agent, and a combination thereof. In some embodiments, the therapeutic agent of the present disclosure is selected from the group consisting of cytotoxic agents, protein toxins, proteins, enzymes, radionuclides, and any combinations thereof. In some embodiments, the diagnostic agent of the present disclosure is selected from the group consisting of a fluorescent moiety, a luminescent moiety, a radioactive moiety, and any combinations thereof.

[0017] In another aspect, the present disclosure further provides a conjugate including a biomolecule conjugated to the scaffold polynucleotide of the present disclosure via the arm linker or the conjugator; and an agent conjugated to the scaffold polynucleotide of the present disclosure via the arm linker or the conjugator. In some embodiments, the scaffold polynucleotide of the present disclosure includes a linear polynucleotide or a branched polynucleotide; and a conjugator bound to the linear polynucleotide or the branched polynucleotide, wherein the branched polynucleotide has an arm linker emanating therefrom via the conjugator.

[0018] In some embodiments, the linear polynucleotide or the branched polynucleotide is a single-stranded form or a double-stranded form.

[0019] In some embodiments, the arm linker emanates from a nucleobase, a nucleosugar, or a backbone of the branched polynucleotide.

[0020] In some embodiments, the conjugator of the present disclosure is selected from the group consisting of a biotin moiety, a thiol group, a succinimide moiety, a maleimide moiety, an amine moiety, an acylhydrazide moiety, a hydrazine moiety, tyrosine moiety, a hydrazone moiety, an azide moiety, a dibenzocyclooctyne group (DBCO), a bicyclononyne (BCN) group, an aminooxy moiety, a hydroxylamine moiety, a terminal alkyne moiety, a strained terminal alkyne moiety, a phosphine moiety, polyethylene glycol (PEG) chains, and any combinations thereof.

[0021] In some embodiments, the biomolecule of the present disclosure is selected from the group consisting of a humanized antibody or antigen binding fragment thereof, a chimeric antibody or antigen binding fragment thereof, a monoclonal antibody or antigen binding fragment thereof, a monovalent Fab’, a divalent Fab2, a single-chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), a camelid antibody, antigen binding fragment thereof, a peptide, and any combinations thereof.

[0022] In some embodiments, the agent of the present disclosure is selected from the group consisting of a therapeutic agent, a diagnostic agent, and a combination thereof.

[0023] In some embodiments, the therapeutic agent of the present disclosure is selected from the group consisting of cytotoxic agents, protein toxins, proteins, enzymes, radionuclides, andany combinations thereof.

[0024] In some embodiments, the diagnostic agent of the present disclosure is selected from the group consisting of a fluorescent moiety, a luminescent moiety, a radioactive moiety, and any combinations thereof.

[0025] In yet another aspect, provided herein is a method of preparing the scaffold polynucleotide of the present disclosure and the method includes providing a target nucleic acid having a plurality of modified nucleobases at 5 ’-end of the target nucleic acid or within the target nucleic acid; providing a mono-functional DNA glycosylase to react with the target nucleic acid to form an intermediate nucleic acid having a plurality of abasic sites at the 5 ’-end of the target nucleic acid or within the target nucleic acid; and incorporating the conjugator to the intermediate nucleic acid to form the linear polynucleotide, provided that the scaffold polynucleotide includes the branched polynucleotide, the method further includes conjugating the arm linker to the linear polynucleotide via the conjugator to form the branched polynucleotide.

[0026] Further, provided herein is a method of preparing a conjugate of the present disclosure, and the method includes conjugating a biomolecule and an agent with the scaffold polynucleotide of the present disclosure.

[0027] The details of one or more embodiments of the present disclosure are set forth in the description below. Other features or advantages of the present disclosure will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0029] FIGs. 1A-1, 1A-2, IB-1 and IB-2 show an example of labeling a fluorescent dye to the 3 ’-end of a polynucleotide via enzymatic synthesis of Atto532-labeled 3'-AZ-dNTP to the 3 '-end of the polynucleotide, followed by the azide-DBCO click conjugation reaction between the incorporated 3'-O-azidomethyl deoxynucleotide monophosphate (3'-AZ-dNMP) and the DBCO-modified maleimide functional moiety allowing to perform the thiol-maleimide reaction with reduced biomolecule. FIGs. 1A-1, 1A-2, IB-1 and IB-2 are images from the different gels, which are 20% Urea polyacrylamide gel and 15% SDS polyacrylamide gel, respectively. FIG. 1C shows an example overview of 3'-end dual labeling of 27-mer ss DNAwith fluorescent dyes (the symbol denotes Atto532-fluorescent dye) and DBCO-maleimide conjugated with anti-HER2 nanobody.

[0030] FIGs. 2A, 2B-1 and 2B-2 show an example of labeling a fluorescent dye to the 5’-end of the polynucleotide via uracil-DNA glycosylase derived from Micrococcus luteus (MluUDG) and an aldehyde-reactive probe (ARP), followed by the 3 ’-end labeling performed with enzymatic synthesis of 3'-AZ-dNTP to the 3 '-end of the polynucleotide, and modified with maleimide by the azide-DBCO click conjugation reaction between the incorporated 3'-AZ-dNMP and DBCO-modified maleimide functional moiety. FIGs. 2B-1 and 2B-2 are images from the different gels, which are 20% Urea polyacrylamide gel and 15% SDS polyacrylamide gel, respectively. FIG. 2C shows an example overview of the dual labeling of 26-mer ss DNA with 5 ’-FAM (the symboldenotes FAM -fluorescent dye) and 3’- DBCO-maleimide conjugated with anti-HER2 nanobody.

[0031] FIGs. 3A, 3B and 3C show examples of dual labeling, a therapeutic agent and a maleimide moiety, of the target polynucleotides. FIGs. 3A, 3B and 3C are images from the different gels. FIGs. 3 A and 3B are 20% Urea polyacrylamide gels. FIG. 3 A shows an example of labeling a therapeutic agent to the 5’-end and a maleimide moiety to the 3’-end, FIG. 3B shows an example of labeling a maleimide moiety to the 5 ’-end and a therapeutic agent to the 3’-end. FIG. 3C was a 15% SDS polyacrylamide gel and shows an example of labeling the biomolecule via the maleimide moiety of the target polynucleotide. FIG. 3D shows an example overview of the dual labeling of 25-mer ss DNA with 5’-MMAE and 3’- DBCO-maleimide conjugated with anti-HER2 nanobody. FIG. 3E shows an example overview of the dual labeling of 45-mer ss DNA with 5’- DBCO-maleimide conjugated with anti-HER2 nanobody and 3’- DM1.

[0032] FIGs. 4A-1 to 4B-3 show examples of labeling a fluorescent dye to the 5’-end of the target polynucleotide via uracil-DNA glycosylase derived from Micrococcus luteus (MluUDG) and an aldehyde-reactive probe (ARP), followed by the 3 ’-end labeling performed with enzymatic synthesis of DY681 -labeled 3'-AZ-dCTP to the 3'-end of the polynucleotide, and modified with maleimide by the azide-DBCO click conjugation reaction between the incorporated 3'-AZ-dCMP and DBCO-modified maleimide functional moiety. FIGs. 4A-1 to 4B-3 are images from the different gels, which are 20% Urea polyacrylamide gel and 15% SDS polyacrylamide gel respectively.

[0033] FIGs. 5 A and 5B show examples of labeling the therapeutic agent at the 5 ’-end and internal respectively of a polynucleotide via hSMUGl and an aldehyde-reactive probe (ARP), followed by the 3 ’-end labeling performed with enzymatic synthesis of 3'-AZ-dATP to the 3 '-end of the polynucleotide, and modified with maleimide by the azide-DBCO clickconjugation reaction between the incorporated 3'-AZ-dAMP and DBCO-modified maleimide functional moiety. FIGs. 5A and 5B are images from the different gels, which are 20% Urea polyacrylamide gel and 15% SDS polyacrylamide gel, respectively. FIG. 5C shows an example overview of the triple labeling of 25-mer ss DNA with 5 ’-DM1, internal MMAE and 3’-DBCO-maleimide conjugated with anti-HER2 nanobody.

[0034] FIG. 6A and FIG. 6B show an example of constructing the polynucleotides conjugated with 5’-end to 5’-end and the analysis results on 20% Urea polyacrylamide gels. FIG. 6A and FIG. 6B depict the modifications of 46-mer and 26-mer target polynucleotides, respectively. FIG. 6C illustrates the oligonucleotides conjugation. FIG. 6D shows an example overview of the branch product, which is the 5’-DBCO-25-mer polynucleotides conjugated with the 5’-Azide-45-mer polynucleotides.FIGs. 7A-1 to FIG. 7C show an example of constructing the branch-formed polynucleotide containing the main chain polynucleotide with the two side chain polynucleotides. FIGs. 7A-1, 7A-2, 7B-1 and 7B-2 illustrate the modification of the main chain polynucleotides and the side chain polynucleotides on 20% Urea polyacrylamide gels. FIG. 7C illustrates the branch construction on 8% TBE polyacrylamide gel. FIG. 7D shows an example overview of the branch product, which consists of the 5’-DBCO- / / / / DBCO-25mer polynucleotides conjugated with two arm linkers (5’-Azide-26mer-3’Atto532 / FAM). The DBCO group functions as the conjugator of the arm linkers in the target polynucleotide. As shown in FIG. 7D, the symbol denotes Atto532-fluorescent dye, while the symboldenotes FAM-fluorescent dye.

[0035] FIGs. 8A-1 to 8B-2 show an example of performing six modifications on the branch-formed polynucleotides containing the main chain polynucleotide with the two side chain polynucleotides. FIGs. 8A-1, 8A-2 and 8A-3 are 20% Urea polyacrylamide gels, which depict the modifications of the main chain polynucleotides. FIGs. 8B-1 and 8B-2 illustrate the branch construction on 8% TBE polyacrylamide gel.

[0036] FIGs. 9A-1 to 9C show an example of constructing of the polynucleotide with two therapeutic agents modified in branch form. FIGs. 9A-1, 9A-2 and 9B illustrate the modifications of the main chain polynucleotide and the control polynucleotide on 20% Urea polyacrylamide gels; FIG. 9C illustrates the modifications of the side chain polynucleotide on 20% Urea polyacrylamide gel; and FIG. 9D illustrates the branch construction and the nanobody-polynucleotide conjugation on 15% SDS- polyacrylamide gel.

[0037] FIGs. 10A to FIG. 10E-4 show an example of performing six modifications on the duplex-formed polynucleotides containing two Biotin moieties, three different fluorescent dyes, and a biomolecule. FIGs. 10A, 10B-1 and 10B-2 illustrate the modifications of the top strand polynucleotide on 20% Urea polyacrylamide gels. FIGs. 10C-1 and 10C-2 illustrate themodifications of the bottom strand polynucleotide on 20% Urea polyacrylamide gel. FIGs. 10D-1, 10D-2 and 10D-3 illustrate the hybridization of the DNA duplex on 12% TBE polyacrylamide gel. FIGs. 10E-1, 10E-2, 10E-3 and 10E-4 illustrate the results of nanobody-polynucleotide conjugation on 15% SDS polyacrylamide gel. FIG. 10F shows an example overview of the top-strand polynucleotide (the 5’-Biotin-zwtBiotin-26-mer-Atto532 / FAM). As shown in FIG. 10F, the symboldenotes Atto532-fluorescent dye and the symboldenotes FAM-fluorescent dye. FIG. 10G shows an example overview of the bottom-strand polynucleotide (the 28-mer-3’Dye681-mal). As shown in FIG. 10G, the symbol “ ♦” denotes Dye681 -fluorescent dye.DETAILED DESCRIPTION OF THE DISCLOSURE

[0038] Those skilled in the art will readily observe that numerous modifications and alterations of the present disclosure may be made while retaining the teachings of the disclosure described herein. Accordingly, the embodiments described are intended to cover the modifications and alterations within the scope of the present disclosure, rather than to limit the present disclosure. The scope of the claims therefore should be accorded the broadest interpretation so as to encompass all such modifications and alterations.

[0039] The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, and immunology, which are well within the purview of a skilled artisan in the art. Such techniques are explained fully in the literature, such as “Molecular Cloning shows A Laboratory Manual,” second edition (Sambrook, et al., 1989), Cold Spring Harbor Press; “Oligonucleotide Synthesis” (M. J. Gait, 1984); “Methods in Molecular Biology,” Humana Press; “Cell Biology shows A Laboratory Notebook” (J. E. Cellis, ed., 1998) Academic Press; “Animal Cell Culture” (R. I. Freshney, ed., 1987); “Handbook of Experimental Immunology” (Weir, 1996); “Introduction to Cell and Tissue Culture” (J. P. Mather and P. E. Roberts, 1998); “Cell and Tissue Culture shows Laboratory Procedures” (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8); “Methods in Enzymology” (Academic Press, Inc.); “Handbook of Experimental Immunology” (D. M. Weir and C. C. Blackwell, eds.); “Gene Transfer Vectors for Mammalian Cells” (J. M. Miller and M. P. Calos, eds., 1987); “Current Protocols in Molecular Biology” (F. M. Ausubel, et al., eds., 1987); “PCR shows The Polymerase Chain Reaction (Mullis, et al., eds., 1994); and “Current Protocols in Immunology” (J. E. Coligan et al., eds., 1991); “Short Protocols in Molecular Biology” (Wiley and Sons, 1999); “Immunobiology” (C. A. Janeway and P. Travers, 1997); “Antibodies” (P. Finch, 1997); “Antibodies shows a practical approach” (D. Catty., ed., IRL Press, 1988-1989); “Monoclonal antibodies shows a practical approach” (P. Shepherd andC. Dean, eds., Oxford University Press, 2000); “Using antibodies shows a laboratory manual” (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995). Particularly useful techniques for particular embodiments will be discussed in the sections that follow. Without further elaboration, it is believed that one skilled in the art can, based on the above descriptions, utilize the present disclosure to its fullest extent. The following embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

[0040] As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms, unless the context clearly indicates otherwise. The terms “includes,” “including,” “comprises,” and “comprising” are used in either the detailed descriptions and / or the claims, and such terms are intended to be inclusive in a manner of not excluding others, such as other components, materials, steps, etc. The terms “sec,” “min,” and “hr” as used herein are abbreviations of “second,” “minute,” and “hour.”

[0041] Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of this disclosure, unless the context clearly dictates otherwise.

[0042] As used herein, the terms “about,” “approximately,” and “around” generally mean within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term “about,” “approximately,” and “around” mean within an acceptable standard error of the mean when considered by one of ordinary skill in the art. Unless otherwise expressly specified, all of the numerical ranges, amounts, values, and percentages such as those for quantities of materials, durations of time periods, temperatures, operating conditions, ratios of amounts, and the likes disclosed herein should be understood as modified in all instances by the terms “about,” “approximately,” or “around.”

[0043] As used herein, the term “derived,” when referring to a biological sample, indicates the sample being obtained from the stated source at some point in time. For example, a biological sample derived from an organism can represent a primary biological sample obtained directly from the organism (i.e., unmodified), or can be modified, e.g., by introduction of a recombinant vector, by culturing under particular conditions, or immortalization.

[0044] As used herein, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of theelements in the list of elements, but not necessarily including at least one of each and every element listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements).

[0045] As used herein, the term “payload” refers to the crucial part with cytotoxic potency for antibody oligonucleotide conjugates (hereinafter referred as AOCs). Non-limiting examples of payloads for AOCs can be small molecules (cellular toxins), protein toxins, proteins, enzymes and radionuclides. In some embodiments, payloads of cellular toxins in clinical trials fall into three categories shows anti-mitotic (tubulin filaments damaging), DNA damaging or transcription inhibitors. For instance, the payloads that disrupt microtubule dynamics, inhibiting cell division and leading to apoptosis of tumor cells include maytansinoids, auristatins, taxol derivatives, monomethyl auristatin-E (MMAE), N2'-Deacetyl-N2'-(3-mercapto-l-oxopropyl)-maytansine (DM1), and monomethyl auristatin-F (MMAF). The payloads that target DNA directly, causing irreversible damage and inhibiting tumor cell proliferation include Calicheamicin, Pyrrolobenzodiazepines, or Duocarmycins. The payloads that inhibit topoisomerase activity, interfering with DNA unwinding and replication include SN-38, Exatecan and Deruxtecan.

[0046] As used herein, an abasic site, also known as an apurinic / apyrimidinic (AP) site, encompasses any chemical structure following removal of a nucleobase portion (including the entire base) with an agent capable of cleaving a base portion of a nucleotide, e.g., by the treatment of a nucleotide (present in a polynucleotide chain) with an agent (e.g., an enzyme, an acidic condition, or a chemical reagent) capable of effecting cleavage of a base portion of a nucleotide. In an embodiment, an AP site is a position in the backbone of nucleic acids such as deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) that lacks a nucleobase, i.e., a deoxyribose of the DNA backbone or a ribose of the RNA backbone without either a purine base, such as adenine (A) or guanine (G), or a pyrimidine base, such as cytosine (C), uracil (U) or thymine (T). An AP site may reside within the polynucleotide sequence of the nucleic acids,at both the 5 ’ - and 3 ’ -ends, at either the 5 ’ -end or the 3 ’ end, or within the internal region of the nucleic acids.

