Cleavable cyclic ring nucleotides for nanopore sequencing

Abstract

In one aspect, the disclosed technology relates to nanopore sequencing with a polynucleotide comprising a plurality of nucleotides wherein each nucleotide comprises a linker construct between two positions of the nucleotide wherein the linker construct optionally comprises a reporter moiety corresponding to the identity of the nucleotide, and wherein the linker construct is part of a cleavable cyclic ring nucleotide comprising a cleavable site. In some embodiments, the nucleotides also comprise capture constructs for slowing or stopping translocation of the polynucleotides through the nanopores.

Description

Background Art

[0001] Some polynucleotide sequencing techniques involve performing a large number of controlled reactions on a support surface or in a predetermined reaction chamber. The controlled reactions can then be observed or detected, and subsequent analysis can help identify the characteristics of the polynucleotides involved in the reaction. Examples of such sequencing techniques include next generation sequencing or massively parallel sequencing involving ligation sequencing, synthesis sequencing, reversible terminator chemistry, or pyrophosphate sequencing methods.

[0002] Some polynucleotide sequencing technologies utilize nanopores, which can provide a path for ionic current. For example, when a polynucleotide passes through a nanopore, the polynucleotide affects the current passing through the nanopore. Each passing nucleotide or series of nucleotides passing through the nanopore will generate a blocking current. These characteristic currents passing through the polynucleotide can be recorded to determine the sequence of the polynucleotide. Summary of the invention

[0003] The nanopore reading head (e.g., the constriction of the nanopore) typically "senses" several bases simultaneously along the sample DNA strand, increasing the challenge of accurate nanopore sequencing due to the many permutations of signals from different sequences. For example, the MspA pore reads about 4 bases at a time, generating at least 4^4 = 256 different signals that need to be deconvoluted and resolved.

[0004] In one aspect, the disclosed technology provides a method that is not directly sequencing sample DNA, but using cyclic cyclic nucleotides to synthesize daughter chains. In some embodiments, each cyclic cyclic nucleotide contains a unique barcoding / reporter region and a cleavable site that is specific to the original base (e.g., A, T, C, or G). The daughter chain is then "extended" by cutting the cleavable site. Therefore, when sequencing the daughter chain, the nanopore can "read" the barcoding / reporter region to identify the base encoded by it. The joint and barcoding construct introduced into the daughter chain by polymerization are designed to completely occupy the reader of the nanopore, so the number of signals is reduced to only four, i.e., one for each core base. Therefore, the disclosed technology allows the decoding based on barcodes of each base. In some embodiments, the cyclic ring contains a non-barcoding joint construct, which allows the daughter chain to extend after cutting the cleavable site. When passing through the nanopore, the non-barcoding joint construct can produce a signal that is distinguishable from the signal of the core base or a distinguishable signal interruption, thereby separating and / or enhancing the recorded signal from the core base. Thus, the disclosed technology allows for improved resolution of recorded signals. In some embodiments, the linker construct may contain a barcoded / reporter region and a non-barcoded linker construct. In some embodiments, the linker construct may be a barcoded / reporter element. In some embodiments, heavy atoms (including, for example, sulfur and selenium) are used to modify nucleotides and oligonucleotides.

[0005] In another aspect, the disclosed technology provides systems, devices, kits and methods that allow for cleavable linkages along the DNA backbone, synthesis of cleavable cyclic cyclic nucleotides, barcodes for single base identification, and polymerase mutations for incorporation of modified nucleotides. Systems can be prepared to allow parallel reading in multiple nanopores (e.g., thousands or millions of nanopores). Thus, the components of any system can be functionally replicated to increase sequencing throughput. Any system can also be adapted for microfluidics or automation.

[0006] The systems, devices, kits, and methods disclosed herein each have several aspects, none of which is solely responsible for its desired properties. Without limiting the scope of the claims, some outstanding features will now be briefly discussed. Many other examples are also contemplated, including examples with fewer, additional, and / or different components, steps, features, objects, benefits, and advantages. The various components, aspects, and steps may also be arranged and ordered in different ways. After considering this discussion, particularly after reading the section entitled "Specific Embodiments," it will be understood how the features of the devices and methods disclosed herein provide advantages over other known devices and methods.

[0007] Additional details of exemplary nanopore sequencing devices that can be used with the disclosed technology and methods of operating the devices can be found in U.S. Provisional Patent Application Nos. 63 / 200868 and 63 / 169041, the entire contents of each of which are incorporated herein by reference.

[0008] Disclosed herein is a compound having one of the following structures:

[0009]

[0010] Wherein X is -O-, -CH2-, -NH-, X' is =N-SO2-, =NH-CO- or Y is -O-, -S-, -NH- or -Se-; L1 is a first linking group; L2 is a second linking group; and SP is a spacer.

[0011] Also disclosed herein is an oligonucleotide comprising one of the following structures:

[0012]

[0013]

[0014] Wherein X is -O-, -CH2-, -NH-, X' is =N-SO2-, =NH-CO- or Y is -O-, -S-, -NH- or -Se-; R 1 , R 2 and R 3 One of them is allyl and the other is H, L1 is the first linking group; L2 is the second linking group; and SP is a spacer.

[0015] In some embodiments, structure (VII) can also be represented by the following structure:

[0016]

[0017] In some embodiments, structure (VIII) can also be represented by the following structure:

[0018]

[0019] In some embodiments, structure (X) can also be represented by the following structure:

[0020]

[0021] Also disclosed herein is an oligonucleotide comprising one of the following structures:

[0022]

[0023] wherein Y is -O-, -S-, -NH- or -Se-; and Y 1 , Y 2 and Y 3 One of them is -S- or -Se-, and the other is -O- or -NH-.

[0024] In some embodiments, the SP comprises one or more of the following moieties: (1) an alkyl chain having 5 to 50 carbons, (2) an oligonucleotide or modified oligonucleotide having 1 to 100 repeating units, (3) a polypeptide having 1 to 100 repeating units, (4) a hydrophilic polymer having 1 to 100 repeating units selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polyacrylamide, polyvinyl pyrrolidone, polystyrene sulfonate and polyethylene imine, and (5) a hydrophobic polymer having 1 to 100 repeating units selected from the group consisting of polylactic acid, polymethyl methacrylate and polystyrene.

[0025] In some embodiments, the hydrophilic polymer is selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polyacrylamide, polyvinyl pyrrolidone, polystyrene sulfonate, and polyethylene imine. In some embodiments, the hydrophobic polymer is selected from the group consisting of polylactic acid, polymethyl methacrylate, and polystyrene.

[0026] In some embodiments, each of L1 and L2 independently comprises a conjugated moiety selected from the group consisting of: amine-NHS ester, amine-imidate, amine-pentafluorophenyl ester, amine-hydroxymethylphosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-hydrazide, aldehyde-alkoxyamine, hydroxy-isocyanate, azide-alkyne, azide-phosphine, trans-cyclooctene-tetrazine, norbornene-tetrazine, azide-cyclooctyne, and azide-norbornene.

[0027] In some embodiments, L1 and L2 independently further comprise a first linker between the conjugated moiety and X / X', and a second linker between the conjugated moiety and SP.

[0028] In some embodiments, the first joint and the second joint are independently selected from the group consisting of the following: hydrophilic polymer, hydrophobic polymer, oligonucleotide, peptide, polypeptide, aliphatic chain (C5 to C50) and their combination. In some embodiments, hydrophilic polymer, hydrophobic polymer, oligonucleotide and polypeptide can each have 1 to 100 repeating units, 2 to 100 repeating units, 5 to 100 repeating units, 10 to 100 repeating units, 2 to 50 repeating units, 2 to 30 repeating units or any range between 1 to 100. The hydrophilic polymer can include polyethylene glycol, polyvinyl alcohol, polyacrylamide, polyvinyl pyrrolidone, polystyrene sulfonate, polyethylene imine or their combination. The hydrophobic polymer can include polylactic acid, polymethyl methacrylate or polystyrene or their combination. In some embodiments, the aliphatic chain can include alkyl, alkenyl, alkynyl or their combination.

[0029] In some embodiments, the SP further comprises an arresting construct. In some embodiments, the base further comprises an arresting construct. In some embodiments, the arresting construct is a linear, branched or cyclic polymer. In some embodiments, the arresting construct comprises a synthetic hydrophobic polymer, a synthetic hydrophilic polymer, an oligonucleotide / polynucleotide, a peptide / polypeptide or a combination thereof.

[0030] In some embodiments, L1, SP and L2 are sub-elements of the cyclic ring. In some embodiments, the cyclic ring is symmetrical. In some embodiments, the cyclic ring is asymmetrical. In some embodiments, the cyclic ring is synthesized using one or more of the following: solid phase synthesis, solution phase synthesis and enzyme synthesis. In some embodiments, the cyclic ring is synthesized using one or more of the following: linear synthesis, branched synthesis or segmented synthesis.

[0031] Also disclosed herein is a method for determining the sequence of a polynucleotide in a nanopore-based sequencing system, the method comprising: providing a polynucleotide comprising a plurality of nucleotides, wherein each nucleotide comprises a linker construct having a first end attached to a first position of the nucleotide and a second end attached to a second position of the nucleotide; cleaving a cleavable bond between the first position and the second position on each of the plurality of nucleotides, thereby extending the polynucleotide to form an extended polymer; applying a voltage to cause the extended polymer to insert into and translocate through a nanopore; and (i) detecting and identifying a reporter portion when the linker construct passes through the nanopore; or (ii) detecting and identifying a base on the nucleotide when the nucleotide passes through the nanopore.

[0032] In some embodiments, the linker construct comprises a first linking group, a second linking group, and a spacer between the first linking group and the second linking group.

[0033] In some embodiments, the spacer comprises an oligonucleotide, a modified oligonucleotide, or a polyphosphate having 1 to 100 repeating units; a polypeptide having 1 to 100 repeating units; an alkyl chain having 5 to 50 carbons; a hydrophilic polymer having 1 to 100 repeating units selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polyacrylamide, polyvinyl pyrrolidone, polystyrene sulfonate, and polyethylene imine; a hydrophobic polymer having 1 to 100 repeating units selected from the group consisting of polylactic acid, polymethyl methacrylate, and polystyrene; and combinations thereof.

[0034] In some embodiments, the spacer comprises a reporter moiety, wherein the reporter moiety corresponds to and identifies a nucleotide.

[0035] In some embodiments, each of the first and second linking groups L1 and L2 independently comprises a conjugated moiety selected from the group consisting of: amine-NHS ester, amine-imidate, amine-pentafluorophenyl ester, amine-hydroxymethylphosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-hydrazide, aldehyde-alkoxyamine, hydroxy-isocyanate, azide-alkyne, azide-phosphine, trans-cyclooctene-tetrazine, norbornene-tetrazine, azide-cyclooctyne and azide--norbornene.

[0036] In some embodiments, the elongated polymer further comprises a capture construct attached to each nucleobase or each linker construct, wherein the capture construct is configured to slow, pause, or stop translocation.

[0037] In some embodiments, the capture construct (i.e., modification) is a linear, branched, or cyclic polymer. In some embodiments, the capture construct comprises a synthetic hydrophobic polymer, a synthetic hydrophilic polymer, an oligonucleotide / polynucleotide, a peptide / polypeptide, or a combination thereof. In some embodiments, the nanopore comprises a constriction having an opening with an inner diameter of about 0.6 nm to about 1.2 nm.

[0038] In some embodiments, the reporter portion comprises one or more sub-reporter portions, wherein the one or more sub-reporter portions correspond to and identify nucleotides or translocation events. In some embodiments, the reporter portion comprises two or more sub-reporter portions, wherein each sub-reporter portion in the two or more sub-reporter portions is distinguishable, reproducible and resolvable. In some embodiments, the two or more sub-reporter portions comprise crown ethers, cucurbiturils, pillar aromatics or cyclodextrins.

[0039] Disclosed herein is a kit for performing a method of determining the sequence of a polynucleotide in a nanopore-based sequencing system, the kit comprising a compound disclosed herein.

[0040] Disclosed herein includes a system for determining the sequence of a polynucleotide, the system being configured to perform the method disclosed herein.

[0041] Disclosed herein includes a system for performing a method of determining the sequence of a polynucleotide comprising a plurality of nucleotides, wherein the nucleotides are selected from any one of the compounds disclosed herein.

[0042] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below are contemplated as being part of the inventive subject matter disclosed herein and can be used to achieve the benefits and advantages described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] By referring to the following detailed description and accompanying drawings, the features of the examples of the present disclosure will become apparent, wherein like reference numerals correspond to similar but possibly different parts. For the sake of brevity, reference numerals or features having previously described functions may or may not be described in conjunction with other drawings in which they appear.

[0044] Figure 1 An example of sequencing an extended polynucleotide is schematically illustrated.

[0045] Figure 2 An example of elongation of a polynucleotide by cleavable cyclic nucleotides is schematically illustrated.

[0046] Figure 3 Examples of cleavable linkages are schematically illustrated.

[0047] Figure 4 An example of using allyl cleavable chemistry in the disclosed sequencing methods is schematically illustrated.

[0048] Figure 5 Schematic illustration of an example of an allyl-based cleavable cyclic nucleotide with 10Ts as the barcoding region, a phosphate-base linked (PBL) cyclic ring and a "modification" on the base.

[0049] Figure 6 An example of sequencing an extended polynucleotide having a modification is schematically illustrated.

[0050] Figure 7 Experimental data showing polymerase incorporation of allyl dTTP.

[0051] Figure 8 Experimental data showing cleavage of the allylic group.

[0052] Fig. 9 An example of a first design of a fully functional cyclic nucleotide (CLN-1) is schematically illustrated.

[0053] Fig.10 An exemplary synthesis process of a fully functional cyclic nucleotide (CLN-1A) is schematically illustrated.

[0054] Fig.11 An exemplary synthesis process of a fully functional cyclic nucleotide (CLN-1B) is schematically illustrated.

[0055] Fig.12 An exemplary synthesis process of a fully functional cyclic nucleotide (CLN-1C) is schematically illustrated.

[0056] Fig.13 An exemplary synthesis process of a fully functional cyclic nucleotide (CLN-1D) is schematically illustrated.

[0057] Fig.14 An example of a second design of a fully functional cyclic nucleotide (CLN-2) is schematically illustrated.

[0058] Fig.15 An exemplary synthesis process of a fully functional cyclic nucleotide (CLN-2A) is schematically illustrated.

[0059] Fig.16 An exemplary synthesis process of a fully functional cyclic cyclic nucleotide (CLN-2B) is schematically illustrated.

[0060] Fig.17 An exemplary synthesis process of a fully functional cyclic cyclic nucleotide (CLN-2C) is schematically illustrated.

[0061] Fig.18 Schematically illustrated is an example of a third design of a fully functional cyclic nucleotide (CLN-3), wherein X can be O, NH, NSO2 or CH2, and Y can be O, S or NH, with an alpha phospho-allyl-linked asymmetric ring and a capture construct on the nucleobase.

[0062] Fig.19 An exemplary synthesis process of a fully functional cyclic cyclic nucleotide (CLN-3A) is schematically illustrated.

[0063] Fig. 20 An exemplary synthesis process of a fully functional cyclic cyclic nucleotide (CLN-3B) is schematically illustrated.

[0064] Fig.21 An exemplary synthesis process of a fully functional cyclic cyclic nucleotide (CLN-3C) is schematically illustrated.

[0065] Fig. 22 Schematically illustrated is an example of a fourth design of a fully functional cyclic nucleotide (CLN-4), wherein X can be O, NH, NSO2 or CH2, and Y can be O, S or NH, having an alpha phospho-allyl linked asymmetric ring and a capture construct on the ring.

[0066] Fig.23 An exemplary synthesis process of a fully functional cyclic cyclic nucleotide (CLN-4A) is schematically illustrated.

[0067] Fig.24 An exemplary synthesis process of a fully functional cyclic cyclic nucleotide (CLN-4B) is schematically illustrated.

[0068] Fig.25 An exemplary synthesis process of a fully functional cyclic cyclic nucleotide (CLN-4C) is schematically illustrated.

[0069] Fig.26 Schematically illustrated is an example of a fifth design of a fully functional cyclic nucleotide (CLN-5), wherein X can be O, NH, NSO2 or CH2, and Y can be O, S or NH, having an asymmetric ring with an alpha phosphate-base linkage and a capture construct on the ring.

[0070] Fig. 27 An exemplary synthesis process of a fully functional cyclic nucleotide (CLN-5A) is schematically illustrated.

[0071] Fig.28 An exemplary synthesis process of a fully functional cyclic cyclic nucleotide (CLN-5B) is schematically illustrated.

[0072] Fig.29An exemplary synthesis process of a fully functional cyclic cyclic nucleotide (CLN-5C) is schematically illustrated.

[0073] Fig.30 An exemplary synthesis process of a fully functional cyclic cyclic nucleotide (CLN-5D) is schematically illustrated.

[0074] Fig.31 Schematically illustrated is an example of a sixth design of a cyclic cyclic nucleotide (CLN-6), wherein X can be O, NH, NSO2 or CH2, and Y can be O, S or NH, having a peptide-based cyclic ring and a capture construct on the nucleobase.

[0075] Fig.32 Schematically illustrated is an example of a seventh design of a cyclic cyclic nucleotide (SNL-1), wherein X can be O, NH, NSO2 or CH2, having a symmetrical nucleoside ring with an alpha phosphate-base linkage and a capture construct on the ring.

