Translocation control elements, reporter codes and other means for translocation control for nanopore sequencing

Abstract

The present invention provides phosphoramidate-based monomers for synthesizing expandable polymers for nanopore-based sensing. Such monomers include a reporter construct comprising a first reporter code, a symmetrical chemical branch with a translocation control element, and a second reporter code, wherein the termini of the reporter construct are connected to phosphoramidate-nucleosides. Related methods and products are also provided.

Description

Technical Field

[0001] The present invention relates generally to novel synthetic reporter constructs, and more specifically to novel nucleotide-free, phosphoramidite-based translocation control elements, reporter codes and other features that produce a unique signal upon passage through a nanopore, and methods of making and utilizing the same, particularly in nanopore-based polymer sequencing methods. Background Art

[0002] The measurement of biomolecules is fundamental to modern medicine and is widely used in medical research, more specifically in diagnostics and therapeutics, as well as in drug development. Nucleic acids encode information necessary for the activity and reproduction of living organisms and are essentially the blueprints of life. Determining such blueprints is useful in both pure research and applied science. In medicine, sequencing can be used to diagnose and develop treatments for a variety of conditions, including cancer, heart disease, autoimmune diseases, multiple sclerosis, and obesity. In industry, sequencing can be used to design improved enzymatic processes or synthetic organisms. In biology, for example, the tool can be used to study the health of ecosystems and therefore has a wide range of uses. Similarly, the measurement of proteins and other biomolecules provides markers and insights into the spread of disease and pathogens.

[0003] An individual's unique DNA sequence provides valuable information about their susceptibility to certain diseases. It also provides patients with the opportunity to be screened for early detection and / or receive preventive treatment. Furthermore, based on a patient's personal blueprint, clinicians will be able to administer personalized therapies to maximize drug efficacy and / or minimize the risk of adverse drug reactions. Similarly, determining the blueprint of a pathogenic organism could provide new treatments for infectious diseases and more robust pathogen surveillance. Low-cost whole-genome DNA sequencing will lay the foundation for modern medicine. To achieve this goal, sequencing technology must continue to advance in throughput, accuracy, and read length.

[0004] In the past decade, a large number of next-generation DNA sequencing technologies have become commercially available, which have greatly reduced the cost of whole genome sequencing. These include sequencing by synthesis ("SBS") platforms (Illumina, Inc., 454 Life Sciences, Ion Torrent, Pacific Biosciences) and similar ligation-based platforms (Complete Genomics, Life Technologies Corporation). Many other technologies are under development, which utilize a variety of sample processing and detection methods. For example, GnuBio (Cambridge, Mass.) uses picoliter reaction vessels to control millions of discreet probe sequencing reactions, and Halcyon Molecular (Redwood City, Calif.) is attempting to develop a technology for direct DNA measurement using a transmission electron microscope.

[0005] Nanopore-based nucleic acid sequencing is an attractive approach that has been extensively studied. Kasianowicz et al. (Proc. Natl. Acad. Sci. USA 93: 13770-13773, 1996) characterized single-stranded polynucleotides as they were electrically translocated through alpha-hemolysin nanopores embedded in lipid bilayers. It has been demonstrated that partial blockage of the nanopore orifice can be measured as a measure of reduced ionic current during polynucleotide translocation. However, polynucleotide sequencing in nanopores suffers from small signal differences immersed in significant background noise due to the need to resolve closely spaced bases (0.34 nm). The measurement challenge of single-base resolution in nanopores becomes more demanding due to the observed rapid translocation rates of polynucleotides, typically on the order of 1 base per microsecond. The translocation rate can be reduced by adjusting operating parameters such as voltage, salt composition, pH, temperature, and viscosity. However, such adjustments cannot reduce the translocation rate to a level that allows single-base resolution.

[0006] Stratos Genomics has developed a method called extended sequencing ("SBX") that uses a biochemical process to transcribe DNA sequences onto measurable polymers called "Xpandomers" (Kokoris et al., U.S. Patent No. 7,939,259 "High Throughput Nucleic Acid Sequencing by Expansion"). The transcribed sequence is encoded in high signal-to-noise ratio reporters along the Xpandomer backbone, which are separated by approximately 10 nm and designed for high signal-to-noise ratio, well-differentiated reactions. These differences provide significant performance enhancements in sequence read efficiency and accuracy of Xpandomers relative to native DNA. Xpandomers can enable a variety of next-generation DNA sequencing detection technologies and are well suited for nanopore sequencing.

[0007] Nanopores have proven to be powerful amplifiers, just like their highly developed predecessor, the Coulter counter. However, contemporary organic nanopores whose mission is base recognition of DNA (such as hemolysin and MspA) are transmembrane proteins that do not interact with DNA in nature. They have no natural function to control DNA translocation. This is a recognized shortcoming that some have tried to correct by adding the functionality of protein motors near the nanopore. For example, Akeson's team added a motor near the α-hemolysin nanopore. The polymerase is used so that the ss-DNA can be fed into the pore at a controlled rate (see GMCherf et al. "Automated forward and reverse ratcheting of DNA in a nanopore at 5-Aprecision," Nat Biotech, vol. advance online publication, February 2012). This approach complicates the assay and forces the separation of the measurement region in α-hemolysin from the positional control in the polymerase, which may introduce additional noise and sequence-dependent variations to the measurement.

[0008] In another approach, called translocation control by hybridization (TCH), nanopore translocation events are paused by using hybridization-generated structures that dissociate to translocate (see, e.g., U.S. Pat. No. 10,457,979 to McRuer and Kokoris). Akeson et al. (U.S. Pat. No. 6,465,193) first demonstrated this by pausing DNA translocation with sequential hairpin double-stranded regions. Translocation stops at the duplex because it is larger than the orifice of the α-hemolysin nanopore. When the duplex is released by random thermal fluctuations, translocation proceeds to the next duplex. During each pause, the DNA region located in the nanopore (adjacent to the duplex) can be measured and identified. When applied to nanopore sequencing, this duplex method for translocation control has limitations, including incomplete duplex formation or hybridization filling rate and randomness of duplex dissociation, which may lead to deletion or insertion events. Insertions and deletions that cannot be localized can severely reduce data quality.

[0009] While significant progress has been made in this area, commercially viable implementation of translocation control, such as using Xpandomers, would benefit from improvements that overcome the limitations imposed by duplexes.The present invention meets these needs and provides further related advantages as described below.

[0010] All subject matter discussed in the background section is not necessarily prior art and should not be assumed to be prior art simply because it is discussed in the background section. Along these lines, any recognition of a problem in the prior art discussed in the background section or related to such subject matter should not be considered prior art unless it is explicitly stated to be prior art. Rather, the discussion of any subject matter in the background section should be considered as part of the inventor's approach to solving a particular problem, which may itself be inventive. Summary of the invention

[0011] Briefly, disclosed are compounds (e.g., XNTPs) and methods for improving nanopore sequencing (e.g., generating sequences with higher read length, accuracy, and / or throughput) of polymer analytes (e.g., Xpandomers), comprising polymer reporters and linker constructs synthesized from a series of novel phosphoramidite monomer units.

[0012] In some embodiments, polymer constructs can be designed to be completely devoid of nucleotides.

[0013] In one aspect, the present disclosure provides a compound (ie, XNTP) having the following structure:

[0014]

[0015] wherein R is OH or H; the nucleobase is adenine, cytosine, guanine, thymine, uracil or a nucleobase analog; the reporter construct is a polymer having a first end and a second end, and comprises a first reporter code, a symmetrical chemical branch with a translocation control element, and a second reporter code connected in series from the first end to the second end; linker A joins the oxygen atom of the alpha phosphoramidate to the first end of the reporter construct; linker B joins the nucleobase to the second end of the reporter construct; and wherein the translocation control element is a polymer as described below.

[0016] In one embodiment, the translocation control element is a polymer comprising two or more repeating units selected from the group consisting of: 1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane (Compound 12b), 1,3-O-bis(phosphodiester)-2-(4-Me-O-PEG3)-1-(Et-O-Ac)-1,2,3-triazole)-propane (Compound 35b), 1,3-O-bis(phosphodiester)-2s-O-mPEG6-propane (Compound 12c), 1,2-O-bis(phosphodiester)-3-O-mPEG2-propane (Compound 16), 2,3-O-bis(phosphodiester)-1-(5-benzofuran)-propane (Compound 20i), 1,2- O-bis(phosphodiester)-3-(4-methylpiperazin-1-yl)-propane (Compound 20j), 2,3-O-bis(phosphodiester)-1-(N1-(2-Me-5-nitroindole)-propane (Compound 20g), 1,8-O-bis(phosphodiester)-N,N-diethylpiperazine (Compound 26h), 1,2-O-bis(phosphodiester)-3-(4-(Me-O-PEG3-O-Bz)-1-(1,2,3-triazole))-propane (Compound 31d), 1,3-O-bis(phosphodiester)-2s-O-(4-(Me-O-PEG2)-1-(Et-OBz)-1,2,3-triazole)-propane (Compound 35a), 1,3-O-bis(phosphodiester)- Phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-O-Ac)-1,2,3-triazole)-propane (Compound 35c), 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG7)-1-(Et-OBz)-1,2,3-triazole)-propane (Compound 35d), 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Me-acetate)-1,2,3-triazole)-propane (Compound 35e), 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Et-2,2,2-tri-(Me-O-Bz))-1,2,3 -triazole)-propane (Compound 37a), 1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-2,2,2-tri-(Me-O-Ac))-1,2,3-triazole)-propane (Compound 37b), 1,3-O-bis(phosphodiester-2S-O-(PEG4-O-Bz)-propane (Compound 38b), 1,3-O-bis(phosphodiester-2,2-bis(Me-O-mPEG2)-propane (Compound 45b), 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG2-O-Me)-1-(Et-O-Bz)-1,2,3-triazole)-propane (Compound 47f ...S-O-(PEG4-O-Bz)-propane (Compound 38b), 1,3-O-bis(phosphodiester-2,2-bis(Me-O-mPEG2)-propane (Compound 45b), 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG2-O-Me)-1-(Et-O-Bz)-1,2,3-triazole)-propane3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG3-O-Me)-1-(Et-O-Bz)-1,2,3-triazole)-propane (Compound 47Gg), 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG3-O-Me)-1-(Et-2,2,2-tri-(Me-O-Bz))-1,2,3-triazole)-propane (Compound 47i) or 1,3-O-bis(phosphodiester-2,2-bis(1-Me-4-(Me-O-PEG2-O-Bz)-1,2,3-triazole)-propane (Compound 52).

[0017] In some embodiments, R is OH.

[0018] In some embodiments, R is H.

[0019] In other embodiments, the nucleobase is adenine, cytosine, guanine, thymine, or uracil.

[0020] In other embodiments, the nucleobase is a nucleobase analog.

[0021] In other embodiments, the symmetric chemical branch is 1,2,3-O-tris-(phosphodiester)-propane, 1,3-bis-(5-O-phosphodiester-pentylamido)-2-O-phosphodiester-propane or 1,4,7-O-tris-(phosphodiester)-heptane.

[0022] In other embodiments, the symmetric chemical branching agent is 1,2,3-O-tris-(phosphodiester)-propane.

[0023] In other embodiments, the translocation control element is a polymer comprising two or more repeating units selected from Table 1A.

[0024] In other embodiments, the translocation control element is a polymer comprising two or more repeating units selected from the group consisting of 1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane (compound 12b) and 1,3-O-bis(phosphodiester)-2-(4-Me-O-PEG3)-1-(Et-O-Ac)-1,2,3-triazole)-propane (compound 35b).

[0025] In yet other embodiments, the translocation control element is a polymer comprising the following sequence: [(1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane (compound 12b))]n1[(1,3-O-bis(phosphodiester)-2-(4-Me-O-PEG3)-1-(Et-O-Ac)-1,2,3-triazole)-propane (compound 35b))]n2, wherein n1 is 0 to 6 and n2 is 6 to 10.

[0026] In other embodiments, the first reporting sub-code and the second reporting sub-code are the same.

[0027] In a further embodiment, the first reporter code and the second reporter code are polymers comprising two or more repeating units selected from the group consisting of hexaethylene glycol (D), ethane (L), triethylene glycol (X), 1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane (compound 12b), 1,3-O-bis(phosphodiester)-2-(4-Me-O-PEG3)-1-(Et-O-Ac)-1,2,3-triazole)-propane (compound 35b), 1,3-O-bis(phosphodiester-2,2-bis(Me-O-mPEG2)-propane (compound 45b), 1,3-O-bis(phosphodiester-2S-O-(PEG4-O-Bz)-propane (compound 54b). Compound 38b), 1,3-O-bis(phosphodiester)-2s-O-mPEG6-propane (Compound 12c), 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Et-2,2,2-tri-(Me-O-Bz))-1,2,3-triazole)-propane (Compound 37a), 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Me-acetate)-1,2,3-triazole)-propane (Compound 35e), 1,3-O-bis(phosphodiester)-2s-O-(4-(Me-O-PEG2)-1-(Et-OBz)-1,2,3-triazole)-propane (Compound 35a), 1,3-O-bis(phosphodiester)-2-(4-Et-1-(Et-O-mPEGl)-1,2,3-triazole)-propane (Compound 31a), 2,3-O-bis(phosphodiester)-1-(1-dimethoxyquinazolinedione)-propane (Compound 20c), 2,3-O-bis(phosphodiester)-1-(N9-(3,6-dimethoxycarbazole)-propane (Compound 20e), 1,1′-O-bis(phosphodiester)-2,2′-(sulfonylbis(benzyl-4-yl))-diethanol (Compound 26d), 1,1′-O-bis(phosphodiester)-2,2′-bipyridyl-4,4′-yl)-dimethanol (Compound 26a), 2,3-O-bis(phosphodiester)-1-(1-dimethoxyquinazolinedione)-propane (Compound 20c), 2,3-O-bis(phosphodiester)-1-(N9-(3,6-dimethoxycarbazole)-propane (Compound 20e), ester)-1-(N1-(4,6-dimethoxy-3-Me-indole)-propane (Compound 20b), 3-(1,2-O-bis(phosphodiester)-propyl)-8,8-dimethylhexahydro-3H-3a,6-methanobenzo[c]isothiazole 2,2-dioxide (Compound 20d), 2,3-O-bis(phosphodiester)-1-(N1-(6-azathymine))-propane (Compound 20f), 1,5-O-bis(phosphodiester)-hexahydrofuro[2,6]furan (Compound 23), 1,1′-O-bis(phosphodiester)-octahydro-2,6-dimethyl-3,8:4,7-dimethano-2,6-naphthyridine-4,8-diyl)-dimethanol (Compound 26e), 2,3-O-bis(phosphodiester)-1-(N1-(2-Me-5-nitroindole)-propane (Compound 20h), 2,3-O-bis(phosphodiester)-1-(N1-(2-Me-5-nitroindole)-propane (Compound 20g), 2,3-O-bis(phosphodiester)-1-(5-benzofuran)-propane (Compound 20i), 1,2-O-bis(phosphodiester)-3-O-mPEG2-propane (Compound 5b), 1,3-O-bis(phosphodiester)-2-(4-Et-1-(Et-O-mPEG3)-1,2,3-triazole)-propane (Compound 31b) and 1,3-O-bis(phosphodiester)-3-O-mPEG4-propane (Compound 5a).

[0028] In other embodiments, the first reporter code and the second reporter code are polymers comprising two or more repeating units selected from hexaethylene glycol, ethane, triethylene glycol, and any compound listed in Table 1A.

[0029] In a further embodiment, the first reporter code and the second reporter code are polymers comprising two or more repeating units selected from hexaethylene glycol, ethane, triethylene glycol and 1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane (Compound 12b).

[0030] In a more specific embodiment, the first reporter code and the second reporter code are polymers comprising sequences selected from the group consisting of: (i) [(hexaethylene glycol)2(ethane)3(hexaethylene glycol)(triethylene glycol)], (ii) [(hexaethylene glycol)2(1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane (compound 12b))2(ethane)(triethylene glycol)3], (iii) [(hexaethylene glycol)2(1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane (compound 12b))3(ethane)2(hexaethylene glycol)(triethylene glycol)] and (iv) [(triethylene glycol)2(ethane)(1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane (compound 12b))6(ethane)7].

[0031] In other embodiments, Linker A and Linker B are polymers comprising two or more repeating units selected from the group consisting of spermine (Q), hexaethylene glycol (D), 2-((4-((3-(benzoyloxy)-2-(((1-(3-(benzoyloxy)-2-((benzoyloxy)methyl)-2-((phosphodiester-oxy)methyl)propyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-2-((benzoyloxy)methyl)propyloxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-2-O-phosphodiester-propane-1,3-diyl dibenzoate (Compound 62), 1,3-O-bis(phosphodiester- 2,2-bis(1-Me-4-(Me-O-PEG2-O-Bz)-1,2,3-triazole)-propane (Compound 52), 1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-O-Ac)-1,2,3-triazole)-propane (Compound 35c), 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG7)-1-(Et-OBz)-1,2,3-triazole)-propane (Compound 35d), 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Et-2,2,2-tri-(Me-O-B z))-1,2,3-triazole)-propane (Compound 37a), 1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-2,2,2-tri-(Me-O-Ac))-1,2,3-triazole)-propane (Compound 37b), 1,2-O-bis(phosphodiester)-3-(4-(Me-O-PEG3-O-Bz)-1-(1,2,3-triazole))-propane (Compound 31d), 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG2-O-Me)-1-(Et-O-Bz)-1,2,3-triazole)-propane (Compound 47f) , 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG3-O-Me)-1-(Et-2,2,2-tri-(Me-O-Bz))-1,2,3-triazole)-propane (Compound 47i), 1,2-O-bis(phosphodiester)-3-(4-methylpiperazine-1-yl)-propane (Compound 20j), 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG3-O-Me)-1-(Et-O-Bz)-1,2,3-triazole)-propane (Compound 47g) and 1,1′-O-bis(phosphodiester)-N(p-tolyl)-diethanolamine (Compound 26b).

[0032] In other embodiments, Linker A and Linker B are polymers comprising two or more repeating units selected from spermine and any of the compounds listed in Table 1A.

[0033] In yet other embodiments, Linker A and Linker B comprise a polymerase enhancing region comprising two spermine repeating units.

[0034] In further embodiments, Linker A and Linker B comprise a metastasis slowing region comprising two or more repeating units selected from the group consisting of 1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-O-Ac)-1,2,3-triazole)-propane (Compound 35c), 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG7)-1-(Et-OBz)-1,2,3-triazole)-propane (Compound 35d), 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Et-2,2,2-tri-(Me-O-Bz))-1,2,3-triazole)-propane (Compound 37a) and 1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-2,2,2-tri-(Me-O-Ac))-1,2,3-triazole)-propane (Compound 37b).

[0035] In more specific embodiments, Linker A and Linker B comprise a metathesis slowing down region comprising a polymer selected from the group consisting of: (i) [((hexaethylene glycol)(1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-O-Ac)-1,2,3-triazole)-propane (Compound 35c))3(hexaethylene glycol)2], (ii) [((hexaethylene glycol)(1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-O-Ac)-1,2,3-triazole)-propane (Compound 35c))3(hexaethylene glycol)2], )4(hexaethylene glycol)2], (iii) [((hexaethylene glycol)(1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG7)-1-(Et-OBz)-1,2,3-triazole)-propane (compound 35d))4(hexaethylene glycol)2] and (iv) [((hexaethylene glycol)(1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-2,2,2-tri-(Me-O-Ac))-1,2,3-triazole)-propane (compound 37b))4(hexaethylene glycol)2].

[0036] In other embodiments, Linker A is joined to the oxygen atom of the alpha phosphoramidate through a triazole-containing linkage, and Linker B is joined to the nucleobase through a triazole-containing linkage.

[0037] In another aspect, the present invention provides a reporter construct comprising a polymer having a first end and a second end, and comprising a first reporter code, a symmetrical chemical branch with a translocation control element, and a second reporter code connected in series from the first end to the second end; and wherein the translocation control element is a polymer comprising two or more repeating units selected from the following: 1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane (compound 12b), 1,3-O-bis(phosphodiester)-2-(4-Me-O-PEG3)-1-(Et-O-Ac)-1,2,3-triazole)-propane (compound 35b), 1,3-O-bis(phosphodiester)-2s-O-mPEG4-propane (compound 12b), 1,3-O-bis(phosphodiester)-2-(4-Me-O-PEG3)-1-(Et-O-Ac)-1,2,3-triazole)-propane (compound 35c), 1,3-O-bis(phosphodiester)-2s-O- mPEG6-propane (Compound 12c), 1,2-O-bis(phosphodiester)-3-O-mPEG2-propane (Compound 16), 2,3-O-bis(phosphodiester)-1-(5-benzofuran)-propane (Compound 20i), 1,2-O-bis(phosphodiester)-3-(4-methylpiperazin-1-yl)-propane (Compound 20j), 2,3-O-bis(phosphodiester)-1-(N1-(2-Me-5-nitroindole)-propane (Compound 20g), 1,8-O-bis(phosphodiester)-N,N-diethylpiperazine (Compound 26h), 1,2-O-bis(phosphodiester)-3-(4-(Me-O-PEG3-O-Bz)-1-(1, 2,3-triazole)-propane (Compound 31d), 1,3-O-bis(phosphodiester)-2s-O-(4-(Me-O-PEG2)-1-(Et-OBz)-1,2,3-triazole)-propane (Compound 35a), 1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-O-Ac)-1,2,3-triazole)-propane (Compound 35c), 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG7)-1-(Et-OBz)-1,2,3-triazole)-propane (Compound 35d), 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3 )-1-(Me-acetate)-1,2,3-triazole)-propane (Compound 35e), 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Et-2,2,2-tris-(Me-O-Bz))-1,2,3-triazole)-propane (Compound 37a), 1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-2,2,2-tris-(Me-O-Ac))-1,2,3-triazole)-propane (Compound 37b), 1,3-O-bis(phosphodiester-2S-O-(PEG4-O-Bz)-propane (Compound 38b), 1,3-O-bis(phosphodiester-2,2-bis(Me-O-mPEG2)-propane (Compound 45b), 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG2-O-Me)-1-(Et-O-Bz)-1,2,3-triazole)-propane (Compound 47f), 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG3-O-Me)-1-(Et-O-Bz)-1,2,3-triazole)-propane alkane (compound 47Gg), 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG3-O-Me)-1-(Et-2,2,2-tri-(Me-O-Bz))-1,2,3-triazole)-propane (compound 47i) or 1,3-O-bis(phosphodiester-2,2-bis(1-Me-4-(Me-O-PEG2-O-Bz)-1,2,3-triazole)-propane (compound 52).