[0047] The terms “nucleic acid,” “nucleic acid sequence,” and “nucleic acid fragment” as used herein refer to a nucleotide sequence in a single-stranded or double-stranded form, of which the sources are not limited herein, and generally, include naturally occurring nucleotides or artificial chemical mimics. The term “nucleotide” as used herein refers to the monomeric unit of nucleic acids or polynucleotides as described hereafter, having a glycoside with or without a nucleobase, and one or more internucleotide linkages, e.g., phosphodiester linkage. In some embodiments, the nucleobase includes naturally occurring bases such as adenine (A), thymine (T), cytosine (C), guanine (G), and uracil (U), non-naturally occurring bases such as xanthine, hypoxanthine, isoguanine, and isocytosine, as well as any analogs or derivatives thereof. In some embodiments, a nucleotide with an abasic site (loss of nucleobase) is also included within the scope of the present disclosure. In some embodiments, the nucleosugars in the glycoside include naturally occurring sugars such as pentose sugars (e.g., deoxyribose and ribose), non-naturally occurring sugars, and the analogs thereof. In some embodiments, nucleotides are linked via internucleotide linkages such as, but not limited to, phosphate, boranephosphate, phosphorothioate, phosphodiester, phosphotriester, H-phosphonate, aminophosphonate, methylphosphonate, phosphonoacetate, sulfur phosphonoacetate, or other variants of the phosphate backbone of natural nucleic acids. The term “nucleotide” as used herein also encompasses structural analogs in place of natural or nonnatural nucleotides, such as modified nucleotides. For example, the term “xeno nucleotide” refers to the nucleotide being modified to have a different sugar moiety than those contained in a natural DNA or RNA. The exemplary nucleic acids having the xeno nucleotide, i.e., xeno-nucleic acids (XNA), include but not limited to peptide nucleic acid (PNA), locked nucleic acid (LN A), 1,5-anhydrohexitol nucleic acid (HNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), cyclohexene nucleic acid (CeNA), and FANA (fluoro arabino nucleic acid).

[0048] As used herein, the term “polynucleotide” refers to a polymer of nucleotides and is generic to any type of nucleic acids such as natural or non-natural DNA or RNA and modified nucleic acids such as xeno-nucleic acids (XNA) as described herein. A polynucleotide can also include any combinations of glycosides with or without a nucleobase and internucleotide linkages. Unless otherwise specified, the polynucleotide featured herein has an intrinsic directionality in terms of the 5 ’-end of one nucleotide to the 3 ’-end of its neighboring nucleotide, where the template-independent synthesis of a polynucleotide provided herein proceeds in a 5’ to 3’ direction.

[0049] In some embodiments, the polynucleotide used herein is not intended to be distinct inlength of nucleotide unit, where the term refers only to the polymeric molecule structure. That is to say, a polynucleotide used herein is interchangeable with the term “oligonucleotide,” and can have a length ranging from a few monomeric nucleotide units to several thousands of monomeric nucleotide units, such as 2 to 5 nucleotides, 5 to 20 nucleotides, 20 to 100 nucleotides, 100 to 1,000 nucleotides, or longer. A polynucleotide can be composed entirely of natural or non-natural occurring, modified or non-modified deoxyribonucleotides, entirely of natural or non-natural occurring, modified or non-modified ribonucleotides, or chimeric mixtures thereof. Nucleobases (also known as nitrogenous bases) contained in a polynucleotide may be, for example, adenine, thymine, cytosine, guanine, uracil, xanthine, hypoxanthine, isocytosine, or isoguanine. In addition, a polynucleotide may contain one or more abasic sites (apurinic / apyrimidinic site), also known as AP sites.

[0050] The terms “nucleic acids” and “polynucleotides” may be used interchangeably herein to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. These terms encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and / or which have similar chemical properties as the reference nucleic acids, and / or which are metabolized in a manner similar to the reference nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. In some embodiments, nucleotides are linked via internucleotide linkages such as, but not limited to, phosphate, boranephosphate, phosphorothioate, phosphodiester, phosphotriester, H-phosphonate, aminophosphonate, methylphosphonate, phosphonoacetate, sulfur phosphonoacetate, or other variants of the phosphate backbone of natural nucleic acids. The term “nucleotide” as used herein also encompasses structural analogs in place of natural or nonnatural nucleotides, such as modified nucleotides. For example, the term “xeno nucleotide” refers to the nucleotide being modified to have a different sugar moiety than those contained in a natural DNA or RNA. The exemplary nucleic acids having the xeno nucleotide, i.e., xeno-nucleic acids (XNA), include, but are not limited to, peptide nucleic acid (PNA), locked nucleic acid (LNA), 1,5-anhydrohexitol nucleic acid (HNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), cyclohexene nucleic acid (CeNA), and fluoro-arabino nucleic acid (FANA).

[0051] As used herein, the term “glycosylase” is an enzyme capable of excising a base portion of a nucleotide and creating an AP site in a nucleic acid, which includes N-glycosylases and is also called as “DNA glycosylase” or “glycosidase,” including, but not limited to, uracil N-glycosylase (UNG) that specifically cleaves deoxyuridine from a nucleic acid and isinterchangeably termed as “uracil DNA glycosylase” (UDG); hypoxanthine-N-glycosylase; hydroxymethyl cytosine-N-glycosylase; 3 -methyladenine DNA glycosylase; 3- or 7-methylguanine DNA glycosylase; hydroxymethyl uracil DNA glycosylase, and T4 endonuclease V. A glycosylase cleaves a base portion of the nucleotide in the middle, or at either, or both, ends of a nucleic acid. As used herein, a 5 ’-end glycosylase excises a base portion of the nucleotide at the 5 ’-end of a nucleic acid.

[0052] As used herein, the term “polymerase” refers to enzyme / protein capable of synthesizing nucleic acids, which is generically a DNA polymerase, an RNA polymerase, or a functionally equivalent enzyme, including naturally occurring enzymes, modified enzymes, enzyme subunits and the derivatives thereof. For example, an amino acid sequence modification (e.g., mutation and functional group substitution) can be applied to these enzymes for desired properties, such as removing the 5’ to 3’ exonuclease activity and enhancing the polymerase activity, to obtain altered enzymes with improved properties, such as thermostability / thermotolerance and catalytic efficiency.

[0053] According to the present disclosure, the polymerase of the present disclosure may be a template-dependent polymerase or a template-independent polymerase. The polymerase may include a family-A DNA polymerase (e.g., T7 DNA polymerase, Pol I, Pol y, 9, and v), a family-B DNA polymerase (e.g., Pol II, Pol B, PolPol a, 5 and a), a family-C DNA polymerase (e.g., Pol III), a family-D DNA polymerase (e.g., PolD), a family-X DNA polymerase (e.g., Pol P, Pol c, Pol X, Pol p and terminal deoxynucleotidyl transferase), a family-Y DNA polymerase (e.g., Pol t, Pol K, Pol q, DinB, Pol IV and Pol V), a reverse transcriptase (e.g., telomerase and hepatitis B virus), and enzymatically active fragments thereof.

[0054] Non-limiting examples of widely employed template-dependent polymerases include T7 DNA polymerase of T7 bacteriophage and T3 DNA polymerase of T3 bacteriophage, which are DNA-dependent DNA polymerases; T7 RNApolymerase of T7 bacteriophage and T3 RNA polymerase of T3 bacteriophage, which are DNA-dependent RNA polymerases; DNA polymerase I or its proteolytic fragment known as the Klenow fragment of Escherichia coli. which is a DNA-dependent DNA polymerase; Thermophilus aquaticus DNA polymerase, Tth DNA polymerase and Vent DNA polymerase, which are thermostable DNA-dependent DNA polymerases; eukaryotic DNA polymerase P, which is a DNA-dependent DNA polymerase; telomerase, which is an RNA-dependent DNA polymerase; and non-protein catalytic molecules, such as modified RNA (ribozymes; Unrau & Bartel, 1998) and DNA with template-dependent polymerase activity.

[0055] Non-limiting examples of the template-independent polymerases include reversetranscriptase, poly A polymerase, DNA polymerase theta (9), terminal deoxynucleotidyl transferase (TdT), and DNA polymerase mu (p). Since polymerases suitable for performing nucleic acid synthesis, nucleotide addition / incorporation, and process of nucleic acid synthesis are within the expertise and routine skills of those skilled in the art, further details thereof are omitted herein for the sake of brevity. Furthermore, the B-family DNA polymerases provided previously by inventors of the present disclosure are also suitable for being used under template-independent conditions, and the US Patent No. 11591629B2 and 12098396B2 are hereby incorporated entirely by reference.

[0056] As used herein, the term “modification” refers to the alteration(s) of the chemical structure of a reactant molecule. When a nucleic acid is used as a reactant molecule, the means of modifications include, but are not limited to, the introduction of an additional chemical group / moiety to the nucleic acid, removal or substitution of an original chemical group / moiety from the nucleic acid, or the combination thereof, regardless of the source of the nucleic acid. Alternatively, the modification(s) may be introduced to a specific sequence of nucleic acids during the de novo nucleic acid synthesis resulting in a direct modification, or modifications, on the nucleic acid. For example, a fhiorophore-labeled nucleotide analogue can be incorporated into a nucleic acid alongside with natural counterparts to become a “fluorescent labeled” nucleic acid. Likewise, a site-specific modification, or modifications, can also be inserted enzymatically into a nucleic acid by incorporating nucleotide(s) carrying desired modification(s). For example, a nucleoside triphosphate having a 3’-O-azidomethyl group can be enzymatically introduced to the 3 ’-end of a nucleic acid, and thus, directly adds an azidomethyl modification to the 3 ’-end of the nucleic acid. Such modifications result in the addition of a nucleotide together with a site-specific chemical group to a target nucleic acid.

[0057] As used herein, the term “exonuclease” refers to any wild-type or variant enzyme, which is capable of cleaving phosphodiester bond(s) linking the end nucleotides of an oligonucleotide or a polynucleotide, such as a 5’ to 3’ exonuclease, a 3’ to 5’ exonuclease, and a poly(A)-specific 3’ to 5’ exonuclease. Non-limiting examples of exonucleases include exonuclease I, exonuclease II, exonuclease III, exonuclease IV, exonuclease V, exonuclease VI, exonuclease VII, exonuclease VII, Xml, and Rati.

[0058] As used herein, the term “5’ to 3’ exonuclease” refers to an exonuclease that breaks phosphodiester bonds at the 5’ end of an oligonucleotide or a polynucleotide. Non-limiting examples of 5’ to 3’ exonucleases include T5 exonuclease (T5 exo), T7 exonuclease (T7 exo), viral alkaline exonuclease, bacterial alkaline exonuclease, phage lambda exonuclease, 5 ’-exonuclease of DNA polymerase I (ExoVI) from, e.g., Streptococcus pneumoniae or Helicobacter pylori, Escherichia coli exonuclease VIII (Exo VIII), Red from, e.g.,Escherichia coli or Deinococcus radiodurans, RecJf derived from RecJ fusion to the maltose-binding protein, Thermus thermophilus (Tth) RecJ, Mycoplasma pneumonia (Mpn) NrnA, human exonuclease 5 (hEXO5), human exonuclease 1 (hEXOl), SNM1 from Saccharomyces cerevisiae. human or bovine SNM1A, human SNM IB / Apollo, bovine SNM1B, SXT-Exo from, e.g., Vibrio cholerae, phospholipase D3 (PLD3), phospholipase D4 (PLD4), Ssol391-Csal from, e.g., Sulfolobus solfataricus, Sto0027-Csal from, e.g., Sulfolobus lokadaii, Ttxl248-Csal from, e.g., Thermoproteus tenax, Ssol451-Csal from, e.g., Sulfolobus solfataricus, Sto2633-Csal from, e.g., Sulfolobus lokadaii. Pful793-Cas4 from, e.g.,Pyrococcus furiosus. Sto2501 from, e.g., Sulfolobus lokadaii. SsoOOOl from, e.g., Sulfolobus solfataricus, Sto2331-Cas4 from, e.g., Sulfolobus tokadaii, Ttxl245-Cas4 from, e.g.,Thermoproteus tenax, Ssol449-Cas4 from, e.g., Sulfolobus solfataricus, Sto2635-Cas4 from, e.g., Sulfolobus tokadaii, Ssol392-Cas4 from, e.g., Sulfolobus solfataricus, Sulfolobus islandicus rod-shaped virus 2 (SIRV2) gpl9, bacterial AddB, and any combination thereof.

[0059] As used herein, the term “3 ’-end” generally refers to a region or position in a polynucleotide or oligonucleotide downstream from the 5 ’-region or position in the same polynucleotide or oligonucleotide.

[0060] As used herein, the term “5 ’-end” generally refers to a region or position in a polynucleotide or oligonucleotide upstream from the 3 ’-region or position in the same polynucleotide or oligonucleotide.

[0061] As used herein, the term “mono-functional DNA glycosylase” refers to a naturally existing mono-functional glycosylase that intrinsically contains only a DNA glycosylase activity. The term “mono-functional DNA glycosylase” may also refer to a mono-functional glycosylase that is derived from a bi-functional DNA glycosylase naturally having both DNA glycosylase and abasic-site lyase (AP lyase) activities by eliminating or inactivating the AP lyase domain of the bi-functional DNA glycosylase.

[0062] In some embodiments, the processes of signal detection are known in the art. Signal detection may be visual or utilize a suitable instrument appropriate for the label used, such as a spectrometer, fluorimeter, luminometer, phosphor imager, Geiger counter, scintillation counter, or microscope. For example, where the label is a radioisotope, detection can be achieved by using, for example, a scintillation counter, or photographic film as in autoradiography. Where a fluorescent label is used, detection may be achieved by exciting the fluorochrome with an appropriate wavelength of light and detecting the emitting fluorescence, such as by a fluorescence microscopy, visual inspection, photographic film, fluorometer, luminometer, charge-coupled device (CCD) cameras, and scanner. Where enzymatic labels are used, detection may be achieved by providing appropriate substrates for the enzyme and detecting theresulting reaction product. For example, many substrates of horseradish peroxidase, such as o-phenylenediamine, give colored products. Instruments suitable for high sensitivity detection are known in the art. Otherwise, the signal amplification strategies can be optionally used to facilitate the detection of low-abundance molecular targets.

[0063] 1. Scaffold Polynucleotide

[0064] In one aspect, the present disclosure provides a scaffold polynucleotide including a linear polynucleotide or a branched polynucleotide; and a conjugator bound to the linear polynucleotide or the branched polynucleotide, wherein the branched polynucleotide has an arm linker emanating therefrom via the conjugator.

[0065] In some embodiments, the linear polynucleotide or the branched polynucleotide of the present disclosure is a single-stranded form or a double-stranded form.

[0066] In some embodiments, the scaffold polynucleotide of the present disclosure has a plurality of arm linkers. In some embodiments, the arm linker of the present disclosure is a linear polynucleotide or a branched polynucleotide. In some embodiments, the arm linker of the present disclosure emanates from a nucleobase, a nucleosugar or a backbone of the branched polynucleotide.

[0067] In some embodiments, the polynucleotide of the present disclosure described herein may contain non-naturally occurring nucleobases, nucleosugars, or covalent internucleoside linkages (backbones). Such a modified polynucleotide confers desirable properties such as enhanced cellular uptake, improved affinity to the target nucleic acid, and increased in vivo stability.

[0068] In some embodiments, the conjugator of the present disclosure is selected from the group consisting of a biotin moiety, a thiol group, a succinimide moiety, a maleimide moiety, an amine moiety, an acylhydrazide moiety, a hydrazine moiety, tyrosine moiety, a hydrazone moiety, an azide moiety, a dibenzocyclooctyne group (DBCO), a bicyclononyne (BCN) group, an aminooxy moiety, a hydroxylamine moiety, a terminal alkyne moiety, a strained terminal alkyne moiety, a phosphine moiety, polyethylene glycol (PEG) chains, and any combinations thereof. In some embodiments, the conjugator of the conjugator is linked to 2’-carbon or 3 ’-carbon of the nucleosugar, or the nucleobase of the polynucleotide.

[0069] In some embodiments, the scaffold polynucleotide of the present disclosure further includes a biomolecule conjugated to the scaffold polynucleotide via the arm linker or the conjugator of the present disclosure. As described herein, a biomolecule with respect to conjugation to a polynucleotide is preferably a targeting molecule which functions in recognizing a particular target (for example, a disease-associated antigen such as a tumor antigen) so as to localize at a target area, enter a target cell and / or bind to a target antigen orreceptor. In some embodiments, the biomolecule of the present disclosure is selected from the group consisting of a humanized antibody or antigen binding fragment thereof, a chimeric antibody or antigen binding fragment thereof, a monoclonal antibody or antigen binding fragment thereof, a monovalent Fab’, a divalent Fab2, a single-chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), a camelid antibody, antigen binding fragment thereof, a peptide, and any combinations thereof.

[0070] As used herein, an antibody (interchangeably used in plural form) is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term “antibody” encompasses not only intact (e.g., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab’, F(ab’)2, and Fv), single-chain antibody (scFv), fusion proteins including an antibody portion, humanized antibodies, chimeric antibodies, diabodies, single domain antibody (e.g., nanobody), single domain antibodies (e.g., a VH only antibody), multi-specific antibodies (e.g., bispecific antibodies) and any other modified configuration of the immunoglobulin molecule that includes an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. An antibody includes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes.

[0071] A typical antibody molecule includes a heavy chain variable region (VH) and a light chain variable region (VL), which are usually involved in antigen binding. The VH and VL regions can be further subdivided into regions of hypervariability, also known as “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, which are known as “framework regions” (“FR”). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order shows FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework region and CDRs can be precisely identified using methodology known in the art.

[0072] The antibodies described herein can be of a suitable origin, for example, murine, rat, Camelidae, or human. Such antibodies are non-naturally occurring, i.e., would not be produced in an animal without human act (e.g., immunizing such an animal with a desired antigen or fragment thereof or isolated from antibody libraries). Any of the antibodies described herein, can be either monoclonal or polyclonal. A “monoclonal antibody” refers to a homogenous antibody population and a “polyclonal antibody” refers to a heterogeneous antibody population.These two terms do not limit the source of an antibody or the manner in which it is made.