[0076] Fig.33 Schematically illustrated is an example of an eighth design of a cyclic cyclic nucleotide (SNNL-1), wherein X can be O, NH or CH2, having a symmetric non-nucleoside ring with an alpha phosphate-base linkage and a capture construct on the ring.

[0077] Fig.34 Schematically illustrated is an example of a ninth design of a cyclic cyclic nucleotide (SPL-1), wherein X can be O, NH, NSO2 or CH2, having a symmetric peptide ring with an alpha phosphate-base linkage and a capture construct on the ring.

[0078] Fig.35 Schematically illustrated is an example of a tenth design of a cyclic cyclic nucleotide (SNL-2), wherein X can be O, NH, NSO2 or CH2, having an alpha phospho-allyl linked symmetrical nucleoside ring and a capture construct on the ring.

[0079] Fig.36 Schematically illustrated is an example of an eleventh design of a cyclic cyclic nucleotide (SNNL-2), wherein X can be O, NH, NSO2 or CH2, having an alpha phospho-allyl linked symmetrical non-nucleoside ring and a capture construct on the ring.

[0080] Fig.37 Schematically illustrated is an example of a twelfth design of a cyclic cyclic nucleotide (SPL-2), wherein X can be O, NH, NSO2 or CH2, having an alpha phospho-allyl linked symmetric peptide ring and a capture construct on the ring.

[0081] Fig.38Schematically illustrated is an example of a thirteenth design of a cyclic cyclic nucleotide (ASL-1), wherein X can be O, NH, NSO2 or CH2, having an asymmetric ring with an alpha phosphate-base linkage and a capture construct on the ring.

[0082] Fig.39 Schematically illustrated is an example of a fourteenth design of a cyclic cyclic nucleotide (ASL-2), wherein X can be O, NH, NSO2 or CH2, having an asymmetric ring with an alpha phospho-C5' allyl linkage and a capture construct on the ring.

[0083] Fig.40 Schematically illustrated is an example of a fifteenth design of a cyclic cyclic nucleotide (ASL-3), wherein X can be O, NH, NSO2 or CH2, having an asymmetric ring with an alpha phospho-C5' allyl linkage and a capture construct on the nucleobase.

[0084] Fig.41 An example of a sixteenth design of a cyclic ring 4-mer oligonucleotide (4-mer-SNL-1) having a 4-mer oligonucleotide with a symmetrical nucleoside ring and a capture construct on the ring is schematically illustrated.

[0085] Fig.42 Schematically illustrated is an example of a seventeenth design of a cyclic ring 4-mer oligonucleotide (4-mer-SNNL-1) having a 4-mer oligonucleotide with a symmetrical non-nucleoside ring and a capture construct on the ring.

[0086] Fig.43 Schematic illustration of an example of an eighteenth design of a cyclic loop 4-mer oligonucleotide (4-mer-SPL-1) having a 4-mer oligonucleotide with a symmetrical peptide loop and a capture construct on the loop.

[0087] Fig.44 Schematically illustrated is an example of a nineteenth design of a cyclic loop 4-mer oligonucleotide (4-mer-ASL-1) having a 4-mer oligonucleotide with an asymmetric loop and a capture construct on the loop.

[0088] Fig.45 An exemplary synthetic process for generating a 4-mer oligonucleotide that can be used to prepare a cleavable circular 4-mer oligonucleotide is schematically illustrated.

[0089] Fig.46 An example of a chemical scheme (Scheme I) for the synthesis of a bridged PS nucleotide is schematically illustrated, the product being nucleotide 1.

[0090] Fig.47ANon-limiting characterization of phosphorothiol (bridged PS) nucleotides prepared according to Scheme I is illustrated.

[0091] Fig.47B Non-limiting characterization of phosphorothiol (bridged PS) nucleotides prepared according to Scheme I is illustrated.

[0092] Fig.48 Scheme II, an alternative method for generating bridged PS nucleotides, is schematically illustrated.

[0093] Fig.49 Shown are experimental data from the incorporation of poly-A tails formed by bridging PS (phosphorothiol) deoxyribonucleotides.

[0094] Fig.50A is the reaction scheme for the cleavage of the phosphorus-sulfur bond by silver nitrate through the bridging PS nucleotide.

[0095] Fig.50B Shown are experimental results obtained from analysis of cleavage products following cleavage of the phosphorus-sulfur bond by a bridging PS nucleotide by silver nitrate.

[0096] Fig.51 Gel results illustrating the cleavage reaction using the bridging PS nucleotide 1 and DNA polymerase Pol1901 on the extended primer.

[0097] Fig.52 Schematic illustration of the synthesis of 5'SDMT phosphoramidite "9" by various methods.

[0098] Fig.53A One embodiment of conjugating a spacer moiety to a 4-mer oligonucleotide is described.

[0099] Fig.53B One embodiment of a cyclic 4-mer oligonucleotide is described.

[0100] Fig.54A -C Description Fig.53B HPLC, IR and MS characterization of the cyclic 4-mer oligonucleotide shown in .

[0101] Fig.55 Non-limiting experimental ligation results illustrating ligation of up to 10 circular 4-mer oligonucleotides.

[0102] Fig.56 Non-limiting experimental results illustrating the sequential ligation and cleavage of certain polynucleotides.

[0103] Fig.57A is a reaction scheme for one embodiment of synthesizing non-bridged PS nucleotides.

[0104] Fig.57Bis a reaction scheme for another embodiment of synthesizing non-bridged PS nucleotides.

[0105] Fig.58 Demonstrates the incorporation of non-bridging PS nucleotides using different polymerases.

[0106] Fig.59 Schematic illustration of the conjugation of a spacer moiety to a functionalized 4-mer oligonucleotide to form a circular 4-mer oligonucleotide.

[0107] Fig.60A -D schematically illustrates Fig.59 Characterization of the 4-mer oligonucleotides shown in .

[0108] Fig.61 For display Fig.59 The ligase performed up to 10 consecutive ligation events on the circular 4-mer oligonucleotide shown in FIG.

[0109] Fig.62A The use of iodine to cleave the bridging PO bond at the phosphonothioate site is demonstrated.

[0110] Fig.62B The characterization before and after PO bond cleavage is illustrated.

[0111] Fig.63 is a reaction scheme for the synthesis of imino-P substituted nucleotides.

[0112] Fig.64 is a reaction scheme for synthesizing cyclic nucleotides having imino-p substitutions according to one embodiment.

[0113] Fig.65 HPLC and LCMS characterization of bifunctional imino-P nucleotides is shown.

[0114] Fig.66 One embodiment of a method for synthesizing imino-P-allyl difunctional nucleotides is schematically illustrated.

[0115] Fig.67 Another embodiment of a method for synthesizing imino-P-allyl difunctional nucleotides is schematically illustrated.

[0116] Fig.68 Another embodiment of a method for synthesizing imino-P-allyl difunctional nucleotides is schematically illustrated.

[0117] Fig.69 Another embodiment of a method for synthesizing imino-P-allyl difunctional nucleotides is schematically illustrated.

[0118] Fig.70AOne embodiment of a method for synthesizing imino-P-allyl difunctional nucleotides is schematically illustrated.

[0119] Fig.70B Exemplary methods for activating αP monophosphate are provided.

[0120] Fig.71 An exemplary synthesis of bifunctional imino-P-allyl nucleotides with different reactive groups is schematically illustrated.

[0121] Fig.72 Schematic illustration of the deprotection of the 3'-OTBDPS group to form the 3'-OH, yielding nucleotide 6.

[0122] Fig.73 HF-TEA and TBAF deprotection methods are demonstrated to evaluate the conversion efficiency and yield of the desired triphosphate product.

[0123] Fig.74A -C illustrates crude HPLC, analytical HPLC and LCMS spectra of the purified nucleotide.

[0124] Fig.75 Schematic illustration of the stereoselective reduction of the carbonyl group.

[0125] Fig.76 Schematic illustration of the kinetic diastereoisomer selection step of the enzyme from the racemic precursor.

[0126] Fig.77 Schematic illustration of the chiral derivatization of isomers that enabled the final column separation.

[0127] Fig.78 Schematic illustration of stereoselective alkyl addition promoted by chiral ligands.

[0128] Fig.79 Schematic illustration of the stereoselective enzymatic synthesis of the triphosphate.

[0129] Fig.80 The method for controlling the chirality at the alpha phosphorus atom is schematically illustrated.

[0130] Fig.81 The synthesis of an exemplary Staudinger variant is shown.

[0131] Fig.82 The synthesis of exemplary azido variants is illustrated.

[0132] Fig.83A -B schematically illustrates an embodiment of a pathway leading to the formation of an exemplary cyclic cyclic nucleotide structure.

[0133] Fig.84A -C Description Fig.83A HPLC, LCMS and FTIR characterization of the cyclic ring structure in.

[0134] Fig.85 Illustrated are the results for the bifunctional nucleotide 6 tested in an incorporation assay using the Dpo4 enzyme compared to native dTTP.

[0135] Fig.86 Illustrated are the results for the bifunctional nucleotide 10 tested in an incorporation assay using the Dpo4 enzyme compared to native dTTP.

[0136] Fig.87 Experimental results on the incorporation kinetics of cyclic nucleotides are shown.

[0137] Fig.88 Stability assays performed on bifunctional nucleotide 6 and cyclic nucleotide 10 are described.

[0138] Fig.89 Stability assays performed on bifunctional nucleotide 6 and cyclic nucleotide 10 are described.

[0139] Fig.90 An exemplary synthesis of a spacer portion with a capture construct is illustrated.

[0140] Fig.91 Another exemplary synthesis of a spacer portion with a capture construct is illustrated.

[0141] Fig.92 Another exemplary synthesis of a spacer portion with a capture construct is illustrated.

[0142] Fig.93 Illustrate an asymmetric annular ring.

[0143] Fig.94 Illustrate a symmetrical annular ring. DETAILED DESCRIPTION

[0144] All patents, applications, published applications, and other publications mentioned herein are incorporated herein by reference in their entirety. If a term or phrase is used herein in a manner that is contrary to or inconsistent with the definition set forth in the patents, applications, published applications, and other publications incorporated herein by reference, the use herein prevails over the definition incorporated herein by reference.

[0145] definition

[0146] All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs, unless specifically defined otherwise.

[0147] As used herein, the singular forms "a," "an," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a sequence" may include a plurality of such sequences, and so forth.

[0148] The terms include, comprise, contain and various forms of these terms are synonymous with each other and are intended to be equally broad. In addition, unless explicitly stated to the contrary, examples that include or have one or more elements with a particular property may include additional elements, whether or not the additional elements have that property.

[0149] As used herein, the term "modified oligonucleotide" refers to a polymeric chain of nucleobases or nucleotides assembled from moieties comprising modified nucleobases, modified sugar rings (e.g., LNA, constrained ethyl, ethylene bridge, TNA, 2'-Ome, 2'F, 2'-MOE), or nucleobases attached to a scaffold (e.g., unlocked, 4'-thio, CeNA, HNA, TNA, GNA, FNA).

[0150] As used herein, the term "phosphoramidite analog" refers to any polymer synthesized using phosphoramidite or related chemistry that results in the formation of phosphodiester, methylphosphonate, or phosphorothioate bonds between the moieties.

[0151] As used herein, the term "modified polyamide" refers to a polymer assembled from separate moieties each having at least one amino group and one carboxylic acid group, resulting in the formation of an amide bond.

[0152] As used herein, the term "nanopore" is intended to represent a hollow structure that is separated from or limited in a film and extends through a film.Nanopore allows ions, electric current and / or fluid to pass through the other side of the film from one side of the film.For example, a film that suppresses ions or water-soluble molecules from passing through may include a nanopore structure, which extends through the film to allow ions or water-soluble molecules to pass through (by extending through the nanoscale openings of the nanopore structure) to the other side of the film from one side of the film.The diameter of the nanoscale openings extending through the nanopore structure can change along its length (that is, from one side of the film to the other side of the film), but at any point on the nanoscale (that is, about 1nm to about 100nm, or to less than 1000nm).The example of nanopore includes, for example, biological nanopores, solid-state nanopores, and biological and solid-state hybrid nanopores.In some embodiments, a refers to a hole having an opening with a diameter of about 0.3nm to about 2nm at its narrowest point. For example, the nanopore can be a solid-state nanopore, a graphene nanopore, an elastomeric nanopore, or can be a naturally occurring or recombinant protein that forms a channel when inserted into a bilayer, a thin film, a membrane, or a solid-state pore (also referred to herein as a protein pore or a protein nanopore (e.g., a transmembrane pore). If the protein is inserted into a membrane, the protein is a channel-forming protein.

[0153] As used herein, the term "diameter" is intended to mean the longest straight line that can be written in the cross-section of the nanoscale opening through the centroid of the cross-section of the nanoscale opening. It should be understood that the nanoscale opening may or may not have a circular or substantially circular cross-section (the cross-section of the nanoscale opening is substantially parallel to the cis / trans electrodes). In addition, the cross-section may be regular or irregular in shape.

[0154] As used herein, "cis" refers to the side of the nanopore opening through which an analyte or modified analyte enters the opening, or across which an analyte or modified analyte migrates.

[0155] As used herein, "trans" refers to the side of the nanopore opening through which the analyte or modified analyte (or fragment thereof) exits the opening, or across which the analyte or modified analyte does not migrate.

[0156] As used herein, the term "biological nanopore" is intended to mean a nanopore whose structure is made in part from materials of biological origin. Biological origin refers to materials derived from or isolated from a biological environment (such as an organism or a cell), or a synthetically manufactured form of a biologically usable structure. Biological nanopores include, for example, polypeptide nanopores and polynucleotide nanopores.

[0157] As used herein, a "part" is one of two or more parts into which something can be divided, such as, for example, individual parts of a tether, molecule, or probe.

[0158] As used herein, "reporters" consist of one or more reporter elements or reporter moieties. Reporters include so-called "labels" and "tags". Extended polymer linker constructs (when including reporter moieties) or nucleobase residues can be considered reporters. Reporters are used to resolve the identification of target nucleic acids. Reporters can include composed sub-reporters, and multiple reporters can be present on a single nucleotide. When present in the reader of a nanopore, reporters provide different and sometimes unique blocking currents at a given read voltage.

[0159] As used herein, "joint" is a molecule or part that connects two molecules or parts and provides a spacing between the two molecules or parts so that they can work in their expected manner. For example, the joint may include a diamine hydrocarbon chain, which is covalently bound to the oligonucleotide analog molecule through a reactive group on one end, and covalently bound to a solid support (such as, for example, a bead surface) through a reactive group on the other end. The coupling of the joint to the nucleotide of interest and the substrate construct can be completed using coupling agents known in the art (see, for example, Efimov et al., Nucleic Acids Res. 27: 4416-4426, 1999). The method of derivatizing and coupling organic molecules is well known in the field of organic and bioorganic chemistry. The joint can also be cleavable or reversible.

[0160] As used herein, the term "heavy atom" refers to any atom that is not hydrogen and is used within a molecular structure. The heavy atom used in a modified oligonucleotide can be bridged (e.g., for connecting multiple oligonucleotides) or non-bridged (e.g., not directly connected to multiple oligonucleotides).

[0161] As used herein, the term "polypeptide nanopore" is intended to mean a protein / polypeptide that extends through a membrane and allows ions, electric current, polymers (such as DNA or peptides) or other molecules and / or fluids of appropriate size and charge to flow from one side of the membrane to the other side of the membrane. The polypeptide nanopore can be a monomer, a homopolymer or a heteropolymer. The structure of the polypeptide nanopore includes, for example, an α-helical bundle nanopore and a β-barrel nanopore. Exemplary polypeptide nanopores include α-hemolysin, Mycobacterium smegmatis porin A (MspA), gramicidin A, maltoporin, OmpF, OmpC, PhoE, Tsx, F-pilus, etc. The protein α-hemolysin is naturally present in the cell membrane, where it acts as a hole for ion or molecule transport in and out of the cell. Mycobacterium smegmatis porin A (MspA) is a membrane porin produced by mycobacteria that allows hydrophilic molecules to enter the bacterium. MspA forms a tightly interconnected octamer and a transmembrane β-barrel that resembles a goblet and contains a central pore.

[0162] As used herein, "peptide" refers to two or more amino acids linked together by amide bonds (i.e., "peptide bonds"). Peptides contain up to or include 50 amino acids. Peptides can be linear or cyclic. Peptides can be alpha, beta, gamma, delta or higher, or mixed. Peptides can contain any mixture of amino acids as defined herein, such as any combination of D, L, alpha, beta, gamma, delta or higher amino acids.

[0163] As used herein, "protein" refers to an amino acid sequence having 51 or more amino acids.

[0164] The polypeptide nanopore may be synthetic. A synthetic polypeptide nanopore includes a protein-like amino acid sequence that does not exist in nature. A protein-like amino acid sequence may include some of the amino acids that are known to exist but do not form the basis of a protein (i.e., non-proteinogenic amino acids). A protein-like amino acid sequence may be artificially synthesized rather than expressed in an organism and then purified / isolated.

[0165] The nanopores disclosed herein may be hybrid nanopores. "Hybrid nanopore" refers to a nanopore that contains materials of both biological and non-biological origin. Examples of hybrid nanopores include polypeptide-solid-state hybrid nanopores and polynucleotide-solid-state nanopores.

[0166] Applying a potential difference across the nanopore can force nucleic acid to translocate through the nanopore. One or more signals corresponding to nucleotide translocations passing through the nanopore are generated. Therefore, when the target polynucleotide or mononucleotide or the probe derived from the target polynucleotide or mononucleotide passes through the nanopore, the transmembrane current changes due to the base dependence (or probe dependence) blocking of the contraction, for example. Any of the various methods can be used to measure the signal from the current change. Each signal is unique for the type of the nucleotide (or the joint construct with the reporter sub-part region) in the nanopore, so that the resulting signal can be used to determine the characteristics of the polynucleotide. For example, the identity of one or more nucleotide (or probe) types that produce characteristic signals can be determined.