[0038] In some embodiments, the symmetric chemical branch is 1,2,3-O-tris-(phosphodiester)-propane, 1,3-bis-(5-O-phosphodiester-pentylamido)-2-O-phosphodiester-propane, or 1,4,7-O-tris-(phosphodiester)-heptane.

[0039] In another embodiment, the symmetric chemical branching agent is 1,2,3-O-tris-(phosphodiester)-propane.

[0040] In other embodiments, the translocation control element is a polymer comprising two or more repeating units selected from Table 1A.

[0041] In yet other embodiments, the translocation control element is a polymer comprising two or more repeating units selected from the group consisting of 1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane (compound 12b) and 1,3-O-bis(phosphodiester)-2-(4-Me-O-PEG3)-1-(Et-O-Ac)-1,2,3-triazole)-propane (compound 35b).

[0042] In a further embodiment, the translocation control element is a polymer comprising the following sequence: [(1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane (compound 12b))]n1[(1,3-O-bis(phosphodiester)-2-(4-Me-O-PEG3)-1-(Et-O-Ac)-1,2,3-triazole)-propane (compound 35b))]n2, wherein n1 is 0 to 6 and n2 is 6 to 10.

[0043] In other embodiments, the first reporting sub-code and the second reporting sub-code are the same.

[0044] In some embodiments, the first reporter code and the second reporter code are polymers comprising two or more repeating units selected from the group consisting of hexaethylene glycol (D), ethane (L), triethylene glycol (X), 1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane (compound 12b), 1,3-O-bis(phosphodiester)-2-(4-Me-O-PEG3)-1-(Et-O-Ac)-1,2,3-triazole)-propane (compound 35b), 1,3-O-bis(phosphodiester-2,2-bis(Me-O-mPEG2)-propane (compound 45b), 1,3-O-bis(phosphodiester-2S-O-(PEG4-O-Bz)-propane (compound 5 Compound 38b), 1,3-O-bis(phosphodiester)-2s-O-mPEG6-propane (Compound 12c), 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Et-2,2,2-tri-(Me-O-Bz))-1,2,3-triazole)-propane (Compound 37a), 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Me-acetate)-1,2,3-triazole)-propane (Compound 35e), 1,3-O-bis(phosphodiester)-2s-O-(4-(Me-O-PEG2)-1-(Et-OBz)-1,2,3-triazole)-propane (Compound 3 5a), 1,3-O-bis(phosphodiester)-2-(4-Et-1-(Et-O-mPEG1)-1,2,3-triazole)-propane (Compound 31a), 2,3-O-bis(phosphodiester)-1-(1-dimethoxyquinazolinedione)-propane (Compound 20c), 2,3-O-bis(phosphodiester)-1-(N9-(3,6-dimethoxycarbazole)-propane (Compound 20e), 1,1′-O-bis(phosphodiester)-2,2′-(sulfonylbis(benzyl-4-yl))-diethanol (Compound 26d), 1,1′-O-bis(phosphodiester)-2,2′-bipyridyl-4,4′-yl)-dimethanol (Compound 26a), 2,3-O-bis(phosphodiester)-1-(1-dimethoxyquinazolinedione)-propane (Compound 20c), 2,3-O-bis(phosphodiester)-1-(N9-(3,6-dimethoxycarbazole)-propane (Compound 20e), ester)-1-(N1-(4,6-dimethoxy-3-Me-indole)-propane (Compound 20b), 3-(1,2-O-bis(phosphodiester)-propyl)-8,8-dimethylhexahydro-3H-3a,6-methanobenzo[c]isothiazole 2,2-dioxide (Compound 20d), 2,3-O-bis(phosphodiester)-1-(N1-(6-azathymine))-propane (Compound 20f), 1,5-O-bis(phosphodiester)-hexahydrofuro[2,6]furan (Compound 23), 1,1′-O-bis(phosphodiester)-octahydro-2,6-dimethyl-3,8:4,7-dimethano-2,6-naphthyridine-4,8-diyl)-dimethanol (Compound 26e), 2,3-O-bis(phosphodiester)-1-(N1-(2-Me-5-nitroindole)-propane (Compound 20h), 2,3-O-bis(phosphodiester)-1-(N1-(2-Me-5-nitroindole)-propane (Compound 20g), 2,3-O-bis(phosphodiester)-1-(5-benzofuran)-propane (Compound 20i), 1,2-O-bis(phosphodiester)-3-O-mPEG2-propane (Compound 5b), 1,3-O-bis(phosphodiester)-2-(4-Et-1-(Et-O-mPEG3)-1,2,3-triazole)-propane (Compound 31b) and 1,3-O-bis(phosphodiester)-3-O-mPEG4-propane (Compound 5a).

[0045] In other embodiments, the first reporter code and the second reporter code are polymers comprising two or more repeating units selected from hexaethylene glycol, ethane, triethylene glycol, and any compound listed in Table 1A.

[0046] In a further embodiment, the first reporter code and the second reporter code are polymers comprising two or more repeating units selected from hexaethylene glycol, ethane, triethylene glycol and 1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane (Compound 12b).

[0047] In a further embodiment, the first reporter code and the second reporter code are polymers comprising sequences selected from the group consisting of: (i) [(hexaethylene glycol)2(ethane)3(hexaethylene glycol)(triethylene glycol)], (ii) [(hexaethylene glycol)2(1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane (compound 12b))2(ethane)(triethylene glycol)3], (iii) [(hexaethylene glycol)2(1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane (compound 12b))3(ethane)2(hexaethylene glycol)(triethylene glycol)] and (iv) [(triethylene glycol)2(ethane)(1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane (compound 12b))6(ethane)7].

[0048] In another aspect, the present invention provides a symmetrical synthetic reporter tether (SSRT), wherein the symmetrical synthetic reporter tether is a polymer having a first end and a second end, and comprises a first linker connected in series from the first end to the second end, a reporter construct according to any one of the above reporter constructs, and a second linker, wherein the first linker and the second linker are the same and are a polymer comprising two or more repeating units selected from the group consisting of spermine (Q), hexaethylene glycol (D), 2-((4-((3-(benzoyloxy)-2-(((1-(3-(benzoyloxy)-2-((benzoyloxy)methyl)-2-((phosphodiester-oxy)methyl)propyl)-1H- 1,2,3-triazol-4-yl)methoxy)methyl)-2-((benzoyloxy)methyl)propoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-2-O-phosphodiester-propane-1,3-diyl dibenzoate (Compound 62), 1,3-O-bis(phosphodiester-2,2-bis(1-Me-4-(Me-O-PEG2-O-Bz)-1,2,3-triazole)-propane (Compound 52), 1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-O-Ac)-1,2,3-triazole)-propane (Compound 35c), 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG7 )-1-(Et-OBz)-1,2,3-triazole)-propane (Compound 35d), 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Et-2,2,2-tri-(Me-O-Bz))-1,2,3-triazole)-propane (Compound 37a), 1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-2,2,2-tri-(Me-O-Ac))-1,2,3-triazole)-propane (Compound 37b), 1,2-O-bis(phosphodiester)-3-(4-(Me-O-PEG3-O-Bz)-1-(1,2,3-triazole))-propane (Compound 31d) , 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG2-O-Me)-1-(Et-O-Bz)-1,2,3-triazole)-propane (Compound 47f), 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG3-O-Me)-1-(Et-2,2,2-tri-(Me-O-Bz))-1,2,3-triazole)-propane (Compound 47i), 1,2-O-bis(phosphodiester)-3-(4-methylpiperazin-1-yl)-propane (Compound 20j), 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG3-O-Me)-1-(Et-O-Bz)-1,2,3-triazole)-propane (Compound 47i).3-triazole)-propane (Compound 47g) and 1,1′-O-bis(phosphodiester)-N(p-tolyl)-diethanolamine (Compound 26b).

[0049] In some embodiments, a symmetrically synthetic reporter tether (SSRT) includes a polymerase enhancer region comprising two spermine repeating units.

[0050] In other embodiments, the symmetrically synthesized reporter tether (SSRT) comprises a translocation slowing region comprising two or more repeating units selected from the group consisting of: 1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-O-Ac)-1,2,3-triazole)-propane (compound 35c), 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG7)-1-(Et-OBz)-1,2,3-triazole )-propane (Compound 35d), 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Et-2,2,2-tri-(Me-O-Bz))-1,2,3-triazole)-propane (Compound 37a) and 1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-2,2,2-tri-(Me-O-Ac))-1,2,3-triazole)-propane (Compound 37b).

[0051] In other embodiments, the symmetrically synthesized reporter tether (SSRT) comprises a metastasis slowing region comprising a polymer selected from the group consisting of: (i) [((hexaethylene glycol)(1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-O-Ac)-1,2,3-triazole)-propane (Compound 35c))3(hexaethylene glycol)2], (ii) [((hexaethylene glycol)(1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-O-Ac)-1,2,3-triazole)-propane (Compound 35c))3(hexaethylene glycol)2], 5c))4(hexaethylene glycol)2], (iii) [((hexaethylene glycol)(1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG7)-1-(Et-OBz)-1,2,3-triazole)-propane (compound 35d))4(hexaethylene glycol)2] and (iv) [((hexaethylene glycol)(1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-2,2,2-tri-(Me-O-Ac))-1,2,3-triazole)-propane (compound 37b))4(hexaethylene glycol)2].

[0052] In still other embodiments, the first and second ends of the symmetrically synthesized reporter tether (SSRT) include a bonding moiety, and in certain embodiments, the bonding moiety is an azido (—N 3 ) group.

[0053] In another aspect, the present invention provides a method for sequencing a target nucleic acid, comprising the following steps: A method for sequencing a target nucleic acid, comprising: a) providing a daughter strand produced by template directed synthesis, the daughter strand comprising a plurality of XNTP subunits coupled in a sequence corresponding to a contiguous nucleotide sequence of all or a portion of the target nucleic acid, wherein each XNTP subunit of the daughter strand comprises a reporter construct, a nucleobase residue, and a selectively cleavable bond, and wherein the reporter construct allows extension of the subunit of the daughter strand upon cleavage of the selectively cleavable bond; b) cleaving the selectively cleavable bond to produce an Xpandomer having a length longer than the plurality of the subunits of the daughter strand, the Xpandomer comprising the reporter construct for resolving genetic information in a sequence corresponding to the contiguous nucleotide sequence of all or a portion of the target nucleic acid; and c) detecting the reporter construct of the Xpandomer.

[0054] In some aspects, the reporter construct for parsing genetic information comprises a reporter code and a translocation control element, wherein the translocation control element provides translocation control by steric hindrance and pauses translocation when the Xpandomer passes through a nanopore subjected to a baseline voltage, wherein the translocation control element engages the reporter code within the orifice of the nanopore, wherein the reporter code is sensed by the nanopore.

[0055] In some embodiments, the Xpandomer resumes translocation through the nanopore by application of a pulse voltage, wherein the pulse voltage is sufficient to allow translocation of the translocation control element while leaving the next reporter construct of the Xpandomer free to engage the nanopore.

[0056] In other embodiments, the translocation control element of the reporter construct engaged with the nanopore is translocated upon each pulse of the pulse voltage by steric hindrance.

[0057] In some embodiments, the target construct is sensed by the nanopore during the time period between pulses of the pulsed voltage.

[0058] In certain embodiments, the baseline voltage is about 55 mV to about 75 mV and the pulse voltage is about 550 mV to about 700 mV.

[0059] In some embodiments, the pulse voltage has a duration of about 5 μs to about 10 μs and a periodicity of about 0.5 ms to 1.5 ms.

[0060] In other embodiments, the nanopore is subjected to an alternating current (AC).

[0061] In further embodiments, one or more of the XNTP subunits comprises a 2' fluoroarabinosyl epimer.

[0062] In another aspect, the present disclosure provides a buffer for controlling the translocation rate of a polymer through a nanopore, comprising at least one salt selected from the group consisting of: NH 4 Cl, MgCl 2 , LiCl, KCl, CsCl, NaCl and CaCl 2 .

[0063] In some embodiments, the buffer further comprises at least one solvent selected from the group consisting of 3-methyl-2-oxazolidinone (MOA), DMF, ACN, DMSO and NMP, wherein the solvent is present in a range of about 1% vol / vol to about 35% vol / vol.

[0064] In other embodiments, the buffer further comprises at least one additive selected from the group consisting of sodium hexanoate (NaHex), EDTA, redox agents, PEG, glycerol, polysucrose, and the like.

[0065] In another aspect, the present disclosure provides a buffer system for controlling the translocation rate of a polymer through a nanopore detector, comprising a cis buffer and a trans buffer, wherein the cis buffer has a first salt concentration and the trans buffer has a second salt concentration, wherein the first salt concentration is lower than the second salt concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1A , Figure 1B , Figure 1C and Figure 1D is a concise schematic diagram illustrating the main features of generalized XNTPs and their function in sequencing by expansion (SBX).

[0067] Figure 2 is a schematic diagram illustrating further details of one embodiment of XNTP.

[0068] Figure 3 is a schematic diagram illustrating one embodiment of an Xpandomer passing through a biological nanopore.

[0069] Figure 4 is a schematic diagram illustrating another embodiment of an Xpandomer passing through a biological nanopore.

[0070] Figure 5A-5D is a schematic diagram illustrating an alternative embodiment of a reporting subcode.

[0071] Figure 6 is a schematic diagram illustrating one example of solid-state synthesis of an SSRT reporter construct.

[0072] Figure 7 Alternative structural examples of SSRT reporter constructs are illustrated.

[0073] Fig. 8A and Figure 8B is a schematic diagram illustrating one example of the cyclization of SSRT and dNTP-2c to form XNTP.

[0074] Fig.9A and Fig. 9B is a schematic diagram illustrating one example of the control of translocation of an Xpandomer through a nanopore.

[0075] Fig.10 is a schematic diagram illustrating one example of a biotin derivative.

[0076] Fig.11 is a schematic diagram illustrating one embodiment of a cleavable extension oligonucleotide.

[0077] Fig.12 is a schematic diagram illustrating one embodiment of a polymer undergoing ratcheting through a nanopore.

[0078] Fig.13A and Fig. 13B is a representative trace describing the characteristics of the reported subcode.

[0079] Fig.14A and Fig. 14B is a histogram display of the aligned read population of the nanopore-derived sequences.

[0080] Fig.15 is a representative trace showing a simple DNA template sequence.

[0081] Fig.16 is a representative trace showing the CAGT repeat DNA template sequence.

[0082] Fig.17 is a representative trace showing the composite DNA template sequence.

[0083] Fig.18 is a representative trace showing the composite DNA 222mer template sequence.

[0084] Fig.19 is a histogram display of the aligned read population of the nanopore-derived sequences.

[0085] Fig. 20A is a schematic diagram illustrating one embodiment of an Xpandomer undergoing ratcheting through a nanopore.

[0086] Fig. 20B is an example of current measurement of a translocation Xpandomer subjected to ratcheting. DETAILED DESCRIPTION

[0087] By referring to the following detailed description of the preferred embodiments of the present invention and the examples included herein, the present invention can be more easily understood. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by ordinary technicians in the field to which the present invention belongs.

[0088] References throughout this specification to "one embodiment," "an embodiment," and variations thereof, mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the phrases "in one embodiment" or "in an embodiment" appearing in different places in this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0089] As used in this specification and the appended claims, the singular forms "a", "an", "the", and "said" include plural referents, i.e., one or more, unless the context clearly dictates otherwise. It should also be noted that the conjunctions "and" and "or" are generally used in the broadest sense to include "and / or", unless the content and context clearly dictate inclusivity or exclusivity as the case may be. Therefore, the use of alternatives (e.g., "or") should be understood to mean any one, both, or any combination of the alternatives. In addition, the compositions of "and" and "or" when referenced herein as "and / or" are intended to cover embodiments that include all relevant items or ideas, as well as one or more other alternative embodiments that include less than all relevant items or ideas.

[0090] Unless the context requires otherwise, throughout the specification and following claims, the word "comprise" and its synonyms and variations, such as "have" and "include," and variations thereof, such as "comprising," are to be interpreted in an open, inclusive sense, e.g., "including but not limited to." The term "consisting essentially of limits the scope of a claim to the specified materials or steps, or those that do not materially affect the basic and novel characteristics of the claimed invention.

[0091] The abbreviation, "for example", is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Therefore, the abbreviation "for example" is synonymous with the term "for example". It should also be understood that the singular forms "a", "an" and "the" used herein and in the appended claims include plural forms, unless the context clearly dictates otherwise, the term "X and / or Y" means "X" or "Y" or "X" and "Y", and the letter "s" after a noun means the plural and singular forms of the noun. In addition, in the case of describing features or aspects of the present invention in terms of Markush groups, it is intended and will be recognized by those skilled in the art that the present invention includes and is therefore also described in terms of any individual member and any member subgroup of the Markush group, and the applicant reserves the right to amend the application or claims to explicitly refer to any individual member or any member subgroup of the Markush group.

[0092] Any headings used in this document are only used to speed up the reader's review and should not be construed as limiting the invention or claims in any way. Therefore, the titles and abstracts of the present disclosure provided herein are only for convenience and do not interpret the scope or meaning of the embodiments.

[0093] Where numerical ranges are provided herein, it is to be understood that each intervening value between the upper and lower limits of the range (based on one-tenth of the unit of the lower limit, unless the context clearly dictates otherwise) and any other stated or intermediate value within the stated range are included within the present invention. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and are also encompassed by the present invention, subject to any explicitly excluded limits within the specified ranges. If the specified range includes one or both limits, ranges excluding either or both of those included limits are also included in the present invention.

[0094] For example, any concentration range, percentage range, ratio range, or integer range provided herein should be understood to include the value of any integer within the range and, where appropriate, its fractional value (e.g., one tenth and one hundredth of an integer), unless otherwise stated. In addition, any numerical range associated with any physical feature (e.g., polymer subunits, size, or thickness) cited herein should be understood to include any integer within the range, unless otherwise stated. As used herein, the term "about" refers to ±20% of the indicated range, value, or structure, unless otherwise stated.

[0095] Extended sequencing

[0096] The "sequencing by amplification" (SBX) protocol developed by Stratos Genomics (see, e.g., Kokoris et al., U.S. Pat. No. 7,939,259, "High Throughput Nucleic Acid Sequencing by Expansion") is based on the polymerization of highly modified, non-natural nucleotide analogs called "XNTPs." In general, SBX uses biochemical polymerization to transcribe the sequence of a DNA template onto a measurable polymer called an "Xpandomer." The transcribed sequence is encoded in high signal-to-noise ratio reporters along the Xpandomer backbone, which are approximately 10 nm apart and designed for high signal-to-noise ratio, well-differentiated reactions. These differences provide significant performance enhancements in sequence read efficiency and accuracy of Xpandomers relative to native DNA. An overview of the SBX process is provided in Figure 1A , Figure 1B , Figure 1C and Figure 1D Described in.

[0097] XNTPs are expandable, 5' triphosphate-modified, non-natural nucleotide analogs that are compatible with template-dependent enzymatic polymerization. Figure 1A , which emphasizes the unique features of these non-natural substrates: XNTP 100 has two different functional regions; namely: a selectively cleavable phosphoramidite bond 110, connecting 5'α-phosphate 115 to a nucleobase 105; and a symmetrically synthesized reporter tether (SSRT) 120 connected to certain positions within the nucleoside triphosphoramidite, which allows controlled unfolding by cleavage of the phosphoramidite bond. SSRT includes linkers 125A and 125B, which are separated by a selectively cleavable phosphoramidite bond. Each linker is connected to one end of the reporter code 130. XNTP100 is described in a "constrained configuration", which is a feature of the subchain product of XNTP substrates and template-dependent polymerization. The constrained configuration of polymerized XNTPs is a precursor to the extended configuration, as seen in Xpandomer products. The transition from the constrained configuration to the extended configuration occurs when the PN bond of the phosphoramidite within the main backbone of the subchain is broken.

[0098] The synthesis of Xpandomer polymers is summarized in Figure 1B and Figure 1CDuring assembly, monomeric XNTP substrates 145 (XATP, XCTP, XGTP, and XTTP) are polymerized at the extendable end of the nascent daughter chain 150 by a template-directed polymerization process using the single-stranded template 140 as a guide. Typically, the process starts with a primer and proceeds in the 5' to 3' direction. Typically, a DNA polymerase or other polymerase is used to form the daughter chain, and conditions are selected so as to obtain a complementary copy of the template chain. After the daughter chain is synthesized, the coupled SSRT forms a constrained Xpandomer that further forms the daughter chain. The SSRT in the daughter chain has a "constrained configuration" of the XNTP substrate. The constrained configuration of the SSRT is a precursor to the unfolded configuration, as seen in the Xpandomer product.