[0073] In some embodiments, the scaffold polynucleotide of the present disclosure further includes an agent conjugated to the scaffold polynucleotide via the arm linker or the conjugator of the present disclosure. In some embodiments, the agent of the present disclosure is selected from the group consisting of a therapeutic agent, a diagnostic agent, and a combination thereof. In some embodiments, the therapeutic agent of the present disclosure is selected from the group consisting of cytotoxic agents, protein toxins, proteins, enzymes, radionuclides, and any combinations thereof. In some embodiments, the diagnostic agent of the present disclosure is selected from the group consisting of a fluorescent moiety, a luminescent moiety, a radioactive moiety, and any combinations thereof.

[0074] As described herein, an agent of interest with respect to conjugation to a polynucleotide generate an agent-polynucleotide conjugate can be a molecule of any type having a desired utility e.g. therapeutic utility, diagnostic utility or cosmetic uses. In some embodiments, the agent of interest is a therapeutic agent, which can be any molecule having therapeutic effects against a target disease or disorder. In some examples, the therapeutic agent may be a small molecule cytotoxic agent such as anti-cancer drugs. In some examples, an agent of interest may be a peptide-based or polypeptide-based molecule such as an antibody or a targeting peptide. For example, anti-cancer antibodies include but are not limited to anti-HER2 antibodies, anti-VEGF antibodies, anti-CD20 antibodies, anti-ErbB2 antibodies and anti-CD30 antibodies.

[0075] In some examples, an agent of interest may be a diagnostic agent, which can be any moiety possessing a property or function which can be used for detection purposes, such as a fluorescent moiety (e.g. polyfluorenes, fluorescein, or Ru, Eu, Pt complexes), a luminescent moiety (e.g. a horseradish peroxidase label) or a radioactive moiety (e.g. tritium (3H), 32P, 35S, or 14C, or covalently bound labels, such as 1251 bound to tyrosine, 18F within fluorodeoxyglucose, or metallo-organic complexes e.g. 99Tc-DTPA). In some embodiments, the diagnostic agent of the present disclosure is selected from the group consisting of a fluorescent moiety, a luminescent moiety, a radioactive moiety, and any combinations thereof.

[0076] In some embodiments, conjugation of the scaffold polynucleotides of the present disclosure to biomolecules or agents of interest may be performed covalently or non-covalently. Methods for covalently or non-covalently conjugation are available in this art. Non-covalent linkage may be performed by ionic interactions such as a protamine charge-force approach and affinity binding such as an avidin-based conjugation approach. In case of a covalent linkage between a biomolecule or an agent moiety and a polynucleotide, a direct reaction of an activated group either on the biomolecule or the agent moiety or on polynucleotide with an functional group on either the polynucleotide or on the biomolecule or the agent moiety or viaan heterobifunctional linker molecule, which is firstly reacted with one and then reacted with the other binding partner. In some embodiments, the conjugator of the present disclosure is selected from the group consisting of a biotin moiety, a thiol group, a succinimide moiety, a maleimide moiety, a hydrazine moiety, tyrosine moiety, a hydrazone moiety, an azide moiety, a dibenzocyclooctyne group (DBCO), a bicyclononyne (BCN) group, a terminal alkyne moiety, a strained terminal alkyne moiety, a phosphine moiety, polyethylene glycol (PEG) chains, and any combinations thereof. The conjugation may occur at the 5 ’-end, internal region, or 3 ’-end of the polynucleotides to biomolecules or agents of interest via the conjugator of the present disclosure.

[0077] In some embodiments, a polynucleotide for use in making the conjugate disclosed herein may be modified to add a functional group, which can react with the biomolecule, the agent of interest, or a conjugator (e.g., a chemical linker) to form a covalent bond. Afunctional group can be any chemical moiety including a functional group that can react with another functional group to form covalent bonds. Exemplary functional groups include, but are not limited to, an aldehyde group (R-CH=O), a hydroxyl group (-OH), a methyl group, a carbonyl group (-C=O), a carboxyl group (-COOH), an amino group (-NH2), a phosphate group, or a thiol group (-SH). The functional group may be added to the 5’-end of the polynucleotides. Alternatively, it may be added to the 3 ’-end of the polynucleotides.

[0078] In some embodiments, conjugating an agent of interest to a polynucleotide would depend on the nature of the agent of interest, e.g., chemical structures thereof. In some examples, the agent of interest includes a functional group that is reactive to the functional group carried by a polynucleotide as disclosed herein. In that case, a direct reaction between the agent of interest and the polynucleotide of the present disclosure carrying the reactive moiety can be taken place to conjugate the agent of interest with the polynucleotide. In other cases, the agent of interest may be modified to add a corresponding reactive moiety that is reactive to the reactive moiety linked to the polynucleotide.

[0079] 2. Conjugate

[0080] In another aspect, the present disclosure further provides a conjugate including a biomolecule conjugated to the scaffold polynucleotide of the present disclosure via the arm linker or the conjugator; and an agent conjugated to the scaffold polynucleotide of the present disclosure via the arm linker or the conjugator. In some embodiments, the scaffold polynucleotide of the present disclosure includes a linear polynucleotide or a branched polynucleotide; and a conjugator bound to the linear polynucleotide or the branched polynucleotide, wherein the branched polynucleotide has an arm linker emanating therefrom via the conjugator.

[0081] In some embodiments, the linear polynucleotide or the branched polynucleotide is a single-stranded form or a double-stranded form.

[0082] In some embodiments, the arm linker is conjugated to the polynucleotide via the conjugator. In some embodiments, the arm linker of the present disclosure is a linear polynucleotide or a branched polynucleotide. In some embodiments, the arm linker emanates from a nucleobase, a nucleosugar, or a backbone of the branched polynucleotide.

[0083] In some embodiments, the conjugator of the present disclosure is selected from the group consisting of a biotin moiety, a thiol group, a succinimide moiety, a maleimide moiety, an amine moiety, an acylhydrazide moiety, a hydrazine moiety, tyrosine moiety, a hydrazone moiety, an azide moiety, a dibenzocyclooctyne group (DBCO), a bicyclononyne (BCN) group, an aminooxy moiety, a hydroxylamine moiety, a terminal alkyne moiety, a strained terminal alkyne moiety, a phosphine moiety, polyethylene glycol (PEG) chains, and any combinations thereof.

[0084] In some embodiments, the biomolecule of the present disclosure is selected from the group consisting of a humanized antibody or antigen binding fragment thereof, a chimeric antibody or antigen binding fragment thereof, a monoclonal antibody or antigen binding fragment thereof, a monovalent Fab’, a divalent Fab2, a single-chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), a camelid antibody, antigen binding fragment thereof, a peptide, and any combinations thereof.

[0085] In some embodiments, the agent of the present disclosure is selected from the group consisting of a therapeutic agent, a diagnostic agent, and a combination thereof. In some embodiments, the therapeutic agent of the present disclosure is selected from the group consisting of cytotoxic agents, protein toxins, proteins, enzymes, radionuclides, and any combinations thereof.

[0086] In some embodiments, the diagnostic agent of the present disclosure is selected from the group consisting of a fluorescent moiety, a luminescent moiety, a radioactive moiety, and any combinations thereof.

[0087] 3. Preparation Method of Scaffold Polynucleotide

[0088] In one aspect, the present disclosure further provides a method of preparing the scaffold polynucleotide and the method includes providing a target nucleic acid having a plurality of modified nucleobases at 5 ’-end of the target nucleic acid or within the target nucleic acid; providing a mono-functional DNA glycosylase to react with the target nucleic acid to form an intermediate nucleic acid having a plurality of abasic sites at the 5 ’-end of the target nucleic acid or within the target nucleic acid; and incorporating the conjugator to the intermediate nucleic acid to form the linear polynucleotide, provided that the scaffold polynucleotideincludes the branched polynucleotide, the method further includes conjugating the arm linker to the linear polynucleotide via the conjugator to form the branched polynucleotide.

[0089] In some embodiments, the arm linker is conjugated to the polynucleotide via the conjugator. In some embodiments, the arm linker of the present disclosure is a linear polynucleotide or a branched polynucleotide. In some embodiments, the arm linker emanates from a nucleobase, a nucleosugar, or a backbone of the branched polynucleotide.

[0090] In some embodiments, as used herein, the modified nucleobase of the present disclosure is selected from the group consisting of hypoxanthine, cytosine, 3 -alkyladenine, 8-oxoguanine (8-oxoG), uracil, 5 -hydroxyuracil, 5 -hydroxymethyluracil, 5 -formyluracil, 5 -fluorouracil, dihydroxyuracil, 5-formylcytosine, 5-carboxylcytosine, 3 -methyladenine (3 -me A), 3-methylguanine, 7-methyladenine, 7-methylguanine, N6-methyladenine, 8-oxo-7,8-dihydroguanine, 5 -hydroxylcytosine, ethenocytosine, ethenoadenine, thymine glycol, cytosine glycol, 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine, a formamidopyrimidine derivative of adenine, and a formamidopyrimidine derivative of guanine.

[0091] In some embodiments, as used herein, the mono-functional DNA glycosylase of the present disclosure is selected from the group consisting of uracil-DNA glycosylase (UDG or UNG), alkyladenine DNA glycosylase (AAG; also referred to as methylpurine DNA glycosylase (MPG)), single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), methyl-binding domain glycosylase 4 (MBD4), thymine DNA glycosylase (TDG), MutY homolog DNA glycosylase (MYH), alkylpurine glycosylase C (AlkC), alkylpurine glycosylase D (AlkD), 8-oxo-guanine glycosylase 1 (OGGI) without an abasic site lyase activity, endonuclease Ill-like glycosylase 1 (NTHL1) without the abasic site lyase activity, endonuclease Vlll-like glycosylase 1 (NEIL1) without the abasic site lyase activity, endonuclease Vlll-like glycosylase 2 (NEIL2) without the abasic site lyase activity, endonuclease Vlll-like glycosylase 3 (NEIL3) without the abasic site lyase activity, enzymatically active fragments thereof, and any combinations thereof.

[0092] In some embodiments, the conjugator of the present disclosure is incorporated to the target nucleic acid by a template-independent enzymatic nucleic acid synthesis. In some embodiments, the template-independent enzymatic nucleic acid synthesis includes employing aDNA polymerase, an RNA polymerase, or a functionally equivalent enzyme thereof. In some embodiments, the DNA polymerase of the template-independent enzymatic nucleic acid synthesis is selected from the group consisting of an A family DNA polymerase, a B family DNA polymerase, an X family DNA polymerase, and any combinations thereof. In at least one embodiment, the DNA polymerase is a B family DNA polymerase or a variant thereof. In atleast one embodiment, the B family DNA polymerase is a Thermococcaceae DNA polymerase. In at least one embodiment, the B family DNA polymerase is a Thermococcus or a Pyrococcus DNA polymerase. In at least an embodiment, the B family DNA polymerase is selected from the group consisting of a B family DNA polymerase of Thermococcus kodakarensis (Kodl), a B family DNA polymerase of Pyrococcus furiosus (Pfu), a B family DNA polymerase of Thermococcus litoralis (Vent), a B family DNA polymerase of Thermococcus sp. 9°N (9°N), and a B family DNA polymerase of Thermococcus gorgonarius (Tgo).

[0093] In some embodiments, the template independent enzymatic nucleic acid synthesis is performed at a reaction temperature of from 10°C to 100°C, such as 10°C to 90°C, 20°C to 90 °C, 30°C to 90°C, 20°C to 80°C, 30°C to 80°C, 40°C to 80 °C, 30°C to 70 °C, 40°C to 70 °C, or 50°C to 70°C.

[0094] In some embodiments, an enzymatic synthesis approach is used to introduce the reactive moiety to the target polynucleotide. For example, a nucleotide analogue, or analogues, is enzymatically added to the 3 ’-hydroxyl (3 ’-OH) end of a single-stranded, nucleic acid initiator (the target polynucleotide) in a template-independent synthesis manner to produce a polynucleotide with a desired reactive moiety at the nucleobase and / or at the nucleosugar. In at least one embodiment, the desired reactive moiety is an azide (N3) or azido group, and a suitable reagent / compound, such as a nucleotide analogue, containing an azido moiety at the nucleosugar, such as a 3’-O-azidomethyl group, may be used to introduce such a modification to the 3 ’-end of polynucleotides. The following examples are further provided based on this scenario.

[0095] In some embodiments, as used herein, the preparation method of the scaffold polynucleotide of the present disclosure further includes reacting a biomolecule or an agent of interest with the conjugator via a bioorthogonal reaction. In some embodiments, the bioorthogonal reaction is a click conjugation, an oxime / hydrazone formation, a Staudinger ligation, a tetrazine ligation, or a quadricyclane ligation. In some embodiments, the click conjugation is selected from the group consisting of copper-catalyzed azide-alkyne cycloaddition (CuAAC), strain-promoted azide-alkyne cycloaddition (SPAAC), isocyanide-based click reaction, and inverse electron demand Diels-Alder reaction (IEDDA).

[0096] In some embodiments, as used herein, the preparation method of the scaffold polynucleotide of the present disclosure further includes providing 5’ to 3’ exonuclease to remove an unreactive target nucleic acid.

[0097] In some embodiments, the method provided by the present disclosure further includes a clean-up or enrichment step to remove unlabeled nucleic acids or polynucleotides, e.g., providing a protein possessing a 3’ to 5’ exonuclease activity to digest the nucleic acids orpolynucleotides with an unsuccessful 3 ’-end nucleotide synthesis by a polymerase or an incomplete coupling reaction to the 3 ’-end of the incorporated nucleotide carrying a reactive moiety.

[0098] 4. Preparation Method of Conjugate

[0099] In a further aspect, the present disclosure further provides a method of preparing a conjugate of the present disclosure, and the method includes conjugating a biomolecule and an agent with the scaffold polynucleotide of the present disclosure.

[0100] In some embodiments, the biomolecule or the agent is conjugated to the scaffold polynucleotide of the present disclosure via the arm linker or the conjugator. In some embodiments, the arm linker of the present disclosure is a linear polynucleotide or a branched polynucleotide. In some embodiments, the conjugator of the present disclosure is selected from the group consisting of a biotin moiety, a thiol group, a succinimide moiety, a maleimide moiety, an amine moiety, an acylhydrazide moiety, a hydrazine moiety, a tyrosine moiety, a hydrazone moiety, an azide moiety, a dibenzocyclooctyne group (DBCO), a bicyclononyne (BCN) group, an aminooxy moiety, a hydroxylamine moiety, a terminal alkyne moiety, a strained terminal alkyne moiety, a phosphine moiety, polyethylene glycol (PEG) chains, and any combinations thereof.

[0101] In some embodiments, the biomolecule of the present disclosure is selected from the group consisting of a humanized antibody or antigen binding fragment thereof, a chimeric antibody or antigen binding fragment thereof, a monoclonal antibody or antigen binding fragment thereof, a monovalent Fab’, a divalent Fab2, a single-chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), a camelid antibody, antigen binding fragment thereof, a peptide and combinations thereof.

[0102] In some embodiments, the agent of the present disclosure is conjugated to the scaffold polynucleotide of the present disclosure via the arm linker or the conjugator. In some embodiments, the agent of the present disclosure is a therapeutic agent or a diagnostic agent. In some embodiments, the therapeutic agent of the present disclosure is selected from the group consisting of cytotoxic agents, protein toxins, proteins, enzymes, radionuclides, and any combinations thereof. In some embodiments, the diagnostic agent of the present disclosure is selected from the group consisting of a fluorescent moiety, a luminescent moiety, a radioactive moiety, and any combinations thereof.

[0103] In some embodiments, the conjugator of the present disclosure is selected from the group consisting of a biotin moiety, a thiol group, a succinimide moiety, a maleimide moiety, a hydrazine moiety, tyrosine moiety, a hydrazone moiety, an azide moiety, a dibenzocyclooctyne group (DBCO), a bicyclononyne (BCN) group, a terminal alkyne moiety, a strained terminalalkyne moiety, a phosphine moiety, polyethylene glycol (PEG) chains, and any combinations thereof.

[0104] In some embodiments, the biomolecule or the agent of the present disclosure is conjugated with the arm linker or the conjugator via a bioorthogonal reaction. In some embodiments, the bioorthogonal reaction is a click conjugation, an oxime / hydrazone formation, a Staudinger ligation, a tetrazine ligation, or a quadricyclane ligation. In some embodiments, the click conjugation is selected from the group consisting of copper-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, isocyanide-based click reaction, and inverse electron demand Diels-Alder reaction.

[0105] In some embodiments, the method as described above further includes preparing the polynucleotide in a solution phase. In other embodiments, the method as described above further includes preparing the polynucleotide in a solid phase, e.g., providing a nucleic acid initiator attached to a solid support. In some embodiments, the solid support is selected from the group consisting of a particle, a polymer, a bead, a resin, a slide, a chip, an array surface, a membrane, a flow cell, a well, a matrix, a chamber, a microfluidic chamber, a channel, a microfluidic channel, and a gel.

[0106] Although the present disclosure is illustrated by specific embodiments and optional features, it is understood that modifications and variations of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of the present disclosure.EXAMPLE

[0107] Exemplary embodiments according to the present disclosure are further described in the following examples, which should not be construed to limit the scope of the present disclosure. The materials and methods used in the following examples are described in detail below. The materials used in the present disclosure but unannotated herein are commercially available.