[0167] As used herein, "nucleotide" includes a nitrogenous heterocyclic base, a sugar, and one or more phosphate groups. Nucleotides are monomeric units of nucleic acid sequences. Examples of nucleotides include, for example, ribonucleotides or deoxyribonucleotides. In ribonucleotides (RNA), the sugar is ribose, and in deoxyribonucleotides (DNA), the sugar is deoxyribose, i.e., a sugar lacking a hydroxyl group present at the 2' position in the ribose. The nitrogenous heterocyclic base can be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G) and their modified derivatives or analogs. Pyrimidine bases include cytosine (C), thymine (T) and uracil (U) and their modified derivatives or analogs. The C-1 atom of deoxyribose is bonded to the N-1 of pyrimidine or the N-9 of purine. The phosphate group can be in the form of monophosphate, diphosphate or triphosphate. These nucleotides are natural nucleotides, but it should be further understood that non-natural nucleotides, modified nucleotides or analogs of the aforementioned nucleotides can also be used.

[0168] As used herein, "nucleobase" is a heterocyclic base, such as adenine, guanine, cytosine, thymine, uracil, inosine, xanthine, hypoxanthine, or a heterocyclic derivative, analog or tautomer thereof. Nucleobases may be naturally occurring or synthetic. Non-limiting examples of nucleobases are adenine, guanine, thymine, cytosine, uracil, xanthine, hypoxanthine, 8-azapurine, purine substituted by methyl or bromine at the 8 position, 9-oxo-N6-methyladenine, 2-aminoadenine, 7-deazaxanthine, 7-deazaguanine, 7-deaza-adenine, N4-ethanolylcytosine, 2,6-diaminopurine, N6-ethanolyl-2,6-diaminopurine, 5-methylcytosine , 5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, thiouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine, 7,8-dimethylalloxazine, 6-dihydrothymine, 5,6-dihydrouracil, 4-methyl-indole, ethanoladenine, and U.S. Patent Nos. 5,432,272 and 6,150,510, and PCT Application No. WO 92 / 002258, WO 93 / 10820, WO 94 / 22892 and WO 94 / 24144, and non-naturally occurring nucleobases described in Fasman (Practical Handbook of Biochemistry and Molecular Biology, pp. 385-394, 1989, CRC Press, Boca Raton, LO), all of which are incorporated herein by reference in their entirety.

[0169] The term "nucleic acid" or "polynucleotide" refers to a deoxyribonucleotide or ribonucleotide polymer in single-stranded or double-stranded form, and unless otherwise limited, encompasses known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides, such as peptide nucleic acids (PNA) and thiol phosphate DNA. Unless otherwise specified, a specific nucleic acid sequence includes its complementary sequence. Nucleotides include, but are not limited to, ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, TTP, dUTP, 5-methyl-CTP, 5-methyl-dCTP, ITP, dITP, 2-amino-adenosine-TP, 2-amino-deoxyadenosine-TP, 2-thiothymidine triphosphate, pyrrolopyrimidine triphosphate and 2-thiocytidine, as well as α-thiotriphosphates of all of the above substances, and 2'-O-methyl-ribonucleotide triphosphates of all of the above bases. Modified bases include, but are not limited to, 5-Br-UTP, 5-Br-dUTP, 5-F-UTP, 5-F-dUTP, 5-propynyl dCTP, and 5-propynyl-dUTP.

[0170] As used herein, the term "signal" is intended to mean an indicator representing information. Signals include, for example, electrical signals and optical signals. The term "electrical signal" refers to an indicator representing the electrical quality of information. The indicator can be, for example, current, voltage, tunneling, resistance, potential, voltage, conductance, or transverse electric effect. "Electronic current" or "electric current" refers to the flow of charge. In an example, the electrical signal can be an electric current passing through a nanopore, and the current can flow when a potential difference is applied across the nanopore.

[0171] As used herein, the term "driving force" is intended to refer to the current that allows the translocation of a polynucleotide through a nanopore. In some embodiments, when a current is applied across a nanopore, a current can flow.

[0172] As used herein, the term "holding force" refers to the resistance that slows down and / or stops the translocation of a polynucleotide through a nanopore. In some embodiments, the holding force is overcome by applying a driving force. Thus, the driving force overcomes / surpasses the resistance that slows down and / or stops the polynucleotide, thereby allowing the polynucleotide to translocate through the nanopore.

[0173] As used herein, the term "modification" refers to the part attached to the nucleotide. Modification can provide resistance (in the form of "holding force"), which slows down and / or stops the polynucleotide from being translocated by the nanopore, unless the resistance caused by the modification is overcome by the "driving force". The resistance provided by the modification is attributed to the characteristics of the modification (e.g., size, geometry and / or non-covalent interaction with the nanopore). Modification can act as a ratchet or brake for polypeptides to be translocated by the nanopore. Modification can be attached to any part of the nucleotide, or it can be attached to the nucleotide at two positions forming a ring. Modification can also be referred to as capture construct.

[0174] The aspects and examples set forth herein and recited in the claims may be understood in light of the above definitions.

[0175] Overview

[0176] A common disadvantage of nanopore sequencers is that the nanopore is sensitive to multiple bases of the DNA chain in the nanopore, which is contrary to reading a single base one at a time. For example, the MspA nanopore has a contraction domain that acts as a reader of at least 4 nucleotides (called "k-mer"), resulting in a minimum of 256 (4^4) different permutations of the 4-mer sequence that needs to be deconvoluted. For a k-mer of 5 bases, the number of possible signals is 4^5=1,024. Longer readers will lead to an exponential increase in the number of signals to be distinguished, which complicates sequencing readout and increases the complexity of base calls, thereby reducing accuracy. Another problem with nanopore sequencers is that the translocation speed of natural single-stranded DNA is about> 10 million nucleotides / second, which is much higher than the rate compatible with electronic devices and detectors.

[0177] In some embodiments, by using cleavable sites along the DNA backbone, adjacent nucleobases are simultaneously connected to the barcoded region, and the disclosed technology allows the distance between adjacent nucleobases to be increased, and there is no need to deconvolute a large amount of signal. Once the backbone is cleaved, the reporter portion of the extended polynucleotide will occupy the entire read head of the nanopore for highly accurate single-molecule sequencing at single-base resolution. It has been observed that DNA backbone cleavage may be affected by the instability caused by the introduction of certain α-phosphate substitutions. Therefore, when synthesizing a daughter oligonucleotide chain, a stable α-phosphate substitution is preferred, particularly when a polynucleotide or oligonucleotide is cleaved to extend the leading chain.

[0178] In some embodiments, the disclosed technology allows 1 nucleobase of the extended polynucleotide to reside in the read head at any point in time, successfully reducing the diversity of the reads to 4 (A, T, C, and G), enabling more accurate sequencing at a lower cost. In some embodiments, the disclosed technology provides high-throughput, cheaper, and more accurate DNA sequencing.

[0179] System and method

[0180] Figure 1Schematically illustrate an embodiment of sequencing the extended polynucleotide. Protein nanopore 101 is deposited in lipid bilayer 102. Extended polynucleotide 103 translocation passes through nanopore 101. Polynucleotide 103 includes a joint construct region between consecutive nucleotides. By introducing a joint construct between consecutive nucleotides, the k-mer length can be reduced to 1, generating only 4 signals (for A, T, C and G), thereby reducing the complexity of base calling. Characteristic joints / barcodes can be assigned to each of 4 separate bases to achieve base recognition. For example, a signal unit or portion 105 includes an "A" nucleotide and a corresponding "joint construct 1", which may contain a reporter serving as a barcode for nucleotide A. The diversity of the reading is reduced to 4, with a single barcode feature for each nucleobase located in the nanopore reader.

[0181] So-called "translocation" refers to one side of the opening of the nanopore where an analyte (e.g., a polynucleotide, such as DNA) enters and moves to the other side of the opening and moves out of it. It is contemplated that any embodiment of translocation herein may refer to electrophoretic translocation or non-electrophoretic translocation, unless otherwise specified. The electric field can move an analyte or a modified analyte. "Interaction" refers to an analyte or a modified analyte moving into and optionally through an opening, wherein "through an opening" (or "translocation") refers to one side of an opening where it enters and moves to the other side of an opening and moves out of it. Optionally, it is contemplated that the method of electrophoretic translocation is not adopted. In some embodiments, physical pressure causes the modified analyte to interact with the opening, enter it, or translocate through it (after changing). In some embodiments, magnetic beads are attached to the analyte or modified analyte on the reverse side, and magnetic force causes the modified analyte to interact with the opening, enter it, or translocate through it (after changing). Other methods for translocation include, but are not limited to, gravity, osmotic pressure, temperature, and other physical forces such as centripetal force.

[0182] In some embodiments, the nanopore may include solid materials such as silicon nitride, modified silicon nitride, silicon, silicon oxide or graphene or a combination thereof. In some embodiments, the nanopore is a protein that forms a channel when inserted into a bilayer, a membrane, a thin film or a solid pore. In some embodiments, the nanopore is contained in a lipid bilayer. In some embodiments, the nanopore is contained in an artificial membrane comprising mycolic acid. The nanopore may be a Mycobacterium smegmatis porin (Msp) having a vestibule and a contraction zone defining a channel. The Msp porin may be a mutant MspA porin. In some embodiments, the amino acids at positions 90, 91 and 93 of the mutant MspA porin are each substituted with asparagine. Some embodiments may include changing the translocation speed or sequencing sensitivity by removing, adding or replacing at least one amino acid of the Msp porin. A "mutant MspA porin" is a multimeric complex that has at least or up to 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% or more identity to its corresponding wild-type MspA membrane porin, or any range derivable therefrom, but less than 100%, and retains channel forming ability. The mutant MspA porin can be a recombinant protein. Optionally, the mutant MspA porin is a mutant MspA porin having a mutation in the constriction zone or vestibule of the wild-type MspA porin. Optionally, the mutation may occur at the edge or outside of the periplasmic loop of the wild-type MspA porin. The mutant MspA porin may be used in any of the embodiments described herein.

[0183] "Vestibule" refers to the tapered portion inside the Msp porin, whose diameter generally decreases from one end to the other along the central axis, with the narrowest portion of the vestibule connected to the constriction. The vestibule may also be referred to as a "cup-shaped". The vestibule and constriction together define the channel of the Msp porin. "Constriction" or "reading head" refers to the narrowest diameter portion of the channel of the Msp porin, which is connected to the vestibule. The length of the constriction may be in the range of from about 0.3 nm to about 2 nm. Optionally, the length is about, at most about, or at least about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or 3 nm, or any range derivable therein. The diameter of the constriction may be in the range of from about 0.3 nm to about 2 nm. Optionally, the diameter is about, at most about, or at least about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, or 3 nm, or any range derivable therefrom. A "channel" refers to the central hollow portion of the Msp porin defined by the vestibule and constriction through which gases, liquids, ions, or analytes can pass. A channel is an example of an opening of a nanopore.

[0184] Various conditions, such as light and the liquid medium contacting the nanopore, including its pH, buffer composition, detergent composition and temperature, can affect the performance of the nanopore, particularly with respect to its conductivity through the channel and the movement of analytes relative to the channel (temporarily or permanently).

[0185] In some embodiments, the disclosed system for nanopore sequencing includes an Msp porin having a vestibule and a constriction defining a channel, wherein the channel is positioned between a first liquid medium and a second liquid medium, wherein at least one of the liquid media includes an analyte polynucleotide, and wherein the system is operable to detect the properties of the analyte. The system can be operated to detect the properties of any analyte, including subjecting the Msp porin to an electric field so that the analyte interacts with the Msp porin. The system can be operated to detect the properties of the analyte, including subjecting the Msp porin to an electric field so that the analyte electrophoretically translocates through the channel of the Msp porin. In some embodiments, the system includes an Msp porin having a vestibule and a constriction defining a channel, wherein the channel is positioned in a lipid bilayer between a first liquid medium and a second liquid medium, and wherein the only point of liquid connection between the first and second liquid media occurs in the channel. In addition, any Msp porin described herein can be included in any system described herein. In some embodiments, the system can also include an amplifier or a data acquisition device. The system can also include one or more temperature regulating devices connected to the first liquid medium, the second liquid medium, or both. The systems described herein may be operated to transfer analytes through Msp porin channels by electrophoresis or other means.

[0186] like Figure 2 As shown in, the extended polynucleotide 203 can be formed by a polynucleotide with modified nucleotides 210, each modified nucleotide 210 comprising a cyclic ring modification 211. The daughter chain polynucleotide 220 can be synthesized by a polymerase from a template DNA using a modified nucleotide (e.g., modified dNTP) 210. During polymerization, the modified dNTP 210 with the cyclic ring 211 is incorporated into the growing daughter chain 220. Once the daughter chain 220 is formed, the polynucleotide backbone is cleaved at the cleavable site, allowing the cyclic ring modification 211 to open and cause the extension of the daughter chain polynucleotide 220. The cyclic ring modification on the modified dNTP becomes a joint construct 206 in the extended polynucleotide 203, which creates a distance between adjacent nucleotides.

[0187] The polymerase used is an enzyme commonly used to connect 3'-OH 5'-triphosphate nucleotides, oligomers and their analogs. The polymerase includes, but is not limited to, DNA-dependent DNA polymerase, DNA-dependent RNA polymerase, RNA-dependent DNA polymerase, RNA-dependent RNA polymerase, T7 DNA polymerase, T3 DNA polymerase, T4 DNA polymerase, T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, DNA polymerase I, Kleno fragment, Thermophilus aquaticus DNA polymerase, Tth DNA polymerase, DNA polymerase (New England Biolabs), Deep DNA polymerase (New England Biolabs), Bst DNA polymerase large fragment, Stoeffel fragment, 90N DNA polymerase, 90N DNA polymerase, Pfu DNA polymerase, TfI DNA polymerase, Tth DNA polymerase, RepliPHIPhi29 polymerase, Tii DNA polymerase, eukaryotic DNA polymerase β, telomerase, Therminator TM Polymerase (New England Biolabs), KOD HiFi TM DNA polymerase (Novagen), KOD1 DNA polymerase, Q-β replicase, terminal transferase, AMV reverse transcriptase, M-MLV reverse transcriptase, Phi6 reverse transcriptase, HIV-1 reverse transcriptase, novel polymerases discovered by bioprospecting, and polymerases cited in US2007 / 0048748, US 6,329,178, US 6,602,695, and US 6,395,524 (incorporated herein by reference). These polymerases include wild-type, mutant isoforms, and genetically engineered variants. "Encoding" or "analysis" is a verb, meaning to convert from one form to another, and refers to converting the genetic information of the target template base sequence into an arrangement of reporters.

[0188] After the polymerization process is completed, the cleavage at the predetermined position opens the ring and increases the distance between adjacent nucleotides. The cleavage of the daughter chain can be designed to occur in any part of the main chain, as long as it occurs in the ring structure between the two positions to which the joint construct and the nucleotide structure are attached. The daughter chain opens the ring along the cleavage of the main chain and extends the daughter chain, thereby leaving the joint construct connecting the main chain phosphate and sugar. In the embodiment in which the joint construct contains the modification that is configured to interact with the nanopore, these modifications can slow down or stop the translocation of the extended polymer and allow nucleotides to be read one at a time by the nanopore.

[0189] In embodiments where a reporter moiety (such as a reporter barcode) is part of the adapter construct, the cleavage product (i.e., the extended polymer) exposes a series of reporter moieties, each reporting the identity of the base to which it corresponds. In embodiments where the adapter construct also contains modifications configured to interact with the nanopore, the extended polymer can be sequenced one barcode at a time in the nanopore.

[0190] Cleavable cyclic nucleotides

[0191] Cleavable cyclic cyclic nucleotides are nucleotides / nucleotide analogs that are modified to include a linker construct attached to two positions of the nucleotide / nucleotide analog structure. Although "cleavable cyclic cyclic nucleotides" are used, it includes modified natural nucleotides and modified nucleotide analogs. Nucleotides and nucleotide analogs that can be used as described herein include the following compounds:

[0192]

[0193] wherein X is NHR, OR or CH2R; R is H, alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl; and the base is selected from the group consisting of adenine, cytosine, guanine, thymine and uracil. The linker construct can be conjugated with a nucleotide / nucleotide analog to form a cleavable cyclic cyclic nucleotide.

[0194] The linker construct can be conjugated to a nucleotide / nucleotide analog at one end through a PN, PO or PC bond or other PX heteroatom bond. The other end can be conjugated to: (i) any position on the nucleobase in all structures above, (ii) the 5'-carbon of the ribose in structures NT-1, NT-3 and NT-4, (iii) the N-atom adjacent to the 5'-carbon in structure NT-4, (iv) the 5'-allyl modification in structure NT-2, or (v) any position on the ribose ring. Examples of covalent conjugation chemistry include amine-NHS esters, amine-imidates, amine-pentafluorophenyl esters, amine-hydroxymethylphosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-hydrazide, aldehyde-alkoxyamine, hydroxyl-isocyanate, azide-alkyne, azide-phosphine, trans-cyclooctene-tetrazines, norbornene-tetrazines, azide-cyclooctene and azide-norbornene. Once the linker construct is conjugated to the nucleotide / nucleotide analog, it forms the cyclic ring portion of the cleavable cyclic cyclic nucleotide. Thus, the cyclic ring comprises -L1-SP-L2- portions.