[0099] like Figure 1C As shown, the transition from the constrained configuration 160 to the expanded configuration 165 is caused by the cleavage of a selectively cleavable phosphoramidate bond (represented by an unshaded ellipse for simplicity) within the primary backbone of the daughter strand. In this embodiment, the SSRT comprises one or more reporters or reporter codes, 130A, 130C, 130G, or 130T, that are specific to the nucleobases to which they are attached, thereby encoding the sequence information of the template. In this way, the SSRT provides a means to expand the length of the Xpandomer and reduce the linear density of the parent strand sequence information.

[0100] Figure 1D Xpandomer 165 is illustrated as translocating from cis reservoir 175 to trans reservoir 185 through nanopore 180. After passing through the nanopore, each reporter code of the linearized Xpandomer (labeled "G," "C," and "T" in this illustration) generates a unique and reproducible electronic signal (represented by overlay trace 190) specific for the nucleobase to which it is attached.

[0101] Figure 2 The general structure of one embodiment of an XNTP is depicted in more detail. XNTP 200 comprises a nucleoside triphosphoramidate 210 having a linker portion 220A and 220B, which are separated by a selectively cleavable phosphoramidate bond 230. SSRTs are attached to the nucleoside triphosphate at bonding groups 250A and 250B, wherein the first SSRT terminus is attached to a heterocycle 260 (represented here by cytosine, although the heterocycle can be any of the four standard nucleobases A, C, G, or T), and the second SSRT terminus is attached to an alpha phosphate 270 of the nucleobase backbone. Those skilled in the art will appreciate that many suitable coupling chemistries known in the art can be used to form the final XNTP substrate product, for example, SSRT conjugation can be accomplished by formation of a triazole bonding group.

[0102] In this embodiment, SSRT 275 includes several functional elements or "features", such as polymerase enhancement regions 280A and 280B, reporter codes 285A and 285B, and translation control elements (TCE) 290A and 290B. In other embodiments, SSRT includes a single TCE. Each of these features performs a unique function during the translocation of Xpandomer through the nanopore to generate a series of unique and repeatable electronic signals. SSRT 275 is designed to control the Xpandomer translocation rate caused by TCE by a combination of steric hindrance and / or electrical repulsion, as further discussed herein. The size of different reporter codes can prevent ions from flowing through the nanopore at different measurable levels. Specific SSRT polymer sequences can be effectively synthesized using phosphoramidite chemistry commonly used for oligonucleotide synthesis. Reporter codes and other features can be designed by selecting the sequence of specific phosphoramidites from commercially available and / or proprietary libraries. Such libraries include, but are not limited to, polyethylene glycols with a length of 1 to 12 or more ethylene glycol units and aliphatic polymers with a length of 1 to 12 or more carbon units. In certain embodiments, the SSRT includes a feature called a "polymerase enhancement region" at the end of the SSRT near the nucleotide triphosphoramidite diester. The polymerase enhancement region may include a positively charged polyamine spacer (e.g., a primary amine, a secondary amine, a tertiary amine, or a quaternary amine) or a triamine spacer (three secondary amines, each separated by three carbons) that promotes the incorporation of XNTP structures by nucleic acid polymerases. In certain embodiments, the polymerase enhancement region includes two spermine repeating units, wherein the spermine portion is provided by a phosphoramidite monomer having the following structure (one skilled in the art will recognize that the trifluoroacetamide protecting group is removed at the end of SSRT synthesis to expose the amine group on the spermine):

[0103]

[0104] As used throughout this disclosure, the term "reporter construct" refers to an element of an SSRT that includes a reporter code, a symmetrical chemical branch, and a translocation control element. In certain embodiments, the reporter construct is a polymer that includes a first reporter code, a symmetrical chemical branch with a translocation control element, and a second reporter code connected in series from a first end to a second end. The term "with" refers to a covalent connection between a symmetrical branch and a translocation control element that produces a favorable orientation of the translocation control element relative to the two reporter codes. As further discussed herein and referenced Figure 6 - Figure 8, symmetric chemical branching can be represented by the letter "Y", where two reporter codes are joined to the arms of the Y and the translocation control element is joined to the stem of the Y. Thus, the two reporter codes are joined in-line by the branch, and the branch carries the translocation control element in a perpendicular orientation relative to the linear, in-line, SSRT.

[0105] As used throughout this disclosure, the terms "Linker A" and "Linker B" refer to SSRT regions that each include a polymerase enhancing region and one or more translocation decelerating features or regions, and in certain embodiments, include a spacer region containing, for example, a PEG6 polymer, which can be customized to adjust the length of the SSRT that passes through the nanopore.

[0106] In certain embodiments, the XNTP may be a compound having the following general structure:

[0107]

[0108] In one embodiment, R can be H, for example, when the compound is used to sequence a DNA template. In another embodiment, R can be OH, for example, when the compound is used to sequence an RNA template.

[0109] In certain embodiments, the nucleobase is adenine, cytosine, guanine, thymine, uracil or a nucleobase analog. It will be appreciated by those skilled in the art that adenine, cytosine, guanine, thymine and uracil are naturally occurring nucleobases. As used herein, the term "nucleobase analog" refers to a non-naturally occurring nucleobase that can form a Watson and Crick base pair with a complementary nucleobase on an adjacent single-stranded nucleic acid template. Exemplary nucleobase analogs include, but are not limited to, 5-fluorouracil; 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactoside, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D- Mannosyl quercetin, 5′-methoxycarboxymethyl uracil, 5-methoxy uracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), whibutoxin, pseudouracil, quercetin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, 2,6-diaminopurine, 3-nitropyrrole, 8-aza-7-deazaguanine, 8-aza-7-deazainosine and 8-aza-7-deazaadenine.

[0110] As discussed herein, the reporter construct is a polymer having a first end and a second end, and comprises a first reporter code connected in series from the first end to the second end, a symmetrical chemical branch with a translocation control element, and a second reporter code. This series of features reflects the symmetrical structure of the reporter construct (and the entire SSRT, including the symmetrical linker, linker A and linker B), in which the sequences of the two reporter codes are identical and connected in series in opposite orientations through symmetrical chemical branches. The entire SSRT includes the synthesis of the reporter construct, which is referred to herein as Figure 6 Further discussion to Figure 8. In short, synthesis proceeds in the 3' to 5' direction, starting from the 3' end of the TCE. The addition of a symmetrical branch to the 5' end of the TCE allows the first and second reporter codes to be polymerized simultaneously from each arm of the branch, and then the linker A and linker B are synthesized simultaneously, terminating at the 5' ends of the first and second ends of the SSRT. It has been found that the online redundancy provided by two identical reporter codes separated by symmetrical branches with translocation control elements provides several advantages in the nanopore sequencing process. For example, the Xpandomer may be read by the nanopore when translocated in either direction, that is, the Xpandomer can be read "forward" or "backward". This flexibility enables the "ratchet" sequencing method discussed further herein and other methods, such as "dental floss" based on the AC mode of voltage application.

[0111] Figure 3 An example of an Xpandomer cleaved during translocation of an α-hemolysin nanopore is shown. The biological nanopore is embedded in a lipid bilayer membrane that separates and electrically isolates two electrolyte reservoirs. A typical electrolyte is 1 molar KCl buffered to a pH of 7.0. When a small voltage (typically 100 mV) is applied across the bilayer, the nanopore restricts the flow of ionic current and is the main resistor in the circuit. The Xpandomer reporter code is designed to provide a specific level of ionic current blockade, and the sequence information can be read by measuring the sequence of ionic current levels as the reporter code sequence translocates the nanopore.

[0112] The α-hemolysin nanopore is usually oriented so that translocation occurs by entering the vestibule side and exiting the stalk side. Figure 3As shown, the nanopore is oriented to capture the Xpandomer from the stem side first. In some cases, this orientation may result in fewer blocking artifacts than when entering the vestibule first. However, according to the present invention, the α-hemolysin nanopore can be oriented in either direction. As the Xpandomer translocates, the reporter enters the stem until its translocation control element stops at the entrance of the stem. The reporter is held in the stem until the TCE is able to enter and pass through the stem, and then translocation proceeds to the next reporter. In this embodiment, entry of the TCE into the stem is achieved by dissociation of the translocation control moiety from the TCE. Advantageously, the inventors have discovered that TCEs constructed from a new class of pendant PEG phosphoramidites provide significantly improved translocation control based on inherent physicochemical and steric properties, thereby avoiding the binding and dissociation of translocation control moieties that rely on trans-acting.

[0113] Novel compounds for SSRT feature design

[0114] Phosphoramidite chemistry, commonly used for automated oligonucleotide synthesis, provides an effective and convenient means for synthesizing polymeric SSRTs. However, the ultimate potential for SSRT feature design is limited by the phosphoramidite monomers (PPAs) available in commercial libraries. Commercial PPAs are primarily based on nucleoside core structures and therefore do not provide the range of physicochemical properties required to design a wider array of functions, which improves the efficiency and accuracy of nanopore readings. In order to address this shortcoming in the art, the inventors designed and synthesized a large number of new PPA monomer compounds. Importantly, these compounds are not based on nucleoside core structures well known in the art, and as described above, feature design is limited.

[0115] As used herein, the abbreviation "PPA" refers to a phosphoramidite that is O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite. It is readily understood by those skilled in the art that the term "phosphoramidite" refers to the structure of a monomer precursor; after PPA is polymerized in-line into SSRT, the monomer is converted into a phosphodiester-linked oligomeric product.

[0116] Other methods for making phosphodiester backbone polymers can be used to synthesize SSRT. Therefore, monomers used with these chemicals can also produce SSRTs with non-nucleoside elements. Other assembly methods may involve the use of automatic or manual assembly strategies completed in solution phase or on a solid support. H-phosphonate synthesis and phosphotriester synthesis are examples known in the art. In addition, methods using enzymatic synthesis can be applicable to the synthesis of SSRT (e.g., those for enzymatic oligonucleotide synthesis). In certain embodiments, the synthesis of SSRT can be based on a combination of any of the above-mentioned synthesis methods.

[0117] The following is a brief, non-limiting summary of certain principles used to guide the design of PPA monomers. 1) Phosphate spacing. The designed compounds maintain C3 (3 atoms) spacing, which mimics the spacing of natural nucleotide backbones. In certain embodiments, other suitable spacings include 2 to 20 atomic spacings. Unexpectedly, it was found that the atomic spacing affects the rate of nanopore translocation, allowing for fine-tuning of translocation control. 2) Hydrophilicity. Compounds are designed to optimize the hydrophilicity of SSRT features as needed for specific functions. Some monomer designs are based on PEG because it can increase water solubility, which is an important property of, for example, reporter codes. The inventors were able to fine-tune the hydrophilicity of PPA monomers by adjusting the length of the PEG polymer, as well as by terminating the PEG polymer with methyl ether or introducing 1,2,3-triazole into the polymer, which had the unexpected effect of further improving water solubility. 3) Spatial volume. Several alternative configurations of linear, branched, cyclic, and dendritic PPA structures were designed and tested to evaluate the effect of spatial volume on the current passing through the nanopore. 4) Chirality in the backbone. The nanopore is a chiral environment. Enantiomeric compounds were designed to determine if key nanopore signal properties were affected in any way. 5) Charge. In addition to the anti-phosphate charge, some compounds carry positive charges, such as tertiary amines, or negative charges, such as carboxylic acids. (6) Aromatics: Compounds composed of a variety of aromatic and heteroaromatic structures were incorporated into the backbone to determine if the interaction with the nanopore resulted in the desired signal properties.

[0118] In addition to attenuating nanopore signal properties such as translocation rate control or current level control, PPA monomer compounds can also affect the physicochemical properties of the Xpandomer. As semi-synthetic polymers, Xpandomers exhibit properties associated with both natural polymers (e.g., DNA) and synthetic polymers. In some embodiments, certain PPA monomers can reduce undesirable interactions between Xpandomers or interactions between Xpanodmers and certain process elements of the SBX workflow. For example, it is possible to reduce Xpandomer self-aggregation, formation of higher-order glass or gelatin, sequestration, passive adsorption to or interaction with container surfaces, nanochannel walls, or surfaces of fabricated devices containing membranes and nanopores.

[0119] A class of compounds that have been demonstrated to provide excellent functionality when incorporated into SSRT features are referred to herein as "pendant PEGs." These structures are based on a molecular core that is capable of attaching one or more PEG-containing polymers in a pendant configuration relative to the core. A structural analogy can be made between the polymer of the pendant PEG compound and a comb, where the phosphodiester bonds between the individual compounds form the base of the comb and the PEG-based polymers form the teeth. Advantageously, several properties of the pendant PEG "teeth" can be customized for specific SSRT features (e.g., one or more of the spacing, length, and composition of the polymer teeth). Structures 1a, 2a, 3a, and 4a below illustrate four exemplary embodiments of pendant PEG core structures.

[0120]

[0121] In certain non-limiting embodiments, X or X′ may represent —CH 2 O-[CH 2 CH 2 O-] m O- wherein m is 1-10 and Y or Y' may represent -H, -CH 3 ,

[0122] Tables 1A-C list a non-limiting collection of novel phosphoramidite monomer compounds for use in, for example, SSRT feature design. The synthesis scheme for each compound is mentioned with reference to the relevant examples, and the specific precursors for each compound are included. Analytical data characterizing the purified synthetic compounds are also listed in Table 1A. These compounds can be used to synthesize any suitable polymeric features, such as SSRT reporter codes, translocation control elements, and translocation deceleration features, as described in further detail herein. Table 1A also provides the names of the compounds with reference to the tandem structures they adopt after incorporation into the synthetic polymer.

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171] Table 1B

[0172] Exemplary Novel Phosphoramidite (PPA) Monomers - Group B

[0173]

[0174]

[0175]

[0176] Table 1C

[0177] Exemplary Novel Phosphoramidite (PPA) Monomers - Group C

[0178]

[0179]

[0180]

[0181]

[0182]

[0183] Translocation Control Element (TCE) Based on Novel Phosphoramidite Monomers.

[0184] As discussed herein, the TCE feature of SSRT is designed to prevent Xpandomer translocation so that the reporter code is positioned within the nanopore orifice for measurement. The availability of the new phosphoramidite monomer compound of the present invention enables the design of the next generation of TCE structures, which control the translocation rate by one or more of steric hindrance, electrical repulsion, and preferential interactions with the nanopore. When positioned at the pore orifice, the resistance of the TCE to the driving force of the ionic current and the increase in the voltage (i.e., voltage pulse) required to overcome the stagnation and restore the translocation can be customized by modulating various properties of the TCE (and in some embodiments, the reporter code and other elements of the SSRT) such as volume, length, and / or charge density. Importantly, since the translocation rate is controlled by the intrinsic properties of the TCE, the translocation control is freed from the burden of relying on prior art strategies, which employ, for example, nucleotide hybridization strategies based on reversible interactions with soluble oligonucleotides.

[0185] In certain embodiments, TCE is a polymer produced by solid phase synthesis using a phosphoramidite method, having a suitable monomer structural unit terminated with a branched structure (i.e., "branch"). Branched phosphoramidites are known in the art, and include commercially available symmetric and asymmetric branches from, for example, Glen Research and ChemGenes. In one embodiment, TCE branches are symmetrical branched CED phosphoramidites, wherein each arm of the branch is connected to a reporter code. Exemplary symmetrical chemical branches include 1,2,3-O-tri-(phosphodiester)-propane, 1,3-bis-(5-O-phosphodiester-pentylamide)-2-O-phosphodiester-propane and 1,4,7-O-tri-(phosphodiester)-heptane.

[0186] Figure 4How TCE blocks Xpandomer translocation by nanopore is illustrated in simplified form.Here, each SSRT of Xpandomer includes reporter subcode 485A and 485B, which is connected to TCE 490 at the arm end of the branch structure 493 of TCE.In this embodiment, TCE 490 includes structure 495, which has a physical volume larger than the physical volume of the reporter subcode.When TCE 490 encounters the pore orifice, Xpandomer translocation is blocked by nanopore barrel 450.In certain embodiments, the volume of TCE and the charge density of the reporter subcode (that is, the local electric field of the blocking site) contribute to translocation retardation.During the pause, the reporter subcode 495A is maintained in the nanopore barrel, and the current is blocked from passing through the pore in a characteristic and detectable manner.In order to overcome the retardation, a voltage pulse is applied to the system, forcing TCE to enter and pass through the pore.Translocation is then restored until the next TCE encounters the pore orifice.

[0187] To tailor translocation control, several structural properties of the TCE (and, in certain embodiments, other features of the SSRT) can be adjusted. For example, one or more of the length, volume, and charge density of the TCE, as well as the spatial positioning of the charged elements within the nanopore barrel, can be modified. In some embodiments, the volume of the TCE is increased by incorporating one or more pendant PEG phosphoramidites into the polymeric structure. For example, the TCE can incorporate 2 to 30, 2 to 20, 3 to 15, or 4 to 14 pendant PEG phosphoramidite compounds. In other embodiments, the TCE can include any suitable number of phosphoramidite compounds listed in Tables 1A-C and combinations thereof. For example, the TCE can include 1 to 10, 2 to 8, or 2 or 3 different phosphoramidite compounds in any order; in certain embodiments, at least one of the phosphoramidite compounds is a pendant PEG phosphoramidite. In certain embodiments, the length of the entire TCE can include 2 to 30, 2 to 20, 3 to 15, or 4 to 14 phosphoramidite compounds. In some embodiments, the formula of the TCE can be (PPA1) n1 (PPA2) n2 , wherein PPA1 and / or PPA2 represent pendant PEG phosphoramidite compounds and n1=1 to 12 and n2=0 to 10. The inventors have found that TCEs based on this formula significantly reduce sequencing errors (e.g., insertion or deletion events) and achieve single-pulse transitions between sequential SSRTs. In certain embodiments, the TCE includes a polymer synthesized from a phosphoramidite compound having the following sequence: [(1-O-DMT-3-O-PPA-2S-O-mPEG4-propane (Compound 12b))] n1[(1-O-DMT-3-O-PPA-2-(4-Me-O-PEG3)-1-(Et-O-Ac)-1,2,3-triazole)-propane (Compound 35b)] n2 , wherein n1 is 0 to 6 and n2 is 6 to 10. In other embodiments, the TCE includes one or more phosphoramidite chromophores detectable by UV radiation, such as benzofuran or triazole-containing PPAs.

[0188] In other embodiments, TCE can include a branch structure with more than two arms. In one embodiment, the phosphoramidite branch can have a terminal branch structure. In this embodiment, the branch has four arms, two of which are connected to the reporter code, and two of which contribute to translocation control. In other embodiments. The branch can be customized to optimize features such as size, polarity and stability. In one embodiment, the branch includes an isocyanate trimer.

[0189] In a further embodiment, the TCE of the present invention may include two branches (i.e., a "double-branched" TCE) where the branches are separated by multiple unbranched phosphoramidites. The branches can be symmetrical or asymmetrical structures. The asymmetrical structure can be a single enantiomer or a racemate. In addition, the racemate and / or a combination of two enantiomers can be used at different positions in the TCE. In this embodiment, each branch promotes a different translocation pause event, the first of which can be referred to as a "code pause", which holds the reporter code in the nanopore, and the second of which can be referred to as a "clock pause", which produces a unique signal indicating that the detection system has "read" the previous reporter code.

[0190] Table 2 lists several exemplary TCE sequences. It should be emphasized that the present invention is not limited to these specific embodiments, as the skilled person will appreciate that, based on the present disclosure, a wide range of different TCE libraries can be designed to accommodate a wide range of experimental requirements. In certain embodiments, any of the TCE sequences listed below can terminate at the distal end of a branch having one or more spacer compounds, including C3, benzofuran or PEG3. The key in Table 2 identifies the compound in its form as a phosphoramidite monomer. It will be apparent to those of ordinary skill in the art that the descriptor "phosphoramidite" applies only to compounds in monomeric form; descriptors applicable to compounds in the "online" form of the polymer are listed in Table 1A.

[0191] Table 2

[0192] Exemplary Translocation Control Elements (TCEs)

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199] Methods and constructs for reducing the incidence of insertion and deletion events during Xpandomer translocation

[0200] In other aspects, the present invention provides a means of controlling translocation by modifying Xpandomers in conjunction with discrete translocation deceleration features (also referred to herein as "D cells") designed into SSRT. As disclosed herein, Xpandomers undergo several processing steps after synthesis, including an amine modification step. During the amine modification process, the Xpandomer is treated with succinic anhydride, which reacts with secondary amine groups on the spermine component of the SSRT polymerase enhancer region (and in some cases, primary amine groups introduced by the acid cleavage step). Succinylation of the amine group results in the introduction of a negatively charged hemisuccinate group. By increasing the degree of succinylation, the degree of negative charge on the spermine-based enhancer is also increased. Each spermine phosphoramidite component has a net charge of (+3); in a standard modification reaction, the charge of each amine moiety changes from (+1) to (-1). The inventors have discovered conditions that produce different degrees of spermine succinylation, such that the net charge of the spermine component can vary from (+3) to (-5). Under certain conditions, it may be desirable to increase the negative charge of the enhancer region in order to increase the rate of Xpandomer translocation when a voltage pulse is applied (referred to herein as enhancer electrical mobility). Notably, such electrical mobility has been found to reduce the percentage of insertion errors during sequence reads and increase overall sequencing throughput.