[0108] Single- Stranded Polynucleotides

[0109] Example 1. 3'-end dual labeling of polynucleotide with fluorescent dyes and nanobody

[0110] In this example, a 26-mer DNA polynucleotide (5’- / U / CACCCCGCATTACGTTTGGTGGACC-3’) (SEQ ID NO: 1) was used as the target polynucleotide for 3 ‘-modification or labeling. To introduce an azidomethyl group to the 3 ’-end of the target polynucleotide, the 3’-O-azidomethyl-deoxynucleoside triphosphate (3‘-AZ-dNTP) was used as a template-independent DNA synthesis substrate for the selective B-family DNA polymerase or its variants. The exemplary DNA polymerase used herein may be an in-house DNA polymerase derived from Vent DNA polymerase. The nucleotideincorporation reaction was performed in the reaction mixture (50 pL) containing 100 pM of the target polynucleotide, 5 mM magnesium chloride (MgCl2), 0.25 mM manganese chloride (MnCl2), and 50 pM of DNA polymerase. The reaction was initiated by the addition of 200 pM of Atto532-labeled 3’-AZ-dATP and then incubated at 37°C for 1 hour. To introduce a maleimide group as a conjugator of biomolecule to the polynucleotide carrying an azide (N3) group at the 3 ‘-end, the reaction was subsequently carried on by the addition of 300 pM of DBCO-maleimide. The azide-DBCO ligation reaction was normally performed at 37°C for 1 hour. The final reaction products were analyzed with 20% Urea-PAGE and visualized by imaging the gel on the Amersham Typhoon Laser Scanner (Cytiva Life Sciences, Marlborough, MA, USA). The modified polynucleotide was purified by using the FavorLight clean-up kit. The clean-up reaction products were further concentrated by ethanol precipitation and dissolved in IxPBS buffer.[OHl] To further preforming the nanobody-oligonucleotide conjugation, TCEP solution (IM in ddH2O) was added to the anti-HER2 nanobody stock solution (100 pL, 50 pM in 50 mM Tris, pH 7.0, 300 mM NaCl, 1 mM DTT, 1 mM EDTA, 5% glycerol) in 50 molar equivalents standing at room temperature for 30 minutes. TCEP was removed from nanobody solution through buffer-exchanging process into IxPBS buffer via a concentrator (Vivaspin® 4 Turbo centrifugal concentrators, 10 kDa MWCO). 27mer-3’Atto532-mal and the reduced nanobody were mixed with 5 molar equivalents, and the reaction was performed with horizontal rotation at 70 rpm at room temperature overnight. The reaction products were analyzed via 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the gel was first stained with lx SYBR gold nucleic acid gel stain solution and visualized by imaging on the Amersham Typhoon Laser Scanner. Secondly, the visualization of the nanobody position on the gel was applied with Coomassie blue staining. Finally, the 3'-end dual labeling of the single-stranded polynucleotide with Atto532-fluorescent dye and DBCO-maleimide conjugated with anti-HER2 nanobody was obtained.

[0112] FIGs. 1A-1, 1A-2, IB-1 and IB-2 show an example of labeling a fluorescent dye to the 3 ’-end of a polynucleotide via enzymatic synthesis of Atto532-labeled 3'-AZ-dNTP to the 3 '-end of the polynucleotide, followed by the azide-DBCO click conjugation reaction between the incorporated 3'-O-azidomethyl deoxynucleotide monophosphate (3'-AZ-dNMP) and the DBCO-modified maleimide functional moiety allowing to perform the thiol-maleimide reaction with reduced biomolecule. FIGs. 1A-1, 1A-2, IB-1 and IB-2 are images from the different gels, which are 20% Urea polyacrylamide gel and 15% SDS polyacrylamide gel, respectively. FIG. 1A-1 depicts the gel electrophoresis of the unlabeled and labeled polynucleotides visualized by staining with SYBR Gold dye. FIG. 1A-2 illustrates theelectrophoretic location of nucleic acids labeled with the Atto532-fluorescent dye after the enrichment step. Lane S shows the electrophoretic location of the target polynucleotide (26-mer single-stranded DNA (ssDNA)). Lane 1 shows the electrophoretic location of the target polynucleotide with the incorporated Atto532-labeled 3'-AZdNMP at the 3'-end. Lane 2 shows the electrophoretic location of the polynucleotide with an incorporated Atto532-labeled 3'AZ-dNMP coupled with a maleimide functional moiety at the 3 '-end after the enrichment step. FIG. IB-1 depicts the gel electrophoresis of the unlabeled and labeled nanobodies visualized by staining with Coomassie blue dye. FIG. IB-2 illustrates the electrophoretic location of nanobodies conjugated with the 3 ’end-modified polynucleotides labeled with the Atto532-fluorescent dye. Lane 3 shows the electrophoretic location of the nanobody; and lane 4 shows the electrophoretic location of the nanobody conjugated with the Atto532- and maleimide-labeled polynucleotide. FIG. 1C shows an example overview of 3 '-end dual labeling of 27-mer ss DNA with fluorescent dyes (the symboldenotes as Atto532-fluorescent dye) and DBCO-maleimide conjugated with anti-HER2 nanobody.

[0113] The components and individual experimental groups of the reaction are summarized in Table 1 below. The results of the reaction corresponding to each experimental group are shown in lanes 1 to 4 in FIGs. 1 A-l, 1A-2, IB-1 and IB-2, respectively. In Tablet, four experimental groups were designed, and the reaction components are shown. In the following Tables 1 to 24 throughout the present disclosure, the symbol “+” indicates the addition of the designated reagent to the reaction in each experimental group, while the symbol indicates the absence of this reagent in the reaction for the experimental group.

[0114] Table 1.

[0115] Example 2. Dual labeling of the polynucleotides with 5 ’-FAM and 3 ’-nanobody

[0116] In this example, a 26-mer DNA polynucleotide (SEQ ID NO: 1) containing an uracilresidue at its 5'-end was used as the target polynucleotide. To carry out the uracil excision and subsequent abasic site labeling, 100 pM of the target polynucleotide was mixed with 50 pM of uracil-DNA glycosylase derived from Micrococcus luteus (MluUDG), 4 rnM of guanidine-FAM, and 100 mM of p-phenylenediamine. The reaction was initiated by the addition of MluUDG and guanidine-FAM and incubated at 37°C for 1 hour. To eliminate the unlabeled ssDNA, 15 pM of phage lambda exonuclease and 15 pM of MutM (formamidopyrimidine glycosylase derived from Escherichia coll) were added and then incubated at 37°C for an additional 1 hour. The modified polynucleotide, the FAM-25mer, was purified by ethanol precipitation and dissolved in ddH2O.

[0117] To introduce a maleimide group as the conjugator of biomolecule to the FAM-25mer, the 3‘-AZ-dNTP was first used as the template-independent DNA synthesis substrate to incorporate to the 3 ’-end of the FAM-25mer. The nucleotide incorporation reaction was performed in the reaction mixture (50 pL) containing 50 pM of FAM-25-mer DNA target polynucleotide, 0.25 mM manganese chloride (MnCl2), and 25 pM of Tgo DNA polymerase. The reaction was initiated by the addition of 100 pM of 3’-AZ-dATP and then incubated at 37°C for 1 hour. Subsequently, 200 pM of DBCO-maleimide was added to the reaction mixture and then incubated at 37°C for an additional 1 hour. The intermediate and final reaction products were analyzed with 20% Urea-PAGE and visualized by imaging the gel on the Amersham Typhoon Laser Scanner (Cytiva Life Sciences, Marlborough, MA, USA). The modified polynucleotide was purified by using the FavorLight clean-up kit. The clean-up reaction products were further concentrated by ethanol precipitation and dissolved in IxPBS buffer.

[0118] To further perform the nanobody-polynucleotide conjugation, the anti-HER2 nanobody which prepared and purified in-house was used, and TCEP solution (1 M in ddH2O) was added to the nanobody and stood at room temperature for 30 minutes. The anti-HER2 nanobody was reduced with TCEP -treated, and then the TCEP was removed via a concentrator (Vivaspin® 4 Turbo centrifugal concentrators, 10 kDa MWCO) while exchanging the buffer into IxPBS buffer. 5’GFAM-26mer-mal was added to the reduced nanobody with 5 molar equivalents and preformed with horizontal rotation at 70 rpm at room temperature overnight. The reaction products of oligonucleotides-conjugated nanobody were analyzed via 15% SDS-PAGE, and the gel was firstly stained with lx SYBR gold nucleic acid gel stain solution and visualized by imaging on the Amersham Typhoon Laser Scanner (Cytiva Life Sciences, Marlborough, MA, USA). Secondly, the visualization of the nanobody position on the gel was applied with Coomassie blue staining. Finally, the dual labeling of the single-stranded polynucleotides with 5 ’-FAM and 3 ’-DBCO-maleimide conjugated with anti-HER2 nanobody was obtained.

[0119] FIGs. 2A, 2B-1 and 2B-2 show an example of labeling a fluorescent dye to the 5’-end ofthe polynucleotide via uracil-DNA glycosylase derived from MluUDG and an aldehyde-reactive probe (ARP), followed by the 3 ’-end labeling performed with enzymatic synthesis of 3'-AZ-dNTP to the 3'-end of the polynucleotide, and modified with maleimide by the azide-DBCO click conjugation reaction between the incorporated 3'-AZ-dNMP and DBCO-modified maleimide functional moiety. FIG. 2A depicts the gel electrophoresis of the unlabeled and labeled polynucleotides visualized by staining with SYBR gold dye, and the labeled fluorescent dye was displayed on the same scanner channel. FIG. 2B-1 depicts the gel electrophoresis of the unlabeled and labeled nanobodies visualized by staining with Coomassie blue dye, and FIG. 2B-2 illustrates the electrophoretic location of nanobodies conjugated with the 3 ’end-modified polynucleotides labeled with the FAM-fluorescent dye. Lane S shows the electrophoretic location of the target polynucleotide (26-mer single-stranded DNA); lane 1 shows the electrophoretic location of the target polynucleotide with the fluorescent dye at the 5 ’-end; lane 2 shows the reaction products from the previous step degrading with the unlabeled polynucleotides via exonuclease; lane 3 shows the reaction products after digestion removing free dye with FavorLight clean-up kit; lane 4 shows the electrophoretic location of the 5 ’-labeled polynucleotide with the incorporated 3'-AZ-dAMP at the 3 '-end; lane 5 shows the electrophoretic location of the 5 ’-labeled polynucleotide with the incorporated 3'AZ-dAMP coupled with the maleimide functional moiety; lane 6 shows the electrophoretic location of the anti-HER2 nanobody; and lane 7 shows the electrophoretic location of the anti-HER2 nanobody conjugated with the 5’FAM- and 3’maleimide-labeled polynucleotide. FIG. 2C shows an example overview of the dual labeling of 26-mer ss DNA with 5 ’-FAM (the symbol denotes FAM -fluorescent dye) and 3’- DBCO-maleimide conjugated with anti-HER2 nanobody.

[0120] The components and individual experimental groups of the reaction are summarized in Table 2 below. The results of the reaction corresponding to each experimental group are shown in lanes 1 to 7 in FIGs. 2 A, 2B-1 and 2B-2, respectively. In Table 2, seven experimental groups were designed, and the reaction components are shown.

[0121] Table 2

[0122] Example 3-1. Dual labeling of the polynucleotides with 5’-MMAE and 3’-nanobody

[0123] In this example, a 26-mer DNA polynucleotide (SEQ ID NO: 1) containing an uracil residue at the 5'-end was used as the target polynucleotide. To carry out the uracil excision and subsequent abasic site labeling, 100 pM of the target polynucleotide was mixed with 20 pM of MluUDG, 4mM of (Aminooxy)acetamide-Val-Cit-PAB-MMAE, and 100 mM of p-phenylenediamine. The reaction was initiated by the addition of MluUDG and (Aminooxy)acetamide-Val-Cit-PAB-MMAE and incubated at 37°C for 1 hour. To eliminate the unlabeled target polynucleotide, 20 pM of phage lambda exonuclease and 30 pM of MutM were added and then incubated at 37°C for an additional 1 hour. The intermediate and final reaction products, 5’-(Aminooxy)acetamide-Val-Cit-PAB-MMAE-25mer, were analyzed with 20% Urea-PAGE and visualized by imaging the gel on the Amersham Typhoon Laser Scanner (Cytiva Life Sciences, Marlborough, MA, USA). The modified polynucleotide was purified by ethanol precipitation and dissolved in ddH2O (20 pL).

[0124] To introduce a maleimide group as the conjugator of biomolecule to the 5’-(Aminooxy)acetamide-Val-Cit-PAB-MMAE-25mer, the 3‘-AZ-dNTP was first used as the functional moiety to incorporate to the 3 ’-end of the polynucleotide via the template-independent DNA synthesis. The nucleotide incorporation reaction was performed in the reaction mixture (80 pL) containing 50 pM of 5’-(Aminooxy)acetamide-Val-Cit-PAB-MMAE-25mer DNA target polynucleotide, 0.25 mM manganese chloride (MnCl2), and 25 pM of Tgo DNA polymerase. The reaction was initiated by the addition of 100 pM of 3’-AZ-dATP and then incubated at 37°C for 1 hour. The modified polynucleotide was purified by using the FavorLight clean-up kit. The clean-up reaction products were further concentrated by ethanol precipitation and dissolved in ddH2O (20 pL). Subsequently, 500 pM of DBCO-maleimide was added and reacted with the polynucleotide carrying an azide group. The azide-DBCO ligation reaction was normally performed at 37°Cfor 1 hour. The intermediate and final reaction products were analyzed with 20% Urea-PAGE and visualized by imaging the gel on the Amersham Typhoon Laser Scanner (Cytiva Life Sciences, Marlborough, MA, USA). The modified polynucleotide was further purified and concentrated by ethanol precipitation and dissolved in IxPBS buffer.To further preform the nanobody-polynucleotide conjugation, the anti-HER2 nanobody which prepared and purified in-house was used, and TCEP solution (1 M in ddH2O) was added to the nanobody and stood at room temperature for 30 minutes to reduce the thiol functional groups on the nanobody. The TCEP was removed via a concentrator (Vivaspin® 4 Turbo centrifugal concentrators, 10 kDa MWCO) while exchanging the buffer into IxPBS buffer. After buffer-exchanging, the reduced nanobody was mixed with 100 molar equivalents of L-ascorbic acid standing at room temperature for 5 hours to perform a single conjugation between one nanobody and one modified polynucleotide. 5’MMAE-26mer-3’mal was added to the pre-treated nanobody with 2.5 molar equivalents, and the reaction was performed with horizontal rotation at 70 rpm at room temperature overnight. The reaction products were analyzed via 15% SDS-PAGE, and the gel was first stained with lx SYBR gold nucleic acid gel stain solution and visualized by imaging on the Amersham Typhoon Laser Scanner. Secondly, the visualization of the nanobody position on the gel was applied with Coomassie blue staining. Finally, the dual labeling of the single-stranded polynucleotides with 5’-MMAE and 3’-DBCO-maleimide conjugated with anti-HER2 nanobody was obtained.

[0125] Example 3-2. Dual labeling of polynucleotide with 5’ - nanobody and 3 ’-DM1

[0126] In this example, a 46-mer DNA polynucleotide (5’- / U / CTCGGCCTGGCACAGGTCCGTTCAGTGCTGCGGCGACCACCGAGG-3’) (SEQ ID NO: 2) containing an uracil residue at the 5'-end was used as the target polynucleotide. To introduce an azidomethyl group to the 3 ’-end of the target polynucleotide, the 3‘-AZ-dNTP was used as the template-independent DNA synthesis substrate for the selective B-family DNA polymerase or its variants. The nucleotide incorporation reaction was performed in the reaction mixture (50 pL) containing 20 pM of the target polynucleotide, 0.25 mM of manganese chloride (MnCl2), and 10 pM of Tgo polymerase. The reaction was initiated by the addition of 100 pM of 3’-AZ-dCTP and then incubated at 37°C for 30 minutes. To introduce a DM1 group as a therapeutic agent to the polynucleotide carrying an azide (N3) group at the 3 ‘-end, the reaction was subsequently carried on by the addition of 200 pM of DM1-PEG4-DBCO. The azide-DBCO ligation reaction was normally performed at 37°C for 1 hour. The modified polynucleotide was concentrated by ethanol precipitation and dissolved in ddH2O (20 pL).

[0127] To carry out the uracil excision and subsequent abasic site labeling, the 3 ’DM1 -labeled target polynucleotide was mixed with 50 pM of MluUDG and 1 mM of Aminooxy-Azide, and20 mM of p-phenylenediamine. The reaction was initiated by the addition of MluUDG and Aminooxy- Azide and incubated at 37°C for 1 hour. The modified target polynucleotide was purified by using the FavorLight clean-up kit. The clean-up reaction products were further concentrated by ethanol precipitation and dissolved in ddH2O (20 pL). Subsequently, 500 pM of DBCO-maleimide was added and reacted with the polynucleotide carrying an azide group. The azide-DBCO ligation reaction was normally performed at 37°C for 1 hour. The intermediate and final reaction products were analyzed with 20% Urea-PAGE and visualized by imaging the gel on the Amersham Typhoon Laser Scanner (Cytiva Life Sciences, Marlborough, MA, USA). The modified polynucleotide was further purified and concentrated by ethanol precipitation and dissolved in IxPBS buffer.

[0128] To further preform the nanobody-polynucleotide conjugation, the anti-HER2 nanobody which prepared and purified in-house was used, and TCEP solution (1 M in ddH2O) was added to the nanobody and then stood at room temperature for 30 minutes to reduce the thiol functional groups on the nanobody. The TCEP was removed via a concentrator (Vivaspin® 4 Turbo centrifugal concentrators, 10 kDa MWCO) while exchanging the buffer into IxPBS buffer. After buffer-exchanging, the reduced nanobody was mixed with 100 molar equivalents of L-ascorbic acid and stood at room temperature for 5 hours to perform a single conjugation between one nanobody and one modified polynucleotide. 5’mal-46mer-3’DMl was added to the pre-treated nanobody with 2.5 molar equivalents, and the reaction was performed with horizontal rotation at 70 rpm at room temperature overnight. The reaction products were analyzed via 15% SDS-PAGE, and the gel was firstly stained with lx SYBR gold nucleic acid gel stain solution and visualized by imaging on the Amersham Typhoon Laser Scanner. Secondly, the visualization of the nanobody position on the gel was applied with Coomassie blue staining. Finally, the dual labeling of the single-stranded polynucleotides with 5 ’-DBCO-maleimide conjugated with anti-HER2 nanobody and 3 ’-DM1 was obtained.