[0195] Depending on the structure and composition of the cyclic ring, several functions or structures may be present, including one or more of the following: conjugation moiety, linker, spacer, reporter (reporter element, barcode), and capture construct.

[0196] The conjugated part in the cyclic ring modification is formed by conjugating the spacer part to one or more nucleotides or conjugating another modification (such as capture construct) to the cyclic ring. The reactive group at each end of the spacer part reacts with the reactive group on the bifunctional nucleotide to form a conjugated part. In some embodiments, one or more capture constructs can also be attached to the cyclic ring by a conjugated part. The capture construct is a part configured to slow down the translocation of the polynucleotide, so that one or more reporter elements can have a longer residence time in the nanopore reader in the presence of a driving voltage, thereby allowing the identification of the reporter, so the corresponding base is identified. The spacer (SP) separates the continuous capture constructs to allow the applied pulse voltage to fully decay before the next capture construct. Once a specific main chain element is cleaved, the spacer can also be used to extend any polynucleotide. Therefore, the cyclic ring is composed of any number of sub-elements that can be used to affect and decay sequencing. In some embodiments, the intensity of the background electric field, the type of nanopore and the characteristics of the extended polynucleotide affect the translocation speed, efficiency and accuracy.

[0197] Examples of cleavable cyclic nucleotides include:

[0198]

[0199] Wherein X is -O-, -CH2-, -NH-, X' is =N-SO2-, =NH-CO- or Y is -O-, -S-, -Se- or -NH-; L1 is a first linking group; L2 is a second linking group; SP is a spacer; and the base is selected from the group consisting of adenine, cytosine, guanine, thymine and uracil.

[0200] Other examples of cleavable cyclic nucleotide compounds that may be included in a nanopore sequencing system or a kit for nanopore sequencing are:

[0201]

[0202] Wherein X is -O-, -CH2-, -NH-, X' is =N-SO2-, =NH-CO- or Y is -O-, -S-, -Se- or -NH-; L1 is a first linking group; L2 is a second linking group; SP is a spacer; and the base is selected from the group consisting of adenine, cytosine, guanine, thymine and uracil.

[0203] In some embodiments, each of the first linking group L1 and the second linking group L2 independently comprises a conjugated moiety selected from the group consisting of: amine-NHS ester, amine-imidate, amine-pentafluorophenyl ester, amine-hydroxymethylphosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-hydrazide, aldehyde-alkoxyamine, hydroxy-isocyanate, azide-alkyne, azide-phosphine, trans-cyclooctene-tetrazine, norbornene-tetrazine, azide-cyclooctyne and azide-norbornene. L1 and L2 may be the same or different.

[0204] In some embodiments, each of the first linking group L1 and the second linking group L2 may also independently include a joint. The first joint may be present between the conjugated moiety and X / X' (α phosphoric acid), and the second joint may be present between the conjugated moiety and SP. In some embodiments, the joint may be selected from the group consisting of the following: hydrophilic polymer (e.g., polyethylene glycol, polyvinyl alcohol, polyacrylamide, polyvinyl pyrrolidone, polystyrene sulfonate, polyethylene imine), hydrophobic polymer (e.g., polylactic acid, polymethyl methacrylate, polystyrene), oligonucleotide, peptide, polypeptide, aliphatic chain (C5 to C50) and combinations thereof. In some embodiments, the first joint and the second joint may independently include peptide, polypeptide, alkyl chain, polyethylene glycol or combinations thereof. In some embodiments, one or more joints may be lacking.

[0205] In some embodiments, the SP comprises one or more of the following moieties: (1) simple aliphatic chains, such as alkyl chains having 5 to 50 carbons, and substituted aliphatic chains (the substituents may include halogenated groups, such as chloro, bromo or fluoro, alkyl groups, such as methyl, ethyl or propyl, or aryl groups, such as phenyl or pyridyl), (2) oligonucleotides, modified oligonucleotides or polyphosphates having 1 to 100 repeating units, (3) polypeptides having 1 to 100 repeating units, (4) hydrophilic polymers having 1 to 100 repeating units, examples of which include polyethylene glycol, polyvinyl alcohol, polyacrylamide, polyvinyl pyrrolidone, polystyrene sulfonate and polyethylene imine, and (5) hydrophobic polymers having 1 to 100 repeating units, examples of which include polylactic acid, polymethyl methacrylate and polystyrene. In some embodiments, the alkyl chain may be substituted or unsubstituted. In some embodiments, the number of repeating units (monomers) in the SP can be, for example, in the range of 1-5, 6-10, 11-15, 16-20, 20-25, 26-50, or 50-100, or a combination of any of the foregoing ranges. In some embodiments, the total number of repeating units in the SP can be 5-100, 10-100, 10-80, 10-70, 5-60, or 5-50.

[0206] The number of repeat units and the length of the spacer SP may depend on the following factors: (1) the choice of repeat units / monomers - shorter / smaller monomers may require more repeats to form similar lengths compared to longer monomers; (2) the steric volume of the spacer - larger spacer monomers will likely result in steric collisions with the nanopore reader and, therefore, slower translocation rates compared to smaller monomers; (3) the interaction of the spacer with the nanopore - spacer monomers that are able to form stronger interactions (e.g., electrostatic interactions, H-bonding) with nanopore residues may experience slower translocation rates compared to monomers that form weaker interactions (e.g., non-polar interactions); (4) the charge of the selected modification - rings with higher net negative charge will experience higher translocation rates when a voltage is applied (compared to rings with lower net negative charge).

[0207] In some embodiments, phosphoramidite analogs can be assembled into polymers (polyphosphates) using oligonucleotide synthesis methods (e.g., phosphoramidite methods). Examples of polyphosphates can include:

[0208]

[0209]

[0210] Where X 1 =O - , OMe or S - , and a is 1-100. In some embodiments, a is 1-5, 6-10, 11-15, 16-20, 20-25, 26-50 or 50-100, or a combination of any of the foregoing ranges.

[0211] In some embodiments, the polypeptide can be a homologous polypeptide or a heterologous polypeptide.

[0212]

[0213] wherein a is 1-100. In some embodiments, a is 1-5, 6-10, 11-15, 16-20, 20-25, 26-50, or 50-100, or a combination of any of the foregoing ranges. The polypeptide may comprise natural and non-natural amino acid residues, including non-exhaustive examples of residues selected from:

[0214] L / D- Natural amino acids

[0215]

[0216] L / D-unnatural amino acids

[0217]

[0218]

[0219] Non-amino acids

[0220]

[0221]

[0222] In some embodiments, the modified oligonucleotides in the SP may include modified nucleotides and / or modified nucleobases. In some embodiments, examples of modified nucleotides and modified nucleobases include, but are not limited to:

[0223] Modified nucleotides

[0224]

[0225] Modified nucleobases

[0226]

[0227] In some embodiments, the polyamide compound can be a homopolyamide or a heteropolyamide.

[0228]

[0229] The polyamide compound may include one or more of the following residues:

[0230] Polyamide

[0231]

[0232]

[0233] In some embodiments, the spacer may include a reporter portion corresponding to a specific nucleobase. In other embodiments, the spacer may not include a reporter portion.

[0234] In some embodiments, the cleavable cyclic cyclic nucleotide can also include a capture construct configured to interact with the nanopore. The capture construct can include a linear, branched or cyclic polymer, wherein the polymer is selected from a synthetic hydrophobic polymer, a synthetic hydrophilic polymer, an oligonucleotide / polynucleotide, a peptide / polypeptide and a combination thereof. In some embodiments, the capture construct can be a side branch attached to a cleavable cyclic cyclic nucleotide. In some embodiments, the capture construct can be a side branch attached to the core base of a nucleotide / nucleotide analog. In some embodiments, the capture construct can be a side branch attached to the cyclic ring on the spacer SP or the linking group L1 or L2. In some embodiments, the capture construct can be integrated into the cyclic ring structure or be a part of the cyclic ring structure. In some embodiments, the capture construct can be adjacent to the reporter in the cyclic ring structure.

[0235] When the capture construct is attached to the spacer SP portion of the cyclic ring, the capture construct can be attached to any portion of the spacer, for example, in the middle of the spacer chain, on either end of the spacer chain, or anywhere between the spacer chains. In embodiments where the capture construct is attached to the middle of the cyclic ring structure, the cyclic ring can be a symmetrical ring. In embodiments where the capture construct is attached to other portions of the cyclic ring structure, the cyclic ring can be an asymmetric ring.

[0236] The capture construct can be attached to the cleavable cyclic cyclic nucleotide via a third linker group L3. In some embodiments, L3 can be a portion selected from the group consisting of: a hydrophilic polymer (polyethylene glycol, polyvinyl alcohol, polyacrylamide, polyvinyl pyrrolidone, polystyrene sulfonate, polyethylene imine), a hydrophobic polymer (polylactic acid, polymethyl methacrylate, polystyrene), an oligonucleotide, a peptide, a polypeptide, an aliphatic chain (C5 to C50), an aromatic group (phenyl or pyridyl), and combinations thereof.

[0237] The cyclic ring comprises one or more sub-elements, including a linker, a conjugated moiety, a spacer, a capture construct and a reporter. The arrangement of the sub-elements of the cyclic ring can be asymmetric (i.e., asymmetric cyclic ring - ACL) or symmetric about the order of the sub-elements (i.e., symmetric cyclic ring - SCL). Fig.93 Illustrate non-limiting examples of ACLs, each cyclic ring comprises any number of conjugated moieties, spacers, reporters, and capture constructs (ARCs). Asymmetric cyclic rings are polymer rings in which the sequence of individual sub-elements or the entire composition is not arranged in a symmetrical manner. When oriented within the read head of a nanopore, ACLs can therefore affect physiochemical properties, residence time, retention, and voltage required for translocation. In contrast, symmetric cyclic rings include cyclic rings in which the subunits are symmetrically arranged. Fig.94Non-limiting examples of SCLs are illustrated, each comprising any number of conjugated moieties (handles), spacers, reporters, and capture constructs (ARCs). ACL and SCL structures may feature any number of the subelements described herein, and specific subelements may be omitted without limitation.

[0238] Model α-phosphate substituted nucleotides: imino-P and other substitutions

[0239] In some embodiments, compared with naturally occurring nucleotides, the constituent groups in the nucleotides may have one or more substitutions. In some embodiments, one or more phosphate groups comprising nucleotides are substituted. In some embodiments, one or more atoms in the nucleotide structure may be substituted by its heteroatom, heteroatom isotope or homoatomic isotope. In some embodiments, stable sulfonyl imino-P substitutions ("imino-P") of nucleotides substituted with α-phosphate are provided herein. The above text shows a bifunctional nucleotide comprising imino-P substitutions at α-phosphate.

[0240]

[0241] Other useful nucleotides having substitutions at the alpha phosphate include:

[0242]

[0243] In some embodiments, the nucleotides described herein are stable for 2 days at 25°C. In some embodiments, the nucleotides described herein are stable for 2 days at -25°C. In some embodiments, the nucleotides described herein are stable for 2 days at -25°C to 25°C. In some embodiments, the nucleotides described herein are stable for 2 days at -25°C to 25°C. In some embodiments, the nucleotides described herein are stable for 2 days at -25°C to 25°C. In some embodiments, the nucleotides described herein are stable for 2 days at -50°C to 50°C. In some embodiments, the nucleotides described herein are stable for 2 days at 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, and 50°C. In some embodiments, the nucleotides described herein are stable for 0 to 2 days at 25°C. In some embodiments, the nucleotides described herein are stable for 0 to 2 days at -25°C. In some embodiments, the nucleotides described herein are stable for 0 to 2 days at -25°C to 25°C. In some embodiments, the nucleotides described herein are stable for 0 to 2 days at -25°C to 25°C. In some embodiments, the nucleotides described herein are stable for 0 to 2 days at -25°C to 25°C. In some embodiments, the nucleotides described herein are stable for 0 to 2 days at -50°C to 50°C. In some embodiments, the nucleotides described herein are stored for 0 to 2 days at 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, and 50°C. In some embodiments, the nucleotides described herein are storage stable at 25°C. In some embodiments, the nucleotides described herein are storage stable at -25°C. In some embodiments, the nucleotides described herein are storage stable between -25°C and 25°C. In some embodiments, the nucleotides described herein are storage stable between -25°C and 25°C. In some embodiments, the nucleotides described herein are storage stable between -50°C and 50°C.In some embodiments, the nucleotides described herein are storage stable at 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, and 50°C.

[0244] Cleavable site

[0245] Cleavable cyclic cyclic nucleotides include certain cleavable sites in the bond, which can be broken under controlled conditions, such as, for example, selective cleavage of thiol phosphorothioate bonds, photocleavable bonds, phosphoramidite bonds, phosphoramide bonds, 3'-OBD-ribofuranosyl-2' bonds, thioether bonds, selenoether bonds, sulfoxide bonds, disulfide bonds, deoxyribosyl-5'-3' phosphodiester bonds or ribosyl-5'-3' phosphodiester bonds and other cleavable bond conditions known in the art. Selective cleavable bonds can be bonds within the tether or between probes or core base residues or internal bonds, or can be bonds formed by hybridization between probes and template strands. Selective cleavable bonds are not limited to covalent bonds, and can be non-covalent bonds or associations, such as those based on hydrogen bonds, hydrophobic bonds, ionic bonds, π-bond ring stacking interactions, van der Waals interactions, etc.

[0246] For example, in some embodiments, the cleavable sites include PY bonds / links in cleavable cyclic nucleotide structures (I) and (II), PN bonds / links in structure (III), and OC (ribose 5'-C) bonds / links in structures (IV), (V), and (XII). These bonds are shown below as bold bonds and can be cleaved under conditions known in the art.

[0247]

[0248] wherein X, X', Y, L1, L2, SP and base are as defined above.

[0249] Figure 3 Schematically illustrate an embodiment of a cleavable connection. The dashed arc represents a possible linker construct conjugated to 2 segments / positions of the same nucleotide. As disclosed herein, there are several attachment points on the nucleotide to which a linker construct (e.g., a cyclic ring) can be conjugated, and the cleavage site must be located within two attachment points.

[0250] There are a variety of cleavable linkages that can be easily incorporated into the existing structure of an oligonucleotide without resulting in a significant increase in the length of the DNA backbone. Figure 3 , the cleavable linkages / bonds in various nucleotides / nucleotide analogs are circled (shown as: ). The following is the cracking Figure 3 The conditions for these bonds for the nucleotides / nucleotide analogs shown in (from left to right):

[0251] 1) Phosphoramidates - The circled bonds can be cleaved under acidic conditions (e.g., 10 mM sodium citrate-HCl, pH 4.0 or 80% acetic acid).

[0252] 2) Thiol phosphorothioates - The circled bonds can be cleaved with 50 mM silver nitrate or iodine in aqueous acetone / pyridine (1:1).

[0253] 3) The allyl-circle bond can be cleaved with Pd(0) salts. In one example, Figure 4 Schematic illustration of the allyl cleavable chemistry.

[0254] 4) The phosphodiester-circled bond can be cleaved by enzymatic cleavage (e.g., endonucleases, exonucleases, or RNases, alkaline conditions in the case of RNA ribose).

[0255] Method for preparing cyclic cyclic nucleotides

[0256] Cyclic cyclic nucleotides can be prepared by conjugating a spacer moiety to a bifunctional nucleotide. In some embodiments, the conjugation of the spacer moiety and the bifunctional nucleotide involves click chemistry. The bifunctional nucleotide design can be derived from the following:

[0257]

[0258] R1 and R2 are reactive groups that can be conjugated to the spacer SP using click chemistry. In some embodiments, R1 and R2 include click chemistry reagents. In some embodiments, R1 and R2 can independently be hydroxyl, thiocyanate, aldehyde, carboxyl, azide (-N3), amine (-NH2), alkyne, bicyclononene (BCN), dibenzocyclooctyne (DBCO), thiol (-SH), tetrazine, trans-cyclooctyne (TCO), NHS ester, imidoester, pentafluorophenyl ester, hydroxymethylphosphine, carbodiimide, maleimide, haloacetyl, pyridyl disulfide, thiosulfonate, vinyl sulfone, hydrazide, alkoxyamine, isocyanate, phosphine and norbornene. In some embodiments, R1 and R2 are the same. In other embodiments, R1 and R2 may be different.

[0259] X can be -O-, -CH2-, -NH-, X' can be =N-SO2-, =NH-CO- or Y can be -O-, -S-, -NH- or -Se-. L' is a joint. In some embodiments, joint L' can be independently selected from the group consisting of the following: hydrophilic polymers, hydrophobic polymers, oligonucleotides, peptides, polypeptides, aliphatic chains (C5 to C50) and combinations thereof. In some embodiments, hydrophilic polymers, hydrophobic polymers, oligonucleotides and polypeptides can each have 1 to 100 repeating units. The hydrophilic polymer can include polyethylene glycol, polyvinyl alcohol, polyacrylamide, polyvinyl pyrrolidone, polystyrene sulfonate, polyethylene imine or a combination thereof. The hydrophobic polymer can include polylactic acid, polymethyl methacrylate or polystyrene or a combination thereof. In some embodiments, joint L' can include a reporter encoding a related nuclear base. In some embodiments, joint L' can also further include a capture construct, which is configured to interact with the nanopore to slow down the translocation of the polynucleotides incorporated therein.