[0201] Thus, in certain embodiments, the Xpandomer treatment includes an amine modification step. Amine (e.g., spermine) modification can be achieved by changing the succinylation reaction conditions, such as one or more of the reaction time, temperature, pH, and / or the concentration of succinic anhydride used in the reaction. In other embodiments, the Xpandomer treatment further includes one or more of the HEPES wash steps after the amine modification step to achieve more complete amine succinylation.

[0202] In another aspect, the present disclosure provides one or more translocation deceleration features or regions (the terms "features" and "regions" are used interchangeably herein) that are permanently charged, such as tertiary or quaternary amines and / or bulky compounds. The translocation deceleration feature can be introduced into the SSRT at a position within or near the polymerase enhancer. The deceleration feature is selected so that it will not change during the Xpandomer modification (e.g., succinylation) reaction. It has been found that the inclusion of one or more deceleration features into the appropriate position in the SSRT reduces the percentage of deletion errors, which is caused by the increase in translocation rate due to excessive modification of the enhancer. Without being bound by theory, it is speculated that most deceleration features generate a "friction" type force that reduces the Xpandomer translocation rate when encountering the nanopore orifice. Typically, the deceleration feature is introduced into the SSRT at a position between the polymerase enhancer and the reporter code (i.e., adjacent to the enhancer).

[0203] The metastasis deceleration features of the present invention can be incorporated into any suitable number and combination of the phosphoramidite compounds listed in Tables 1A-1C or commercially available phosphoramidites. In some embodiments, the deceleration feature comprises a combination of 1 to 4 different monomer units. In other embodiments, the deceleration feature may comprise 1 or 2 different monomer units. The entire length of the deceleration feature can be 1 to 15 monomer units, or in other embodiments, 4 to 12 or 6 to 10 monomer units. Table 3 lists non-limiting examples of alternative metastasis deceleration features. The key in Table 3 identifies the compound in its form as a phosphoramidite monomer. It will be apparent to those of ordinary skill in the art that the descriptor "phosphoramidite" applies only to compounds in monomeric form; descriptors applicable to compounds in the "online" form of the polymer are listed in Table 1A.

[0204] Table 3

[0205] Exemplary Translocation Slowdown Features

[0206]

[0207]

[0208]

[0209] Report subcode

[0210] Each SSRT uses TCE to locate the reporter code within a nanopore region with high ionic current resistance. In α-hemolysin, this region is the stem. In this region, the sizes of different reporters are designed to block ion flow at different measurable levels. Reporters can be designed by selecting specific phosphoramidite sequences from a collection of phosphoramidite monomer compounds set in Tables 1A-1C and / or commercially available libraries. Suitable monomer compounds are also disclosed in Applicant's U.S. Patent No. 10,457,979, which is incorporated herein by reference in its entirety, including PEG3, PEG6, and C2.

[0211] Each constituent monomer compound contributes to the net resistance depending on its location in the nanopore, its displacement, its charge, its interaction with the nanopore, its chemical and thermal environment, and other factors.

[0212] Reporter code design is guided by balancing measurement features including: (i) normalized ion current (I / I o ): where I is the ionic current, and I o is the open channel current; (ii) ionic current noise: including multi-state responses, blockade, random spikes, etc.; and / or (iii) the release time of the control moiety or the time that the TCE is stalled at the stem entrance.

[0213] Figures 5A-5D Illustrated is how different reporter codes can be designed to maintain similar charge density along the backbone while providing characteristic levels of pore blockage due to the different volumes occupied by each unique monomer component. In these insets, the nanopore is depicted in cross-section with the barrel and vestibule portions indicated. The reporter codes are depicted in simplified form with black circles representing the charged phosphodiester moieties introduced by the phosphoramidite component of the code. Figure 5A A linear code is illustrated, identified in this embodiment as a "zero PEG code," but the invention is not intended to be so limited, for example, in certain embodiments, a linear code may include a PEG portion. Figure 5B , Figure 5C and 5D It is illustrated how a unique code can be constructed from a repeating unit of a single branched monomer compound, showing exemplary PEG-based structures, for example, pendant PEG compounds. In these embodiments, the branched portions of the three different codes occupy different volumes in the pore channel and thereby produce unique signals that can be distinguished from each other. In these embodiments, the charge density along the backbone is the same for each reporter code. However, as further discussed herein, in other embodiments, the reporter code can be designed to have a charge density gradient along the backbone.

[0214] The reporter ion current blockade and its duplex release time are also modulated by the measurement conditions, such as: (i) voltage; (ii) electrolyte; (iii) temperature; (iv) pressure; and / or (v) pH, as further described herein.

[0215] In some embodiments, the TCE associated with the reporter also contributes to ion flow blockage.

[0216] For a given set of measurement conditions, reporters can be designed to define minimum and maximum I / Io levels that define the dynamic range of the measurement. Other reporters can be designed to have different I / Io levels within the dynamic range. Since each reporter is suspended in the nanopore, the measured I / Io levels must remain stable for a long enough time and have low enough noise so that the reporter type can be uniquely distinguished. The dynamic range is maximized by choosing a backbone of low impedance molecules (reporter code polymers), typically those with a small physical cross-section and low linear mass density.

[0217] Table 4 lists exemplary reporter codes, but it should be understood that the present invention contemplates any suitable combination and amount of reporter codes incorporated into the phosphoramidite compounds disclosed herein. The key in Table 4 identifies the compounds in their form as phosphoramidite monomers. It will be apparent to one of ordinary skill in the art that the descriptor "phosphoramidite" applies only to compounds in monomeric form; descriptors applicable to compounds in multimeric "in-line" form are listed in Table 1A.

[0218] Table 4

[0219] Example Reporting Subcodes

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227] Synthesis of SSRT and XNTP

[0228] As disclosed herein, symmetrically synthesized reporter tethers (SSRTs) were synthesized using standard automated oligonucleotide synthesis protocols. Figure 6 The SSRT synthesis is illustrated in simplified form. In step A, the phosphoramidite is immobilized on a solid support; in step B, the phosphoramidite monomer is polymerized on the support using phosphoramidite coupling; in step C, symmetric branches are added to the growing SSRT structure; in step D, symmetric phosphoramidite branches are polymerized on each arm of the symmetric branch; in step E, a terminal azide group is added to enable conjugation of SSRT to dNTP-2c via a click reaction; in step F, SSRT is released from the substrate in its final form.

[0229] In one embodiment, SSRT synthesis utilizes a four-step iterative process that includes 1) synthesizing SSRT polymer on a solid support controlled pore glass bead (reflected in the cartoons of steps AD). In this step, SSRT is synthesized using MerMade TM The reporter constructs were synthesized one at a time on a 1 μM scale using a MerMade 12 synthesizer (commercially available from BioAutomation). TM The sequence manager then prepares the phosphoramidites (e.g., prepares a 0.067 M solution of each phosphoramidite). The appropriate coupling time for each phosphoramidite is programmed into the synthesizer. The SSRT synthesis cycle is based on a traditional four-step process: detritylation (using a solvent such as 3% DCA in dichloromethane), monomer coupling (using a solvent such as 0.25 M ETT in acetonitrile), capping (using solvents such as THF / lutidine / Ac2O (CAP A) and 16% methylimidazole in THF (CAP B)), and oxidation (using a solvent such as 0.02 M I2 in THF / pyridine / H 2 O). Step 2) Functionalization of the 5′ end by manual conversion, replacing Br with azide (reflected in the cartoon of step E), i.e., “azide modification”. In this step, the synthesis column is washed with 1 mL of DCM and transferred to a 2 mL tube; an azide conversion solution (100 mM sodium iodide and 100 mM sodium azide in DMF) is prepared and 1.6 mL is added to the tube and incubated for 2 hours at room temperature; the support is then rinsed with 1 mL of DMF and transferred to the column; the column is rinsed with 2 mL of DMF, then 3 mL of ACN and 1 mL of DCM. Step 3) Removal of the cyanoethyl protecting group. In this step, a 10% solution of DEA is prepared in ACN, which may contain 0.1 M nitromethane; a steady stream of this solution is passed through the column under vacuum for at least 10′; the column is then rinsed with 2 mL of ACN and then 1 mL of DCM. Step 4) Cleavage completely deprotects SSRT from the solid support (reflected in the cartoon of step F). At this step, the support was transferred to a 2 ml tube and 500 μL of 30% NH 4OH (which may contain 100 mM nitromethane) was added to the tube and incubated at 55°C for 30'; the tube was then cooled in a refrigerator for 5'; 500 μL of 40% methylamine was added to the tube and incubated at 65°C for 1 hour. The sample was then frozen in the refrigerator for 5 minutes; then the column was drained and washed with 15 mL of HO. 2 The sample was desalted by washing with 0.5% TEAA; SSRT was then eluted from the column with 100 mM TEAA and quantified.

[0230] Figure 7 Four exemplary SSRT products are described that can be used as reporter constructs to form XATP, XCTP, XGTP, and XTTP, each of which is designed to produce a unique electronic signal when passing through a nanopore. The chemical structures of these specific phosphoramidites are critically described because they are present in the final SSRT reporter constructs. As used throughout this disclosure, R represents hexaazide, Q represents spermine, D represents PEG6, X represents PEG3, L represents C2 spacer, 4 represents pendant PEG4, Y represents symmetrical chemical branches, and 5 represents benzofuran. In this embodiment, R provides an azide conjugation feature, QQ polymers provide enhancer features, Y444444444444 polymers provide TCE features, and DDLLLDX, DDD44LXXX, DDD4444LLDX, and XXL444444LLLLLLL polymers provide four unique reporter code features.

[0231] Fig. 8A The cyclization of exemplary XNTPs via SSRT and dNTP-2c is summarized, and the formation is performed via a copper-catalyzed click reaction. As used throughout this disclosure, "dNTP-2c" refers to a cleavable nucleoside triphosphoramidate analog, which includes a 1,7-octadiynyl linker conjugated to the heterocyclic portion of the nucleoside and a 5-hexynyl linker conjugated to the alpha phosphoramidate portion of the triphosphoramide. This is a three-step process comprising the following steps: 1) click reaction; in one embodiment, SSRT and dNTP-2c are added to a 0.2 mM CuSO 4 / 0.6mM THPTA / 1.2mM NaAsc click reaction solution and incubated for 30'; 2) standard HPLC purification; and 3) desalting using methods recognized in the art. More details on the structure and synthesis of dNTP-2c compounds are disclosed in U.S. Patent No. 10,301,345 issued to the applicant, which is incorporated herein by reference in its entirety. Figure 8B Exemplary XNTPs illustrating detailed chemical structures.

[0232] In certain embodiments, the nucleobase of the XNTP can be a non-natural analog, such as 7-deazaadenine, 7-deazaguanine, and the like.

[0233] Additional translocation control measures

[0234] I. Translocation control by reversible binding of the translocation control moiety

[0235] exist Fig.9A and Fig. 9B This example of translocation control is illustrated in simplified form in . In this example, the translocation rate is controlled by the binding and dissociation of a soluble translocation binding moiety to the TCE of the Xpandomer. Fig.9A As shown, the binding of the first soluble translocation binding moiety to the first TCE element of the first reporter code of the Xpandomer forms a "code translocation control complex". This reversible interaction stops, pauses or blocks the first reporter code in the nanopore (for simplicity, these terms are used interchangeably herein) and produces a current change unique to the first code. These code signals are used to identify each monomer unit of the Xpandomer. Subsequently, as Fig. 9B As shown, the first translocation binding moiety dissociates from the first TCE, allowing the first reporter code to pass, i.e. exit, the nanopore on the opposite side of the pore where it enters. Then, the translocation event is stopped by the binding of the second translocation binding moiety to the second TCE distal to the first reporter code in the Xpandomer. The second translocation control complex is referred to herein as the "clock translocation control complex". This interruption of translocation will produce a change in the current (i.e., the "clock signal"), indicating the complete translocation of the first code region through the nanopore. In certain embodiments, the clock signals of each unit of the Xpandomer may be identical or almost indistinguishable from each other. It will be appreciated by those skilled in the art that the code signal itself is sufficient to determine the sequence information of the Xpandomer.

[0236] According to the present invention, any suitable set of reversible binding partners can be used for translocation control. In one embodiment, the TCE comprises a biotin derivative and the translocation control portion is provided by streptavidin. In this embodiment, the biotin derivative can be engineered to bind streptavidin with a lower affinity than natural biotin. An example of a suitable biotin derivative is desthiobiotin (DTB), such as Fig.10 In other embodiments, the biotin-SA TCE system can be controlled by, for example, using other biotin analogs that form weaker biotin-SA complexes and / or using SA mutants that form weaker complexes.

[0237] II. Translocation control by adjusting operating conditions

[0238] It has been found that fine-tuning various conditions used in nanopore-based detection systems can improve the accuracy of Xpandomer translocation control and code reading. Therefore, in other aspects, the present disclosure provides means to increase the rate of polymer translocation through a nanopore by modifying one or more of the following operating conditions:

[0239] A. Voltage parameters

[0240] The flow of ions from the cis chamber to the trans chamber of the nanopore-based detection system described herein is the result of a voltage potential applied across the membrane, which is interchangeably referred to as a "read voltage" or "baseline voltage". In one embodiment of the invention, the Xpandomer translocation rate is adjusted by changing the baseline voltage. In some embodiments, the baseline voltage may be in the range of from about 40mV to about 150mV. In other embodiments, the baseline voltage may be in the range of from about 90mV to about 110mV. In yet other embodiments, the baseline voltage may be in the range of from about 55mV to about 75mV. In some embodiments, a higher baseline voltage may be required to capture the reporter code read at a higher rate.

[0241] As discussed herein, when the TCE near the reporter code encounters the orifice of the pore, the Xpandomer translocation is blocked. The reporter code remains in the pore until a sufficiently strong voltage pulse is applied to overcome the resistance provided by the TCE structure maintained in the pore. Therefore, in another embodiment, the Xpandomer translocation rate is adjusted by changing the intensity of the pulse voltage. In some embodiments, the pulse voltage is in the range of from about 250mV to about 2000mV. In other embodiments, the pulse voltage is in the range of from about 550mV to about 700mV. Similarly, the duration of the voltage pulse can affect the rate of Xpandomer translocation. In some embodiments, the duration of the voltage pulse is in the range of from about 1μs to about 50μs. In other embodiments, the duration of the voltage pulse is in the range of from about 5μs to about 10μs. In another embodiment, the periodicity of the pulse voltage can be optimized. In some embodiments, the periodicity is in the range of from about 0.5ms to about 20ms. In other embodiments, the period of the pulse voltage is from about 0.5ms to about 1.5ms. Those skilled in the art will appreciate that the strength, duration and periodicity of the optimum voltage pulses will depend on many factors, such as the force of the TCE.

[0242] B. Salt

[0243] The rate of current flowing through the nanopore-based detection systems described herein can be affected by the salt composition of the buffers filling the cis and trans chambers of the system. Thus, in certain embodiments, the rate at which the Xpandomer translocates through the pore can be modulated by the salt composition. In these embodiments, salts containing any suitable monovalent or divalent cations can be used. In some embodiments, suitable salts include, but are not limited to, NH 4 Cl, MgCl 2 , LiCl, KCl, CsCl, NaCl and CaCl 2 In other embodiments, suitable salts include salts in which the anion is acetate. Under conditions where a slower current is desired, salts with lower ion mobility, such as LiCl, may be advantageous. In some embodiments, the trans chamber contains 2M NH 4 Cl and a second optional salt having a suitable molar concentration of about 0.2 M, and the cis chamber contains NH having a suitable molar concentration ranging from about 0.4 M to about 1 M 4 Cl and a second optional salt having a suitable molar concentration ranging from about 0.2 M to about 0.8 M. In other embodiments, other molar concentrations outside of these ranges and / or other combinations of salts may be desired.

[0244] C. Chaotropic agent

[0245] In certain other aspects, the cis chamber of the nanopore-based detection system of the present invention may include one or more chaotropic agents to improve the translocation of a single polymer analyte, such as a linearized Xpandomer. Any suitable chaotropic agent may be used, such as urea and / or guanidine hydrochloride (GuCl). In some embodiments, the buffer composition of the cis chamber includes GuCl and / or urea in a range of about 200 mM to about 2 M.

[0246] D. Osmotic gradient

[0247] In other aspects, the present invention provides a nanopore-based detection system in which an osmotic gradient is established across the membrane to affect the rate at which the Xpandomer translocates through the pore. Without being bound by theory, it is assumed that a gradient in which the concentration of salt and / or other additives in the trans chamber is higher relative to the cis chamber produces a water flow toward the nanopore, thereby pulling the Xpandomer toward the pore. Under these conditions, an increase in the rate of event frequency (e.g., code reading) can be observed at a lower operating voltage. Thus, in some embodiments, the operating conditions include establishing an osmotic gradient of about 50% across the membrane; for example, a salt (and / or other additive) concentration of about 1 M in the cis chamber and a salt (and / or other additive) concentration of about 2 M in the trans chamber. In further embodiments, any other suitable osmotic gradient may be employed.

[0248] E. Solvents

[0249] It has been found that certain solvents can enhance the solubility of the Xpandomer and increase the translocation rate through the nanopore. Therefore, in certain embodiments, the sample buffer of the present invention includes one or more organic solvents. Suitable solvents include, but are not limited to, 3-methyl-2-oxazolidinone (MOA), DMF, ACN, DMSO, and NMP used in a range of about 1% to about 25%.

[0250] F. Buffer, Additive add Agents and other operating conditions

[0251] Suitable buffers for the present invention include, but are not limited to, 20mM-100mM HEPES with a pH value of about 7.4 and a double tris-propane buffer with a molar concentration ranging from about 25mM to about 250mM and a pH value ranging from about 6 to about 10. In other aspects, the buffer of the present invention may include certain detergent additives, such as sodium hexanoate (NaHex), to increase the rate of Xpandomer translocation. In certain embodiments, the sample buffer of the present invention includes about 20mM NaHex. Other suitable additives include, but are not limited to, stabilizers, such as EDTA and redox reagents. The viscosity of any buffer may also be changed by additives such as PEG, glycerol, polysucrose, etc.

[0252] In other aspects, the translocation rate of the Xpandomer can be regulated by temperature. In some embodiments, the operating temperature can be in the range of from about 4°C to about 40°C. In other embodiments, the operating temperature can be in the range of about 16°C to about 22°C.

[0253] Cleavable extension oligonucleotide

[0254] In another aspect, the present disclosure provides cleavable extended oligonucleotides (EO) for Xpandomer synthesis. The cleavable design feature enables the EO to be removed from the Xpandomer after synthesis and before nanopore analysis, i.e., cleaved from the Xpandomer. This feature provides advantages when it is not desired that the polynucleotide sequence be translocated through the nanopore. The synthesis, processing and nanopore sequence analysis of Xpandomers are described in, for example, PCT Patent Application No. PCT / US18 / 67763 of the applicant, which is incorporated herein by reference in its entirety.

[0255] One example of a cleavable extension oligonucleotide is shown in simplified form at Fig.11The 3′ end of the EO is modified to include a cleavable bond, for example, an acid-cleavable phosphoramidate bond, represented herein by “P-NH”. The nucleobase attached to the EO via a cleavable linker provides a free 3′ hydroxyl group for Xpandomer synthesis, the direction of which is indicated by the dashed arrow. The base portion of the same nucleobase is modified to provide other features required for nanopore translocation, such as a leader group, also referred to herein as an “overhanging leader sequence”. Thus, after the EO is extended to form the Xpandomer, acid treatment releases the oligonucleotide primer, while the leader group and other joining features remain associated with the Xpandomer. Advantageously, the inventors have found that Xpandomer synthesis is not affected by the addition of an overhanging leader sequence to the cleavable extension oligonucleotide.

[0256] ratchet

[0257] One disadvantage of nanopore-based detection systems implemented in the art is the current consumption over time due to electrolyte depletion that occurs during the continuous application of a DC voltage. For example, where the electrolyte circuit is based on a ferrocyanide-ferrocyanide redox pair, each pore in the nanopore array has a finite volume and therefore contains a finite number of these redox ion species. Under a DC voltage, one species will convert to another and will cause the current to drop. In order to overcome this problem of current consumption and maintain a more balanced current over time, the present disclosure provides a means for detecting polymer analytes using a nanopore-based detection system that instead relies on an alternating current (AC) mode of voltage application. This mode is referred to herein as a "ratchet." A general overview of a ratchet is shown in Fig.12 middle. Fig.12 The top panel of illustrates an exemplary pattern of voltage application; in this example, a +70mV "forward" read voltage is applied, and in the middle, the system is subjected to a brief (5μs) pulse voltage of +500mV. The "forward" read voltage is then followed by a -70mV "reverse" read voltage, and this cycle of +70mV forward read / 500mV pulse / +70mV forward read / -70mV reverse read is repeated until the entire polymerized analyte passes through the nanopore. A significant advantage of the ratchet protocol is that the electrolyte distribution is replenished in each forward read-reverse read cycle.