[0129] FIG. 3A depicts the gel electrophoresis of the unlabeled and labeled polynucleotides visualized by staining with SYBR gold dye. In FIG. 3A, lane S shows the electrophoretic location of the target polynucleotide (26-mer ssDNA, SEQ ID NO: 1); lane 1 shows the electrophoretic location of the target polynucleotide with the therapeutic agent, (Aminooxy)acetamide-Val-Cit-PAB-MMAE at the 5’-end; lane 2 shows the reaction products from previous step degrading with the unlabeled polynucleotides via exonuclease; lane 3 shows the electrophoretic location of the 5 ’-labeled polynucleotide with the incorporated 3'-AZ-dAMP at the 3 '-end; and lane 4 shows the electrophoretic location of the 5 ’-labeled polynucleotide with the incorporated 3'AZ-dAMP coupled with the maleimide functional moiety.

[0130] The components and individual experimental groups of the reaction are summarized in Table 3 below. The results of the reaction corresponding to each experimental group are shown in lanes 1 to 4 in FIG. 3 A. In Table 3, four experimental groups were designed, and the reaction components are shown.

[0131] Table 3

[0132] FIG. 3B depicts the gel electrophoresis of the unlabeled and labeled polynucleotides visualized by staining with SYBR gold dye. In FIG. 3B, lane S shows the electrophoretic location of the target polynucleotide (46-mer ssDNA, SEQ ID No: 2); lane 1 shows the electrophoretic location of the target polynucleotide with the incorporated 3’-AZ-dCMP at the 3 ’-end; lane 2 shows the electrophoretic location of the polynucleotide with the incorporated 3’-AZ-dCMP coupled with the therapeutic agent, DM1-PEG4-DBCO at the 3 ’-end; lane 3 shows the electrophoretic location of the 3 ’-labeled target polynucleotide performed 5 ’uracil excision; lane 4 shows the electrophoretic location of the 3 ’-labeled target polynucleotide with the azide moiety at the 5 ’-end; and lane 5 shows the electrophoretic location of the 3 ’-labeled target polynucleotide with the azide moiety coupled with the maleimide functional moiety.

[0133] The components and individual experimental groups of the reaction are summarized in Table 4 below. The results of the reaction corresponding to each experimental group are shown in lanes 1 to 5 in FIG. 3B. In Table 4, five experimental groups were designed, and the reaction components are shown.

[0134] Table 4

[0135] FIG. 3C illustrates the gel electrophoresis of the unlabeled and labeled nanobody visualization by staining with Coomassie blue dye. In FIG. 3C, lane 1 shows the electrophoretic location of the anti-HER2 nanobody; lane 2 shows the electrophoretic location of the anti-HER2 nanobody conjugated with the 5’maleimide- and 3’DMl-labeled polynucleotide; and lane 3 shows the electrophoretic location of the anti-HER2 nanobody conjugated with the 5’MMAE- and 3’maleimide-labeled polynucleotide. FIG. 3D shows an example overview of the dual labeling of 25-mer ss DNAwith 5’-MMAE and 3’-DBCO-maleimide conjugated with anti-HER2 nanobody. FIG. 3E shows an example overview of the dual labeling of 45-mer ss DNAwith 5’-DBCO-maleimide conjugated with anti-HER2 nanobody and 3 ’-DM1.

[0136] The components and individual experimental groups of the reaction are summarized in Table 5 below. The results of the reaction corresponding to each experimental group are shown in lanes 1 to 3 in FIG. 3C. In Table 5, three experimental groups were designed, and the reaction components are shown.

[0137] Table 5

[0138] Example 4. Triple labeling of the polynucleotide with 5 ’-FAM, 3 ’Dye 681 and nanobody

[0139] In this example, a 46-mer DNA polynucleotide (46-mer ssDNA, SEQ ID NO: 2) containing an uracil residue at the 5'-end was used as the target polynucleotide. To carry out the uracil excision and subsequent abasic site labeling, 100 pM of the target polynucleotide was mixed with 50 pM of MluUDG, 4 mM of guanidine-FAM, and 100 mM of p-phenylenediamine. The reaction was initiated by the addition of MluUDG and guanidine-FAM and incubated at 37°C for 1 hour. To eliminate the unlabeled target polynucleotide, 15 pM of Kpn exonuclease (5’ exonuclease derived from Klebsiella pneumoniae) and 15 pM of MutM were added and then incubated at 37°C for an additional 1 hour. The modified polynucleotide, 5’-FAM-45-mer DNA, was purified by ethanol precipitation and dissolved in ddH2O (20 pL).

[0140] To introduce the azidomethyl group to the 3’-end of 5’-FAM-45-mer DNA, the 3‘-AZ-dNTP was used as the template-independent DNA synthesis substrate for the selective B-family DNA polymerase or its variants. The nucleotide incorporation reaction was performed in the reaction mixture (50 pL) containing 20 pM of the 5’-FAM-45-mer DNA target polynucleotide, 0.25 mM manganese chloride (MnCl2), and 10 pM of Tgo DNA polymerase. The reaction was initiated by the addition of 100 pM of Dye681 -labeled 3’-AZ-dCTP and then incubated at 37°C for 1 hour. The modified polynucleotide was purified by using the QIAquick Nucleotide Removal Kit (Qiagen, Germantown, MD, USA). The clean-up reaction products were concentrated by ethanol precipitation and dissolved in ddH2O (20 pL).

[0141] To introduce a maleimide group as the conjugator of biomolecule to the target polynucleotide carrying the azide (N3) group at the 3 ‘-end, the reaction was subsequently carrying on by the addition of 200 pM of DBCO-maleimide. The azide-DBCO ligation reaction was normally performed at 37°C for 1 hour. The intermediate and final reaction products were analyzed with 20% Urea-PAGE and visualized by imaging the gel on the Amersham Typhoon Laser Scanner (Cytiva Life Sciences, Marlborough, MA, USA). The modified polynucleotide, 5’FAM-46mer-3’DY681-mal, was purified by using the FavorLight clean-up kit. The clean-up reaction products were further concentrated by ethanol precipitation and dissolved in IxPBS buffer.

[0142] As previously performing the nanobody-polynucleotide conjugation, the anti-HER2 nanobody was reduced via TCEP -treated, and then a concentrator was used to exchange the nanobody buffer into IxPBS buffer and remove the TCEP. The reduced nanobody was mixed with 100 molar equivalents of L-ascorbic acid and stood at room temperature for 5 hours. The 5’FAM-46mer-3’DY681-mal were added to the pre-treated nanobody with 2.5 molarequivalents, and the reaction was performed with horizontal rotation at 70 rpm at room temperature overnight. The reaction products were analyzed via 15% SDS-PAGE, and the gel was first stained with lx SYBR gold nucleic acid gel stain solution and visualization by imaging on the Amersham Typhoon Laser Scanner. Secondly, the visualization of the nanobody position on the gel was applied with Coomassie blue staining. Finally, the triple labeling of the single-stranded polynucleotides with 5 ’-FAM, 3 ’-Dye 681 and 3’-DBCO-maleimide conjugated with anti-HER2 nanobody was obtained.

[0143] FIGs. 4A-1 to 4B-3 show examples of labeling a fluorescent dye to the 5’-end of the target polynucleotide via MluUDG and an aldehyde-reactive probe (ARP), followed by the 3’-end labeling performed with enzymatic synthesis of DY681 -labeled 3'-AZ-dCTP to the 3 '-end of the polynucleotide, and modified with maleimide by the azide-DBCO click conjugation reaction between the incorporated 3'-AZ-dCMP and DBCO-modified maleimide functional moiety. FIG. 4A-1 depicts the gel electrophoresis of the unlabeled and labeled polynucleotides visualized by staining with SYBR gold dye, and FIGs. 4A-2 and 4A-3 illustrate the electrophoretic location of nucleic acids labeled with the FAM- and DY681 -fluorescent dye after the enrichment step. FIG. 4B-1 depicts the result of the unlabeled and labeled nanobodies visualized by staining with Coomassie blue dye, and FIG. 4B-2 and FIG. 4B-3 illustrate the electrophoretic location of nanobodies conjugated with the modified polynucleotides labeled with the FAM- and DY681 -fluorescent dye. Lane S shows the electrophoretic location of the target polynucleotide (46-mer ssDNA, SEQ ID No: 2); lane 1 shows the electrophoretic location of the target polynucleotide with the FAM fluorescent dye at the 5 ’-end; lane 2 shows the electrophoretic location of the 5 ’-labeled polynucleotide with the incorporated DY681 -labeled 3'-AZ-dCMP at the 3 '-end; lane 3 shows the electrophoretic location of the 5 ’-labeled polynucleotide with the incorporated DY681 -labeled 3'-AZ-dCMP coupled with the maleimide functional moiety; lane 4 shows the electrophoretic location of the anti-HER2 nanobody; and lane 5 shows the electrophoretic location of the anti-HER2 nanobody conjugated with the 5’-FAM-, 3’-DY681 and 3’-maleimide-labeled polynucleotide.

[0144] The components and individual experimental groups of the reaction are summarized in Table 6 below. The results of the reaction corresponding to each experimental group are shown in lanes 1 to 5 in FIGs. 4A-1 to 4B-3. In Table 6, five experimental groups were designed, and the reaction components are shown.

[0145] Table 6

[0146] Example 5. Triple labeling of polynucleotide with 5’ DM1, internal MMAE and 3 ’ nanobody

[0147] In this example, a 27-mer DNA polynucleotide (5’- / U / CACCCCGCAT / U / TACGTTTGGTGGACC-3’) (27-mer ssDNA, SEQ ID NO: 3) containing two uracil residues, one at the 5'-end and one internally, was used as the target polynucleotide. To carry out the internal modification at the internal uracil site, the uracil excision and subsequent abasic site labeling were performed in the reaction mixture (100 pL) containing 50 pM of the target nucleotide, 1 unit of hSMUGl (purchased from New England Biolabs), 1 mM of (Aminooxy)acetamide-Val-Cit-PAB-MMAE, and 10 mM of p-phenylenediamine. The reaction was initiated by the addition of hSMUGl and (Aminooxy)acetamide-Val-Cit-PAB-MMAE and incubated at 37°C for 1 hour. To eliminate the unlabeled target nucleotide, 4 pM of PfuEndoQ (endonuclease Q derived from Pyrococcus furiosus) was first added and then incubated at 55°C for 1 hour followed by the addition of 4 pM of Kpn exonuclease and then incubated at 37°C for an additional Ihour. The modified polynucleotide, internal-MMAE -labeled polynucleotide, was purified by ethanol precipitation.

[0148] To introduce a DM1-PEG4-DBCO drug to the 5’-end of the target polynucleotide, the azide group was first used as a functional moiety to label to the 5 ’-end of the target polynucleotide via abasic site labeling. To carry out the 5 ’-end uracil excision and subsequent abasic site labeling, 90 pM of the internal -MMAE-labeled polynucleotide was mixed with 30 pM of MluUDG, 4 mM of guanidine-azide, and 100 mM of p-phenylenediamine. The reactionwas initiated by the addition of MluUDG and guanidine- Azide and incubated at 37°C for 1 hour. Subsequently, 3 mM of DM1-PEG4-DBC0 was added to the reaction mixture. The azide-DBCO ligation reaction was normally performed at 37°C for 1 hour. To eliminate the 5 ’-end unlabeled target polynucleotide, 10 pM of Kpn exonuclease and 10 pM of MutM were added and then incubated at 37°C for an additional 1 hour. The modified polynucleotide, 5’ -DM1 - / / z / MMAE -25-mer DNA target polynucleotide, was purified by ethanol.

[0149] To introduce the maleimide group as the conjugator of biomolecule to the 3 ’-end of the target polynucleotide, the 3‘-AZ-dNTP was first used as a functional moiety to incorporate to the 3 ’-end of the target polynucleotide via the template-independent DNA synthesis. The nucleotide incorporation reaction was performed in the reaction mixture (80 pL) containing 40 pM of 5’-DMl-z>?tMMAE-25-mer DNA target polynucleotide, 0.25 mM manganese chloride (MnCh), and 20 pM of Tgo DNA polymerase. The reaction was initiated by the addition of 200 pM of 3’-AZ-dATP and then incubated at 37°C for 1 hour. The modified polynucleotide was purified by using the FavorLight clean-up kit. The clean-up reaction products were further concentrated by ethanol precipitation and dissolved in ddH2O (20 pL). Subsequently, 200 pM of DBCO-maleimide was added and reacted with 3 ’-end of the polynucleotide carrying an azide group. The azide-DBCO ligation reaction was normally performed at 37°C for 1 hour. The intermediate and final reaction products were analyzed with 20% Urea-PAGE and visualized by imaging the gel on the Amersham Typhoon Laser Scanner (Cytiva Life Sciences, Marlborough, MA, USA). The modified polynucleotide, 5’DMl-MMAE-26mer-3’mal, was further purified and concentrated by ethanol precipitation and dissolved in IxPBS buffer.

[0150] To perform the nanobody-polynucleotide conjugation, the reduced anti-HER2 nanobody was mixed with 100 molar equivalents of L-ascorbic acid and stood at room temperature for 5 hours. 5’DMl-MMAE-26mer-3’mal were added to the pre-treated nanobody with 2.5 molar equivalents, and the reaction was performed with horizontal rotation at 70 rpm at room temperature overnight. The reaction products were analyzed via 15% SDS-PAGE, and the gel was first stained with lx SYBR gold nucleic acid gel stain solution and visualization by imaging on the Amersham Typhoon Laser Scanner. Secondly, the visualization of the nanobody position on the gel was applied with Coomassie blue staining. Finally, the triple labeling of the single-stranded polynucleotides with 5 ’-DM1, internal MMAE and 3 ’-DBCO-maleimide conjugated with anti-HER2 nanobody was obtained.

[0151] FIGs. 5 A and 5B show examples of labeling the therapeutic agent at the 5 ’-end and internal respectively of a polynucleotide via hSMUGl and an aldehyde-reactive probe (ARP), followed by the 3 ’-end labeling performed with enzymatic synthesis of 3'-AZ-dATP to the 3 '-end of the polynucleotide, and modified with maleimide by the azide-DBCO clickconjugation reaction between the incorporated 3'-AZ-dAMP and DBCO-modified maleimide functional moiety. FIG. 5A depicts the gel electrophoresis of the unlabeled and labeled polynucleotides visualized by staining with SYBR gold dye, and FIG. 5B illustrates the gel electrophoresis of the unlabeled and labeled nanobodies visualized by staining with Coomassie blue dye. Lane S shows the electrophoretic location of the target polynucleotide (27-mer ssDNA, SEQ ID NO: 3); lane 1 shows the electrophoretic location of the target polynucleotide with the therapeutic agent, (Aminooxy)acetamide-Val-Cit-PAB-MMAE as an internal labeling; lane 2 shows the electrophoretic location of the ternal labeled polynucleotide with the azide moiety at the 5 ’-end; lane 3 shows the electrophoretic location of the ternal labeled polynucleotide with the azide moiety at the 5 ’-end following with the therapeutic agent clicked; lane 4 shows the electrophoretic location of the ternal labeled polynucleotide with the azide moiety at the 5 ’-end following with the therapeutic agent clicked with the incorporated 3'-AZ-dAMP at the 3 ’-end; lane 5 shows the electrophoretic location of the ternal labeled polynucleotide with the azide moiety at the 5 ’-end following with the therapeutic agent clicked with the incorporated 3'-AZ-dAMP coupled with the maleimide functional moiety; lane 6 shows the electrophoretic location of the anti-HER2 nanobody; and lane 7 shows the electrophoretic location of the anti-HER2 nanobody conjugated with the 5’DM1-, internal MMAE and 3’maleimide-labeled polynucleotide. FIG. 5C shows an example overview of the triple labeling of 25-mer ss DNA with 5 ’-DM1, internal MMAE and 3’-DBC0-maleimide conjugated with anti-HER2 nanobody.

[0152] The components and individual experimental groups of the reaction are summarized in Table 7 below. The results of the reaction corresponding to each experimental group are shown in lanes 1 to 7 in FIGs. 5 A and 5B. In Table 7, seven experimental groups were designed, and the reaction components are shown.

[0153] Table 7

[0154] Example 6. Construction of single-stranded polynucleotide with one arm linker

[0155] In this example, a 26-mer DNA polynucleotide (5’- / U / CACCCCGCATTACGTTTGGTGGACC-3’) (SEQ ID NO: 1) containing an uracil residue at the 5'-end and a 46-mer DNA polynucleotide (5’- / U / CTCGGCCTGGCACAGGTCCGTTCAGTGCTGCGGCGACCACCGAGG-3’) (SEQ ID NO: 2) containing an uracil residue at the 5'-end were used as the target polynucleotides. The method used here to conjugate with polynucleotide involves the DBCO-Azide click reaction. To carry out the uracil excision and subsequent abasic site labeling, 100 pM of the target DNA polynucleotide was mixed with 10 pM of MluUDG, 4 mM of guanidine-Azide (or guanidine-DBCO) and 100 mM of p-phenylenediamine. In this example, Azide functional group was labeled in 46-mer DNA polynucleotide and DBCO functional group was labeled in 26-mer DNA polynucleotide. The reaction was initiated by the addition of MluUDG and guanidine-Azide (or guanidine-DBCO) and incubated at 37°C for 1 hour. To eliminate the unlabeled DNA polynucleotide, 30 pM of Kpn exonuclease and 30 pM of MutM were added and then incubated at 37°C for an additional hour. The modified polynucleotide was purified by using the FavorLight clean-up kit. The clean-up reaction products were further concentrated by ethanol precipitation.