[0260] Representative examples of bifunctional nucleotides include:

[0261]

[0262] In order to be conjugated with the spacer part to form a cyclic cyclic nucleotide, the spacer part includes a spacer SP and a reactive group R1' and R2' at both ends of the spacer part that needs to be conjugated with a bifunctional nucleotide. In some embodiments, R1' and R2' can be selected from the group consisting of the following: hydroxyl, thiocyanate, aldehyde, carboxyl, azide (-N3), amine (-NH2), alkynes, bicyclononene (BCN), dibenzocyclooctyne (DBCO), thiol (-SH), tetrazine, trans-cyclooctyne (TCO), N-hydroxysuccinimide (NHS) ester, imidoester, pentafluorophenyl ester, hydroxymethyl phosphine, carbodiimide, maleimide, haloacetyl, pyridyl disulfide, thiosulfonate, vinyl sulfone, hydrazide, alkoxyamine, isocyanate, phosphine and norbornene. In some embodiments, R1' and R2' are identical. In other embodiments, R1' and R2' can be different.

[0263] In some embodiments, the spacer portion may also be included on one or both sides of the SP, such as a linker L" between the SP and R1' and / or between the SP and R2'. In some embodiments, each of the linkers L" may be independently selected from the group consisting of: a hydrophilic polymer, a hydrophobic polymer, an oligonucleotide, a peptide, a polypeptide, an aliphatic chain (C5 to C50), and a combination thereof. In some embodiments, the hydrophilic polymer, the hydrophobic polymer, the oligonucleotide, and the polypeptide may each have 1 to 100 repeating units. The hydrophilic polymer may comprise polyethylene glycol, polyvinyl alcohol, polyacrylamide, polyvinyl pyrrolidone, polystyrene sulfonate, polyethylene imine, or a combination thereof. The hydrophobic polymer may comprise polylactic acid, polymethyl methacrylate, or polystyrene, or a combination thereof. In some embodiments, the spacer portion may also comprise a capture construct designed to slow down the translocation of the polynucleotide through the nanopore.

[0264] To form a cyclic cyclic nucleotide, R1 and R2 of the bifunctional nucleotide react with R1' and R2' of the SP, respectively, to form a conjugated moiety. In some embodiments, the conjugated moieties (e.g., R1-R1' and R2-R2') can be independently selected from a non-exhaustive list of chemical substances, such as: amine-NHS esters, amine-imidates, amine-pentafluorophenyl esters, amine-hydroxymethylphosphines, carboxyl-carbodiimides, thiol-maleimides, thiol-haloacetyl groups, thiol-pyridyl disulfides, thiol-thiosulfonates, thiol-vinyl sulfones, aldehyde-hydrazides, aldehyde-alkoxyamines, hydroxy-isocyanates, azide-alkynes, azide-phosphines, trans-cyclooctenes-tetrazines, norbornene-tetrazines, azide-cyclooctynes, and azide-norbornene. Examples of reactive groups R1 / R2, R1' / R2' and the resulting conjugated moieties formed by R1-R1' or R2-R2' are shown in the table below. As shown in this table, R and R' indicate two groups connected by a reaction between R1 and R1' or R2 and R2'. One of the two groups may represent a bifunctional nucleotide, and the other group may represent a spacer moiety.

[0265] Table 1

[0266]

[0267] The spacer part can be produced by one or more synthesis schemes, including solid phase, solution / liquid phase and enzyme-based synthesis. Exemplary synthesis methods include polymers synthesized using phosphoramidite chemistry, peptide synthesis, click chemistry and other bioconjugation methods known in the art, which can result in the formation of phosphodiester, methylphosphonate or thiophosphate bonds between each part. In some embodiments, the synthesis of the spacer part can be carried out linearly. In some embodiments, the synthesis of the spacer part can be carried out by branching. In some embodiments, the synthesis of the spacer part is carried out by the connection of a specific segment, which includes one or more sub-elements. In linear synthesis, the sub-element (monomer) of composition can be added to the growing chain of the sub-element including the spacer part. In some embodiments, the start of synthesis starts at or near one end of the spacer part, wherein the first reactive group is configured to be attached to a bifunctional nucleotide, and ends at or near the other end of the spacer part, wherein the second reactive group is configured to be attached to a bifunctional nucleotide. In some embodiments, the first reactive group and the second reactive group are chemically different. In some embodiments, the first reactive group and the second reactive group are chemically identical.

[0268] In contrast, branched synthesis (ie, branching) includes synthetic schemes in which two or more branches (ie, arms) of a spacer moiety are generated from any starting subunit to produce the resulting spacer moiety on a solid or liquid support.

[0269] In some embodiments, solid phase synthesis is used to synthesize the spacer part. In solid phase synthesis, the polymerization spacer part is synthesized on a solid support in a stepwise manner, and the subsequent sub-element (monomer) is added to the growing polymer chain. In some embodiments, solution phase synthesis is used to synthesize the spacer part. In solution phase synthesis, the spacer part is gradually synthesized in solution, and the sub-element (monomer) is assembled on the growing polymer chain. In some embodiments, enzymatic synthesis is used to synthesize the spacer part. In enzymatic synthesis, enzymes are used to synthesize the polymerization spacer part in a stepwise method. Enzymes can be used to connect specific sub-elements (monomers) to specific chemical bonds. In some embodiments, the spacer part is synthesized using a combination of one or more of the following: solid phase synthesis, solution phase synthesis and enzyme synthesis.

[0270] In some embodiments, the Fig.90 The solid phase synthesis route shown in is used to synthesize an exemplary symmetric spacer portion with a capture construct. In some embodiments, a solid phase synthesis route such as Fig.91 An exemplary asymmetric spacer portion with a capture construct was synthesized using the solid phase synthesis route shown in .

[0271] In some embodiments, an exemplary symmetrical spacer moiety with a capture construct can be synthesized by linking the segments in a 2-step process, where a polymer chain compound 1 containing a cyclic ring is attached to the capture construct mPEG2 in step 1, and an azido conjugate moiety is attached in step 2 to produce compound 3 (see Fig.92 ).

[0272] Subchain

[0273] Compounds (Ia), (IIa), (IIIa), (IVa), (Va) and (XIIa) can be used as cyclic cyclic nucleotides for synthesizing daughter chains. The daughter chain comprises at least one of compounds (I), (II), (III), (IV), (V) and (XII). In some embodiments, the daughter chain is formed by connecting multiple compounds (I), multiple compounds (II), multiple compounds (III), multiple compounds (IV), multiple compounds (V) or multiple compounds (XII) together into a polynucleotide or oligonucleotide. The daughter chain comprises one of the following structures:

[0274]

[0275]

[0276] wherein X, X', Y, L1, L2, SP and bases are as defined above, and adjacent bases on a daughter strand may be different or the same.

[0277] In some embodiments, structure (VII) can also be represented by the following structure:

[0278]

[0279] In some embodiments, structure (VIII) can also be represented by the following structure:

[0280]

[0281] In some embodiments, structure (X) can also be represented by the following structure:

[0282]

[0283] Cleavage of cyclic cyclic nucleotides

[0284] The daughter strand can be further subjected to conditions disclosed herein suitable for cleaving cyclic cyclic nucleotides, thereby extending the daughter strand to form an extended polynucleotide:

[0285]

[0286]

[0287] wherein X, X', Y, L1, L2, SP and base are as defined above, and adjacent bases on the extended polymer chain may be different or the same.

[0288] Cleavable Circular 4-mer Oligonucleotide

[0289] In some embodiments, the two ends of the joint construct can be conjugated with 2 core bases of a 4-mer oligonucleotide to form a cleavable cyclic ring 4-mer oligonucleotide. The cleavable bond of the allyl group can be located at any position along the oligonucleotide backbone between two conjugation points, and the cyclic ring is allowed to open when cracked. In some embodiments, the conjugation point can be the first base and the second base, the first base and the third base, the first base and the fourth base, the second base and the third base, the second base and the fourth base or the third base and the fourth base. For example, when the conjugation point is the first base and the second base, the allyl cleavage site can be located on the main chain between the first base and the second base. When the conjugation point is the first base and the third base, the allyl cleavage site can be located on the main chain between the first base and the third base. When the conjugation point is the first base and the fourth base, the allyl cleavage site can be located on the main chain between the first base and the third base. When the conjugation point is the first base and the fourth base, the allyl cleavage site can be located on the main chain between the first base and the fourth base. When the conjugation point is the second base and the third base, the allylic cleavage site can be located on the main chain between the second base and the third base. When the conjugation point is the second base and the fourth base, the allylic cleavage site can be located on the main chain between the second base and the fourth base. When the conjugation point is the third base and the fourth base, the allylic cleavage site can be located on the main chain between the third base and the fourth base.

[0290] In some embodiments, when the conjugation point is the first base and the second base, the allyl group can be located at C5' of the second nucleotide. When the conjugation point is the first base and the third base, the allyl group can be located at C5' of the second nucleotide or the third nucleotide. When the conjugation point is the first base and the fourth base, the allyl group can be located at C5' of the second nucleotide, the third nucleotide or the fourth nucleotide. When the conjugation point is the second base and the third base, the allyl group can be located at C5' of the third nucleotide. When the conjugation point is the second base and the fourth base, the allyl group can be located at C5' of the third nucleotide or the fourth nucleotide. When the conjugation point is the third base and the fourth base, the allyl group can be located at C5' of the fourth nucleotide. In some embodiments, heavy atom-substituted nucleotides can be used to produce the resulting 4-mer oligonucleotides.

[0291] Examples of cleavable circular 4-mer oligonucleotides include:

[0292]

[0293]

[0294] Wherein L1 is the first linking group; L2 is the second linking group; R 1 , R 2 and R 3 One of the R is allyl and the other is H; SP is a spacer; and the base is selected from the group consisting of adenine, cytosine, guanine, thymine and uracil.

[0295] According to the conjugation chemistry on the nucleotide, the cyclic ring can be designed symmetrically or asymmetrically. In some embodiments, each of the first linking group L1 and the second linking group L2 independently comprises a conjugated portion selected from the group consisting of the following: amine-NHS ester, amine-imidate, amine-pentafluorophenyl ester, amine-hydroxymethylphosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-hydrazide, aldehyde-alkoxyamine, hydroxyl-isocyanate, azide-alkyne, azide-phosphine, trans-cyclooctene-tetrazine, norbornene-tetrazine, azide-cyclooctyne and azide-norbornene. L1 and L2 can be the same or different.

[0296] In some embodiments, each of the first linking group L1 and the second linking group L2 may also independently include a joint. The first joint may be present between the base in the conjugated moiety and the 4-mer oligonucleotide, and the second joint may be present between another base in the conjugated moiety and the 4-mer oligonucleotide. In some embodiments, the joint may be selected from the group consisting of the following: hydrophilic polymer (polyethylene glycol, polyvinyl alcohol, polyacrylamide, polyvinyl pyrrolidone, polystyrene sulfonate, polyethylene imine), hydrophobic polymer (polylactic acid, polymethyl methacrylate, polystyrene), oligonucleotide, peptide, polypeptide, aliphatic chain (C5 to C50) and their combination. In some embodiments, the first joint and the second joint may independently include peptide, polypeptide, alkyl chain, polyethylene glycol or their combination. In some embodiments, one or more joints may be lacking.

[0297] In some embodiments, the SP (spacer) comprises a polymer. The spacer within the cyclic ring provides a buffer distance between continuous cyclic rings or continuous sub-elements comprising one or more cyclic rings. In some embodiments, the polymer in the SP comprises an oligonucleotide, a modified oligonucleotide, a hydrophilic polymer (polyethylene glycol, polyvinyl alcohol, polyacrylamide, polyvinyl pyrrolidone, polystyrene sulfonate, polyethylene imine), a hydrophobic polymer (polylactic acid, polymethyl methacrylate, polystyrene), a polypeptide, an aliphatic chain (C5 to C50), a substituted aliphatic chain (small molecules, such as chlorine, bromine or fluorine, an alkyl group, such as methyl, ethyl or propyl, or an aromatic group, such as phenyl or pyridyl), or a combination thereof. In some embodiments, the modified oligonucleotide may include an oligonucleotide that does not have a base attached to a sugar, such as In some embodiments, the modified oligonucleotides may include phosphoramidite analogs such as It can be assembled into a polymer using oligonucleotide synthesis methods. In some embodiments, the spacer may include a reporter portion corresponding to a specific nucleobase. In other embodiments, the spacer may not include a reporter portion (barcode).

[0298] In some embodiments, the cyclic ring may contain one or more barcodes or reporters, including reporter moieties, sub-reporters, reporter elements, and sub-reporter elements. Examples include, but are not limited to, nucleobases, non-nucleobases, peptides or other synthetic polymers, such as polyethylene glycol, polyvinyl alcohol, polyacrylamide, polyvinyl pyrrolidone, polyethyleneimine, etc. In some embodiments, the reporter element may include a macromolecule, such as a crown ether, cucurbituril, pillararene, or cyclodextrin. It is possible to conjugate these macromolecules to the cyclic ring structure through covalent conjugation chemistry, such as amine-NHS esters, amine-imidates, amine-pentafluorophenyl esters, amine-hydroxymethylphosphines, carboxyl-carbodiimides, thiol-maleimides, thiol-haloacetyl groups, thiol-pyridyl disulfides, thiol-thiosulfonates, thiol-vinyl sulfones, aldehyde-hydrazides, aldehyde-alkoxyamines, hydroxy-isocyanates, azide-alkynes, azide-phosphines, trans-cyclooctene-tetrazines, norbornene-tetrazines, azide-cyclooctene, azide-norbornene. In some embodiments, the reporter can be selected from any moiety, including spacers, conjugated moieties, and capture constructs. In some embodiments, the reporter can comprise multiple modifications ranging in length from 1-10, or 11-15, or 16-20, or 21-25, 26-50, or 50-100 units, as long as the reporter provides a reproducible signal at a given voltage waveform when residing in the read head of a nanopore.

[0299] In some embodiments, the advancement of each nucleotide carrying one or more barcodes or reporter moieties corresponds to a translocation event. In some embodiments, the reporter moiety comprises one or more sub-reporter moieties, which are configured to identify translocation events and generate signals when passing through the reader of the nanopore. In some embodiments, the reporter moiety comprises two or more sub-reporter moieties, wherein each of the two or more sub-reporter moieties is distinguishable, reproducible and resolvable. In some embodiments, the cyclic ring may comprise a first set of reporter moieties and a second set of reporter moieties, wherein the first set of reporter moieties is configured to generate signals to identify specific nucleotides passing through the reader, and wherein the second set of reporter moieties is configured to generate signals to identify the passage of each nucleotide, regardless of the nucleotide identity.

[0300] Heavy atom substituted oligonucleotides

[0301] In some embodiments, heavy atoms (e.g., atoms that are not hydrogen) can be substituted into modified nucleotides. Nucleotides based on naturally occurring triphosphates and phosphodiester bonds may be restrictive for various enzymatic processes (e.g., connection, incorporation). Heavy atoms (including, for example, sulfur and selenium) as replacements in nucleotides can allow new biochemistry and applications to crack, connect, incorporate or otherwise manipulate oligonucleotides. Heavy atom-modified nucleotides can be incorporated into or connected to the resulting modified polynucleotides.

[0302] In some embodiments, thiol phosphorothioate (bridging PS) and phosphorothioate (non-bridging PS) substitutions and methods for producing such substitutions are described. Table 2 illustrates various cleavage agents known to cleave phosphorus-sulfur bonds, including iodine and silver nitrate. In some embodiments, selenium can also be used to replace sulfur when heavy atom-modified nucleotides or oligonucleotides are produced. Selenium-modified nucleic acid analogs can also be potentially synthesized and cleaved according to the embodiments provided herein. Therefore, disclosed herein include the following oligonucleotides:

[0303]

[0304] wherein Y is -O-, -S-, -NH- or -Se-; Y 1 , Y 2 and Y 3 one of them is -S- or -Se-, and the other is -O- or -NH-,

[0305] Table 2: Phosphorus-sulfur bond cleavage conditions

[0306]

[0307] Some examples of heavy atom substituted nucleotides:

[0308]

[0309] The number of bridged PS substitutions of any arbitrary K-mer oligonucleotide can be any number between 1 and K-1. For example, for a 4-mer oligonucleotide, there can be 1 to 3 bridged PS modifications. In some embodiments, the number of bridged PS substitutions that can be introduced is 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90 and 1-100 substitutions, or any value between the values ​​of the aforementioned ranges. In some embodiments, for an oligonucleotide with a length of K, the number of bridged PS substitutions can be between 0 and K-1.

[0310] For phosphorothioates (non-bridging PS), iodine can selectively cleave the bridged PO bond at the site of phosphorothioates in the presence of nucleophiles (including, for example, amines). However, subsequent bridging PO bond cleavage is not specific, and 5' or 3' ends may be potentially cleaved. In addition, the efficiency of cleavage may be affected by the possible conversion of phosphorothioates to phosphates.

[0311] Example

[0312] Figure 5 Schematically illustrates an embodiment of an allyl-based cleavable cyclic cyclic nucleotide 501 having 10Ts as a spacer region 502, a phosphate-base linked (PBL) cyclic ring, and a capture construct 509 attached to the spacer region 502. One end of the spacer region 502 is attached to the alpha phosphate group of the nucleotide through a first linking group 503, and the other end of the spacer region 502 is attached to the base of the nucleoside through a second linking group 504. The capture construct 509 is attached to the spacer region 502 through a third linking group 505.