[0258] Fig.12 The bottom panel of illustrates how the directionality of polymer translocation changes during ratcheting. In this example, each unit of the polymer analyte includes two identical reporter subcodes (e.g., 1210A and 1210B) separated by a translocation control element, such as TCE 1215. Fig.12As shown, applying a +70mM forward running voltage causes the polymer to move from the cis side of the membrane to the trans side through the pore until the reporter code 1210A is blocked in the pore by the translocation pause induced by TCE 1215. The current change through the pore caused by the blocked reporter code 1210A is read as a signal "L1+". A 500mV pulse is applied to force TCE 1215 and reporter code 1210B to pass through the pore. After the 5μs pulse, the reading voltage returns to +70mV, and then the next reporter code 1220A in series is blocked in the pore by its corresponding TCE 1225. The current change flowing through the pore caused by the blocked reporter code 1220A is read as a signal "L2+". Next, the voltage is reversed by applying a -70mM reverse reading voltage to the system, resulting in a reversal of the polymer translocation direction. During this period, the reporter code 1220A is pushed back through the pore to the cis side of the membrane. Translocation continues until TCE 1215 encounters a pore, whereupon translocation is blocked, positioning reporter code 1210B in the pore to produce a current change measured as level "L1-". Next, the voltage is restored to the +70mM forward running voltage, so the direction of polymer translocation is reversed back to the cis to trans direction until blockade occurs due to the ITC effect of TCE 1225, positioning reporter code 1220A in the pore. The resulting current change is read as level "L2+". During this ratchet protocol, the reporter code characterizing each unit in the polymer is read three times (reporter code "A" is read twice and reporter code "B" is read once). This redundancy provides a means of quality control for sequence reads and improves the accuracy of the resulting sequence data. Insertion and deletion errors are easily identified as deviations from the expected pattern, such as L1+ / L2+ / L1- / L2+.

[0259] although Fig.12 The ratchet pattern depicted in FIG. 1 shows a pulse voltage applied during the "middle" of the forward read voltage, but other pulse patterns are contemplated by the present invention. For example, in some embodiments, the pulse may be applied before the forward read voltage is applied, while in other embodiments, the pulse may be applied at the end of the forward read voltage, just before the reverse read voltage is applied.

[0260] In other embodiments, the ratchet provides a means for compensating or correcting for one or both of the current draw due to pulses and asymmetries in the resistance of different reporting subcodes. For example, in some embodiments, the reverse read voltage may be increased to compensate for the current loss due to pulses applied during the forward read voltage. When different reporting subcodes have different inherent resistances, the percentage increase in the reverse read voltage may also be adjusted to balance the current.

[0261] In other variations of the ratchet scheme, the order of the forward, pulse, and reverse voltages can be changed. For example, in one embodiment, the ratchet cycle can be run as follows: (forward read voltage), (forward read voltage), (reverse read voltage). In another embodiment, the ratchet cycle can be run as follows: (forward read)n (reverse read)n, where "n" represents the total number of monomer units in the polymer measured by the nanopore.

[0262] In a related idea, "flossing" has been proposed, in which DNA can be read along its entire length in a nanopore, stopped, and then read in the other direction by reversing the voltage polarity (see, e.g., Kasianowicz, John J. "Nanopores: Flossing with DNA." Nature Materials 3, no. 6 (2004): 355-56. https: / / doi.org / 10.1038 / nmat1143). This is a less efficient approach in arrays because DNA polymers are not arrested or stopped synchronously, whereas ratcheting is a continuous forward process on these time scales.

[0263] SBX-based diagnostic methods and kits

[0264] In another aspect, the present invention discloses methods and kits for detecting and diagnosing genetic changes / mutations in target samples, which may be solid tissues or body fluids. The genetic changes may be germline or somatic mutations. The present invention may be used for detection and diagnosis of cancer, autoimmune diseases, organ transplant rejection, inherited fetal abnormalities, pathogens, and other suitable conditions.

[0265] Examples

[0266] Materials and methods

[0267] Unless otherwise noted, the following materials (with indicated abbreviations) were obtained from the above sources in the U.S.A. 2-phenyl-1,3-dioxan-5-ol, TBDPS-Cl (tert-butyldiphenylchlorosilane), DMAP (4-dimethylaminopyridine), (R)-(+)-glycidol, (+)-2,3-O-isopropylidene-L-threitol, isosorbide, 4,4′-bis(hydroxymethyl)-2,2′-bipyridine, TBTA (tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine) were from TCI America (Porrland, OR). NaH (sodium hydride), MeOH (methanol), toluene, THF (tetrahydrofuran), TBAF (tetrabutylammonium fluoride), DCM (dichloromethane), HCl (concentrated hydrochloric acid), DMSO (dimethyl sulfoxide), Na ascorbic acid (sodium ascorbate), sodium bicarbonate, copper sulfate, propargyl dimethylmalonate, lithium borohydride and acetic acid were obtained from Sigma-Aldrich (St. Louis, MO). DMT-Cl (4,4'-dimethoxytrityl chloride) and PPA-Cl (N,N-diisopropylaminocyanoethyl phosphoramide) were from ChemGenes Corporation (Wilmington, MA). TEA (triethylamine), hexane, ethyl acetate, EDTA (ethylenediaminetetraacetic acid), ether were from EMD Millipore (Billerica, MA). m-PEG4-Tos was prepared from m-PEG4-OH (Cat. No. BP-23742). Fluoro[3,2-c]pyridin-4(5h)-one (Combi-Blocks, San Diego, CA).

[0268] High performance liquid chromatography (HPLC) was performed on a ProStar Helix from Agilent Technologies, Inc. (Santa Clara, CA). TM The HPLC system consisted of two pumps (ProStar 210 solvent delivery module) with 10 ml titanium pump heads, a column oven (ProStar 510 Air Oven), and a UV detector (ProStar 320 UV / Vis detector) set at 292 nm. The system was controlled by Star Chromatography Workstation software (version 6.41). The columns used were Cadenza Guard Column System CD-C18 (2.0 mm x 5 mm) from Imtakt USA (Porrland, OR). The buffers used were buffer A (100 mM triethylammonium acetate, pH 7.0) and buffer B (100 mM triethylammonium acetate, pH 7.0, containing 95% by volume acetonitrile). Automated solid phase phosphoramidite synthesis was performed on a MerMadeTM 12 synthesizer (Bioautomation Corp, Plano, TX). MerMade TM The synthetic solution was purchased from Glen Research (Sterling, VA).

[0269] Example 1

[0270] Synthesis of DMT phosphoramidites of racemic 2-(3,6-dioxaheptyloxy)-1,3-propanediol Pendant Code 2-Gly OLEYL PEG-2 PHOSPHORAMIDE [RACEME]

[0271] 2-phenyl-1,3-dioxane-5-ol (1,2.7g, 15mmol) is dissolved in 30mL anhydrous THF. Sodium hydride (1.08g, 27mmol) is added to produce alcohol salt. When bubbling stops, mPEG4-Tos (4.94g, 18mmol) is dissolved in 10mL THF and added in batches. The reaction is warmed to 40°C and incubated for 3 hours under stirring, and then allowed to cool to room temperature overnight. Excess NaHH is quenched with 1mL MeOH, then diluted with water and extracted with DCM. The combined organic layers are concentrated under reduced pressure. The residue is resuspended in toluene, separated from the remaining salt, and 2 is obtained in a yield of 73% by flash chromatography.

[0272] Benzylpyridinium protected 2b (3.05 g, 10.8 mmol) was dissolved in 10 mL MeOH and HCl (0.2 mL, 2.3 mmol) was added. The solution was incubated for 20 minutes, then neutralized with sodium bicarbonate (200 mg) and dried under reduced pressure. The residue was resuspended in DCM and purified by flash chromatography to give diol 3 in 72% yield.

[0273] Diol 3 (1.52 g, 7.8 mmol) was dissolved in 20 mL DCM and TEA (2.17 mL, 15.6 mmol). A solution of DMT-Cl (1.85 g, 5.46 mmol) in 10 mL DCM was added in portions over 90 minutes to maximize the monotritylation. MeOH (1 mL) was added and the reaction was dried under reduced pressure. The residue was resuspended in toluene and separated from the salt, and then purified by flash chromatography to obtain the monotrityl product 4 in 48% yield and the recovered starting diol.

[0274] Monotrityl 4 (1.84 g, 3.7 mmol) was dissolved in 10 mL DCM and TEA (1.03 mL 7.4 mmol). PPA-Cl (1.05 g, 4.4 mmol) was added and the reaction was incubated for 15 minutes. The reaction was dried under reduced pressure and resuspended in toluene containing 1% TEA and then purified by flash chromatography. The phosphoramidite 5 (2.03 g, 2.9 mmol) was obtained in 79% yield and confirmed by 1H and 31P NMR.

[0275]

[0276] Example 2

[0277] The DMT phosphoramidite (pendent code 2-glyceryl) enantiomer of 2-(3,6-dioxaheptyloxy)-1,3-propanediol (12b) Synthesis of Oily PEG-2 Phosphoramidite [Enantiomer]

[0278] 2,3-Isopropylidene-sn-glycerol 6 was dissolved in anhydrous DCM and TEA. DMAP and TBDPS-Cl were added. The reaction was extracted from water with DCM and purified by flash chromatography to give product 7.

[0279] Silyl ether 7 was dissolved in MeOH and HCl was added. The solution was incubated for 20 min, then neutralized with sodium bicarbonate and dried under reduced pressure. The residue was resuspended in DCM and purified by flash chromatography to give diol 8.

[0280] Diol 8 was dissolved in DCM and TEA. DMT-Cl in DCM was added in portions. MeOH was added and the reaction was dried under reduced pressure. The residue was resuspended in toluene and separated from the salt, and then purified by flash chromatography to give product 9 as a monotrityl group.

[0281] Secondary alcohol 9 was dissolved in anhydrous THF. Sodium hydride was added to produce the alkoxide. When bubbling ceased, mPEG4-Tos was dissolved in THF and added in portions. The reaction was warmed to 40 °C and incubated with stirring for 3 hours, then allowed to cool to room temperature overnight. Excess NaH was quenched with 1 mL of MeOH, then diluted with water and extracted with DCM. The combined organic layers were dried under reduced pressure. The residue was resuspended in toluene, separated from the remaining salts, and purified by flash chromatography to give 10.

[0282] The mPEG4 ether 10b was resuspended in THF and TBAF was added. The reaction was concentrated under reduced pressure and purified by flash chromatography to give 11.

[0283] DMT PEG4 alcohol 11b was dissolved in DCM and TEA. PPA-Cl was added and the reaction was incubated for 15 minutes. The reaction was dried under reduced pressure and resuspended in toluene containing 1% TEA and then purified by flash chromatography. Phosphoramidite 12 was isolated and confirmed by 1H and 31P NMR.

[0284]

[0285] Example 3

[0286] Synthesis of DMT phosphoramidites of enantiomers of 1-(3,6-dioxaheptyloxy)-2,3-propanediol (16) Pendant code 1-Glyceryl PEG-2 phosphoramidite

[0287] 2,3-isopropylidene-sn-glycerol 6 was dissolved in anhydrous THF. Sodium hydride was added to produce an alkoxide. When bubbling stopped, mPEG2-Tos (Broadpharm Cat. No. BP-2-982) was dissolved in THF and added in batches. The reaction was incubated for 24 hours. Excess NaH was quenched with MeOH, then diluted with water and extracted with DCM. The combined organic layers were concentrated under reduced pressure. The residue was resuspended in toluene, separated from the remaining salt, and purified by flash chromatography to give 13.

[0288] The PEG2 product 13 was dissolved in MeOH and HCl was added. The solution was incubated for 20 minutes, then neutralized with sodium bicarbonate and dried under reduced pressure. The residue was resuspended in DCM and purified by flash chromatography to give diol 14.

[0289] Diol 14 was dissolved in DCM and TEA. DMT-C1 in DCM was added in portions. MeOH was added and the reaction was dried under reduced pressure. The residue was resuspended in toluene and separated from the salts and then purified by flash chromatography to give product 15 as a monotrityl group.

[0290] Mono-DMT 15 was dissolved in DCM and TEA. PPA-Cl was added and the reaction was incubated for 15 min. The reaction was dried under reduced pressure and resuspended in toluene containing 1% TEA and then purified by flash chromatography. Phosphoramidite 16 was isolated and confirmed by 1H and 31P NMR.

[0291]

[0292] Example 4

[0293] Synthesis of DMT phosphoramidite of 1-(5H-furo[3,2-c]pyridin-4-one)-2,3-propanediol (20) as pendant 1-Glyceryl heterocyclic phosphoramidite

[0294] (R)-(+)-glycidol 17 was dissolved in DCM and TEA. DMT-Cl in DCM was added in batches. MeOH was added and the reaction was dried under reduced pressure. The residue was resuspended in toluene and separated from the salt, and then purified by flash chromatography to give DMT ether 18.

[0295] DMT ether 18 was dissolved in anhydrous DMF. Sodium hydride was added to produce the alkoxide. When bubbling stopped, fluoro[3,2-c]pyridine-4(5h)-one was dissolved in THF and added in portions. The reaction was brought to 100°C and incubated for 12 hours under stirring. Excess NaH was quenched with MeOH, then diluted with water and extracted with DCM. The combined organic layers were concentrated under reduced pressure. The residue was resuspended in toluene, separated from the remaining salt, and purified by flash chromatography to give 19.

[0296] Secondary alcohol 19 was dissolved in DCM and TEA. PPA-Cl was added and the reaction was incubated for 15 minutes. The reaction was dried under reduced pressure and resuspended in toluene containing 1% TEA and then purified by flash chromatography. Phosphoramidite 20 was isolated and confirmed by 1H and 31P NMR.

[0297]

[0298] Example 5

[0299] Online code for isosorbide (23) Synthesis of DMT phosphoramidite Di-secondary alcohol code main chain

[0300] Isosorbide 21 was dissolved in DCM and TEA. DMT-Cl in DCM was added in portions. MeOH was added and the reaction was dried under reduced pressure. The residue was resuspended in toluene and separated from the salt, and then purified by flash chromatography to give monotrityl 22.

[0301] Monotrityl 22 was dissolved in DCM and TEA. PPA-C1 was added and the reaction was incubated for 15 min. The reaction was dried under reduced pressure and resuspended in toluene containing 1% TEA and then purified by flash chromatography. Phosphoramidite 23 was isolated and confirmed by 1H and 31P NMR.

[0302]

[0303] Example 6

[0304] Online code for bipyridine (26) Synthesis of DMT phosphoramidite Bis-primary alcohol code backbone

[0305] 4,4'-Bis(hydroxymethyl)-2,2'-bipyridine 24 was dissolved in DCM and TEA. DMT-Cl in DCM was added in portions. MeOH was added and the reaction was dried under reduced pressure. The residue was resuspended in toluene and separated from the salts and then purified by flash chromatography to give the monotrityl 25.

[0306] Monotrityl 25 was dissolved in DCM and TEA. PPA-C1 was added and the reaction was incubated for 15 min. The reaction was dried under reduced pressure and resuspended in toluene containing 1% TEA and then purified by flash chromatography. Phosphoramidite 26 was isolated and confirmed by 1H and 31P NMR.

[0307]

[0308] Example 7

[0309] Synthesis of 2-Alkyltriazole Pendant Code DMT Phosphoramidite PEG-21,3-Propanediol (31a). Pendant Triazole PEG Code

[0310] Propargyl dimethyl malonate 27 is added dropwise to a cold suspension of lithium borohydride in ether. The reaction is then warmed to room temperature and incubated overnight. The reaction is quenched with methanol, then water and acetic acid. The solution is extracted with ether and the combined organic layers are concentrated under reduced pressure. The crude material is purified by flash chromatography to obtain diol 28.

[0311] Diol 28 was dissolved in DCM and TEA. DMT-C1 in DCM was added in batches. MeOH was added and the reaction was dried under reduced pressure. The residue was resuspended in toluene and separated from the salt, and then purified by flash chromatography to give DMT alcohol 29.

[0312] DMT alcohol 29 is dissolved in DMSO and azide (Cat.No.BP-20988Broadpharm) is added. Separately, TBTA is dissolved in DMSO and sodium ascorbate and copper sulfate are merged. Under stirring, TBTA solution is added to alkyne / azide solution in batches. After incubation for 45 minutes, the reaction is quenched with EDTA. The solution is diluted with water and extracted with ethyl acetate, then the organic layer is concentrated under reduced pressure and purified by flash chromatography to obtain 30.

[0313] 1,2,3-Triazole 30a was dissolved in DCM and TEA. PPA-Cl was added and the reaction was incubated for 15 minutes. The reaction was dried under reduced pressure and resuspended in toluene containing 1% TEA and then purified by flash chromatography. Phosphoramidite 31 was isolated and confirmed by 1H and 31P NMR.

[0314]

[0315] Example 8

[0316] Synthesis of pendant PEG-2, PEG-4 and PEG-6 phosphoramidites [isomeric purity] (35a)

[0317] 1-O-TBDPS-3-O-DMTr-propane-1,2,3-triol 9 (from Example 2) was dissolved in anhydrous THF. Sodium hydride was added to produce alcohol salt. When bubbling stopped, toluenesulfonate (prepared by tosylation of Cat.No.BP-21657, Broadpharm) was added in batches. The reaction was incubated for 48 hours under stirring. Excess NaH was quenched with water, and the solution was then transferred to a separatory funnel and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was resuspended in toluene, separated from the remaining salt, and 32 was obtained by flash chromatography purification.

[0318] The alkyne 32a was resuspended in THF and TBAF was added. The reaction was concentrated under reduced pressure and purified by flash chromatography to give 33.

[0319] To a solution of alkyne 33a in DMSO was added ethyl 2-azidoacetate. In a separate vial, sodium ascorbate was dissolved in water and DMSO was added, followed by 1 M CuSO 4 , to prepare the catalyst mixture. The catalyst mixture was added dropwise to the alkyne / azide solution over 10 minutes. After completion, it was quenched with 0.5M EDTA and stirred for 15 minutes. It was diluted with water and extracted three times with ethyl acetate. The combined organic extracts were washed with brine and dried over sodium sulfate. The residue was resuspended in toluene and purified by flash chromatography to give 34a.

[0320] Triazole 34a was dissolved in DCM and TEA. PPA-Cl was added and the reaction was incubated for 15 min. The reaction was dried under reduced pressure and resuspended in toluene containing 1% TEA and then purified by flash chromatography. Phosphoramidite 35 was isolated and confirmed by 1H and 31P NMR.

[0321]

[0322] Example 9

[0323] Synthesis of PEG-based reporter code (37b)

[0324] To a solution of product 33 in DMSO was added 2-(acetoxymethyl)-2-(azidomethyl)propane-1,3-diacetic acid diester (prepared by dissolving 2-(bromomethyl)-2-(hydroxymethyl)-1,3-propanediol in DMF and then adding NaN3). The reaction was incubated at 110°C, concentrated and purified by flash chromatography. After the product was isolated, the residue was reacted with acetic anhydride and purified). In a separate vial, sodium ascorbate was dissolved in water and DMSO was added, followed by 1 M CuSO 4 , to prepare the catalyst mixture. The catalyst mixture was added dropwise to the alkyne / azide solution over 10 minutes. Upon completion, it was quenched with 0.5M EDTA and stirred for 15 minutes. It was diluted with water and extracted three times with ethyl acetate. The combined organic extracts were washed with brine and dried over sodium sulfate. The residue was resuspended in toluene and purified by flash chromatography to give 36.

[0325] Primary alcohol 36b was dissolved in DCM and TEA. PPA-Cl was added and the reaction was incubated for 15 min. The reaction was dried under reduced pressure and resuspended in toluene containing 1% TEA and then purified by flash chromatography. Phosphoramidite 37 was isolated and confirmed by 1H and 31P NMR.

[0326]

[0327] Example 10

[0328] Synthesis of PEG-based reporter code (40)

[0329] 1-O-TBDPS-3-O-DMTr-propane-1,2,3-triol 9 (from Example 2) was dissolved in anhydrous THF. Sodium hydride was added to produce an alkoxide. When bubbling stopped, tosylate (prepared by sequential tosylation and silyl protection of Cat.No.BP-21036, Broadpharm) was dissolved in THF and added in batches. The reaction was incubated overnight under stirring. Excess NaH was quenched with water and extracted with DCM. The combined organic layers were dried under reduced pressure. The residue was resuspended in toluene, separated from the remaining salt, and 38 was obtained by flash chromatography purification.

[0330] The bissilyl ether 38 was resuspended in THF and TBAF was added. The reaction was concentrated under reduced pressure and purified by flash chromatography. The purified material was resuspended in DCM and TEA was added. BzCl was added dropwise. The reaction was stirred at room temperature until completion. The reaction was concentrated under reduced pressure and purified by flash chromatography to give alcohol 39.

[0331] Alcohol 39 was dissolved in DCM and TEA. PPA-Cl was added and the reaction was incubated for 15 minutes. The reaction was dried under reduced pressure and resuspended in toluene containing 1% TEA and then purified by flash chromatography. Phosphoramidite 40 was isolated and confirmed by 1H and 31P NMR.

[0332]

[0333] Example 11

[0334] Synthesis of bis-PEG-based reporter codes (45a-d and 47e-i)

[0335] O,O'-benzylidene pentaerythritol (41, Cat.No.B2682, TCI) was dissolved in anhydrous THF. Sodium hydride was added to produce the alkoxide. When bubbling stopped, the tosylate (prepared by tosylation of Cat.No.BP-21397, Broadpharm or Cat.No.BP-21657, Broadpharm) was dissolved in THF and added in batches. The reaction was incubated overnight with stirring. Excess NaH was quenched with water and extracted with DCM. The combined organic layers were dried under reduced pressure. The residue was resuspended in toluene, separated from the remaining salt, and purified by flash chromatography to give 42a-h.

[0336] The products 42a-h were dissolved in MeOH and HCl was added. The reaction was incubated at room temperature overnight and then neutralized with sodium bicarbonate. It was concentrated under reduced pressure and purified by flash chromatography to give 43a-h.

[0337] The product 43a-h was dissolved in DCM and TEA. DMT-C1 in DCM was added in portions. The reaction was dried under reduced pressure. The residue was resuspended in toluene and separated from the salt, and then purified by flash chromatography to give the monotrityl 44a-h.