[0156] To perform the polynucleotides conjugation, the 5’-Azide-45-mer and 5’-DBCO-25-mer polynucleotides were mixed at a 1 showsl molar ratio in IxPBS buffer. The Azide-DBCO click reaction was then incubated at 60°C for 6 hours. The reaction products were analyzed using 20% Urea-PAGE and visualized by imaging the gel with the Amersham Typhoon Laser Scanner (Cytiva Life Sciences, Marlborough, MA, USA). Finally, the branchproduct, consisting of the 5’-DBCO-25-mer polynucleotides conjugated with the 5’-Azide-45-mer polynucleotides, was obtained, with the DBCO group functioning as the conjugator of the arm linkers in the target polynucleotide. As shown in FIG. 6D, the branch product is a single-stranded polynucleotide with one arm linker at its 5 ’-end.

[0157] FIG. 6A and FIG. 6B show an example of constructing the polynucleotides conjugated with 5 ’-end to 5 ’-end. FIG. 6 A depicts the gel electrophoresis of the unlabeled and the labeled polynucleotides visualized by staining with SYBR gold dye. Lane S shows the electrophoretic location of the target polynucleotide (46-mer ssDNA, SEQ ID NO: 2); lane 1 shows the electrophoretic location of the target polynucleotide with the Azide functional moiety at the 5 ’-end; lane 2 shows the elimination of the unlabeled polynucleotide with Kpn exonuclease. FIG. 6B depicts the gel electrophoresis of the unlabeled and the labeled polynucleotides visualized by staining with SYBR gold dye. Lane S shows the electrophoretic location of the target polynucleotide (26-mer ssDNA, SEQ ID NO: 1); lane 1 shows the electrophoretic location of the target polynucleotide with an DBCO functional moiety at the 5 ’-end; and lane 2 shows the elimination of the unlabeled polynucleotide with Kpn exonuclease.

[0158] The components and individual experimental groups of the 5’-Azide-45-mer and 5’-DBCO-25-mer are summarized in Table 8 and Table 9 below. The results of the labeling reaction corresponding to each experimental group are shown in lanes 1 to 2 in FIG. 6A and FIG. 6B, respectively. In Table 8 and Table 9, four experimental groups were designed, and the reaction components are shown.

[0159] Table 8

[0160] Table 9

[0161] FIG. 6C depicts the gel electrophoresis of the target polynucleotides and the conjugating products visualized by staining with SYBR gold dye. Lane S shows the electrophoretic location of the marker U-U-60-mer (SEQ ID NO: 5); lane 1 shows the electrophoretic location of the 5’-DBCO-25-mer polynucleotide; lane 2 shows the electrophoretic location of the 5’-Azide-45-mer polynucleotide; and lane 3 shows the electrophoretic location of the conjugating product, e.g., branch product, which is the 5’-DBCO-25-mer polynucleotide conjugated with the 5’-Azide-45-mer polynucleotide. FIG. 6D shows an example overview of the branch product, which is the 5’-DBCO-25-mer polynucleotide conjugated with the 5’-Azide-45-mer polynucleotide.

[0162] The components and individual experimental groups of the conjugates are summarized in Table 10 below. The results of the labeling reaction corresponding to each experimental group are shown in lanes 1 to 3 in FIG. 6C. In Table 10, three experimental groups were designed, and the reaction components are shown.

[0163] Table 10

[0164] Example 7. Construction of single-stranded polynucleotide with two arm linkers

[0165] In this example, a 27-mer DNA polynucleotide (5’- / U / CACCCCGCAT / U / TACGTTTGGTGGACC-3’) (SEQ ID NO : 3) containing two uracilresidues, one at the 5'-end and one internally, was used as the target polynucleotide. To introduce two DBCO groups as a conjugator of the arm linkers in the target polynucleotide, both uracil excision and subsequent abasic site labeling were performed in the reaction mixture (10 pL) containing 100 pM of the ssDNA mixed with 10 pM of MluUDG, 4 rnM of guanidine-DBCO and 100 mM of p-phenylenediamine. The reaction was initiated by the addition of MluUDG and guanidine-DBCO and incubated at 37°C for 1 hour. To eliminate the unlabeled ssDNA, 5 pM of Pfu Endonuclease Q (hereinafter referred as “Pfu Endo Q”) was first added and then incubated at 55°C for 1 hour. The reaction was followed by the addition of 5 pM of Kpn exonuclease and 5 pM of MutM and then incubated at 37°C for an additional hour. To confirm that the polynucleotide was dual-labeled with guanidine-DBCO, a control reaction was carried out in parallel. In this reaction, 50 pM of the target polynucleotide was mixed with 1 unit of hSMUGl, 1 mM of guanidine-DBCO, and 20 mM of p-phenylenediamine, then incubated at 37°C for 1 hour. Similarly, to eliminate the unlabeled ssDNA, 4 pM of Pfu Endo Q was added and then incubated at 55°C for 1 hour, followed by the addition of 5 pM of Kpn exonuclease and incubated at 37°C for an additional hour. The intermediate and final reaction products were analyzed with 20% Urea-PAGE and visualized by imaging the gel on the Amersham Typhoon Laser Scanner (Cytiva Life Sciences, Marlborough, MA, USA). The modified polynucleotide was purified and concentrated by ethanol precipitation and dissolved in ddH2O (20 pL).

[0166] To produce the arm linkers, a 26-mer DNA polynucleotide (5’- / U / CACCCCGCATTACGTTTGGTGGACC-3’) (SEQ ID NO: 1) containing an uracil residue at the 5 ’-end of the polynucleotide was used as the side chain polynucleotide. To introduce two different fluorescent dyes to the 3 ’-end of the polynucleotide, the 3‘-AZ-dNTP with base-labeled fluorescent dye was first used to incorporate to the 3 ’-end of the polynucleotide via template-independent DNA synthesis. The nucleotide incorporation reaction was performed in the reaction mixture (50 pL) containing 50 pM of 26-mer DNA polynucleotide, 0.25 mM manganese chloride (MnCl2), and 25 pM of Tgo DNA polymerase. The reaction was initiated by the addition of 100 pM of Atto532-labeled 3’-AZ-dATP and then incubated at 37°C for 1 hour. Subsequently, 400 pM of DBCO-FAM was added to the reaction mixture and then incubated at 37°C for an additional hour. The modified polynucleotide was purified by ethanol precipitation and dissolved in ddH2O (50 pL). Subsequently, the azidomethyl group was introduced to the 5 ’-end of 27-mer-3’-Atto532 / FAM. The uracil excision and subsequent abasic site labeling were performed in the reaction mixture (80 pL) containing 100 pM of the ssDNA mixed with 20 pM of MluUDG, 2 mM of guanidine-Azide and 20 mM of p-phenylenediamine. The reaction was initiated by the addition of MluUDG andguanidine- Azide and incubated at 37°C for 1 hour. To eliminate the unlabeled polynucleotide, 15 pM of Kpn exonuclease, 15 pM of MutM, and 15 pM of hTREXl were added and then incubated at 37°C for 1 hour. The intermediate and final reaction products were analyzed with 20% Urea-PAGE and visualized by imaging the gel on the Amersham Typhoon Laser Scanner (Cytiva Life Sciences, Marlborough, MA, USA). The modified polynucleotide was purified by using the FavorLight clean-up kit. The clean-up reaction products were further concentrated by ethanol precipitation and dissolved in ddH2O (30pL).

[0167] To construct the 5’-DBCO-z>7ZDBCO-25mer polynucleotide (the main chain polynucleotide) with two arm linkers, 5’-Azide-26mer-3’Atto532 / FAM (the side chain polynucleotide), both of the polynucleotides were mixed at a 1 :2 molar ratio in the IxPBS buffer. The Azide-DBCO click reaction was incubated at 60°C for 6 hours. The reaction products were analyzed with 8% TBE-Native-PAGE and visualized by imaging the gel on the Amersham Typhoon Laser Scanner (Cytiva Life Sciences, Marlborough, MA, USA). Finally, the branch product, consisting of the 5’-DBCO-zwZDBCO-25mer polynucleotides conjugated with two arm linkers, 5’-Azide-26mer-3’Atto532 / FAM, was obtained, with the DBCO group functioning as the conjugator of the arm linkers in the target polynucleotide. As shown in FIG. 7D, the branch product is a single-stranded polynucleotide with two arm linkers.

[0168] FIGs. 7A-1 to FIG. 7C show an example of constructing the branch-formed polynucleotide containing the main chain polynucleotide with the two side chain polynucleotides. FIGs. 7A-1 and 7A-2 depict the gel electrophoresis of the unlabeled and labeled polynucleotides visualized by staining with SYBR gold dye. Lane S shows the electrophoretic location of the target polynucleotide (27-mer ssDNA, SEQ ID NO: 3); lane 1 shows the electrophoretic location of the target polynucleotide with a DBCO functional moiety as an internal labeling; lane 2 shows the electrophoretic location of the target polynucleotide with two DBCO functional moieties at the 5 ’-end and internally; lane 3 shows the elimination of the unlabeled polynucleotide by the internally cleaving with Pfu Endo Q; and lane 4 shows the elimination of the unlabeled polynucleotide by the internally cleaving with Pfu Endo Q, followed by the unlabeled nucleotides removal with Kpn exonuclease.

[0169] The components and individual experimental groups of the conjugates are summarized in Table 11 below. The results of the labeling reaction corresponding to each experimental group are shown in lanes 1 to 4 in FIGs. 7A-1 and 7A-2. In Table 11, four experimental groups were designed, and the reaction components are shown.

[0170] Table 11

[0171] FIG. 7B-1 depicts the gel electrophoresis of the unlabeled and labeled polynucleotides visualized by staining with SYBR gold dye, FIG. 7B-2 illustrates the electrophoretic location of nucleic acids labeled with the Atto532-fluorescent dye. Lane S shows the electrophoretic location of the target polynucleotide (26-mer ssDNA, SEQ ID NO: 1); lane 1 shows the electrophoretic location of the target polynucleotide with an incorporated Atto532-labeled 3’-AZ-dAMP at the 3 ’-end; lane 2 shows the electrophoretic location of the target polynucleotide with an incorporated Atto532-labeled 3’-AZ-dAMP coupled with a FAM-fluorescent dye at the 3 ’-end; lane 3 shows the electrophoretic location of the dual-fluorescent-labeled polynucleotide with an azidomethyl group at the 5 ’-end; and lane 4 shows the elimination of the unlabeled polynucleotide with 3’- to 5’- DNA exonuclease hTREXl and Kpn exonuclease.

[0172] The components and individual experimental groups of the conjugates are summarized in Table 12 below. The results of the labeling reaction corresponding to each experimental group are shown in lanes 1 to 4 in FIGs. 7B-1 and 7B-2. In Table 12, four experimental groups were designed, and the reaction components are shown.

[0173] Table 12

[0174] FIG. 7C depicts the gel electrophoresis of the main chain polynucleotide and the side chain polynucleotide visualized by staining with SYBR gold dye. Lane 1 shows the electrophoretic location of the control main chain polynucleotide (5’-U-zwZDBCO-26mer); lane 2 shows the electrophoretic location of the side chain polynucleotide (5’-Azide-26mer-3’Atto532 / FAM); lane 3 shows the electrophoretic location of the branch product, which is the control main chain polynucleotide coupled with the side chain polynucleotide as an internally labeling; lane 4 shows the electrophoretic location of the main chain polynucleotide (5’-DBCO- / / / / DBCO-25mer); and lane 5 shows the electrophoretic location of the branch products, which are the main chain polynucleotide coupled with one or two side chain polynucleotide, e.g., Branch product 1 and Branch product 2. FIG. 7D shows an example overview of the branch product, which consists of the 5’-DBCO- / / / / DBCO-25mer polynucleotides conjugated with two arm linkers (5’-Azide-26mer-3’Atto532 / FAM). The DBCO group functions as the conjugator of the arm linkers in the target polynucleotide. As shown in FIG. 7D, the symboldenotes Atto532-fluorescent dye, while the symboldenotes FAM-fluorescent dye.

[0175] The components and individual experimental groups of the conjugates are summarized in Table 13 below. The results of the labeling reaction corresponding to each experimental group are shown in lanes 1 to 6 in FIG. 7C. In Table 13, six experimental groups were designed, and the reaction components are shown.

[0176] Table 13

[0177] Example 8. Construction of polynucleotides with six modified groups in branch form

[0178] In this example, a 27-mer DNA polynucleotide (5’- / U / CACCCCGCAT / U / TACGTTTGGTGGACC-3’) (SEQ ID NO: 3) containing two uracil residues, one at the 5'-end and one internally, was used as the target polynucleotide. To introduce two different fluorescent dyes to the 3 ’-end of the polynucleotide, the 3‘-AZ-dNTP with a base-labeled fluorescent dye was first used to incorporate to the 3 ’-end of the polynucleotide via the template-independent DNA synthesis. The nucleotide incorporation reaction was performed in the reaction mixture (20 pL) containing 50 pM of the 27-mer DNA target polynucleotide, 0.25 mM manganese chloride (MnCl2), and 25 pM of Tgo DNA polymerase. The reaction was initiated by the addition of 100 pM of Atto532-labeled 3’-AZ-dATP and then incubated at 37°C for 1 hour. Subsequently, 400 pM of DBCO-Cy5 was added to the reaction mixture and then incubated at 37°C for an additional hour. To remove the unlabeled DNA strand, 20 pM of hTREXl was added in the labeling mixture at 37°C for 1 hour. The modified polynucleotide was purified by ethanol precipitation and dissolved in ddH2O (20 pL).

[0179] To introduce two DBCO groups as a conjugator of the arm linkers in the 28-mer-3’-Atto532 / Cy5 polynucleotide containing two uracil residues, one at the 5'-end and one internally, both of the uracil excision and the subsequent abasic site labeling were performed in the reaction mixture (10 pL) containing 10 pM of the ssDNA mixed with 5 pM of MluUDG, 1 mM of guanidine-DBCO and 20 mM of p-phenylenediamine. The reaction was initiated by the addition of MluUDG and guanidine-DBCO and incubated at 37°C for 1 hour. To confirm the polynucleotide was dual-labeled with guanidine-DBCO, a control reaction was carried out in parallel. In this reaction, 10 pM of the target polynucleotide was mixed with 1 unit of hSMUGl, 1 mM of guanidine-DBCO and 20 mM of p-phenylenediamine and then incubated at 37°C for 1 hour. The intermediate and final reaction products were analyzed with 20% Urea-PAGE and visualized by imaging the gel on the Amersham Typhoon Laser Scanner (Cytiva Life Sciences, Marlborough, MA, USA). The modified polynucleotide was purified and concentrated by ethanol precipitation and dissolved in ddH2O.

[0180] To construct the 5’-DBCO-7wtDBCO-26mer-3’Atto532 / Cy5 polynucleotide (the main chain polynucleotide) with two arm linkers, 5’-Azide-26mer-3’Atto532 / FAM (the side chainpolynucleotide), both of the polynucleotides were mixed at a molar ratio of 1 :2 in the l x PBS buffer. The side chain polynucleotide was prepared as in the previous description from example 2. The Azide-DBCO click reaction was incubated at 60°C for 16 hours. The reaction products were analyzed with 8% TBE-Native-PAGE and visualized by imaging the gel on the Amersham Typhoon Laser Scanner (Cytiva Life Sciences, Marlborough, MA, USA). Finally, the branch product, consisting of the 5’-DBCO- / «ZDBCO-26mer-3’Atto532 / Cy5 polynucleotides conjugated with two arm linkers, 5’-Azide-26mer-3’Atto532 / FAM, was obtained, with the DBCO group functioning as the conjugator of the arm linkers in the target polynucleotide.

[0181] FIGs. 8A-1 to 8B-2 show an example of performing six modifications on the branch-formed polynucleotides containing the main chain polynucleotide with the two side chain polynucleotides. FIG. 8A-1 depicts the gel electrophoresis of the unlabeled and labeled polynucleotides visualized by staining with SYBR gold dye. FIGs. 8 A-2 and 8 A- 3 illustrate the electrophoretic location of the polynucleotides labeled with the Atto532- and Cy5-fluorescnet dye, respectively. Lane S shows the electrophoretic location of the target polynucleotide (27-mer ssDNA, SEQ ID NO: 3); lane 1 shows the electrophoretic location of the target polynucleotide with the incorporated Atto532-labeled 3’-AZ-dAMP at the 3 ’-end; lane 2 shows the electrophoretic location of the target polynucleotide with the incorporated Atto532-labeled 3’-AZ-dAMP coupled with a Cy 5 -fluorescent dye at the 3 ’-end; lane 3 shows the elimination of the unlabeled polynucleotide with 3’- to 5’- DNA exonuclease hTREXl; lane 4 shows the reaction clean-up via ethanol precipitation; lane 5 shows the electrophoretic location of the dual-fluorescent-labeled polynucleotide with a DBCO functional moiety as an internal labeling; and lane 6 shows the electrophoretic location of the dual-fluorescent-labeled polynucleotide with two DBCO functional moieties at the 5 ’-end and internally.

[0182] The components and individual experimental groups of the conjugates are summarized in Table 14 below. The results of the labeling reaction corresponding to each experimental group are shown in lanes 1 to 6 in FIGs. 8A-1, 8 A-2 and 8A-3. In Table 14, six experimental groups were designed, and the reaction components are shown.