[0313] The capture construct 509 can be composed of one or more durable, water-soluble or solvent-soluble polymers, including but not limited to one or more of the following segments: polyethylene glycol, polyglycol, polypyridine, polyisonitrile, polyisocyanate, poly(triarylmethyl)methacrylate, polyaldehyde, polypyrrolidone, polyurea, polyglycol phosphodiester, polyacrylate, polymethacrylate, polyacrylamide, polyvinyl ester, polystyrene, polyamide, polyurethane, polycarbonate, polybutyrate, polybutadiene, polybutyrolactone, polypyrrolidone, polyvinylphosphonate, Polyacetamide, polysaccharide, polyhyaluronate, polyamide, polyimide, polyester, polyethylene, polypropylene, polystyrene, polycarbonate, polyterephthalate, polysilane, polyurethane, polyether, polyamino acid, polyglycine, polyproline, N-substituted polylysine, polypeptide, side chain N-substituted peptide, poly-N-substituted glycine, peptoid, side chain carboxyl substituted peptide, homotypic peptide, oligonucleotide, ribonucleic acid oligonucleotide, deoxynucleic acid oligonucleotide, modified to prevent Watson-Crick base pairing (Watson-Crick oligonucleotides, oligonucleotide analogs, polycytidylic acid, polyadenylic acid, polyuridylic acid, polythymidine, polyphosphates, polynucleotides, polyribonucleotides, polyethylene glycol-phosphodiester, peptide polynucleotide analogs, threonyl-polynucleotide analogs, diol-polynucleotide analogs, morpholino-polynucleotide analogs, locked nucleotide oligomer analogs, polypeptide analogs, branched polymers, comb polymers, star polymers, dendrimers, random, gradient and block copolymers, anionic polymers, cationic polymers, stem-loop forming polymers, rigid segments and flexible segments.

[0314] Figure 6 An example of sequencing an extended polymer 603 with a capture construct is schematically illustrated. Figure 5 As shown in FIG. 5 , an extended polymer 603 is formed after cleavage of the OC bond 506 of the allyl-based cleavable cyclic nucleotide 501. Figure 6 In the embodiment, a protein nanopore 601 is deposited in a lipid bilayer 602. An extended polymer 603 translocates through the nanopore 601. The polynucleotide 603 includes a spacer region 502 between consecutive nucleotides. In this embodiment, the spacer region 502 comprises a reporter portion, such as a reporter barcode (10Ts) corresponding to the nucleotide "T". When the extended polymer 603 translocates through the nanopore 601, when an interaction occurs between the capture construct 509 and the nanopore 601, the capture construct 509 slows down or temporarily stops translocation, thereby allowing the reader to read the reporter barcode, thereby identifying the nucleotide.

[0315] In other embodiments, the capture construct may be attached to different positions on the joint construct (such as attached to the first linking group or the second linking group, or a core base). The position to which the capture construct is attached, the type / size of the capture construct, the length of the spacer region / joint construct may affect which region of the polymer extended when the translocation slows down or stops is present in the nanopore contraction. In some embodiments, when the translocation slows down or stops, the core base may be located in the contraction. Therefore, the reader may also read the core base itself, regardless of whether the reporter portion is included in the spacer.

[0316] Fig. 9 Schematically illustrated is an example of a first design of a fully functional cyclic nucleotide (CLN-1), wherein X can be O, NH, NSO2 or CH2, and Y can be O, S, Se or NH, having a symmetric ring with alpha phosphate-base linkages and a capture construct on the ring. Fig.10 Schematically illustrate Fig. 9 An exemplary synthesis of a fully functional cyclic nucleotide (CLN-1A) is shown in FIG. 1 , wherein X=O and Y=O. Fig.11 Schematically illustrate Fig. 9 An exemplary synthesis of a fully functional cyclic nucleotide (CLN-1B) wherein X=NH and Y=O is shown in FIG. Fig.12 Schematically illustrate Fig. 9 An exemplary synthesis of a fully functional cyclic nucleotide (CLN-1C) wherein X=CH2 and Y=O is shown in FIG. Fig.13 Schematically illustrate Fig. 9 An exemplary synthesis of a fully functional cyclic nucleotide (CLN-1D) shown in FIG. 5 , wherein X═O and Y═S, which is compatible with phosphorothiol cleavage.

[0317] Fig.14 Schematically illustrated is an example of a second design of a fully functional cyclic nucleotide (CLN-2), wherein X can be O, NH, NSO2 or CH2, and Y can be O, S or NH, having a symmetric ring with an alpha phospho-C5' allyl linkage and a capture construct on the ring. Fig.15 Schematically illustrate Fig.14 An exemplary synthesis of a fully functional cyclic nucleotide (CLN-2A) is shown in FIG. Fig.16 Schematically illustrate Fig.14 An exemplary synthesis of a fully functional cyclic nucleotide (CLN-2B) wherein X=NH and Y=O is shown in FIG. Fig.17 Schematically illustrate Fig.14 An exemplary synthesis of a fully functional cyclic nucleotide (CLN-2C) wherein X=CH2 and Y=O is shown in FIG.

[0318] Fig.18 Schematically illustrated is an example of a third design of a fully functional cyclic nucleotide (CLN-3), wherein X can be O, NH, NSO2 or CH2, and Y can be O, S or NH, with an alpha phospho-allyl-linked asymmetric ring and a capture construct on the nucleobase. Fig.19 Schematically illustrate Fig.18 An exemplary synthesis of a fully functional cyclic nucleotide (CLN-3A) shown in FIG. 1 , wherein X=O and Y=O. Fig. 20 Schematically illustrate Fig.18 An exemplary synthesis of a fully functional cyclic nucleotide (CLN-3B) wherein X=NH and Y=O is shown in FIG. Fig.21 Schematically illustrate Fig.18 An exemplary synthesis of a fully functional cyclic nucleotide (CLN-3C) wherein X=CH2 and Y=O is shown in FIG.

[0319] Fig. 22 Schematically illustrated is an example of a fourth design of a fully functional cyclic nucleotide (CLN-4), wherein X can be O, NH, NSO2 or CH2, and Y can be O, S or NH, having an alpha phospho-allyl linked asymmetric ring and a capture construct on the ring. Fig.23 Schematically illustrate Fig. 22 An exemplary synthesis of a fully functional cyclic nucleotide (CLN-4A) shown in FIG. 4 , wherein X=O and Y=O. Fig.24 Schematically illustrate Fig. 22 An exemplary synthesis of a fully functional cyclic nucleotide (CLN-4B) wherein X=NH and Y=O is shown in FIG. Fig.25 Schematically illustrate Fig. 22 An exemplary synthesis of a fully functional cyclic nucleotide (CLN-4C) wherein X=CH2 and Y=O is shown in FIG.

[0320] Fig.26 Schematically illustrated is an example of a fifth design of a fully functional cyclic nucleotide (CLN-5), wherein X can be O, NH, NSO2 or CH2, and Y can be O, S or NH, having an asymmetric ring with an alpha phosphate-base linkage and a capture construct on the ring. Fig. 27 Schematically illustrate Fig.26 An exemplary synthesis process of a fully functional cyclic nucleotide (CLN-5A) is shown in FIG. 5 , wherein X=O and Y=O. Fig.28 Schematically illustrate Fig.26 An exemplary synthesis of a fully functional cyclic nucleotide (CLN-5B) wherein X=NH and Y=O is shown in FIG. Fig.29Schematically illustrate Fig.26 An exemplary synthesis of a fully functional cyclic nucleotide (CLN-5C) wherein X=CH2 and Y=O is shown in FIG. Fig.30 Schematically illustrate Fig.26 An exemplary synthesis of a fully functional cyclic nucleotide (CLN-5D) shown in FIG. 5 , wherein X═O and Y═S, which is compatible with phosphorothiol cleavage.

[0321] Fig.31 Schematically illustrates an embodiment of a sixth design of a cyclic cyclic nucleotide (CLN-6), wherein X can be O, NH, NSO2 or CH2, and Y can be O, S or NH, having a peptide-based cyclic ring and a capture construct on a nucleobase. The peptide-based cyclic ring can contain various amino acid residues, such as arginine, histidine, lysine, glutamic acid, aspartic acid, cysteine, tyrosine, asparagine, tryptophan, leucine and alanine. The amino acid side chain Z on the cyclic ring is depicted in Fig.31 middle.

[0322] Some other embodiments below relate to symmetrical cyclic cyclic nucleotides and cyclic k-mer oligonucleotides, which may include nucleoside bases, non-nucleoside residues, sp9, sp18, abasic nucleotides, C3 moieties or peptide residues. Some other embodiments below relate to asymmetrical cyclic cyclic nucleotides and cyclic k-mer oligonucleotides, which may include nucleoside bases and non-nucleoside residues.

[0323] Fig.32 Schematically illustrates an embodiment of a seventh design of a cyclic cyclic nucleotide (SNL-1), wherein X can be O, NH, NSO2, or CH2, having a symmetrical nucleoside ring linked by an alpha phosphate-base and a capture construct on the ring. In some embodiments, the cyclic ring may contain one or more different nucleobases. For example, nucleosides may include inosine, nitroindole, etc. In some embodiments, other nucleobases with modified sugars (e.g., locked nucleic acids (LNA), 2'-Ome, or 2'-F) may be used in the cyclic ring. The structures of non-limiting nucleobases are also depicted in Fig.32 middle.

[0324] Fig.33 Schematically illustrates an embodiment of the eighth design of a cyclic cyclic nucleotide (SNNL-1), wherein X can be O, NH or CH2, having a symmetric non-nucleoside ring with an alpha phosphate-base connection and a capture construct on the ring. The cyclic ring may include one or more of the following parts:

[0325] Alkyl (C3-C12) phosphates, polyethylene glycol phosphates, other modified non-nucleoside moieties (eg, spermine, phosphorothioates, or methylphosphonates) may be used in the ring in some embodiments.

[0326] Fig.34 Schematically illustrates an embodiment of a ninth design of a cyclic cyclic nucleotide (SPL-1), wherein X can be O, NH, NSO2 or CH2, having a symmetric peptide ring with an alpha phosphate-base connection and a capture construct on the ring. Other modified amino acid residues or moieties suitable for peptide synthesis (e.g., PEG2, 6, 11, 12 or Lys(FITC)) can be used in some embodiments.

[0327] Fig.35 Schematically illustrates an embodiment of the tenth design of a cyclic cyclic nucleotide (SNL-2), wherein X can be O, NH, NSO2 or CH2, having a symmetrical nucleoside ring linked by an alpha phosphate-allyl group and a capture construct on the ring. In some embodiments, other modified nucleoside bases such as inosine, nitroindole, LNA, 2'-Ome or 2'-F can be used.

[0328] Fig.36 Schematically illustrate an embodiment of the eleventh design of a cyclic cyclic nucleotide (SNNL-2), wherein X can be O, NH, NSO2 or CH2, having a symmetrical non-nucleoside ring connected by an α-phospho-allyl group and a capture construct on the ring. In some embodiments, other modified non-nucleoside moieties, such as alkyl (C3-C12), spermine, phosphorothioate or methylphosphonate, can be used.

[0329] Fig.37 Schematically illustrates an example of a twelfth design of a cyclic cyclic nucleotide (SPL-2), wherein X can be O, NH, NSO2 or CH2, having an α-phospho-allyl linked symmetrical peptide ring and a capture construct on the ring. Other modified amino acid residues or moieties suitable for peptide synthesis (e.g., PEG2, 6, 11, 12 or Lys(FITC)) can be used in some embodiments.

[0330] Fig.38Schematically illustrates an embodiment of the thirteenth design of a cyclic cyclic nucleotide (ASL-1), wherein X can be O, NH, NSO2 or CH2, having an asymmetric ring connected by an alpha phosphate-base and a capture construct on the ring. The asymmetric ring can be formed by a combination of nucleoside bases, non-nucleoside moieties and amino acid residues, as shown in the figure. In some embodiments, other modified nucleoside bases such as inosine, nitroindole, LNA, 2'-Ome or 2'-F can be used. In some embodiments, other modified non-nucleoside moieties such as alkyl (C3-C12), spermine, thiophosphate or methylphosphonate can be used. Other modified amino acid residues or moieties suitable for peptide synthesis (e.g., PEG2, 6, 11, 12 or Lys (FITC)) can be used in some embodiments.

[0331] Fig.39 Schematically illustrates an example of a fourteenth design of a cyclic cyclic nucleotide (ASL-2), wherein X can be O, NH, NSO2 or CH2, having an asymmetric ring with an α-phospho-C5' allyl linkage and a capture construct on the ring. The asymmetric ring can be formed by a combination of nucleoside bases, non-nucleoside moieties and amino acid residues, such as Fig.39 In some embodiments, other modified nucleoside bases such as inosine, nitroindole, LNA, 2'-Ome or 2'-F can be used. In some embodiments, other modified non-nucleoside moieties such as spC12, spermine, thiophosphate or methylphosphonate can be used. Other modified amino acid residues or moieties suitable for peptide synthesis (e.g., PEG2, 6, 11, 12 or Lys (FITC)) can be used in some embodiments.

[0332] Fig.40 Schematically illustrates an embodiment of a fifteenth design of a cyclic cyclic nucleotide (ASL-3), wherein X can be O, NH, NSO2 or CH2, having an asymmetric ring with an α-phospho-C5' allyl linkage and a capture construct on a nucleobase. The asymmetric ring can be formed by a combination of nucleobases, non-nucleoside moieties and amino acid residues, such as Fig.40 In some embodiments, other modified nucleoside bases such as inosine, nitroindole, LNA, 2'-Ome or 2'-F can be used. In some embodiments, other modified non-nucleoside moieties such as alkyl (C3-C12), spermine, thiophosphate or methylphosphonate can be used. Other modified amino acid residues or moieties suitable for peptide synthesis (e.g., PEG2, 6, 11, 12 or Lys (FITC)) can be used in some embodiments.

[0333] Fig.41Schematically illustrating an embodiment of a sixteenth design of a cyclic ring 4-mer oligonucleotide (4-mer-SNL-1) having a 4-mer oligonucleotide with a symmetrical nucleoside ring and a capture construct on the ring. The ring conjugation position can be 1-2, 1-3, 1-4, 2-3, 2-4, or 3-4 bases; the cleavage chemistry can be RNA cleavage or allyl cleavage; and the cleavage position can be the 2nd, 3rd, or 4th base.

[0334] Fig.42 Schematically illustrating an embodiment of a seventeenth design of a cyclic ring 4-mer oligonucleotide (4-mer-SNNL-1) having a 4-mer oligonucleotide with a symmetrical non-nucleoside ring and a capture construct on the ring. The ring conjugation position can be 1-2, 1-3, 1-4, 2-3, 2-4, or 3-4 bases; the cleavage chemistry can be RNA cleavage or allyl cleavage; and the cleavage position can be the 2nd, 3rd, or 4th base.

[0335] Fig.43 Schematic illustration of an eighteenth design example of a cyclic ring 4-mer oligonucleotide (4-mer-SPL-1) having a 4-mer oligonucleotide with a symmetrical peptide ring and a capture construct on the ring. The ring conjugation position can be 1-2, 1-3, 1-4, 2-3, 2-4, or 3-4 bases; the cleavage chemistry can be RNA cleavage or allyl cleavage; and the cleavage position can be the 2nd, 3rd, or 4th base.

[0336] Fig.44 An embodiment of a nineteenth design of a cyclic ring 4-mer oligonucleotide (4-mer-ASL-1) is schematically illustrated, having a 4-mer oligonucleotide with an asymmetric ring and a capture construct on the ring. The asymmetric ring can be formed by a combination of nucleoside bases, non-nucleoside moieties, and amino acid residues. The ring conjugation position can be 1-2, 1-3, 1-4, 2-3, 2-4, or 3-4 bases; the cleavage chemistry can be RNA cleavage or allyl cleavage; and the cleavage position can be the 2nd, 3rd, or 4th base.

[0337] Fig.45 An exemplary synthesis process of one embodiment of a 4-mer oligonucleotide that can be used to prepare a cleavable circular 4-mer oligonucleotide is schematically illustrated.

[0338] Example 1: Synthesis and incorporation of nucleotides with 5'-allyl capture constructs into modified polynucleotides middle

[0339] The synthesis of nucleotides with 5'-allyl capture constructs is described, and the subsequent demonstration that the modified nucleotides can be incorporated by a polymerase followed by cleavage of the allyl group.

[0340] Figure 7 Unexpected and successful experimental results showing polymerase incorporation of allyl dTTP. Figure 7 The polymerase incorporates "allyl dTTP" and "extallyl dTTP" into the template and primer combination up to 10 times in succession, as indicated by the arrows in FIG. The reaction conditions and the definitions of "allyl dTTP" and "extallyl dTTP" are described in Figure 7 In addition, "A" and "B" refer to the optically pure diastereomers generated from the C5' allylic carbon center.

[0341] Figure 7 Twelve lanes in the gel image are shown. Lanes 1 (from the left), Lane 3, Lane 5, Lane 7, Lane 9, and Lane 11 were loaded with reactions run for one hour; and Lanes 2, Lane 4, Lane 6, Lane 8, Lane 10, and Lane 12 were loaded with reactions run for 22 hours. Lanes 1 and 2 show: controls with native dTTP without any capture constructs (note that a band above +10 is observed due to the use of a homopolymeric A template, which is a common observation). Lanes 3 and 4 show: use of an isomer of allyl dTTP ("Allyl A dTTP") that was generated at the C-stereocenter (at Figure 7 ). Up to +7 incorporations were observed after 1 hour. The extended duration of 22 hours shows exo-activity. Lanes 5 and 6 show: One isomer of Ext Allyl dTTP was used ("Ext Allyl A dTTP"). Up to +7 incorporations were observed after 22 hours. The slower incorporation may be due to the increased space. Lanes 7 and 8 show: Another isomer of Allyl dTTP was used ("Allyl B dTTP"). Up to +10 incorporations were observed after 22 hours. Lanes 9 and 10 show: Another isomer of Ext Allyl dTTP was used ("Ext Allyl B dTTP"). Up to +7 incorporations were observed after 22 hours. Lanes 11 and 12 show: Negative control without any nucleotide added.