[0338] The products 44a-d were dissolved in DCM and TEA. PPA-C1 was added and the reaction was incubated for 15 minutes. The reaction was dried under reduced pressure and resuspended in toluene containing 1% TEA and then purified by flash chromatography to give phosphoramidites 45a-d.

[0339] To a solution of product 44 (eh) in DMSO was added ethyl 2-azidoacetate. In a separate vial, sodium ascorbate was dissolved in water and DMSO was added, followed by 1 M CuSO 4, to prepare the catalyst mixture. The catalyst mixture was added dropwise to the alkyne / azide solution within 10 minutes. After completion, it was quenched with 0.5M EDTA and stirred for 15 minutes. It was diluted with water and extracted three times with ethyl acetate. The combined organic extracts were washed with brine and dried over sodium sulfate. The residue was resuspended in toluene and purified by flash chromatography to give 46e-h.

[0340] The product 46e-h was dissolved in DCM and TEA. PPA-Cl was added and the reaction was incubated for 15 minutes. The reaction was dried under reduced pressure and resuspended in toluene containing 1% TEA and then purified by flash chromatography to give phosphoramidites 47e-h.

[0341]

[0342] Example 12

[0343] Synthesis and testing of PEG-based reporter code (52)

[0344] 2,2-Bis(bromomethyl)-1,3-propanediol (48, Cat. No. D1808, TCI) was dissolved in DMF and NaN3 was added. The reaction was incubated at 110°C, concentrated and purified by flash chromatography to give the product 49.

[0345] To a solution of 49 in DMSO was added 2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl benzoate (prepared by benzoylation of a commercial alcohol precursor). In a separate vial, sodium ascorbate was dissolved in water and DMSO was added, followed by 1 M CuSO 4 , to prepare the catalyst mixture. The catalyst mixture was added dropwise to the alkyne / azide solution over 10 minutes. After completion, it was quenched with 0.5M EDTA and stirred for 15 minutes. It was diluted with water and extracted three times with ethyl acetate. The combined organic extracts were washed with brine and dried over sodium sulfate. The residue was resuspended in toluene and purified by flash chromatography to give 50.

[0346] The product 50 was dissolved in DCM and TEA. DMT-Cl in DCM was added in portions. The reaction was dried under reduced pressure. The residue was resuspended in toluene and separated from the salt, and then purified by flash chromatography to give 51.

[0347] The product 51 was dissolved in DCM and TEA. PPA-Cl was added and the reaction was incubated for 15 minutes. The reaction was dried under reduced pressure and resuspended in toluene containing 1% TEA and then purified by flash chromatography to give phosphoramidite 52.

[0348]

[0349] Example 13

[0350] Synthesis and testing of PEG-based reporter codes (62)

[0351] 2-(Bromomethyl)-2-(hydroxymethyl)-1,3-propanediol (53, Cat. No. B4057, TCI) was dissolved in DMF and NaN3 was added. The reaction was incubated at 110°C, concentrated and purified by flash chromatography to give the product 54.

[0352] Product 54 was dissolved in DCM and TEA. Benzoyl chloride was added. The solution was incubated overnight at room temperature. The reaction was extracted from water with DCM and purified by flash chromatography to give product 55, bis-Bz, which was separated and partitioned from any mono- or tri-protected species.

[0353] A portion of the product 55 was dissolved in DCM and TEA. DMT-Cl in DCM was added in portions. MeOH was added and the reaction was dried under reduced pressure. The residue was resuspended in toluene and separated from the salts and then purified by flash chromatography to give tritylated 56.

[0354] 41 was dissolved in anhydrous THF. Sodium hydride was added to produce the alkoxide. When bubbling ceased, propargyl bromide was dissolved in THF and added in portions. Excess NaH was quenched with 1 mL of MeOH, then diluted with water and extracted with DCM. The combined organic layers were dried under reduced pressure. The residue was resuspended in toluene, separated from the remaining salt, and purified by flash chromatography to give 57.

[0355] The product 57 was dissolved in MeOH and HCl was added. The reaction was incubated at room temperature overnight and then neutralized with sodium bicarbonate. It was concentrated under reduced pressure and purified by flash chromatography to give 58.

[0356] Product 58 was dissolved in DCM and TEA. Benzoyl chloride was added and the reaction was incubated for 60 minutes. The reaction was extracted from water with DCM and purified by flash chromatography to give product 59.

[0357] Products 56 and 59 were dissolved in 9:1 DMSO:H2O. Solutions of TBTA, sodium ascorbate and copper sulfate were added and the reaction was incubated for 60 minutes. The reaction was extracted from water with DCM and purified by flash chromatography to give product 60.

[0358] Products 60 and 55 were dissolved in 9:1 DMSO:H2O. Solutions of TBTA, sodium ascorbate and copper sulfate were added and the reaction was incubated for 60 minutes. The reaction was extracted from water with DCM and purified by flash chromatography to give product 61.

[0359] The product 61 was dissolved in DCM and TEA. PPA-Cl was added and the reaction was incubated for 15 minutes. The reaction was dried under reduced pressure and resuspended in toluene containing 1% TEA and then purified by flash chromatography. Phosphoramidite 62 was isolated and confirmed by 1H and 31P NMR.

[0360]

[0361] Example 14

[0362] Enantiomers (9S, 10S)-2,5,8,11,14,17-hexaoxaoctadecane-9,10-diol (pendent code C2 di- Synthesis of PEG-2 phosphoramidite (enantiomer) (67) DMT phosphoramidite

[0363] A solution of (+)-2,3-O-isopropylidene-L-threitol 63 in anhydrous DMF was slowly added to a mixture of NaH in anhydrous DMF (note: H2 gas was vigorously released). When bubbling stopped, mPEG2-Tos (Broadpharm Cat. No. BP-20983) was dissolved in DMF and added in batches to stir overnight at ambient temperature. The reaction mixture was poured into water, extracted with ethyl acetate and purified by flash chromatography to obtain 64.

[0364] The product 64 was dissolved in MeOH and HCl was added. The solution was incubated for 20 minutes, then neutralized with sodium bicarbonate and dried under reduced pressure. The residue was resuspended in ethyl acetate and purified by flash chromatography to give 65.

[0365] The product 65 was dissolved in DCM and TEA. DMT-C1 in DCM was added in portions. The reaction was dried under reduced pressure. The residue was resuspended in toluene and separated from the salt, and then purified by flash chromatography to give 66.

[0366] The product 66 was dissolved in DCM and TEA. PPA-Cl was added and the reaction was incubated for 15 minutes. The reaction was dried under reduced pressure and resuspended in toluene containing 1% TEA and then purified by flash chromatography. The phosphoramidite 67 was confirmed by 1H and 31P NMR.

[0367]

[0368] Example 15

[0369] Synthesis of 1-O-DMT-3-O-PPA-2,2-bis(Me-O-mPEG4)-propane (72)

[0370] To a solution of pentaerythritol (68, Cat. No. P0039, TCI) in DMF was added p-toluenesulfonic acid. The reaction was neutralized with triethylamine, concentrated and purified by flash chromatography to give 69.

[0371] The product 69 was added to a stirred solution of EDC-HCl, DMAP and levulinic acid in THF and stirred overnight at ambient temperature. The solution was concentrated and purified by flash chromatography to give 70.

[0372] The product 70 was dissolved in DCM and TEA. DMT-Cl in DCM was added in portions. The reaction was dried under reduced pressure. The residue was resuspended in toluene and separated from the salt, and then purified by flash chromatography to give 71.

[0373] The product 71 was dissolved in DCM and TEA. PPA-Cl was added and the reaction was incubated for 15 minutes. The reaction was dried under reduced pressure and resuspended in toluene containing 1% TEA and then purified by flash chromatography. The phosphoramidite 72 was confirmed by 1H and 31P NMR.

[0374]

[0375] Example 16

[0376] Synthesis and testing of PEG-based reporter codes

[0377] In this example, the reporter codes were synthesized using PEG-based phosphoramidites; notably, these codes do not contain nucleotides. Four exemplary reporter codes are listed in Table 5.

[0378] Table 5

[0379] Code Indicator sequence L1 DDDLLLL L2 DXXX3LL3XXLL L3 DXLL44XXXLLLL L4 DXXL4444LLXXLL

[0380] Key: D = PEG6: X = PEG3; L = C2; 3 = Click PEG-2; 4 = Pendant PEG-4

[0381] The level discrimination (i.e., distinguishable electronic signals) and translocation time of each code were evaluated by synthesizing 100mer Xpandomer copies of sequences derived from the HIV-2 genome, incorporating XNTPs, wherein each of the four XNTPs contained a unique code from the group listed in Table 4. Xpandomer synthesis, processing, and nanopore sequence analysis were performed as described in Applicant's PCT Patent Application No. PCT / US18 / 67763, which is incorporated herein by reference in its entirety. As a control, Xpandomer copies of the same HIV-2 sequence incorporating different known codes were sequenced in parallel. Fig.13A Representative traces illustrating the level distinction and translocation timing for each code are shown in 13A (control - old code) and 13B (test - new, PEG-based control).

[0382] To assess the accuracy of the Xpandomer sequence information, the sequence data was analyzed by displaying a histogram display of the sequence read population from the SBX reaction. The analysis software aligns each sequence read to the template sequence and trims the sequence range at the ends of the reads that do not align to the correct template sequence. A representative histogram of SBX sequencing of a 100mer template is presented in Fig.14A (control) and Fig. 14B (New, PEG-based code). Notably, Xpandomers incorporating nucleotide-free PEG-based codes generated highly accurate sequence reads for this template.

[0383] Example 17

[0384] Translocation control of PEG-based TCE-2 code levels

[0385] In this example, translocation control of TCEs incorporating pendant PEG phosphoramidites was evaluated by sequencing a simple 60mer template consisting of TG dinucleotide repeats using the SBX protocol. Both XATP and XCTP substrates were designed to incorporate the following TCEs: Y22222222222255, where "Y" represents a symmetric phosphoramidite branch; "2" represents pendant PEG2; and "5" represents benzofuran. The XATP substrate was designed to incorporate the following reporter code: DDDDDDLLLL, where "D" represents PEG6, and "L" represents C2. The XCTP substrate was designed to incorporate the following reporter code: DDDDXX44XXDL, where "X" represents PEG3, and "4" represents pendant PEG4.

[0386] To generate Xpandomer copies of a 60mer template, a primer extension reaction was performed using 4 pm of the extension oligonucleotide and 250 pm of each XNTP. A 10 μL extension reaction included the following reagents: 50 mM TrisCl, pH 8.84, 200 mM NH 4 OAc, 20% PEG8K, 5% NMS, 0.75 nmol polyphosphate PP-60.20, 2 μg SSB, 0.5 M urea, 5 mM PEM additive (suitable polymerase enhancing molecules are disclosed in the applicant's pending PCT patent application No. PCT / US18 / 67763, which is incorporated herein by reference in its entirety) and 1.2 μg purified recombinant DNA polymerase (suitable engineered variants of DPO4 polymerase are disclosed in the applicant's PCT patent application Nos. WO2017 / 087281, PCT / US2018 / 030972 and PCT / US1864794, which are incorporated herein by reference in their entirety). The extension reaction was carried out at 42°C for 30 minutes.

[0387] Next, the Xpandomer products of the extension reaction were sequenced using the SBX protocol. Briefly, the constrained Xpandomer product was cleaved to produce a linearized Xpandomer. This was achieved by first quenching the extension reaction and amine-modifying the Xpandomer with 2M succinic anhydride. The phosphoramidate bond of the Xpandomer was then cleaved by treating the sample with 11.7M DCl at 23°C for 30 minutes. The linearized Xpandomer was purified by ethanol precipitation and resuspended in a buffer supplemented with 34% ACN and 15% DMF.

[0388] For sequencing, Xpandomer was added to 2.8 M NH 4 Cl, 1.2 M GuanCl, 20 mM NaHex, 10% DMF, 2 mM EDTA, and 20 mM HEPES pH 7.4. Protein nanopores were prepared by inserting α-hemolysin into the DPhPE / hexadecane bilayer members in a buffer containing 2 M NH 4 Cl and 100 mM HEPES, pH 7.4. This experiment used 0.4 M NH 4 Cl, 600mM GuanCl and 100mM HEPES, pH 7.4 buffer, and 2M NH4Cl and 100mM HEPES, pH 7.4 were used in the trans well of the detection system. The Xpandomer sample was heated to 70°C for 2 minutes, cooled completely, and then 2μL of sample was added to the cis well. The voltage parameters of the run were as follows: 60mV / 300mV / 10μs / 2ms (read voltage / pulse voltage / pulse voltage duration / pulse frequency). Data were acquired via Labview acquisition software. A representative trace of this run is shown in Fig.15 shown.

[0389] like Fig.15 As shown by the number of levels superimposed above the trace in , the detection system in this experiment correctly read all reporter codes, demonstrating 100% accuracy. The absence of deletion or insertion errors validates the efficacy of the pendant PEG-based TCE as a structure capable of tightly regulating Xpandomer translocation. Notably, with the pendant PEG-based TCE used in this experiment, a transition between two code levels was observed after a single voltage pulse. The ability to switch between sequence reporter codes of an Xpandomer with a single voltage pulse is a significant advance in the art and enables higher sequencing throughput.

[0390] Example 18

[0391] Sequencing by amplification of TCE-4 code levels based on pendant PEG

[0392] In this example, translocation control of TCEs incorporating pendant PEG phosphoramidites was evaluated by sequencing a 60mer template consisting of the sequence repeat CATG using the SBX protocol. All XNTP substrates were designed to incorporate the following TCEs: Y444444444444455, where "Y" represents a symmetric phosphoramidite branch; "4" represents pendant PEG4; and "5" represents benzofuran. XATP substrates were designed to incorporate the following reporter code: DDDDDDLLDX; XCTP substrates were designed to incorporate the following reporter code: DDDDDDLLLL; XTTP substrates were designed to incorporate the following reporter code: DDDDDD44LXXX; and XGTP substrates were designed to incorporate the following reporter code: DDDXXL444444XLLLL, where "D" represents PEG6, "L" represents C2, "X" represents PEG3, and "4" represents pendant PEG4.

[0393] To generate Xpandomer copies of a 60mer template, a primer extension reaction was performed using 4 pm of an extension oligonucleotide and 1000 pm of each XNTP end. The 10 μL extension reaction included the following reagents: 50 mM TrisCl, pH 8.84, 200 mM NH4OAc, 20% PEG8K, 10% NMP, 3 nmol polyphosphate PP-60.20, 2 μg SSB, 1 M urea, 10 mM PEM additive, and 1.8 μg purified recombinant DNA polymerase. The extension reaction was performed at 37°C for 30 minutes.

[0394] Next, the Xpandomer products of the extension reaction were sequenced using the SBX protocol. Briefly, the constrained Xpandomer product was cleaved to produce a linearized Xpandomer. This was achieved by first quenching the extension reaction and amine-modifying the Xpandomer with 2M succinic anhydride. The phosphoramidate bond of the Xpandomer was then cleaved by treating the sample with 11.7M DCl at 23°C for 30 minutes. The linearized Xpandomer was purified by ethanol precipitation and resuspended in a buffer supplemented with 34% ACN and 15% DMF.

[0395] For sequencing, Xpandomer was added to a sample buffer of 0.8M NH4Cl, 1.2M GuanCl and 200mM HEPES, pH 7.4. Protein nanopores were prepared by inserting α-hemolysin into a DPhPE / hexadecane bilayer member in a buffer containing 2M NH4Cl and 100mM HEPES, pH 7.4. The experiment used a buffer of 0.4M NH4Cl, 600mM GuanCl and 100mM HEPES, pH 7.4 in the cis-well, and 2M NH4Cl and 100mM HEPES, pH 7.4 in the trans-well. The Xpandomer sample was heated to 70°C for 2 minutes, cooled completely, and then 2 μL of the sample was added to the cis-well. The voltage parameters for the run were as follows: 70mV / 650mV / 6μs / 1.5ms (reading voltage / pulse voltage / pulse voltage duration / pulse frequency). Data was acquired using Labview acquisition software. A representative trace of this run is shown in Fig.16 shown.

[0396] Fig.16 The level numbers superimposed above the traces in correspond to the signals generated by the XCTP code (L1), XTTP code (L2), XATP code (L3), and XGTP code (L4). In this experiment, the detection system correctly read all reporter codes, demonstrating 100% accuracy. The absence of deletion and insertion errors emphasizes the efficacy of the overhanging PEG-based TCE in transiently pausing the reporter code in the pore channel to produce accurate signal reads during the "read voltage" period and allowing translocation to resume during the "pulse voltage". Similarly, using the overhanging PEG-based TCE used in this experiment, switching between sequential reporter codes was achieved with a single voltage pulse. Notably, the same level of accuracy was observed in different runs of aliquots of the same Xpandomer sample using the following voltage parameters: 65 / 700 / 6 / 1.5; 65 / 650 / 6 / 1.5; 60 / 650 / 6 / 1.5 and 60 / 600 / 6 / 1.5. It was further observed that the flux of code reads was affected by various voltage parameters.

[0397] Example 19

[0398] Sequencing by amplification of complex templates with pendant PEG-based TCEsIn this example, the translocation control of TCEs incorporating pendant PEG phosphoramidites was evaluated by sequencing a complex 100mer template using the SBX protocol. Each XNTP substrate was synthesized with the following TCE: Y22222222222255, where "Y" represents a symmetrical phosphoramidite branch; "2" represents pendant PEG2; and "5" represents benzofuran. XNTP substrates were synthesized with the following reporter codes: (XC) DDDDDDLLLL, where "D" represents PEG6 and "L" represents C2; (XT) DDDDDD44LDX, where "X" represents PEG3 and "4" represents pendant PEG4; (XA) DDDDXX44XXDL; and (XG) DDDXXL.

[0399] To generate Xpandomer copies of a 100mer template, a solid-state primer extension reaction was performed using 1 pmol of XATP and XCTP and 1.5 pmol of XGTP and XTTP (the solid-state Xpandomer synthesis in which the extension oligonucleotides are covalently bound to the chip substrate is described in the applicant's provisional patent application No. 62 / 826,805, which is incorporated herein by reference in its entirety). The 50 μL extension reaction included the following reagents: 50 mM TrisCl, pH 8.84, 200 mM NH4OAc, 20% PEG8K, 10% NMP, 15 pmol polyphosphate PP-60.20, 10 μg SSB, 1 M urea, 10 mM PEM additive, and 9 μg purified recombinant DNA polymerase. The extension reaction was performed at 37° C. for 30 minutes.

[0400] Next, the Xpandomer product was sequenced using the SBX protocol. In short, the constrained Xpandomer product was cut to produce a linearized Xpandomer. This was achieved by first quenching the extension reaction and amine-modifying the Xpandomer with succinic anhydride. The phosphoramidate bond of the Xpandomer was then cut by treating the sample with 7.5M DCl at 23°C for 30 minutes. The linearized Xpandomer was released from the chip substrate by photocleavage of the extended oligonucleotide and recovered in an elution buffer supplemented with 15% ACN and 5% DMSO (20% final solvent).

[0401] For sequencing, Xpandomer was added to 0.8 M NH 4 Cl, 1.2 M GuCl, 200 mM HEPES; pH 7.4 sample buffer. 4Protein nanopores were prepared in DPhPE / hexadecane bilayer members in a buffer of 100 mM HEPES, pH 7.4 and 0.4 M NH4Cl, 600 mM GuanCl, 100 mM HEPES; pH 7.4 for the cis pores, and 2 M NH4Cl, 100 mM HEPES; pH 7.4 for the trans pores. The Xpandomer samples were heated to 70 °C for 2 minutes, cooled completely and vortexed, and then 2 μL aliquots were added to the cis pores. The voltage parameters for the run were as follows: 60 mV / 600 mV / 6 μs / 1.5 ms (read voltage / pulse voltage / pulse voltage duration / pulse frequency). Data were acquired via Labview acquisition software. Representative traces of this run are shown in Figure 2. Fig.17 shown.

[0402] like Fig.17 As shown, in this experiment, the detection system correctly read the sequence of all reporter codes, demonstrating 100% accuracy. The absence of deletion or insertion errors once again verified the efficacy of the TCE based on the pendant PEG as a structure that can read the polymer template sequence with high reliability. Importantly, the run of homopolymers was accurately sequenced under these conditions (see, for example, the 4x L1 code sequence near the beginning of the trace shown in Figure 14), thereby highlighting the accuracy of SBX with TCE based on the pendant PEG.

[0403] Example 20

[0404] Sequencing by amplification of a complex 222mer template with TCFs with pendant PEG

[0405] In this example, translocation control of pendant PEG-based TCEs was evaluated by sequencing a complex 222mer template using the SBX protocol. Each XNTP substrate was synthesized to contain the following TCEs: Y44444444444455, where "Y" represents a symmetrical phosphoramidite branch; "4" represents pendant PEG-4; and "5" represents benzofuran. XNTP substrates were synthesized with the following reporter codes: (XC) DDDDDDLLLDX; (XT) DDDDDD44LXXX; (XA) DDDDDD444LLDX; and (XG) DDDXXL444444XLLLL, where "D" represents PEG-6, "L" represents C2, "X" represents PEG-3, and "4" represents pendant PEG-4.

[0406] To generate Xpandomer copies of the 222mer template, a solid-state primer extension reaction was performed using 1.25 pmol of each XNTP, 10 pmol of template, and 20 pmol of E-oligo primer (solid-state Xpandomer synthesis in which the extension oligonucleotide is covalently attached to the chip substrate is described in applicant's provisional patent application No. 62 / 826,805, which is incorporated herein by reference in its entirety). The 50 μL extension reaction included the following reagents: 50 mM TrisCl, pH 8.84, 200 mM NH 4 OAc, 20% PEG8K, 8% NMP, 15 nmol polyphosphate PP-60.20, 10 μg SSB, 1 M urea, 5 mM PEM additive and 9 μg purified recombinant DNA polymerase. The extension reaction was carried out at 37°C for 30 minutes.