[0183] Table 14

[0184] FIGs. 8B-1 and 8B-2 depict the gel electrophoresis of the labeled main chain polynucleotide and the labeled side chain polynucleotides with Cy5- and Atto532-fluorescent dye. Lane 1 shows the electrophoretic location of the main chain polynucleotide (5’-DBCO- / / / / DBCO-26mer-3’ Atto532-Cy5 ssDNA); lane 2 shows the electrophoretic location of the side chain polynucleotide (5’-gAzide-26mer-3’ Atto532-FAM ssDNA); and lane 3 shows the electrophoretic location of the branch products, e.g., Branch product 1 and Branch product 2, which are the main chain polynucleotide coupled with one or two side chain polynucleotides.

[0185] The components and individual experimental groups of the conjugates are summarized in Table 15 below. The results of the labeling reaction corresponding to each experimental group are shown in lanes 1 to 3 in FIGs. 8B-1 and 8B-2. In Table 15, three experimental groups were designed, and the reaction components are shown.

[0186] Table 15

[0187] Example 9. Construction of polynucleotides with two therapeutic agents modified in the branch form

[0188] In this example, a 27-mer DNA polynucleotide (5’- / U / CACCCCGCAT / U / TACGTTTGGTGGACC-3’) (SEQ ID NO: 3) containing two uracil residues, one at the 5'-end and one internally, was used as the target polynucleotide. To carry out the uracil excision, 100 pM of the target DNA polynucleotide was mixed with 10 pM of MluUDG in the reaction mixture (20 pL). To introduce a maleimide group as a conjugator of the biomolecule to the 3 ’-end of the polynucleotide containing two abasic sites, the3‘-AZ-dNTP was first used as a functional moiety to incorporate to the 3 ’-end of polynucleotide via the template-independent DNA synthesis. The nucleotide incorporation reaction was performed in the reaction mixture (40 pL) containing 50 pM of the target polynucleotide, 0.25 mM manganese chloride (MnCl2), and 25 pM of Tgo DNA polymerase. The reaction was initiated by the addition of 200 pM of 3’-AZ-dATP and then incubated at 37°C for 1 hour. The modified polynucleotide was purified by using the FavorLight clean-up kit. The clean-up reaction products were further concentrated by ethanol precipitation and dissolved in ddH2O (10 pL). Subsequently, 500 pM of DBCO-maleimide was added to the reaction mixture and the Azide-DBCO click reaction was incubated at 37°C for 1 hour. The modified polynucleotide was purified by ethanol precipitation and dissolved in ddH2O. To introduce two DBCO groups as a conjugator of the arm linkers in the target polynucleotide containing two abasic sites, 4 mM of guanidine-DBCO was added and then incubated at 37°C for 1 hour. The modified polynucleotide was purified and concentrated by ethanol precipitation and dissolved in ddH2O.

[0189] To confirm the polynucleotide was dual-labeled with guanidine-DBCO, a control reaction was carried out in parallel. In this reaction, 50 pM of the ssDNA was mixed with 1 unit of hSMUGl, 1 mM of guanidine-DBCO and 20 mM of p-phenylenediamine and then incubated at 37°C for 1 hour. Similarly, the 3’-O-azidomethyl-deoxynucleoside triphosphate (3‘-AZ-dNTP) was used as a functional moiety to incorporate to the 3 ’-end of the polynucleotide via the template-independent DNA synthesis. The nucleotide incorporation reaction was performed in the reaction mixture (20 pL) containing 25 pM of the 5’U-int / abasic / -26mer target polynucleotide, 0.25 mM manganese chloride (MnCl2), and 12.5 pM of Tgo DNA polymerase. The reaction was initiated by the addition of 100 pM of 3’-AZ-dATP and then incubated at 37°C for 1 hour. The modified polynucleotide was purified by using the FavorLight clean-up kit.

[0190] The clean-up reaction products were further concentrated by ethanol precipitation and dissolved in ddH2O (20 pL). Subsequently, 80 pM of DBCO-maleimide was added to the reaction mixture and the Azide-DBCO click reaction was incubated at 37°C for 1 hour. The modified polynucleotide was purified by ethanol precipitation and dissolved in ddH2O. To introduce DBCO group in the target polynucleotide containing one internal abasic site, 1 mM of guanidine-DBCO was added and then incubated at 37°C for 1 hour. The intermediate and final reaction products were analyzed with 20% Urea-PAGE and visualized by imaging the gel on the Amersham Typhoon Laser Scanner (Cytiva Life Sciences, Marlborough, MA, USA).

[0191] To perform the arm linkers, a 26-mer DNA polynucleotide (5’- / U / CACCCCGCATTACGTTTGGTGGACC-3’) (SEQ ID NO: 1) containing an uracilresidue at the 5 ’-end was used as the side chain polynucleotide. To carry out the uracil excision and subsequent abasic site labeling, 100 pM of the target polynucleotide was mixed with 20 pM of MluUDG, 4 mM of (Aminooxy)acetamide-Val-Cit-PAB-MMAE, and 100 mM of p-phenylenediamine. The reaction was initiated by the addition of MluUDG and (Aminooxy)acetamide-Val-Cit-PAB-MMAE and incubated at 37°C for 1 hour. To eliminate the unlabeled polynucleotide, 20 pM of Kpn exonuclease and 20 pM of MutM were added and then incubated at 37°C for an additional hour. The modified polynucleotide was purified by ethanol precipitation and dissolved in ddH2O (50 pL). Subsequently, the azidomethyl group was introduced to the 3’-end of 5’-MMAE-25-mer. the 3‘-AZ-dNTP was used to incorporate to the 3 ’-end of polynucleotide via the template-independent DNA synthesis. The nucleotide incorporation reaction was performed in the reaction mixture (90 pL) containing 100 pM of 5 ’-MMAE -25-mer DNA target polynucleotide, 0.25 mM manganese chloride (MnCl2), and 50 pM of Tgo DNA polymerase. The reaction was initiated by the addition of 100 pM of 3’-AZ-dATP and then incubated at 37°C for 1 hour. The modified polynucleotide was purified by using the FavorLight clean-up kit. The clean-up reaction products were further concentrated by ethanol precipitation and dissolved in ddH2O (30 pL).

[0192] To further perform the nanobody-polynucleotide conjugation, the anti-HER2 nanobody which prepared and purified in-house was used. Firstly, 5’DBCO-7wZDBCO-26mer-3’mal was added to the nanobody with 1 molar equivalents, and the reaction was performed with horizontal rotation at 70 rpm at room temperature for 24 hours. Secondly, the arm strand 5’MMAE-26mer-3’Azide was added to the half of the reaction products with 2 molar equivalents and performed with horizontal rotation at 70 rpm at room temperature for an additional 24 hours. The reaction products were analyzed via 15% SDS-PAGE, and the gel was first stained with lx SYBR gold nucleic acid gel stain solution and visualized by imaging on the Amersham Typhoon Laser Scanner. Secondly, the visualization of the nanobody position on the gel was applied with Coomassie blue staining.

[0193] To construct the 5’-DBCO-7wZDBCO-26-mer-3’maleimide polynucleotide (the main chain polynucleotide) with two arm linkers, 5’-MMAE-26-mer-3’Azide (the side chain polynucleotide), both of the polynucleotides were mixed at a molar ratio of 1 :2 in the l x PBS buffer. The Azide-DBCO click reaction was incubated at 60°C for 16 hours. The reaction products were analyzed with 8% TBE-Native-PAGE and visualized by imaging the gel on the Amersham Typhoon Laser Scanner (Cytiva Life Sciences, Marlborough, MA, USA).

[0194] FIGs. 9A-1 to 9C show an example of constructing of the polynucleotide with two therapeutic agents modified in branch form. FIGs. 9A-1 and 9A-2 depict the gel electrophoresis of the labeled main chain polynucleotide. Lane S shows the electrophoretic location of thetarget polynucleotide (27-mer ssDNA, SEQ ID NO: 3); lane 1 shows the electrophoretic location of the target polynucleotide performed the uracil excision at the 5 ’-end and internally; lane 2 shows the electrophoretic location of the dual uracil excised polynucleotide with the incorporated 3’-AZ-dAMP at the 3 ’-end; lane 3 shows the electrophoretic location of the dual uracil excised polynucleotide with the incorporated 3’-AZ-AMP coupled with a DBCO-maleimide at the 3 ’-end; and lane 4 shows the electrophoretic location of the target polynucleotide with the incorporated 3’-AZ-AMP coupled with a DBCO-maleimide at the 3 ’-end, followed by with dual DBCO moieties via the abasic sites at 5 ’-end and internally.

[0195] The components and individual experimental groups of the conjugates are summarized in Table 16 below. The results of the labeling reaction corresponding to each experimental group are shown in lanes 1 to 4 in FIGs. 9A-1 and 9A-2. In Table 16, four experimental groups were designed, and the reaction components are shown.

[0196] Table 16

[0197] FIG. 9B depicts the gel electrophoresis of the labeled main chain polynucleotide. Lane S shows the electrophoretic location of the target polynucleotide (27-mer ssDNA, SEQ ID NO: 3); lane 1 shows the electrophoretic location of the target polynucleotide performed the internal uracil excision; lane 2 shows the electrophoretic location of the internal uracil excised polynucleotide with the incorporated 3’-AZ-dAMP at the 3 ’-end; lane 3 shows the electrophoretic location of the internally uracil excised polynucleotide with the incorporated 3’-AZ-AMP coupled with a DBCO-maleimide at the 3 ’-end; lane 4 shows the electrophoretic location of the target polynucleotide with the incorporated 3’-AZ-AMP coupled with the DBCO-maleimide at the 3 ’-end, following with the DBCO moiety via the internal abasic site; and lane 5 shows the electrophoretic location of the main chain polynucleotide(5’-DBCO-intDBCO-26mer-3’mal).

[0198] The components and individual experimental groups of the conjugates are summarized in Table 17 below. The results of the labeling reaction corresponding to each experimental group are shown in lanes 1 to 5 in FIG. 9B. In Table 17, five experimental groups were designed, and the reaction components are shown.

[0199] Table 17

[0200] FIG. 9C depicts the gel electrophoresis of the labeled main chain polynucleotide. Lane S shows the electrophoretic location of the target polynucleotide (26-mer ssDNA, SEQ ID NO: 1); lane 1 shows the electrophoretic location of the target polynucleotide with the therapeutic agent, (Aminooxy)acetamide-Val-Cit-PAB-MMAE at the 5’-end; lane 2 shows the elimination of the unlabeled polynucleotide with Kpn exonuclease; and lane 3 shows the electrophoretic location of the 5 ’-labeled polynucleotide with the incorporated 3'-AZ-dAMP at the 3 '-end.

[0201] The components and individual experimental groups of the conjugates are summarized in Table 18 below. The results of the labeling reaction corresponding to each experimental group are shown in lanes 1 to 3 in FIG. 9C. In Table 18, three experimental groups were designed, and the reaction components are shown.

[0202] Table 18

[0203] FIG. 9D illustrates the branch construction and the nanobody-polynucleotide conjugation on 15% SDS- polyacrylamide gel. Lane 1 shows the electrophoretic location of the anti-HER2 nanobody; lane 2 shows the electrophoretic location of the main chain polynucleotide (5’DBCO-z«ZDBCO-26mer-3’mal), which ran out of the gel; lane 3 shows the electrophoretic location of the anti-HER2 nanobody conjugated with the main chain polynucleotide; lane 4 shows the electrophoretic location of the side chain polynucleotide (5’MMAE-26mer-3’Azide), which ran out of the gel; and lane 5 shows the electrophoretic location of the anti-HER2 nanobody conjugated with the main chain polynucleotide and then preformed the branch construction with the side chain polynucleotide, e.g., Branch product 1 and Branch product 2.

[0204] The components and individual experimental groups of the conjugates are summarized in Table 19 below. The results of the labeling reaction corresponding to each experimental group are shown in lanes 1 to 5 in FIG. 9D. In Table 19, five experimental groups were designed, and the reaction components are shown.

[0205] Table 19

[0206] Double Strand Polynucleotides

[0207] Example 10. Construction of polynucleotide with six modified groups in duplex form

[0208] The top strand polynucleotide used here is a 27-mer DNA polynucleotide (5’- / U / CACCCCGCAT / U / TACGTTTGGTGGACC-3’) (SEQ ID NO: 3) containing two uracil residues, one at the 5'-end and one internally. To carry out the uracil excision and the subsequent abasic site labeling, 100 pM of the ssDNA was mixed with 10 pM of MluUDG, 4 mM of guanidine-Biotin and 100 mM of p-phenylenediamine. The reaction (50 pL) was initiated by the addition of MluUDG and guanidine-Biotin and incubated at 37°C for 1 hour. To eliminate the unlabeled ssDNA, 15 pM of Pfu Endo Q was first added and then incubated at 55°C for 1 hour. The reaction was followed by the addition of 15 pM of Kpn exonuclease and 15 pM of MutM and then incubated at 37°C for an additional hour. The modified polynucleotide was purified by ethanol precipitation and dissolved in ddH2O (25 pL). To confirm the polynucleotide was dual-labeled with guanidine-Biotin, a control reaction was carried out in parallel. In this reaction, 25 pM of 27-mer DNA polynucleotide was mixed with 1 unit of hSMUGl, 1 mM of guanidine-Biotin, and 20 mM of p-phenylenediamine. Similarly, to eliminate the unlabeled ssDNA, 4 pM of Pfu Endo Q was first added and then incubated at 55°C for 1 hour, followed by the addition of 4 pM of Kpn exonuclease and then incubated at 37°C for an additional hour.

[0209] To introduce two different fluorescent dyes to the 3 ’-end of polynucleotide, the 3‘-AZ-dNTP with base-labeled fluorescent dye was first used to incorporate to the 3 ’-end of polynucleotide via template-independent DNA synthesis. The nucleotide incorporation reaction was performed in the reaction mixture (40 pL) containing 50 pM of 5’-Biotin-z>?tBiotin-25-mer target DNA polynucleotide, 0.25 mM of manganese chloride (MnCl2), and 25 pM of Tgo DNA polymerase. The reaction was initiated by the addition of 100 pM of Atto532-labeled 3’-AZ-dATP and then incubated at 37°C for 1 hour. Subsequently, 400 pM of DBCO-FAM was added to the reaction mixture and then incubated at 37°C for an additional hour. To remove the unlabeled DNA polynucleotide, 13 pM of hTREXl was added in the labeling mixture at 37°C for 1 hour. The intermediate and final reaction products were analyzed with 20% Urea-PAGE and visualized by imaging the gel on the Amersham Typhoon Laser Scanner (Cytiva Life Sciences, Marlborough, MA, USA). The modified polynucleotide was purified and concentrated by ethanol precipitation and dissolved in ddH2O.

[0210] The bottom strand polynucleotide, a 27-mer DNA polynucleotide (5’- GGGTCCACCAAACGTAATGCGGGGTGA-3’) (SEQ ID NO: 4) was used to perform the dual modifications at the 3 ’-end of the polynucleotide. To introduce a fluorescent dye and a maleimide group to the 3 ’-end of the polynucleotide, the 3’-O-azidomethyl-deoxynucleoside triphosphate (3‘-AZ-dNTP) with base-labeled Dye681 fluorescent dye was incorporated to the 3 ’ -end of the polynucleotide via the template-independent DNA synthesis by using the selective B-family DNA polymerase or its variants. The nucleotide incorporation reaction was performedin the reaction mixture (50 pL) containing 50 pM of 27-mer bottom strand polynucleotide, 0.25 mM of manganese chloride (MnCl2), and 25 pM of Tgo DNA polymerase. The reaction was initiated by the addition of 100 pM of Dye681 -labeled 3’-AZ-dCTP and then incubated at 37°C for 1 hour. The modified polynucleotide was purified by using the FavorLight clean-up kit. The clean-up reaction products were further concentrated by ethanol precipitation and dissolved in ddH2O (20 pL). Subsequently, 500 pM of DBCO-maleimide was added and reacted with the polynucleotide carrying an azide group. The azide-DBCO ligation reaction was normally performed at 37°C for 1 hour. The intermediate and final reaction products were analyzed with 20% Urea-PAGE and visualized by imaging the gel on the Amersham Typhoon Laser Scanner (Cytiva Life Sciences, Marlborough, MA, USA). The modified polynucleotide was further purified and concentrated by ethanol precipitation and dissolved in ddH2O.

[0211] To hybrid the 5’-Biotin-zzztBiotin-26-mer-Atto532 / FAM (the top strand polynucleotide) and the 28-mer-3’Dye681-mal (the bottom strand polynucleotide), the two polynucleotides were mixed at a molar ratio of 1 : 1 in the U Tris-EDTA (TE) buffer consisting of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 100 mM NaCl. The DNA annealing reaction was performed in a thermal cycler by heating up the DNA mixture to 95°C for 3 minutes, followed by gradually cooling down (e.g., 1 minute for every 5°C) to 4°C. The resulting duplex DNA was analyzed with 12% TBE-Native-PAGE and visualized by imaging the gel on the Amersham Typhoon Laser Scanner (Cytiva Life Sciences, Marlborough, MA, USA).

[0212] To further perform the nanobody-polynucleotide conjugation, the anti-HER2 nanobody which prepared and purified in-house was mixed with 100 molar equivalents of L-ascorbic acid and stood at room temperature for 5 hours. The duplex modified DNA polynucleotide was added to the pre-treated nanobody with 2.5 molar equivalents, and the reaction was performed with horizontal rotation at 70 rpm at room temperature overnight. The reaction products were analyzed via 15% SDS-PAGE, and the gel was first visualized by imaging on the Amersham Typhoon Laser Scanner. Secondly, the visualization of the nanobody position on the gel was applied with Coomassie blue staining. Finally, the double-stranded polynucleotide, formed by hybridizing the 5’-Biotin-zzztBiotin-26-mer-Atto532 / FAM (the top-strand polynucleotide) with the 28-mer-3’Dye681-mal (the bottom-strand polynucleotide), was obtained.