[0342] Figure 8Unexpected and successful experimental results showing the cleavage of the allyl group. After incorporation of nucleotides, the samples were treated with 100mM Pd / THP solution at 37°C for 1 hour to cleave the allylic bond. Lanes 1, 3, 5, 7 and 9 were loaded with samples after incorporation, but not treated with Pd / THP. Lanes 2, 4, 6, 8 and 10 were loaded with samples after treatment with Pd / THP. Lanes 1 and 2 show that there is no allyl group, so there is no change with or without Pd / THP treatment. Lane 3 compared to Lane 4, Lane 5 compared to Lane 6, Lane 7 compared to Lane 8 and Lane 9 compared to Lane 10 show that the higher band was reduced back to the baseline with Pd / THP treatment, proving the successful cleavage of the allyl group.

[0343] Example 2: Generation of modified polynucleotides using bridging PS nucleotides and subsequent cleavage

[0344] The synthesis of modified nucleotides with bridging PS substitutions is described, and it is subsequently demonstrated that the modified nucleotides can be incorporated by a polymerase followed by cleavage of the phosphorus-sulfur bond.

[0345] a. Synthesis of bridged PS nucleotides

[0346]

[0347] according to Fig.46 Heavy atom substituted nucleotides with bridging PS substitutions were prepared according to the synthetic scheme shown in . Fig.47A and Fig.47B Mass spectra and NMR spectra of examples of such nucleotides are shown.

[0348]

[0349] Bifunctional nucleotides with bridging PS substitutions can also be prepared according to Fig.48 Prepared according to the reaction scheme.

[0350] b. Incorporation of heavy atom substituted nucleotides to generate modified polynucleotides

[0351] Fig.49 Results of incorporation of a poly-A tail formed by bridging PS (phosphorothiol) deoxyribonucleotides (nucleotide 1) to generate a DNA oligonucleotide with a continuous phosphorothiol backbone are shown. Specifically, incorporation was tested using a variety of polymerases including Pol812, 1901, hPrimpol, and BSU. For each polymerase tested, a positive signal was observed for each heavy atom substituted nucleotide incorporated into the oligonucleotide chain.

[0352] c. Cleavage of phosphorothiol heavy atom modified nucleotides

[0353] Fig.50Ais a reaction scheme for cleavage of the phosphorus-sulfur bond by silver nitrate of the bridged PS nucleotide 1. Silver nitrate is added to nucleotide 1, and 15 minutes later DTT is added and incubated for 1 hour. The cleavage by silver nitrate is specific for the PS bond, thereby generating a 5'-S nucleoside (e.g. Fig.50A ). Fig.50B Illustrate HPLC chromatograms of nucleotides (control) and cleavage products after DTT addition and ESI-MS data of cleavage products.

[0354] The cleavage reaction described above was applied to the primers extended with the bridging PS nucleotide 1 and the DNA polymerase Pol1901 (see Fig.51 Lane 4). Gel analysis showed that silver nitrate and silver nitrate + DTT were able to cleanly cleave the oligonucleotides, respectively. Fig.51 The results are shown in lanes 5 and 6.

[0355] Example 3: Generation of 4-mer oligonucleotides with bridging PS substitutions

[0356] The synthesis and characterization of oligonucleotides with PS-bridging substitutions are described.

[0357] a. Synthesis of bridging PS oligonucleotides

[0358] The bridge PS substitutions found in 4-mer oligonucleotides are structurally similar to the bridging PS nucleotide positioning. The number of bridge PS substitutions that can be introduced into the growing chain at a time by a k-mer oligonucleotide can be any number between 1 and k-1. For example, for a 4-mer oligonucleotide, there can be 1 to 3 bridge PS substitutions. PS substitutions are introduced into 4-mer oligonucleotides by 5'SDMT phosphoramidite 9. Fig.52 The synthesis of 5'SDMT phosphoramidite 9 by three methods is schematically illustrated. Then 5'SDMT phosphoramidite 9 is introduced into a 4-mer oligonucleotide and ligated, thereby generating a substitution of the 4-mer oligonucleotide by 5'SDMT phosphoramidite 9. Fig.53A As shown in , post-synthetic modifications can be added, such as conjugation of a cyclic loop on the modified oligonucleotide product. A 4-mer oligonucleotide with a bridging PS substitution and a cyclic loop attached to the first and last nucleobases of the 4-mer oligonucleotide (oligonucleotide 10) is shown in Fig.53B middle. Fig.54A -C illustrates the HPLC, IR and MS characterization of the resulting cyclic 4-mer oligonucleotide.

[0359] b. Ligation / cleavage of bridging PS oligonucleotides

[0360] Wild-type ligase Ame was used to ligate up to 10 cyclic-loop-modified 4-mer oligonucleotides10. Fig.55This indicates that the wild-type ligase Ame is able to ligate up to 10 circular 4-mer oligonucleotides 10 after 2.5 hours, with improved ligation efficiency at 22 hours. The signal from the ligated oligonucleotide is most strongly observed in lane 6 corresponding to 22 hours.

[0361] Fig.56 This suggests that upon successive ligation, cleavage of newly synthesized chains produces longer chains that run slower (higher in position) in the gel due to the influence of the incorporated PEG ring (and extension following selective cleavage of the polynucleotide backbone).

[0362] Thus, Example 3 illustrates the successful synthesis of a cyclic 4-mer oligonucleotide 10, and the cyclic ring remains closed during the ligation process. In addition, the ligation successfully produces a new synthetic chain, and finally, the new synthetic chain can be selectively and efficiently cleaved using silver nitrate to produce an extended chain.

[0363] Example 4: Generation of modified polynucleotides using non-bridging PS nucleotides and subsequent cleavage

[0364] a. Synthesis of non-bridging PS nucleotides

[0365]

[0366] Prior to enzyme incorporation, the modified nucleotide must be synthesized. Fig.57A The phosphorothioate (non-bridged PS) triphosphate nucleotides shown above were prepared according to the synthetic scheme in. Fig.57B The synthetic scheme in prepares phosphorothioate (non-bridging PS) nucleotide triphosphates functionalized at the alpha phosphate.

[0367] b. Incorporation of non-bridging PS nucleotides

[0368] Non-bridging PS nucleotides are incorporated by several wild-type polymerases, such as Fig.58 As shown in , including 812, Dpo4, DbH, DinB, Asfy, Mu, lambdai, 8k beta, human PrimPoli, sulfoPrimPol, 300 cyanoPrimPol, 190 cyanoPrimPol, BSU, RB49 and PolC.

[0369] Example 5: Generation of 4-mer polynucleotides with non-bridging PS substitutions

[0370] Described herein is the synthesis and characterization of polynucleotides having PS non-bridging substitutions.

[0371] a. Synthesis of non-bridging PS oligonucleotides

[0372] Fig.59 and Fig.60A-D schematically illustrates a 4-mer oligonucleotide 13, which was synthesized using a sulfurizing agent such as DDT to generate a phosphorothioate modification at the site of interest, wherein the resulting diastereomers were separated by HPLC.

[0373] b. Ligation of non-bridging PS oligonucleotides

[0374] Fig.61 Wild-type Ame and PsyA ligases were shown to be able to perform up to 10 consecutive ligation events with the circular 4-mer oligonucleotide 13 in 20 hours.

[0375] c. Cleavage of non-bridging PS oligonucleotides

[0376] Fig.62A This shows that iodine is used to selectively cleave the bridged PO bond at the site of the phosphorothioate in the presence of a nucleophile such as an amine. However, the subsequent cleavage of the bridged PO bond is not specific, and either the 5' or 3' end can potentially be cleaved, resulting in four potential products ( Fig.62B ).

[0377] Example 6: Nucleotides with imino-P substitution at the alpha phosphate

[0378] Fig.63 Schematic illustration of a model synthesis of imino-P substitution on the alpha phosphate of a nucleotide. The imino-P nucleotides were stable in 50 mM KH2PO4 buffer at pH 5.5 and 7.5 over a 2-day period at 25°C. No degradation of the imino-P was observed after 2 days at both pH 5.5 and 7.5.

[0379] Fig.64 The synthesis of cyclic cyclic nucleotides with imino-p substitution according to one embodiment of the present invention is schematically illustrated. Three stages are illustrated: precursor synthesis (introduction of an allyl cleavable group), imino-P synthesis and deprotection of triphosphate nucleotides, followed by exemplary cyclic ring conjugation using a bisazido PEG20 linker. Imino-P nucleotides are stable in 50 mM KH2PO4 buffer at pH 5.5 and 7.5 at 25°C for 2 days. Fig.65 HPLC and LCMS characterization of imino-P nucleotides is illustrated.

[0380] Example 7: Obtaining imino-P-allyl difunctional triphosphoric acid

[0381] Various methods for obtaining imino-p-difunctional triphosphates were evaluated.

[0382] Fig.66One embodiment of a method for synthesizing imino-P-allyl bifunctional nucleotides is schematically illustrated. 5'-OH allyl nucleoside 1 is activated by a phosphitylation agent to form a first intermediate 2, which is then activated and coupled with pyrophosphate to form a second phosphite triester intermediate 3. Staudinger reaction is performed with an azide moiety to convert the unstable phosphite triester 3 into a third imino-P-phospho triester intermediate 4. Deprotection is then performed to remove all orthogonal protecting groups to form imino-P-allyl bifunctional triphosphate 6.

[0383] Fig.67 One embodiment of a method for synthesizing imino-P-allyl bifunctional nucleotides is schematically illustrated. 5'-OH allylic nucleoside 1 is coupled with an activated phosphitylation agent to form a first cyclic phosphite triester intermediate 2, which is then subjected to a Staudinger reaction with an azide functional group to form a second imino-P-phosphotriester intermediate 4. Deprotection is then performed to remove all orthogonal protecting groups, thereby forming imino-P-allyl bifunctional triphosphate 6.

[0384] Fig.68 An embodiment of a method for synthesizing imino-P-allyl bifunctional nucleotides is schematically illustrated. 5'-OH allyl nucleoside 1 is coupled with a phosphitylation agent and hydrolyzed to form a first H-phosphonate intermediate 2. It is subjected to a continuous BSA treatment and a Staudinger reaction with an azide functional group to form a second imino-P-phosphotriester intermediate 3. It is then coupled with pyrophosphate to form a third intermediate 4. Then deprotection is performed to remove all orthogonal protecting groups to form imino-P-allyl bifunctional triphosphate 6.

[0385] Fig.69 An embodiment of the method for synthesizing imino-P allyl difunctional nucleotide is schematically illustrated. Activate 5'-OH allyl nucleoside 1 and couple with phosphitylation agent to form first phosphite triester intermediate 2. Then subject it to Staudinger reaction with azide functional group to form second imino-P phosphotriester intermediate 3. Deprotect to form third intermediate 4, which is activated again (see Table 2) and coupled with pyrophosphate moiety to form triphosphate intermediate 5. Then deprotect to remove all orthogonal protecting groups, thereby form imino-P allyl difunctional triphosphate 6.

[0386] Figure 70 schematically illustrates an embodiment of the method for synthesizing imino-P-allyl difunctional nucleotide.This method relates to the activation (opposite to nucleoside monophosphate) of pyrophosphate moiety, thereby exchanges the effect of nucleophile and electrophile.The activation of pyrophosphate 7 forms the first reactive pyrophosphate intermediate 8, which is then coupled with nucleoside monophosphate 3, thereby forms the second nucleotide triphosphate intermediate 5.Then deprotection is carried out to remove all orthogonal blocking groups, thereby forms imino-P-allyl difunctional triphosphate 6.

[0387] Fig.71 An exemplary synthesis of bifunctional imino-P-allyl nucleotides with different reactive groups is schematically illustrated.

[0388] Fig.72 Schematically illustrates the deprotection of the 3'-OTBDPS group to form 3'-OH, yielding nucleotide 6. Additionally, deprotection methods can be used to remove protecting groups, such as 3'-OTBDPS, including those selected from the group consisting of HF, TEA, HF-pyridine, TBAF, TAST, DBU, acetyl chloride / anhydrous MeOH, selectfluor, or lithium acetate.

[0389] Fig.73 is a table illustrating HF-TEA and TBAF deprotection methods to evaluate the conversion efficiency and yield of the desired triphosphate product. Using the optimized HF-TEA deprotection conditions in entry 3, approximately 77% conversion of 3'-OTBDPS difunctional nucleotide 5 to 3'-OH difunctional nucleotide 6 was achieved.

[0390] use Fig.73 The crude HPLC, analytical HPLC and LCMS spectra of the purified nucleotide produced from entry 3 of the table are shown in Fig.74A -C. Deprotection of 3'-OTBDPS with 1 M TBAF at 4°C for 2 hours was also successful. The recovered nucleotide 6 was similarly characterized by LCMS and HPLC to confirm identity and purity.

[0391] Example 8: Selecting a diastereomeric mixture .

[0392]

[0393] Due to the desymmetrization of the substituents around the α phosphorus and 5'-carbon atoms, two new chiral centers at the α P and 5' C atoms (indicated by the * symbol) are introduced, which results in the possible formation of 4 diastereomers. Methods for controlling chirality and selecting specific isomers are described herein.

[0394] Fig.75 Schematic illustration of the stereoselective reduction of the carbonyl group, thus controlling the chirality at the 5'-carbon atom.

[0395] Fig.76 Schematic illustration of the kinetic diastereoisomer selection step of the enzyme from the racemic precursor.

[0396] Fig.77 Schematic illustration of the chiral derivatization of isomers that enabled the final column separation.

[0397] Fig.78 Chiral ligand-facilitated stereoselective alkyl addition (eg, nucleophilic addition to an aldehyde group) is schematically illustrated, along with representative examples of chiral ligands.

[0398] Fig.79 Schematic illustration of the stereoselective enzymatic synthesis of the triphosphate.

[0399] Fig.80 Schematic illustration of methods for controlling the chirality at the alpha phosphorus atom, in particular the stereoselective Staudinger reaction induced by a chiral auxiliary. Fig.81 is an exemplary list of Staudinger variants. Several variants of the Staudinger reaction can be considered to modify substituents at the alpha phosphorus center. One embodiment described herein produces an "imino-P" substitution involving a Staudinger reaction between a phosphite and a sulfonyl azide moiety. One skilled in the art will be able to extend the Staudinger reaction to other variants, such as phosphites with aryl azides, phosphites with guanidinium azides, and H-phosphonates with acyl azides. Other exemplary synthetic methods for preparing azido variants are provided in Fig.82 middle.

[0400] Example 9: Cyclic cyclic nucleotide synthesis pathway

[0401] Fig.83A and Fig.83B Schematically illustrated are various pathways leading to the formation of an exemplary cyclic nucleotide structure 10 using three alternative pathways: Pathway A: 3→11→9→10, Pathway B: 3→5→9→10, and Pathway C: 3→5→6→10. Fig.84A -C shows HPLC, LCMS and FTIR characterization data for the characterization of cyclic nucleotide structure 10. LCMS confirmed the quality of cyclic nucleotide 10, the HPLC spectrum indicated the purity of the isolated product, and FTIR confirmed the absence of unreacted azide (which would appear at about 2100 cm-1, corresponding to -N=N=N stretching).

[0402] Example 10: Preliminary incorporation assay of cyclic alpha phosphate-substituted nucleotides

[0403] Fig.85Illustrated are the results for the bifunctional nucleotide 6 tested in an incorporation assay using the Dpo4 enzyme compared to native dTTP. The results indicate that the bifunctional nucleotide 6 was incorporated at least twice under the conditions evaluated.

[0404] Similarly, cyclic nucleotide 10 was evaluated, as Fig.86 As shown in . Compared with the incorporation of nucleotide 6, the incorporation of cyclic nucleotide 10, which has a large steric volume, is slower. By increasing the concentration of cyclic nucleotide 10 by 5 times (from 20 μM to 100 μM), increasing the catalytic Mn 2+ By increasing the concentration of 5-fold (from 2 mM to 10 mM), increasing the concentration of Dpo4 by 2-fold (from 1 μM to 2 μM), and increasing the temperature (from rt to 37°C), it can be seen that the incorporation of cyclic nucleotide 10 is improved, as shown in Fig.87 as shown in .

[0405] Example 11: Preliminary stability data of cyclic alpha phosphate substituted nucleotides

[0406] Figures 88-89 Stability assays performed on bifunctional nucleotide 6 and cyclic nucleotide 10 are illustrated, subjecting them to stability testing in 50 mM Tris pH 7.5 at 25°C and 60°C. Nucleotide 6 and nucleotide 10 were stable for up to 2 days at 25°C, while about 55% and about 74% of nucleotide 6 and 10, respectively, degraded at 60°C. The αP-C modified nucleotides were degraded in 50 mM Tris pH 7.5 at 25°C for 2 days, indicating that about 72% of the imino-P modification was able to significantly stabilize the α-phosphate substituted triphosphate nucleotides. This enhanced stability is critical for the manufacture, scalability, and production of similar nucleotides for sequencing purposes.

[0407] Additional Notes

[0408] It should be understood that all combinations of the foregoing concepts and additional concepts discussed in more detail below (assuming such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. Specifically, all combinations of the claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. It should also be understood that terms explicitly adopted herein that may also appear in any disclosure incorporated by reference should be given a meaning most consistent with the specific concepts disclosed herein.