[0407] The Xpandomer products were then sequenced using the SBX protocol. Briefly, the constrained Xpandomer products were sequenced in buffer B.001 (1% Tween-20 / 3% SDS / 5 mM HEPES, pH 8.0 / 100 mM NaPO 4 / 15% DMF) and cleaved by adding 200 μl of buffer C.001 (7.5M DCl) and incubating at 23°C for 30 minutes to produce a linearized Xpandomer. The sample was then neutralized by adding 1000 μl of buffer B.001. The Xpandomer sample was then amine-modified by adding 666 μmol succinic anhydride and incubating at 23°C for 5 minutes. The sample was then washed in buffer D.094 (50% ACN) and the Xpandomer was released from the substrate by photocleavage and stored in buffer AG497 (0.8M NH4Cl / 1.2M GuanCl / 200mM HEPES, pH 7.4).

[0408] Protein nanopores were prepared by inserting α-hemolysin into DPhPE / hexadecane bilayer members in a buffer of 2M NH4Cl and 100mM HEPES, pH 7.4. The cis pores were perfused with a buffer containing 0.4M NH4Cl, 600mM GuanCl, 100mM HEPES; pH 7.4, and the trans pores were perfused with a buffer containing 2M NH4Cl, 100mM HEPES; pH 7.4. The Xpandomer samples were heated to 70°C for 2 minutes, cooled completely and vortexed, and then 2μL aliquots were added to the cis pores. The voltage parameters were run as follows: 60mV / 650mV / 6μs / 1.0ms (read voltage / pulse voltage / pulse voltage duration / pulse frequency). Data were acquired via Labview acquisition software. Representative traces of this run are shown in Fig.18 shown.

[0409] like Fig.18 As shown, in this experiment, the detection system correctly read the sequence of all reporter codes, demonstrating 100% accuracy. The absence of deletion or insertion errors reaffirmed the efficacy of the overhanging PEG-based TCE as a structure capable of reading polymeric template sequences with high reliability. Likewise, homopolymer runs were accurately sequenced under these conditions, highlighting the accuracy of SBX with overhanging PEG-based TCE.

[0410] Example 21

[0411] Sequencing by amplification of a complex 222mer template with pendant PEG-based TCE and D cells

[0412] In this example, translocation control of pendant PEG-based TCEs and D-cell signatures was evaluated by sequencing a complex 222mer template using the SBX protocol. Each XNTP substrate was synthesized to contain the following TCE: Y(32)(32)(32)(32)(32)(32)(61)(61)(61)(61)(61)(61), where "Y" represents a symmetrical phosphoramidite branch; "32" represents pendant mPEG4(PPA032); and "61" represents pendant PEG(PPA061). Each XNTP also included the following D-cell signature: D(63)D(63)D(63)DD where "D" represents PEG6, and "63" represents pendant mPEG4(PPA032). ” indicates pendant PEG (PPA063). XNTP substrates were synthesized using the following reporter codes: (XC)DDLLLX; (XT)LXXX; (XA)DD(32)(32)(32)LLLLLLL; and (XG)XXL(32)(32)(32)(32)(32)(32)LLLLLLL, where “D” indicates PEG-6, “L” indicates C2, “X” indicates PEG-3, and “32” indicates pendant mPEG-4 (PPA032).

[0413] To generate Xpandomer copies of the 222mer template, a solid-state primer extension reaction was performed using 5000 pmol of each XNTP, 4 pmol of template, and 20 pmol of E-oligo primer (the solid-state Xpandomer synthesis in which the extension oligonucleotide is covalently attached to the chip substrate is described in the applicant's provisional patent application No. 62 / 826,805, which is incorporated herein by reference in its entirety). The 50 μL extension reaction included the following reagents: 50 mM TrisCl, pH 8.84, 200 mM NH4OAc, 50 mM GuCl 20% PEG8K, 10% NMP, 15 nmol polyphosphate PP-60.23, 2.5 μg Kod SSB, 0.1 M urea, 15 mM PEM additive, and 13 μg purified recombinant DNA polymerase (a variant of DPO4 polymerase). The extension reaction was performed at 37° C. for 60 minutes.

[0414] Next, the Xpandomer product was sequenced using the SBX protocol. In brief, the constrained Xpandomer product was washed in buffer B.064 (1% Tween-20 / 3% SDS / 5mM HEPES, pH 8.0 / 100mM NaPO4 / 15% DMF) and cut to produce linearized Xpandomer by adding 200 μl buffer C.001 (7.5M DCl) and incubating at 23°C for 30 minutes. The sample was then neutralized by adding 2000 μl buffer B.064 and incubating at room temperature for 2'. The Xpandomer sample was then amine-modified by adding 500 μmol succinic anhydride in buffer B.065 and incubating at 23°C for 5 minutes. The sample was then washed in buffer D.102 (50% ACN), and the Xpandomer was released from the substrate by photocleavage and eluted in 60 μl elution buffer.

[0415] Protein nanopores were prepared by inserting α-hemolysin into DPhPE / hexadecane bilayer members in a buffer of 2M NH4Cl and 100mM HEPES, pH 7.4. Buffer AG242 containing 0.4M NH4Cl, 600mM GuanCl, 100mM HEPES; pH 7.4 and 5% glycerol was perfused for the cis pores, and buffer AB080 containing 0.4M NH4Cl, 600mM GuanCl, 5% ethyl acetate, 10mM HEPES; pH 7.4 was perfused for the trans pores. Xpandomer samples were heated to 70°C for 2 minutes, cooled completely and vortexed, and then 2μL aliquots were added to the cis pores. Voltage parameters were run as follows: 70mV / 625mV / 6μs / 1.0ms (reading voltage / pulse voltage / pulse voltage duration / pulse frequency). Data were acquired via Labview acquisition software.

[0416] To assess the accuracy of the Xpandomer sequence information, the sequence data was analyzed by displaying a histogram display of the sequence read population from the SBX reaction. The analysis software aligns each sequence read to the template sequence and trims the sequence ranges at the ends of the reads that do not align to the correct template sequence. A representative histogram of SBX sequencing of a 222mer template is presented in Fig.19 As can be seen, the SBX experiment produced highly accurate reads for the 222mer template. Notably, the throughput of this experiment was excellent. These results suggest that SSRTs incorporating pendant PEG-based TCE and D-cell features can control the translocation of Xpandomers through nanopores with high accuracy and efficiency.

[0417] Example 22

[0418] ratchet

[0419] In this ratchet example, a single hemolysin nanopore was prepared in a lipid bilayer with a vestibule on the trans side and a reagent mix consisting of 0.4M NH4Cl, 600mM GuanCl, 100mM HEPES; pH 7.4 in the cis reservoir; and a reagent mix consisting of 2M NH4Cl, 100mM HEPES; pH 7.4 in the trans reservoir cis. The current passing between the Ag / AgCl electrodes located in each reservoir was measured by an Axopatch 200B amplifier and digitized at a rate of 100k samples / second. To drive current through the nanopore, a 50% square wave with a duty cycle alternating between +70mV and -50mV was applied to the trans reservoir, while a 6μs pulse of +600mV was applied between transitions from positive to negative voltages (all voltages were referenced to the cis reservoir potential). With this applied pulse sequence, assuming an ideal translocation with no deletions or insertions, two reporters for each XNTP incorporated into the Xpandomer are measured, one at +70 mV and the other at -50 mV. Making two measurements for each base provides redundancy, thereby providing higher confidence in the match result, and also helps to identify deletions and insertions in non-homopolymer sequences. Fig. 20A Illustrates how cycling of +70mV / 600mV pulses / -50mV affects translocation of the Xpandomer through the nanopore and results in two measurements of the C code, followed by two measurements of the A code (and the 1st (one) measurement of the G code). The pattern code shows how the 2 reporter measurements per base can be used to identify insertions and deletions in this non-homopolymer sequence. Insertions and deletions result when the Xpandomer does not advance to the next reporter (in the nanopore) or it skips the next reporter.

[0420] Using the SBX synthesis and purification protocol, Xpandomer samples generated from synthetic DNA templates of known sequence were introduced into the cis reservoir and measured. An example of current measurement of Xpandomer translocation is shown in Fig. 20BThe graph is scaled so that the four reporter current levels for the +70mV measurements (14, 20, 27, and 35pA) and the 4 (four) reporter current levels for the -50mV measurements (-12, -20, -25, and -29pA) are shown as horizontal dashed lines. The data is aligned to the expected DNA template sequence and represented by the sequence of numbers on the graph. Each of the four numbers represents a base. The blue sequence number represents a confirmed base match to the template. Arrows represent errors. Arrows represented by the number 1 are non-homopolymer insertions because the base calls indicate that the Xpandomer did not advance and therefore these insertions can be identified. Arrows marked with the number 2 represent unidentified homopolymer insertions because the base calls are all at the same level.

[0421] Example 24

[0422] Synthesis of Fluoroarabinosyl XNTP Dimers

[0423] This example describes the synthesis of the 2' fluorine (F) epimers (2'FANA XNTPs) of each XNTP. These epimers are based on fluorinated nucleosides and are called "fluoroarabinonucleotides" (FANA). It is expected that the 2'F epimer will show increased stability during acid treatment, which is a key step in the synthetic pathway to produce linearized Xpandomer products. The following is a synthetic scheme to produce each 2'FANA XNTP.

[0424] A.

[0425] How to make 2′FANA XTTP

[0426]

[0427] In the first step, fluorouracil (Compound 1, available from TCI America) is coupled with 1-8 octanediyne via a head reaction (see, e.g., Bag, S., Jana, S., and Kasula, M. (2018). Sonogashira Cross-Coupling: Alkyne-Modified Nucleosides and Their Applications. In Palladium-Catalyzed Modification of Nucleosides, Nucleosides, and Oligonucleotides (pp. 75-146). Elsevier). In the second step, Compound 2 is treated with about one equivalent of DMTrCL in pyridine to produce Compound 3. In the third step, Compound 3 is converted to a triphosphate amide according to U.S. Patent No. 10,301,345 to Kokoris et al., entitled “Phosphoramidate esters and use and synthesis thereof,” which is incorporated herein by reference in its entirety.

[0428] B.

[0429] Method for making 2′FANA XCTP

[0430]

[0431] In the first step, fialcitabine (compound 5, available from TRC Canada) is coupled with 1-8 octadiyne via a mycelial reaction (as described above) to produce compound 6. In the second step, compound 6 is treated with about one equivalent of DMTrCL in pyridine to produce compound 7. In the third step, the exocyclic amine of compound 7 is protected by an acetyl group (see, e.g., Fan, Y., Gaffney, B., and Jones, Gen (2004). Transient Silylation of the Guanosine O6 and the Amino Groups Faciltates N-Acylation. Organic Letters, 6, 15, 2555-2557.), and then converted to the amide triphosphate 8 as described in U.S. Pat. No. 10,301,345 to Kokoris et al.

[0432] C.

[0433] Method for making 2′FANA XGTP

[0434] Commercially available from Granlen

[0435]

[0436] In the first step, 7-deaza-7-iodoguanosine (compound 9 available from Granlen; CAS: 444020-71-7) is treated with 1 equivalent of 1,3-dichloro-1,1,3,3-tetramethyldisiloxane to provide compound 10 (see, e.g., Markiewicz, WT and Wiewiorowski, M. (1978) A new type of silyl protecting groups in nucleoside chemistry. Nuc. Acids Res. 5, s185-ss190). In the second step, compound 10 is converted to compound 11 by using a fluorinating agent DAST (see, e.g., Pankiewicz, K., Kreminski, J., Ciszewski, L., Ren, W., and Watanabe, K. (1992). A synthesis of 9-(2-deoxy-2-fluoro-BD-arabinofuranosyl)adenine and-hypoxanthine. An effect of C3'-endo to C2'-endoconformational shift on the reaction course of 2'-hydroxyl group with DAST. J. of Organic Chem. 57, 2, 553-559.). In the third step, the exocyclic amine in compound 11 is protected with a phenoxyacetyl group as described above. In the fourth step, the resulting compound 12 is coupled with 1-8 octanediyne by the above-described head reaction to give compound 13. In the fifth step, the siloxane group is deprotected as described above to give compound 14. In the sixth step, compound 14 is treated with 1 equivalent of DMTrCl in pyridine to produce compound 15. In the seventh step, compound 15 is converted to guanosine amide triphosphate 16 as described in US Pat. No. 10,301,345 to Kokoris et al.

[0437] The same scheme can be used to synthesize the following ATP analogs:

[0438]

[0439] From the starting compound 7-deaza-7-iodoadenosine (available from Granlen, CAS: 24386-93-4).

[0440] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patents, and non-patent publications mentioned in this specification and / or listed in the application data sheet, including but not limited to U.S. Provisional Patent Application No. 62 / 852,262 filed on May 23, 2019, U.S. Provisional Patent Application No. 62 / 877,183 filed on July 22, 2019, and U.S. Provisional Patent Application No. 62 / 885,746 filed on August 12, 2019, are incorporated herein by reference in their entirety. For the purpose of describing and disclosing materials and methods such as those described in publications, these documents may be incorporated by reference, which may be used in conjunction with the invention currently described. The publications discussed above and throughout the text are provided only for their disclosure prior to the filing date of this application. Nothing herein should be construed as admitting that the inventor has no right to rely on prior inventions earlier than any cited publications.

Claims

1. A compound having the following structure: in R is OH or H; The nucleobase is adenine, cytosine, guanine, thymine, uracil, or a nucleobase analog, wherein "nucleobase analog" refers to a non-naturally occurring nucleobase that is capable of forming Watson and Crick base pairs with a complementary nucleobase on an adjacent single-stranded nucleic acid template; The reporter construct is a polymer having a first end and a second end, and comprises a first reporter code, a symmetrical chemical branch with a translocation control element, and a second reporter code in series from the first end to the second end; Linker A joins the oxygen atom of the alpha phosphoramidate to the first end of the reporter construct; Linker B joins the nucleobase to the second end of the reporter construct; And among them The translocation control element is a polymer comprising two or more repeating units selected from the group consisting of 1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane, 1,3-O-bis(phosphodiester)-2-(4-Me-O-PEG3)-1-(Et-O-Ac)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester)-2s-O-mPEG6-propane, 1,2-O-bis(phosphodiester)-3-O-mPEG2-propane, 2,3-O-bis(phosphodiester)-1-(5-benzofuran)-propane, 1,2-O-bis(phosphodiester)-3-(4-methylpiperazin-1-yl)-propane, 2,3-O-bis(phosphodiester)-1-(N1 -(2-Me-5-nitroindole)-propane, 1,8-O-bis(phosphodiester)-N,N-diethylpiperazine, 1,2-O-bis(phosphodiester)-3-(4-(Me-O-PEG3-O-Bz)-1-(1,2,3-triazole))-propane, 1,3-O-bis(phosphodiester)-2s-O-(4-(Me-O-PEG2)-1-(Et-OBz)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-O-Ac)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG7)-1-(Et-O Bz)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Me-acetate)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Et-2,2,2-tris-(Me-O-Bz))-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-2,2,2-tris-(Me-O-Ac))-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2S-O-(PEG4-O-Bz)-propane, 1, 3-O-bis(phosphodiester-2,2-bis(Me-O-mPEG2)-propane, 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG2-O-Me)-1-(Et-O-Bz)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG3-O-Me)-1-(Et-O-Bz)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG3-O-Me)-1-(Et-2,2,2-tri-(Me-O-Bz))-1,2,3-triazole)-propane or 1,3-O-bis(phosphodiester-2,2-Bis(1-Me-4-(Me-O-PEG2-O-Bz)-1,2,3-triazole)-propane, wherein Linker A and Linker B are polymers comprising two or more repeating units selected from the group consisting of spermine (Q), hexaethylene glycol (D), 2-((4-((3-(benzoyloxy)-2-(((1-(3-(benzoyloxy)-2-((benzoyloxy)methyl)-2-((phosphodiester-oxy)methyl)propyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-2-((benzoyloxy)methyl)propyloxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-2-((benzoyloxy)methyl)propyloxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-2-(phosphodiester-1,3-diyl dibenzoate, ,3-O-bis(phosphodiester-2,2-bis(1-Me-4-(Me-O-PEG2-O-Bz)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-O-Ac)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG7)-1-(Et-OBz)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Et-2 ,2,2-tris-(Me-O-Bz))-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-2,2,2-tris-(Me-O-Ac))-1,2,3-triazole)-propane, 1,2-O-bis(phosphodiester)-3-(4-(Me-O-PEG3-O-Bz)-1-(1,2,3-triazole))-propane, 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG2-O-Me)-1-(Et-O-Bz)-1,2,3 -triazole)-propane, 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG3-O-Me)-1-(Et-2,2,2-tri-(Me-O-Bz))-1,2,3-triazole)-propane, 1,2-O-bis(phosphodiester)-3-(4-methylpiperazin-1-yl)-propane, 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG3-O-Me)-1-(Et-O-Bz)-1,2,3-triazole)-propane and 1,1'-O-bis(phosphodiester)-N(p-tolyl)-diethanolamine; wherein the first end and the second end comprise a connecting portion; wherein the first reporter code and the second reporter code are polymers comprising two or more repeating units selected from the group consisting of hexaethylene glycol (D), ethane (L), triethylene glycol (X), 1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane, 1,3-O-bis(phosphodiester)-2-(4-Me-O-PEG3)-1-(Et-O-Ac)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2,2-bis(Me-O-mPEG2)-propane, 1,3-O-bis(phosphodiester-2S-O-(PEG4-O-Bz)-propane, 1,3-O-bis(phosphodiester)-2s-O-mPEG6-propane 、1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Et-2,2,2-tris-(Me-O-Bz))-1,2,3-triazole)-propane、1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Me-acetate)-1,2,3-triazole)-propane、1,3-O-bis(phosphodiester)-2s-O-(4-(Me-O-PEG2)-1-(Et-OBz)-1,2,3-triazole)-propane、1,3-O-bis(phosphodiester)-2-(4-Et-1-(Et-O-mPEG1)-1,2,3-triazole)-propane、2,3-O- Bis(phosphodiester)-1-(1-dimethoxyquinazolinedione)-propane, 2,3-O-bis(phosphodiester)-1-(N9-(3,6-dimethoxycarbazole)-propane, 1,1'-O-bis(phosphodiester)-2,2'-(sulfonylbis(benzyl-4-yl))-diethanol, 1,1'-O-bis(phosphodiester)-2,2'-bipyridyl-4,4'-yl)-dimethanol, 2,3-O-bis(phosphodiester)-1-(N1-(4,6-dimethoxy-3-Me-indole)-propane, 3-(1,2-O-bis(phosphodiester)-propyl)-8,8-dimethylhexahydro-3H-3a,6-methanobenzo[c]isothiazole 2,2-dioxide, 2,3-O- Bis(phosphodiester)-1-(N1-(6-azathymine))-propane, 1,5-O-bis(phosphodiester)-hexahydrofurano[2,6]furan, 1,1'-O-bis(phosphodiester)-octahydro-2,6-dimethyl-3,8:4,7-dimethoxy-2,6-naphthyridine-4,8-diyl)-dimethanol, 2,3-O-bis(phosphodiester)-1-(N1-(2-Me-5-nitroindole)-propane, 2,3-O-bis(phosphodiester)-1-(N1-(2-Me-5-nitroindole)-propane, 2,3-O-bis(phosphodiester)-1-(5-benzofuran)-propane, 1,2-O-bis(phosphodiester)-3-O-mPEG2-propane, 1,3-O-bis(phosphodiester)-2-(4-Et-1-(Et-O-mPEG3)-1,2,3-triazole)-propane and 1,3-O-bis(phosphodiester)-3-O-mPEG4-propane; The symmetrical chemical branch is selected from 1,2,3-O-tris-(phosphodiester)-propane, 1,3-bis-(5-O-phosphodiester-pentylamido)-2-O-phosphodiester-propane and 1,4,7-O-tris-(phosphodiester)-heptane.

2. The compound according to claim 1, wherein R is OH.

3. The compound according to claim 1, wherein R is H.

4. The compound according to any one of claims 1 to 3, wherein the nucleobase is adenine.

5. The compound according to any one of claims 1 to 3, wherein the nucleobase is cytosine.

6. The compound according to any one of claims 1-3, wherein the nucleobase is guanine.

7. The compound according to any one of claims 1 to 3, wherein the nucleobase is thymine.

8. The compound according to any one of claims 1-3, wherein the nucleobase is uracil.

9. The compound according to any one of claims 1-3, wherein the nucleobase is a nucleobase analog.

10. The compound of any one of claims 1-3, wherein the symmetrical chemical branch is 1,2,3-O-tris-(phosphodiester)-propane.

11. The compound of any one of claims 1-3, wherein the translocation control element is a polymer comprising two or more repeating units selected from the group consisting of 1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane and 1,3-O-bis(phosphodiester)-2-(4-Me-O-PEG3)-1-(Et-O-Ac)-1,2,3-triazole)-propane.

12. A compound according to any one of claims 1-3, wherein the translocation control element is a polymer comprising the following sequence: [(1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane)]n1[(1,3-O-bis(phosphodiester)-2-(4-Me-O-PEG3)-1-(Et-O-Ac)-1,2,3-triazole)-propane)]n2, wherein n1 is 0 to 6 and n2 is 6 to 10.