[0213] FIGs. 10A to FIG. 10E-4 show an example of performing six modifications on the duplex-formed polynucleotides containing two Biotin moieties, three different fluorescent dyes, and a biomolecule. FIG. 10A depicts the gel electrophoresis of the unlabeled and labeled polynucleotides visualized by staining with SYBR gold dye. Lane S shows the electrophoretic location of the target polynucleotide (27-mer ssDNA, SEQ ID NO: 3); lane 1 shows the electrophoretic location of the target polynucleotide with a Biotin moiety as an internallabeling; lane 2 shows the elimination of the unlabeled polynucleotide by cleaving the internal uracil of the polynucleotide via Pfu Endo Q; lane 3 shows the elimination of the unlabeled polynucleotide by the internal cleaving with Pfu Endo Q, followed by the unlabeled nucleotides removal with Kpn exonuclease; lane 4 shows the internal labeled polynucleotide with another Biotin moiety at the 5 ’-end; lane 5 shows the second elimination of the unlabeled polynucleotide by the internal cleaving with Pfu Endo Q; and lane 6 shows the second elimination of the unlabeled polynucleotide by the internal cleaving with Pf Endo Q, followed by the unlabeled nucleotides removal with Kpn exonuclease.

[0214] The components and individual experimental groups of the conjugates are summarized in Table 20 below. The results of the labeling reaction corresponding to each experimental group are shown in lanes 1 to 6 in FIG. 10 A. In Table 20, six experimental groups were designed, and the reaction components are shown.

[0215] Table 20

[0216] FIG. 10B-1 depicts the gel electrophoresis of the unlabeled and labeled polynucleotides visualized by staining with SYBR gold dye. FIG. 10B-2 illustrates the electrophoretic location of the polynucleotide labeled with the Atto532-fluorescnet dye. Lane S shows the electrophoretic location of the target polynucleotide (5’-Biotin-z>?tBiotin-25-mer ssDNA); lane 1 shows the electrophoretic location of the Biotin-labeled polynucleotide with the incorporated Atto532-labeled 3’-AZ-dAMP at the 3 ’-end; lane 2 shows the electrophoretic location of the Biotin-labeled polynucleotide with the incorporated Atto532-labeled 3’-AZ-dAMP coupled with the FAM fluorescent dye at the 3 ’-end; and lane 3 shows the elimination of the unlabeled polynucleotide with 3’ - to 5’- DNA exonuclease hTREXl.

[0217] The components and individual experimental groups of the conjugates are summarized in Table 21 below. The results of the labeling reaction corresponding to each experimental group are shown in lanes 1 to 3 in FIGs. 10B-1 and 10B-2. In Table 21, three experimental groups were designed, and the reaction components are shown.

[0218] Table 21

[0219] FIG. 10C-1 depicts the gel electrophoresis of the unlabeled and labeled polynucleotides visualized by staining with SYBR gold dye. FIG. 10C-2 illustrates the electrophoretic location of nucleic acids labeled with the DY681-fluorescnet dye. Lane S shows the electrophoretic location of the target polynucleotide (27-mer ssDNA, SEQ ID NO: 3); lane 1 shows the electrophoretic location of the target polynucleotide with an incorporated Dye681 -labeled 3’-AZ-dCMP at the 3 ’-end; and lane 2 shows the electrophoretic location of the target polynucleotide with the incorporated Dye681 -labeled 3’-AZ-dCMP coupled with the maleimide functional moiety.

[0220] The components and individual experimental groups of the conjugates are summarized in Table 22 below. The results of the labeling reaction corresponding to each experimental group are shown in lanes 1 to 2 in FIGs. 10C-1 and 10C-2. In Table 22, two experimental groups were designed, and the reaction components are shown.

[0221] Table 22

[0222] FIGs. 10D-1, 10D-2 and 10D-3 depict the gel electrophoresis of the top strand polynucleotide and the bottom stand polynucleotides under the imaging of the FAM-, Atto532-, and Dye681 -fluroescent dye, respectively. Lane 1 shows the electrophoretic location of the top strand polynucleotide (5 ’-Biotin-z>?tBiotin-26-mer-Atto532-FAM DNA polynucleotide); lane 2 shows the electrophoretic location of the bottom strand polynucleotide (28-mer-DY681-mal DNA polynucleotide); and lane 3 shows the electrophoretic location of the hybridized DNA duplex with the top strand and the bottom strand polynucleotides.

[0223] The components and individual experimental groups of the conjugates are summarized in Table 23 below. The results of the labeling reaction corresponding to each experimental group are shown in lanes 1 to 3 in FIGs. 10D-1, 10D-2 and 10D-3. In Table 23, three experimental groups were designed, and the reaction components are shown.

[0224] Table 23

[0225] FIGs. 10E-1, 10E-2, 10E-3 and 10E-4 depict the gel electrophoresis of the nanobody-polynucleotide conjugation. The unlabeled and labeled nanobody are visualized by staining with Coomassie blue dye and illustrate at the far-left part. The remaining parts illustrate the images of the FAM-, Atto532-, and Dye681 -fluorescent dye on the labeled polynucleotides in 4E-1, 4E-2, 4E-3 and 4E-4, respectively. Lane 1 shows the electrophoretic location of the anti-HER2 nanobody; lane 2 shows the electrophoretic location of the six-compounds-labeled hybridized DNA duplex; lane 3 shows the electrophoretic location of the anti-HER2 nanobody conjugated with the hybridized DNA duplex; lane 4 shows the electrophoretic location of the anti-HER2 nanobody conjugated with the bottom strand polynucleotide (28-mer-DY681-mal DNA polynucleotide); and lane 5 shows the electrophoretic location of the bottom strand polynucleotide; and lane 6 shows theelectrophoretic location of the 60-mer DNA polynucleotide as a standard marker. FIG. 10F shows an example overview of the top-strand polynucleotide (the 5’-Biotin-zwtBiotin-26-mer-Atto532 / FAM). As shown in FIG. 10F, the symboldenotes Atto532-fluorescent dye and the symboldenotes FAM-fluorescent dye. FIG. 10G shows an example overview of the bottom-strand polynucleotide (the 28-mer-3’Dye681-mal). As shown in FIG. 10G, the symboldenotes Dye681 -fluorescent dye.

[0226] The components and individual experimental groups of the conjugates are summarized in Table 24 below. The results of the labeling reaction corresponding to each experimental group are shown in lanes 1 to 5 in FIGs. 10E-1, 10E-2, 10E-3 and 10E-4. In Table 24, five experimental groups were designed, and the reaction components are shown.

[0227] Table 24

[0228] The present disclosure has been described with embodiments thereof, and it is understood that various modifications, without departing from the scope of the present disclosure, are in accordance with the embodiments of the present disclosure. Hence, the embodiments described are intended to cover the modifications within the scope of the present disclosure, rather than to limit the present disclosure. The scope of the claims therefore should be accorded the broadest interpretation so as to encompass all such modifications.

Claims

CLAIMSWhat is claimed is:

1. A scaffold polynucleotide, comprising: a linear polynucleotide or a branched polynucleotide; and a conjugator bound to the linear polynucleotide or the branched polynucleotide, wherein the branched polynucleotide has an arm linker emanating therefrom via the conjugator.

2. The scaffold polynucleotide of claim 1, wherein the linear polynucleotide or the branched polynucleotide is a single-stranded form or a double-stranded form.

3. The scaffold polynucleotide of claim 1, having a plurality of the arm linkers.

4. The scaffold polynucleotide of claim 1, wherein the arm linker emanates from a nucleobase, a nucleosugar, or a backbone of the branched polynucleotide via the conjugator.

5. The scaffold polynucleotide of claim 1, wherein the conjugator is selected from the group consisting of a biotin moiety, a thiol group, a succinimide moiety, a maleimide moiety, an amine moiety, an acylhydrazide moiety, a hydrazine moiety, a tyrosine moiety, a hydrazone moiety, an azide moiety, a dibenzocyclooctyne group, a bicyclononyne group, an aminooxy moiety, a hydroxylamine moiety, a terminal alkyne moiety, a strained terminal alkyne moiety, a phosphine moiety, polyethylene glycol chains, and any combinations thereof.

6. The scaffold polynucleotide of claim 1, further comprising a biomolecule conjugated to the scaffold polynucleotide via the arm linker or the conjugator.

7. The scaffold polynucleotide of claim 6, wherein the biomolecule is selected from the group consisting of a humanized antibody or antigen binding fragment thereof, a chimeric antibody or antigen binding fragment thereof, a monoclonal antibody or antigen binding fragment thereof, a monovalent Fab’, a divalent Fab2, a single-chain variable fragment, a diabody, a minibody, a nanobody, a single-domain antibody, a camelid antibody, antigen binding fragment thereof, a peptide, and any combinations thereof.

8. The scaffold polynucleotide of claim 1, further comprising an agent conjugated to the scaffold polynucleotide via the arm linker or the conjugator.

9. The scaffold polynucleotide of claim 8, wherein the agent is selected from the group consisting of a therapeutic agent, a diagnostic agent, and a combination thereof.

10. The scaffold polynucleotide of claim 9, wherein the therapeutic agent is selected from the group consisting of cytotoxic agents, protein toxins, proteins, enzymes, radionuclides, and any combinations thereof.

11. The scaffold polynucleotide of claim 9, wherein the diagnostic agent is selected from the group consisting of a fluorescent moiety, a luminescent moiety, a radioactive moiety, and any combinations thereof.

12. A conjugate, comprising: the scaffold polynucleotide of claim 1 ; a biomolecule conjugated to the scaffold polynucleotide of claim 1 via the arm linker or the conjugator; and an agent conjugated to the scaffold polynucleotide of claim 1 via the arm linker or the conjugator.

13. The conjugate of claim 12, wherein the linear polynucleotide or the branched polynucleotide is a single-stranded form or a double-stranded form.

14. The conjugate of claim 12, wherein the arm linker emanates from a nucleobase, a nucleosugar, or a backbone of the branched polynucleotide.

15. The conjugate of claim 12, wherein the conjugator is selected from the group consisting of a biotin moiety, a thiol group, a succinimide moiety, a maleimide moiety, an amine moiety, an acylhydrazide moiety, a hydrazine moiety, a tyrosine moiety, a hydrazone moiety, an azide moiety, a dibenzocyclooctyne group, a bicyclononyne group, an aminooxy moiety, a hydroxylamine moiety, a terminal alkyne moiety, a strained terminal alkyne moiety, a phosphine moiety, polyethylene glycol chains, and any combinations thereof.

16. The conjugate of claim 12, wherein the biomolecule is selected from the group consisting of a humanized antibody or antigen binding fragment thereof, a chimeric antibody or antigen binding fragment thereof, a monoclonal antibody or antigen binding fragment thereof, a monovalent Fab’, a divalent Fab2, a single-chain variable fragment, a diabody, a minibody, a nanobody, a single-domain antibody, a camelid antibody, antigen binding fragment thereof, a peptide, and any combinations thereof.

17. The conjugate of claim 12, wherein the agent is selected from the group consisting of a therapeutic agent, a diagnostic agent, and a combination thereof.

18. The conjugate of claim 17, wherein the therapeutic agent is selected from the group consisting of cytotoxic agents, protein toxins, proteins, enzymes, radionuclides, and any combinations thereof.

19. The conjugate of claim 17, wherein the diagnostic agent is selected from the group consisting of a fluorescent moiety, a luminescent moiety, a radioactive moiety, and any combinations thereof.

20. A method of preparing the scaffold polynucleotide of any one of claims 1 to 11, comprising: providing a target nucleic acid having a plurality of modified nucleobases at 5 ’-end of the target nucleic acid or within the target nucleic acid; providing a mono-functional DNAglycosylase to react with the target nucleic acid to form an intermediate nucleic acid having a plurality of abasic sites at the 5 ’-end of the target nucleic acid or within the target nucleic acid; and incorporating the conjugator to the intermediate nucleic acid to form the linear polynucleotide, provided that the scaffold polynucleotide comprises the branched polynucleotide, the method further comprises conjugating the arm linker to the linear polynucleotide via the conjugator to form the branched polynucleotide.

21. The method of claim 20, wherein the modified nucleobase is selected from the group consisting of hypoxanthine, cytosine, 3 -alkyladenine, 8-oxoguanine, uracil, 5 -hydroxyuracil, 5 -hydroxymethyluracil, 5 -formyluracil, 5 -fluorouracil, dihydroxyuracil, 5-formylcytosine, 5-carboxylcytosine, 3 -methyladenine, 3-methylguanine,7-methyladenine, 7-methylguanine, N6-methyladenine, 8-oxo-7,8-dihydroguanine, 5-hydroxylcytosine, ethenocytosine, ethenoadenine, thymine glycol, cytosine glycol, 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine, a formamidopyrimidine derivative of adenine, and a formamidopyrimidine derivative of guanine.

22. The method of claim 20, wherein the mono-functional DNA glycosylase is selected from the group consisting of uracil-DNA glycosylase, alkyladenine DNA glycosylase, single-strand-selective mono functional uracil-DNA glycosylase 1, methyl-binding domain glycosylase 4, thymine DNA glycosylase, MutY homolog DNA glycosylase, alkylpurine glycosylase C, alkylpurine glycosylase D, 8-oxo-guanine glycosylase 1 without an abasic site lyase activity, endonuclease Ill-like glycosylase 1 without the abasic site lyase activity, endonuclease Vlll-like glycosylase 1 without the abasic site lyase activity, endonuclease Vlll-like glycosylase 2 without the abasic site lyase activity, endonuclease Vlll-like glycosylase 3 without the abasic site lyase activity, enzymatically active fragments thereof, and any combinations thereof.

23. The method of claim 20, wherein the conjugator is incorporated to the target nucleic acid by a template-independent enzymatic nucleic acid synthesis.

24. The method of claim 23, wherein the template-independent enzymatic nucleic acid synthesis comprises employing a DNA polymerase, an RNA polymerase, or a functionally equivalent enzyme thereof.

25. The method of claim 24, wherein the DNA polymerase is selected from the group consisting of an A family DNA polymerase, a B family DNA polymerase, an X family DNA polymerase, and any combinations thereof.

26. The method of claim 25, wherein the DNA polymerase is a B family DNA polymerase or a variant thereof.

27. The method of claim 26, wherein the B family DNA polymerase is a Thermococcaceae DNA polymerase.

28. The method of claim 26, wherein the B family DNA polymerase is a Thermococcus DNA polymerase or a Pyrococcus DNA polymerase.

29. The method of claim 26, wherein the B family DNA polymerase is selected from the group consisting of a B family DNA polymerase of Thermococcus kodctkarensis. a B family DNA polymerase of Pyrococcus furiosus, a B family DNA polymerase of Thermococcus litoralis, a B family DNA polymerase of Thermococcus sp. 9°N, and a B family DNA polymerase of Thermococcus gorgonarius.

30. The method of claim 20, further comprises reacting a biomolecule or an agent with the conjugator via a bioorthogonal reaction.

31. The method of claim 30, wherein the bioorthogonal reaction is a click conjugation, an oxime / hydrazone formation, a Staudinger ligation, a tetrazine ligation, or a quadricyclane ligation.

32. The method of claim 31, wherein the click conjugation is selected from the group consisting of copper-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, isocyanide-based click reaction, and inverse electron demand Diels-Alder reaction.

33. The method of claim 20, further comprising: providing 5’ to 3’ exonuclease to remove an unreactive target nucleic acid.

34. A method of preparing a conjugate of any one of claims 12 to 19, comprising: conjugating a biomolecule and an agent with the scaffold polynucleotide of any one of claims 1 to 11.

35. The method of claim 34, wherein the biomolecule or the agent is conjugated to the scaffold polynucleotide via an arm linker or a conjugator.

36. The method of claim 35, wherein the conjugator is selected from the group consisting of a biotin moiety, a thiol group, a succinimide moiety, a maleimide moiety, a hydrazine moiety, tyrosine moiety, a hydrazone moiety, an azide moiety, a dibenzocyclooctyne group, a bicyclononyne group, a terminal alkyne moiety, a strained terminal alkyne moiety, a phosphine moiety, polyethylene glycol chains, and any combinations thereof.

37. The method of claim 34, wherein the biomolecule or the agent is conjugated with the arm linker or the conjugator via a bioorthogonal reaction.

38. The method of claim 37, wherein the bioorthogonal reaction is click conjugation, oxime / hydrazone formation, Staudinger ligation, tetrazine ligation, or quadricyclane ligation.

39. The method of claim 38, wherein the click conjugation is selected from the group consisting of copper-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, isocyanide-based click reaction, and inverse electron demand Diels-Alder reaction.

40. The method of claim 34, wherein the biomolecule is selected from the group consisting of a humanized antibody or antigen binding fragment thereof, a chimeric antibody or antigen binding fragment thereof, a monoclonal antibody or antigen binding fragment thereof, a monovalent Fab’, a divalent Fab2, a single-chain variable fragment, a diabody, a minibody, a nanobody, a single-domain antibody, a camelid antibody, antigen binding fragment thereof, a peptide, and any combinations thereof.

41. The method of claim 34, wherein the agent is a therapeutic agent or a diagnostic agent.

42. The method of claim 41, wherein the therapeutic agent is selected from the group consisting of cytotoxic agents, protein toxins, proteins, enzymes, radionuclides, and any combinations thereof.

43. The method of claim 41, wherein the diagnostic agent is selected from the group consisting of a fluorescent moiety, a luminescent moiety, a radioactive moiety, and any combinations thereof.

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