[0409] The references to "one example", "another example", "an example", etc. throughout this specification mean that the specific elements (e.g., features, structures, and / or characteristics) described in conjunction with the example are included in at least one example described herein, and may or may not be present in other examples. In addition, it should be understood that the elements used in any example may be combined in various examples in any suitable manner, unless the context clearly indicates otherwise.

[0410] It should be understood that the ranges provided herein include the specified range and any value or sub-range within the specified range, as if such value or sub-range was explicitly listed. For example, a range of about 2 nm to about 20 nm should be interpreted as including not only the explicitly listed limits of about 2 nm to about 20 nm, but also individual values, such as about 3.5 nm, about 8 nm, about 18.2 nm, etc., and sub-ranges, such as about 5 nm to about 10 nm, etc. In addition, when "about" and / or "substantially" are used to describe a value, this means that a small variation (up to + / - 10%) of the stated value is included.

[0411] While several examples have been described in detail, it is to be understood that modifications may be made to the disclosed examples. Therefore, the above description should be considered non-limiting.

[0412] Although certain examples have been described, these examples are presented only as examples and are not intended to limit the scope of the present disclosure. In fact, the novel methods and systems described herein may be embodied in various other forms. Moreover, various omissions, substitutions, and changes may be made in the systems and methods described herein without departing from the spirit of the present disclosure. The attached claims and their equivalents are intended to cover such forms or modifications that will fall within the scope and spirit of the present disclosure.

[0413] Features, materials, characteristics or groups described in conjunction with a particular aspect or example are to be understood to be applicable to any other aspect or example described in this section or elsewhere in this specification unless incompatible therewith. All features disclosed in this specification (including any accompanying claims, abstract and drawings) and / or all steps of any method or process so disclosed may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. Protection is not limited to the details of any foregoing examples. Protection extends to any novel feature or any novel combination thereof of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel step or any novel combination thereof of the steps of any method or process so disclosed.

[0414] Moreover, certain features described in the present disclosure in the context of separate embodiments may also be implemented in combination in a single embodiment. Conversely, various features described in the context of a single embodiment may also be implemented in multiple embodiments, either individually or in any suitable subcombination. Furthermore, although features may be described above as functioning in certain combinations, in some cases one or more features from the combination may be deleted from a claimed combination, and the combination may be claimed as a subcombination or a variation of a subcombination.

[0415] In addition, although operations may be depicted in the drawings or described in the specification in a particular order, it is not necessary to perform such operations or perform all operations in the particular order shown or in sequence to achieve the desired result. Other operations not depicted or described may be incorporated into the example methods and processes. For example, one or more additional operations may be performed before, after, at the same time, or between any described operations. In addition, these operations may be rearranged or reordered in other embodiments. It will be understood by those skilled in the art that in some examples, the actual steps taken in the process shown and / or disclosed may be different from the actual steps shown in the figure. Depending on the example, some of the above steps may be removed, or other steps may be added. In addition, the features and attributes of the specific examples disclosed above may be combined in different ways to form additional examples, all of which fall within the scope of the present disclosure. Moreover, the separation of various system components in the embodiments described above should not be understood as requiring such separation in all embodiments, and it should be understood that the components and systems described may generally be integrated together in a single product or packaged into multiple products. For example, any of the components of the energy storage system described herein may be provided separately, or integrated together (e.g., packaged together or attached together) to form an energy storage system.

[0416] For purposes of the present disclosure, certain aspects, advantages, and novel features are described herein. Not all of these advantages may be achieved according to any particular example. Thus, for example, one skilled in the art will recognize that the present disclosure may be implemented or performed in a manner that achieves one advantage or group of advantages taught herein without necessarily achieving other advantages that may be taught or suggested herein.

[0417] Unless specifically stated otherwise or otherwise understood within the context of use, conditional language such as "may," or "could" is generally intended to convey that some examples include and other examples do not include certain features, elements, and / or steps. Thus, such conditional language generally does not mean that one or more examples require the features, elements, and / or steps in any way, or that one or more examples must include logic for making decisions, with or without user input or prompting, whether or not features, elements, and / or steps are included in a particular example or are to be performed in any particular example.

[0418] Unless specifically stated otherwise, linking language such as the phrase "at least one of X, Y, and Z" is generally understood in context to convey that an item, term, etc. can be X, Y, or Z. Thus, such linking language is generally not intended to imply that certain examples require the presence of at least one of X, at least one of Y, and at least one of Z.

[0419] As used herein, language of degree, such as the terms "approximately," "about," "generally," and "substantially" represent a value, amount, or characteristic that approaches the stated value, amount, or characteristic and still performs the intended function or achieves the intended result.

[0420] The scope of the present disclosure is not intended to be limited by the specific disclosure of preferred examples in this section or elsewhere in this specification, and may be limited by the claims presented in this section or elsewhere in this specification or presented in the future. The language of the claims should be interpreted broadly based on the language employed in the claims, and not limited to the examples described in this specification or during the prosecution of the application, which examples should be understood to be non-exclusive.

[0421] Although the foregoing invention has been described according to certain preferred embodiments, other embodiments will be apparent to those of ordinary skill in the art. In addition, other combinations, omissions, substitutions and modifications will be apparent to those skilled in the art in view of the disclosure herein. Therefore, the present invention is not intended to be limited by the description of the preferred embodiments, but is defined by reference to the appended claims. All references cited herein are incorporated by reference in their entirety.

[0422] The terms used in the description presented herein are not intended to be interpreted in any limited or restrictive manner, and unless otherwise indicated, refer to the common meaning that will be understood by those of ordinary skill in the art in view of the specification. In addition, the embodiment may include several novel features, be composed of several novel features, be substantially composed of several novel features, and no single novel feature in these several novel features is only responsible for its desired attribute or is considered to be necessary for practicing the embodiments described herein. As used herein, the section title is only used for organizational purposes and should not be interpreted as limiting the described subject matter in any way. All documents and similar materials (including but not limited to patents, patent applications, articles, books, papers and Internet web pages) cited in this application are clearly incorporated by reference in their entirety for any purpose. When the definition of the term in the reference incorporated seems to be different from the definition provided in this teaching, the definition provided in this teaching shall prevail. It should be understood that there is an implicit "about" before the temperature, concentration, time, etc. discussed in this teaching, so that slight and non-substantial deviations are within the scope of this teaching herein.

[0423] Although the present disclosure is in the context of certain embodiments and examples, it will be understood by those of ordinary skill in the art that the present disclosure extends beyond the specifically disclosed embodiments to other alternative embodiments and / or uses of the embodiments and obvious modifications and equivalents thereof. In addition, although several variations of the embodiments have been shown and described in detail, other modifications within the scope of the present disclosure will be apparent to those of ordinary skill in the art based on the present disclosure. It is also contemplated that various combinations or sub-combinations of specific features and aspects of the embodiments may be performed and still fall within the scope of the present disclosure. It should be understood that the various features and aspects of the disclosed embodiments may be combined or substituted with each other to form different modes or embodiments of the present disclosure. Therefore, the scope of the present disclosure disclosed herein should not be limited by the above-mentioned specifically disclosed embodiments.

Claims

1. A compound having one of the following structures: in X is -O-, -CH2-, -NH-, X' is =N-SO2-, =NH-CO- or Y is -O-, -S-, -NH- or -Se-; L1 is the first linking group; L2 is a second linking group; and SP is a spacer.

2. The compound according to claim 1, wherein SP comprises one or more of the following moieties: (1) an alkyl chain having 5 to 50 carbon atoms, (2) oligonucleotides, modified oligonucleotides or polyphosphates having 1 to 100 repeating units, (3) a polypeptide having 1 to 100 repeating units, (4) a hydrophilic polymer having 1 to 100 repeating units, and (5) A hydrophobic polymer having 1 to 100 repeating units.

3. The compound of claim 2, wherein the hydrophilic polymer is selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polyacrylamide, polyvinyl pyrrolidone, polystyrene sulfonate and polyethylene imine.

4. The compound of claim 2, wherein the hydrophobic polymer is selected from the group consisting of polylactic acid, polymethyl methacrylate, and polystyrene.

5. The compound of any one of claims 1 to 4, wherein each of L1 and L2 independently comprises a conjugated moiety selected from the group consisting of amine-NHS esters, amine-imidates, amine-pentafluorophenyl esters, amine-hydroxymethylphosphines, carboxyl-carbodiimides, thiol-maleimides, thiol-haloacetyl groups, thiol-pyridyl disulfides, thiol-thiosulfonates, thiol-vinyl sulfones, aldehyde-hydrazides, aldehyde-alkoxyamines, hydroxy-isocyanates, azide-alkynes, azide-phosphines, trans-cyclooctene-tetrazines, norbornene-tetrazines, azide-cyclooctyne, and azide-norbornene.

6. The compound of claim 5, wherein each of L1 and L2 independently further comprises a first linker between the conjugated moiety and X / X', and a second linker between the conjugated moiety and SP.

7. The compound of claim 6, wherein the first linker and the second linker are independently selected from the group consisting of a hydrophilic polymer, a hydrophobic polymer, an oligonucleotide, a peptide, a polypeptide, an aliphatic chain (C5 to C50), and combinations thereof.

8. The compound according to any one of claims 1 to 7, wherein the SP further comprises a capture construct.

9. The compound according to any one of claims 1 to 7, wherein the base further comprises a capture construct.

10. The compound of claim 8 or 9, wherein the capture construct is a linear, branched or cyclic polymer.

11. The compound of claim 10, wherein the capture construct comprises a synthetic hydrophobic polymer, a synthetic hydrophilic polymer, an oligonucleotide / polynucleotide, a peptide / polypeptide, or a combination thereof.

12. An oligonucleotide comprising one of the following structures: in X is -O-, -CH2-, -NH-, X' is =N-SO2-, =NH-CO- or Y is -O-, -S-, -NH- or -Se-; R 1 , R 2 and R 3 One of them is allyl and the other is H; L1 is the first linking group; L2 is a second linking group; and SP is a spacer.

13. The oligonucleotide according to claim 12, wherein structure (VII) can also be represented by the following structure:

14. The oligonucleotide according to claim 12, wherein structure (VIII) can also be represented by the following structure:

15. The oligonucleotide according to claim 12, wherein structure (X) can also be represented by the following structure:

16. An oligonucleotide comprising one of the following structures: in Y is -O-, -S-, -NH-, or -Se-; and Y 1 , Y 2 and Y 3 One of them is -S- or -Se-, and the other is -O- or -NH-.

17. The oligonucleotide according to any one of claims 12 to 16, wherein the SP comprises one or more of the following moieties: (1) an alkyl chain having 5 to 50 carbon atoms, (2) oligonucleotides, modified oligonucleotides or polyphosphates having 1 to 100 repeating units, (3) a polypeptide having 1 to 100 repeating units, (4) a hydrophilic polymer having 1 to 100 repeating units, the hydrophilic polymer being selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polyacrylamide, polyvinyl pyrrolidone, polystyrene sulfonate and polyethylene imine, and (5) A hydrophobic polymer having 1 to 100 repeating units, the hydrophobic polymer being selected from the group consisting of polylactic acid, polymethyl methacrylate and polystyrene.

18. The oligonucleotide of any one of claims 12 to 16, wherein each of L1 and L2 independently comprises a conjugated moiety selected from the group consisting of amine-NHS esters, amine-imidates, amine-pentafluorophenyl esters, amine-hydroxymethylphosphines, carboxyl-carbodiimides, thiol-maleimides, thiol-haloacetyl groups, thiol-pyridyl disulfides, thiol-thiosulfonates, thiol-vinyl sulfones, aldehyde-hydrazides, aldehyde-alkoxyamines, hydroxy-isocyanates, azide-alkynes, azide-phosphines, trans-cyclooctene-tetrazines, norbornene-tetrazines, azide-cyclooctyne, and azide-norbornene.

19. The oligonucleotide of claim 18, wherein each of L1 and L2 independently further comprises a first linker between the conjugated moiety and X, and a second linker between the conjugated moiety and SP.

20. The oligonucleotide of claim 19, wherein the first linker and the second linker are independently selected from the group consisting of: a hydrophilic polymer (e.g., polyethylene glycol, polyvinyl alcohol, polyacrylamide, polyvinyl pyrrolidone, polystyrene sulfonate, polyethylene imine), a hydrophobic polymer (e.g., polylactic acid, polymethyl methacrylate, polystyrene), an oligonucleotide, a peptide, a polypeptide, an aliphatic chain (C5 to C50), and combinations thereof.

21. The oligonucleotide according to any one of claims 12 to 20, wherein the SP further comprises a capture construct.

22. The oligonucleotide of any one of claims 12 to 20, wherein the base further comprises a capture construct.

23. The oligonucleotide of claim 21 or 22, wherein the capture construct is a linear, branched or circular polymer.

24. The oligonucleotide of claim 23, wherein the capture construct comprises a synthetic hydrophobic polymer, a synthetic hydrophilic polymer, an oligonucleotide / polynucleotide, a peptide / polypeptide, or a combination thereof.

25. The oligonucleotide according to any one of claims 12 to 24, wherein L1, SP and L2 are subelements of a cyclic loop.

26. The oligonucleotide of claim 25, wherein the cyclic ring is symmetrical or asymmetrical.

27. The oligonucleotide of claim 26, wherein the cyclic loop is synthesized using one or more of: solid phase synthesis, solution phase synthesis, and enzymatic synthesis.

28. The oligonucleotide of claim 26, wherein the cyclic loop is synthesized using one or more of: linear synthesis, branched synthesis, or segmented synthesis.

29. A method for determining the sequence of a polynucleotide in a nanopore-based sequencing system, the method comprising: providing a polynucleotide comprising a plurality of nucleotides, wherein each nucleotide comprises a linker construct having a first end attached to a first position of the nucleotide and a second end attached to a second position of the nucleotide; cleaving a cleavable bond between the first position and the second position on each of the plurality of nucleotides, thereby extending the polynucleotide to form an extended polynucleotide; applying a voltage to cause the elongated polymer to insert into and translocate through the nanopore; as well as (i) detecting and identifying a reporter moiety when the linker construct passes through the nanopore; or (ii) detecting and identifying the base on the nucleotide as it passes through the nanopore.

30. The method of claim 29, wherein the linker construct comprises a first linking group, a second linking group, and a spacer between the first linking group and the second linking group.

31. The method of claim 29, wherein the spacer comprises an oligonucleotide having 1 to 100 repeating units; a modified oligonucleotide having 1 to 100 repeating units; a polyphosphate having 1 to 100 repeating units; a polypeptide having 1 to 100 repeating units; an alkyl chain having 5 to 50 carbons; a hydrophilic polymer having 1 to 100 repeating units, the hydrophilic polymer being selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polyacrylamide, polyvinyl pyrrolidone, polystyrene sulfonate, and polyethylene imine; a hydrophobic polymer having 1 to 100 repeating units, the hydrophobic polymer being selected from the group consisting of polylactic acid, polymethyl methacrylate, and polystyrene; and their combinations.

32. The method of claim 29, wherein the spacer comprises the reporter moiety, wherein the reporter moiety corresponds to and identifies a nucleotide.

33. The method of any one of claims 29 to 32, wherein each of the first linking group and the second linking group independently comprises a conjugated moiety selected from the group consisting of: amine-NHS esters, amine-imidates, amine-pentafluorophenyl esters, amine-hydroxymethylphosphines, carboxyl-carbodiimides, thiol-maleimides, thiol-haloacetyl groups, thiol-pyridyl disulfides, thiol-thiosulfonates, thiol-vinyl sulfones, aldehyde-hydrazides, aldehyde-alkoxyamines, hydroxy-isocyanates, azide-alkynes, azide-phosphines, trans-cyclooctene-tetrazines, norbornene-tetrazines, azide-cyclooctyne, and azide-norbornene.

34. The method of any one of claims 29 to 33, wherein the extended polymer further comprises a capture construct attached to each nucleobase or each linker construct, wherein the capture construct is configured to slow, pause or stop the translocation.

35. The method of claim 34, wherein the capture construct is a linear, branched or circular polymer.

36. The method of claim 34, wherein the capture construct comprises a synthetic hydrophobic polymer, a synthetic hydrophilic polymer, an oligonucleotide / polynucleotide, a peptide / polypeptide, or a combination thereof.

37. The method of any one of claims 35 to 36, wherein the nanopore comprises a constriction having an opening with an inner diameter of about 0.6 nm to about 1.2 nm.

38. The method of claim 29, wherein the polynucleotide comprises one of the oligonucleotides of any one of claims 12 to 24.

39. The method of claim 29, wherein each of the plurality of nucleotides is selected from the compounds of any one of claims 1 to 11.

40. The method of claim 29, wherein the reporter moiety comprises two or more sub-reporter moieties, wherein each of the two or more sub-reporter moieties is distinguishable, reproducible, and resolvable.

41. The method of claim 40, wherein the two or more sub-reporter moieties comprise crown ethers, cucurbiturils, pillararenes, or cyclodextrins.

42. A kit for performing a method for determining the sequence of a polynucleotide in a nanopore-based sequencing system, the kit comprising a compound according to any one of claims 1 to 11.

43. A system for determining the sequence of a polynucleotide, the system being configured to perform the method according to any one of claims 29 to 34.

44. A system for performing a method for determining the sequence of a polynucleotide comprising a plurality of nucleotides, wherein the nucleotides are selected from any one of the compounds according to claims 1 to 11.

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