13. The compound of any one of claims 1-3, wherein the first reporter code and the second reporter code are the same.

14. The compound of any one of claims 1-3, wherein the first reporter code and the second reporter code are polymers comprising two or more repeating units selected from hexaethylene glycol, ethane, triethylene glycol, and 1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane.

15. A compound according to any one of claims 1-3, wherein the first reporter code and the second reporter code are polymers comprising a sequence selected from the following: (i) [(hexaethylene glycol)2(ethane)3(hexaethylene glycol)(triethylene glycol)], (ii) [(hexaethylene glycol)2(1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane)2(ethane)(triethylene glycol)3], (iii) [(hexaethylene glycol)2(1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane)3(ethane)2(hexaethylene glycol)(triethylene glycol)] and (iv) [(triethylene glycol)2(ethane)(1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane)6(ethane)7].

16. The compound according to any one of claims 1 to 3, wherein Linker A and Linker B are polymers comprising two or more repeating units selected from spermine.

17. The compound of any one of claims 1-3, wherein Linker A and Linker B comprise a polymerase enhancing region comprising two spermine repeating units.

18. The compound according to any one of claims 1 to 3, wherein Linker A and Linker B comprise a metastasis slowing region comprising two or more repeating units selected from the group consisting of 1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-O-Ac)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG7)-1-(Et-O-Ac)-1,2,3-triazole)-propane Bz)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Et-2,2,2-tris-(Me-O-Bz))-1,2,3-triazole)-propane and 1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-2,2,2-tris-(Me-O-Ac))-1,2,3-triazole)-propane.

19. The compound of any one of claims 1-3, wherein Linker A and Linker B comprise a metathesis slowing down region comprising a polymer selected from the group consisting of: (i) [((hexaethylene glycol)(1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-O-Ac)-1,2,3-triazole)-propane)3(hexaethylene glycol)2], (ii) [((hexaethylene glycol)(1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-O-Ac)-1,2,3 -triazole)-propane)4(hexaethylene glycol)2], (iii) [((hexaethylene glycol)(1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG7)-1-(Et-OBz)-1,2,3-triazole)-propane)4(hexaethylene glycol)2] and (iv) [((hexaethylene glycol)(1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-2,2,2-tris-(Me-O-Ac))-1,2,3-triazole)-propane)4(hexaethylene glycol)2].

20. The compound of any one of claims 1-3, wherein linker A is joined to the oxygen atom of the alpha phosphoramidate through a triazole-containing linkage.

21. The compound of any one of claims 1-3, wherein linker B is joined to the nucleobase via a triazole-containing linkage.

22. A reporter construct comprising a polymer having a first end and a second end, and comprising a first reporter code, a symmetrical chemical branch with a translocation control element, and a second reporter code connected in series from the first end to the second end; and wherein the translocation control element is a polymer comprising two or more repeating units selected from the following: 1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane, 1,3-O-bis(phosphodiester)-2-(4-Me-O-PEG3)-1-(Et-O-Ac)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester)-2s-O-mPEG6-propane, 1,2-O-bis(phosphodiester)-3-O- mPEG2-propane, 2,3-O-bis(phosphodiester)-1-(5-benzofuran)-propane, 1,2-O-bis(phosphodiester)-3-(4-methylpiperazin-1-yl)-propane, 2,3-O-bis(phosphodiester)-1-(N1-(2-Me-5-nitroindole)-propane, 1,8-O-bis(phosphodiester)-N,N-diethylpiperazine, 1,2-O-bis(phosphodiester)-3-(4-(Me-O-PEG3-O-Bz)-1-(1,2,3-triazole))-propane, 1,3-O-bis(phosphodiester)-2s-O-(4-(Me-O-PEG2)-1-(Et-OBz)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester)- (phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-O-Ac)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG7)-1-(Et-OBz)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Me-acetate)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Et-2,2,2-tri-(Me-O-Bz))-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2- (4-(Me-O-PEG5)-1-(Et-2,2,2-tris-(Me-O-Ac))-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2S-O-(PEG4-O-Bz)-propane, 1,3-O-bis(phosphodiester-2,2-bis(Me-O-mPEG2)-propane, 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG2-O-Me)-1-(Et-O-Bz)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG3-O-Me)-1-(Et-O-Bz)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG3-O-Me)-1-(Et-2,2,2-tri-(Me-O-Bz))-1,2,3-triazole)-propane or 1,3-O-bis(phosphodiester-2,2-bis(1-Me-4-(Me-O-PEG2-O-Bz)-1,2,3-triazole)-propane, wherein Linker A and Linker B are polymers comprising two or more repeating units selected from the group consisting of spermine (Q), hexaethylene glycol (D), 2-((4-((3-(benzoyloxy)-2-(((1-(3-(benzoyloxy)-2-((benzoyloxy)methyl)-2-((phosphodiester-oxy)methyl)propyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-2-((benzoyloxy)methyl)propyloxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-2-((benzoyloxy)methyl)propyloxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-2-(phosphodiester-1,3-diyl dibenzoate, ,3-O-bis(phosphodiester-2,2-bis(1-Me-4-(Me-O-PEG2-O-Bz)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-O-Ac)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG7)-1-(Et-OBz)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Et-2 ,2,2-tris-(Me-O-Bz))-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-2,2,2-tris-(Me-O-Ac))-1,2,3-triazole)-propane, 1,2-O-bis(phosphodiester)-3-(4-(Me-O-PEG3-O-Bz)-1-(1,2,3-triazole))-propane, 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG2-O-Me)-1-(Et-O-Bz)-1,2,3 -triazole)-propane, 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG3-O-Me)-1-(Et-2,2,2-tri-(Me-O-Bz))-1,2,3-triazole)-propane, 1,2-O-bis(phosphodiester)-3-(4-methylpiperazin-1-yl)-propane, 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG3-O-Me)-1-(Et-O-Bz)-1,2,3-triazole)-propane and 1,1'-O-bis(phosphodiester)-N(p-tolyl)-diethanolamine; wherein the first end and the second end comprise a connecting portion; wherein the first reporter code and the second reporter code are polymers comprising two or more repeating units selected from the group consisting of hexaethylene glycol (D), ethane (L), triethylene glycol (X), 1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane, 1,3-O-bis(phosphodiester)-2-(4-Me-O-PEG3)-1-(Et-O-Ac)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2,2-bis(Me-O-mPEG2)-propane, 1,3-O-bis(phosphodiester-2S-O-(PEG4-O-Bz)-propane, 1,3-O-bis(phosphodiester)-2s-O-mPEG6-propane 、1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Et-2,2,2-tris-(Me-O-Bz))-1,2,3-triazole)-propane、1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Me-acetate)-1,2,3-triazole)-propane、1,3-O-bis(phosphodiester)-2s-O-(4-(Me-O-PEG2)-1-(Et-OBz)-1,2,3-triazole)-propane、1,3-O-bis(phosphodiester)-2-(4-Et-1-(Et-O-mPEG1)-1,2,3-triazole)-propane、2,3-O- Bis(phosphodiester)-1-(1-dimethoxyquinazolinedione)-propane, 2,3-O-bis(phosphodiester)-1-(N9-(3,6-dimethoxycarbazole)-propane, 1,1'-O-bis(phosphodiester)-2,2'-(sulfonylbis(benzyl-4-yl))-diethanol, 1,1'-O-bis(phosphodiester)-2,2'-bipyridyl-4,4'-yl)-dimethanol, 2,3-O-bis(phosphodiester)-1-(N1-(4,6-dimethoxy-3-Me-indole)-propane, 3-(1,2-O-bis(phosphodiester)-propyl)-8,8-dimethylhexahydro-3H-3a,6-methanobenzo[c]isothiazole 2,2-dioxide, 2,3-O- Bis(phosphodiester)-1-(N1-(6-azathymine))-propane, 1,5-O-bis(phosphodiester)-hexahydrofurano[2,6]furan, 1,1'-O-bis(phosphodiester)-octahydro-2,6-dimethyl-3,8:4,7-dimethoxy-2,6-naphthyridine-4,8-diyl)-dimethanol, 2,3-O-bis(phosphodiester)-1-(N1-(2-Me-5-nitroindole)-propane, 2,3-O-bis(phosphodiester)-1-(N1-(2-Me-5-nitroindole)-propane, 2,3-O-bis(phosphodiester)-1-(5-benzofuran)-propane, 1,2-O-bis(phosphodiester)-3-O-mPEG2-propane, 1,3-O-bis(phosphodiester)-2-(4-Et-1-(Et-O-mPEG3)-1,2,3-triazole)-propane and 1,3-O-bis(phosphodiester)-3-O-mPEG4-propane; The symmetrical chemical branch is selected from 1,2,3-O-tris-(phosphodiester)-propane, 1,3-bis-(5-O-phosphodiester-pentylamido)-2-O-phosphodiester-propane and 1,4,7-O-tris-(phosphodiester)-heptane.

23. The reporter construct of claim 22, wherein the symmetric chemical branch is selected from 1,2,3-O-tris-(phosphodiester)-propane, 1,3-bis-(5-O-phosphodiester-pentylamido)-2-O-phosphodiester-propane and 1,4,7-O-tris-(phosphodiester)-heptane.

24. The reporter construct of claim 22, wherein the symmetric chemical branch is 1,2,3-O-tris-(phosphodiester)-propane.

25. A reporter construct according to any one of claims 22-24, wherein the translocation control element is a polymer comprising two or more repeating units selected from the group consisting of 1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane and 1,3-O-bis(phosphodiester)-2-(4-Me-O-PEG3)-1-(Et-O-Ac)-1,2,3-triazole)-propane.

26. A reporter construct according to any one of claims 22-24, wherein the translocation control element is a polymer comprising the following sequence: [(1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane)]n1[(1,3-O-bis(phosphodiester)-2-(4-Me-O-PEG3)-1-(Et-O-Ac)-1,2,3-triazole)-propane)]n2, wherein n1 is 0 to 6 and n2 is 6 to 10.

27. The reporter construct of any one of claims 22-24, wherein the first reporter code and the second reporter code are the same.

28. The reporter construct of any one of claims 22-24, wherein the first reporter code and the second reporter code are polymers comprising two or more repeating units selected from the group consisting of hexaethylene glycol (D), ethane (L), triethylene glycol (X), 1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane, 1,3-O-bis(phosphodiester)-2-(4-Me-O-PEG3)-1-(Et-O-Ac)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2,2-bis(Me-O-mPEG2)-propane, 1,3-O-bis(phosphodiester-2S-O-(PEG4-O-Bz)-propane, 1,3-O- Bis(phosphodiester)-2s-O-mPEG6-propane, 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Et-2,2,2-tris-(Me-O-Bz))-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Me-acetate)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester)-2s-O-(4-(Me-O-PEG2)-1-(Et-OBz)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester)-2-(4-Et-1-(Et-O-mPEG1)-1,2,3 -triazole)-propane, 2,3-O-bis(phosphoric acid diester)-1-(1-dimethoxyquinazolinedione)-propane, 2,3-O-bis(phosphoric acid diester)-1-(N9-(3,6-dimethoxycarbazole)-propane, 1,1'-O-bis(phosphoric acid diester)-2,2'-(sulfonylbis(benzyl-4-yl))-diethanol, 1,1'-O-bis(phosphoric acid diester)-2,2'-bipyridyl-4,4'-yl)-dimethanol, 2,3-O-bis(phosphoric acid diester)-1-(N1-(4,6-dimethoxy-3-Me-indole)-propane, 3-(1,2-O-bis(phosphoric acid diester)-propyl)-8,8-dimethylhexahydro-3H-3a,6-methanobenzo[c]isothiazole 2,2-dioxide 、2,3-O-bis(phosphodiester)-1-(N1-(6-azathymine))-propane、1,5-O-bis(phosphodiester)-hexahydrofurano[2,6]furan、1,1'-O-bis(phosphodiester)-octahydro-2,6-dimethyl-3,8:4,7-dimethoxy-2,6-naphthyridine-4,8-diyl)-dimethanol、2,3-O-bis(phosphodiester)-1-(N1-(2-Me-5-nitroindole)-propane、2,3-O-bis(phosphodiester)-1-(N1-(2-Me-5-nitroindole)-propane、2,3-O-bis(phosphodiester)-1-(5-benzofuran)-propane、1,2-O-bis(phosphodiester)-3-O-mPEG2-propane、1,3-O-bis(phosphodiester)-2-(4-Et-1-(Et-O-mPEG3)-1,2,3-triazole)-propane and 1,3-O-bis(phosphodiester)-3-O-mPEG4-propane.

29. The reporter construct of any one of claims 22-24, wherein the first reporter code and the second reporter code are polymers comprising two or more repeating units selected from hexaethylene glycol, ethane, triethylene glycol, and 1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane.

30. A reporter construct according to any one of claims 22-24, wherein the first reporter code and the second reporter code are polymers comprising a sequence selected from the following: (i) [(hexaethylene glycol)2(ethane)3(hexaethylene glycol)(triethylene glycol)], (ii) [(hexaethylene glycol)2(1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane)2(ethane)(triethylene glycol)3], (iii) [(hexaethylene glycol)2(1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane)3(ethane)2(hexaethylene glycol)(triethylene glycol)] and (iv) [(triethylene glycol)2(ethane)(1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane)6(ethane)7].

31. A symmetrically synthesized reporter tether (SSRT), wherein the symmetrically synthesized reporter tether is a polymer having a first end and a second end, and comprises a first linker connected in series from the first end to the second end, a reporter construct according to any one of claims 22-30, and a second linker, wherein the first linker and the second linker are the same and are a polymer comprising two or more repeating units selected from the group consisting of spermine (Q), hexaethylene glycol (D), 2-((4-((3-(benzoyloxy)-2-(((1-(3-(benzoyloxy)-2-((benzoyloxy)methyl)-2-((phosphodiester-oxy)methyl)propyl 1,3-O-bis(phosphodiester-2,2-bis(1-Me-4-(Me-O-PEG2-O-Bz)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-O-Ac)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG7)-1- (Et-OBz)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Et-2,2,2-tris-(Me-O-Bz))-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-2,2,2-tris-(Me-O-Ac))-1,2,3-triazole)-propane, 1,2-O-bis(phosphodiester)-3-(4-(Me-O-PEG3-O-Bz)-1-(1,2,3-triazole))-propane, 1,3-O-bis(phosphodiester-2,2-bis(4-(Me -O-PEG2-O-Me)-1-(Et-O-Bz)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG3-O-Me)-1-(Et-2,2,2-tri-(Me-O-Bz))-1,2,3-triazole)-propane, 1,2-O-bis(phosphodiester)-3-(4-methylpiperazin-1-yl)-propane, 1,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG3-O-Me)-1-(Et-O-Bz)-1,2,3-triazole)-propane and 1,1'-O-bis(phosphodiester)-N(p-tolyl)-diethanolamine, wherein the first end and the second end comprise a connecting portion; wherein the first reporter code and the second reporter code are polymers comprising two or more repeating units selected from the group consisting of hexaethylene glycol (D), ethane (L), triethylene glycol (X), 1,3-O-bis(phosphodiester)-2S-O-mPEG4-propane, 1,3-O-bis(phosphodiester)-2-(4-Me-O-PEG3)-1-(Et-O-Ac)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2,2-bis(Me-O-mPEG2)-propane, 1,3-O-bis(phosphodiester-2S-O-(PEG4-O-Bz)-propane, 1,3-O-bis(phosphodiester)-2s-O-mPEG6-propane 、1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Et-2,2,2-tris-(Me-O-Bz))-1,2,3-triazole)-propane、1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Me-acetate)-1,2,3-triazole)-propane、1,3-O-bis(phosphodiester)-2s-O-(4-(Me-O-PEG2)-1-(Et-OBz)-1,2,3-triazole)-propane、1,3-O-bis(phosphodiester)-2-(4-Et-1-(Et-O-mPEG1)-1,2,3-triazole)-propane、2,3-O- Bis(phosphodiester)-1-(1-dimethoxyquinazolinedione)-propane, 2,3-O-bis(phosphodiester)-1-(N9-(3,6-dimethoxycarbazole)-propane, 1,1'-O-bis(phosphodiester)-2,2'-(sulfonylbis(benzyl-4-yl))-diethanol, 1,1'-O-bis(phosphodiester)-2,2'-bipyridyl-4,4'-yl)-dimethanol, 2,3-O-bis(phosphodiester)-1-(N1-(4,6-dimethoxy-3-Me-indole)-propane, 3-(1,2-O-bis(phosphodiester)-propyl)-8,8-dimethylhexahydro-3H-3a,6-methanobenzo[c]isothiazole 2,2-dioxide, 2,3-O- Bis(phosphodiester)-1-(N1-(6-azathymine))-propane, 1,5-O-bis(phosphodiester)-hexahydrofurano[2,6]furan, 1,1'-O-bis(phosphodiester)-octahydro-2,6-dimethyl-3,8:4,7-dimethoxy-2,6-naphthyridine-4,8-diyl)-dimethanol, 2,3-O-bis(phosphodiester)-1-(N1-(2-Me-5-nitroindole)-propane, 2,3-O-bis(phosphodiester)-1-(N1-(2-Me-5-nitroindole)-propane, 2,3-O-bis(phosphodiester)-1-(5-benzofuran)-propane, 1,2-O-bis(phosphodiester)-3-O-mPEG2-propane, 1,3-O-bis(phosphodiester)-2-(4-Et-1-(Et-O-mPEG3)-1,2,3-triazole)-propane and 1,3-O-bis(phosphodiester)-3-O-mPEG4-propane; The symmetrical chemical branch is selected from 1,2,3-O-tris-(phosphodiester)-propane, 1,3-bis-(5-O-phosphodiester-pentylamido)-2-O-phosphodiester-propane and 1,4,7-O-tris-(phosphodiester)-heptane.

32. The symmetrical synthetic reporter tether (SSRT) of claim 31, wherein the first linker and the second linker comprise a polymerase enhancer region comprising two spermine repeating units.

33. The symmetrically synthesized reporter tether (SSRT) of claim 31 or 32, comprising a translocation slowing region comprising two or more repeating units selected from the group consisting of: 1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-O-Ac)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG7)-1-(Et-O-Ac)-1,2,3-triazole)-propane Bz)-1,2,3-triazole)-propane, 1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-1-(Et-2,2,2-tris-(Me-O-Bz))-1,2,3-triazole)-propane and 1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-2,2,2-tris-(Me-O-Ac))-1,2,3-triazole)-propane.

34. The symmetrically synthesized reporter tether (SSRT) of claim 31 or 32, comprising a metastasis slowing region comprising a polymer selected from the group consisting of: (i) [((hexaethylene glycol)(1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-O-Ac)-1,2,3-triazole)-propane)3(hexaethylene glycol)2], (ii) [((hexaethylene glycol)(1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-O-Ac)-1,2,3-triazole)-propane)3(hexaethylene glycol)2] 3-triazole)-propane)4(hexaethylene glycol)2], (iii) [((hexaethylene glycol)(1,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG7)-1-(Et-OBz)-1,2,3-triazole)-propane)4(hexaethylene glycol)2] and (iv) [((hexaethylene glycol)(1,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-1-(Et-2,2,2-tri-(Me-O-Ac))-1,2,3-triazole)-propane)4(hexaethylene glycol)2].

35. The symmetrically synthesized reporter tether (SSRT) of claim 31 or 32, wherein the linking moiety comprises an azido (-N3) group.

36. A method for sequencing a target nucleic acid, comprising: a) providing a daughter chain produced by template directed synthesis, the daughter chain comprising a plurality of subunits of the compound according to claim 1, i.e., XNTP subunits, the plurality of XNTP subunits being coupled in a sequence corresponding to a contiguous nucleotide sequence of all or a portion of the target nucleic acid, wherein each XNTP subunit of the daughter chain comprises a reporter construct, a nucleobase residue, and a selectively cleavable bond, and wherein the reporter construct allows extension of the subunit of the daughter chain upon cleavage of the selectively cleavable bond; b) cleaving the selectively cleavable bond to produce an Xpandomer having a length longer than the plurality of subunits of the daughter chain, the Xpandomer comprising the reporter construct for resolving genetic information in a sequence corresponding to the contiguous nucleotide sequence of all or a portion of the target nucleic acid; and c) detecting the reporter construct of the Xpandomer.

37. The method of claim 36, wherein the reporter construct for parsing the genetic information comprises a reporter code and a translocation control element, wherein the translocation control element provides translocation control by steric hindrance and pauses translocation when the Xpandomer passes through a nanopore subjected to a baseline voltage, wherein the translocation control element engages the reporter code within the orifice of the nanopore, wherein the reporter code is sensed by the nanopore.

38. The method of claim 37, wherein the Xpandomer resumes translocation through the nanopore by application of a pulse voltage, wherein the pulse voltage is sufficient to allow translocation of the translocation control element while leaving the next reporter construct of the Xpandomer free to engage the nanopore.

39. The method of claim 38, wherein the translocation control element of the reporter construct engaged with the nanopore translocates upon each pulse of the pulse voltage by steric hindrance.

40. The method of claim 38, wherein the target construct is sensed by the nanopore during the time period between pulses of the pulsed voltage.

41. The method of claim 37, wherein the baseline voltage is 55 mV to 75 mV.

42. The method of claim 38, wherein the pulse voltage is 550 mV to 700 mV.

43. The method of claim 38, wherein the pulse voltage has a duration of 5 μs to 10 μs.

44. The method according to claim 38, wherein a period of the pulse voltage is 0.5 ms to 1.5 ms.

45. The method of claim 37, wherein the nanopore is subjected to an alternating current (AC).

46. ​​The method of any one of claims 36-45, wherein one or more of the plurality of XNTP subunits comprises a 2' fluoroarabinosyl epimer.

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