Nucleosides and nucleotides having a 3'-hydroxyblocking group, and their use in polynucleotide sequencing methods.

Abstract

To provide a reversible protecting group for use in polynucleotide sequencing methods, which exhibits long-term stability, is efficiently incorporated by a polymerase enzyme, causes blocking of secondary or further nucleotide incorporation, and has the ability to be removed under mild conditions that do not damage the polynucleotide structure.SOLUTION: Provided are methods for preparing nucleotide and nucleoside molecules having an acetal or thiocarbamate 3'-OH blocking group, and the use of fully functionalized nucleotides containing a 3'-OH blocking group for sequencing applications.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (background) (Field) This disclosure generally refers to nucleotides, nucleosides, and nucleotides containing a 3'-hydroxy protecting group. This refers to the use of oligonucleotides and polynucleotides in sequencing methods. Regarding use. 3'-hydroxyprotected nucleotides, nucleosides, or oligonucleotides. A method for preparing tide is also disclosed. [Background technology]

[0002] (Explanation of related technologies) Advances in molecular research are used, in part, to characterize molecules or their biological reactions. This has been driven by improvements in the technologies used. In particular, research on nucleic acids, DNA and RNA, Benefiting from the development of techniques used in column analysis and hybridization event research. I am receiving it.

[0003] An example of a technology that has improved nucleic acid research is the development of arrays containing immobilized nucleic acids. These arrays typically consist of high-density polynucleotides immobilized on a solid support material. It consists of tricks. For example, Fodor et al., Trends Biote See ch. 12: 19-26, 1994. This is protected by a mask. However, to enable the attachment of appropriately modified nucleotide phosphoramidites Nucleic acids are assembled using a chemically sensitized glass surface exposed in a defined region. The method of assembly is described. The manufactured array also uses known polynucleotides as predetermined. It can be manufactured by a technique that "spots" the solid support at a specific position (for example, Stimpson et al., Proc. Natl. Acad. Sci. 92: 6379-6383, 1995).

[0004] One method for determining the nucleotide sequence of nucleic acids bound to an array is "synthetic sequencing." This technique, called "sequencing" or "SBS," is used to determine the sequence of DNA. Ideally, the correct complementary nucleotide on the opposite side of the sequenced nucleic acid is controlled. It requires uptake (i.e., one at a time). This means that each nucleotide residue Since the bases are sequenced one at a time, nucleotides can be added in multiple cycles. This enables precise sequencing and prevents uncontrolled series of captures from occurring. The incorporated nucleotides undergo removal of the labeled portion and subsequent sequencing. It is read using the appropriate label attached to it beforehand. [Overview of the project] [Problems that the invention aims to solve]

[0005] To ensure that only a single incorporation occurs, structural modifications ("protecting group" or " A blocking group is included in each labeled nucleotide added to the growth chain, and one nucleus This ensures that only ocides are incorporated after a nucleotide with a protecting group is added. Next, under reaction conditions that do not impair the integrity of the sequenced DNA, the protecting group is removed. The sequencing cycle then incorporates the next protected labeled nucleotide. It can be continued.

[0006] To be useful in DNA sequencing, nucleotides are usually triphosphates. When a base is added to a nucleotide, it is used to incorporate it into the polynucleotide chain. To prevent the polymerase used from continuing to replicate, the 3'-hydroxy protecting group is generally removed. It is necessary. It can be added to nucleotides, but there are still many limitations on the appropriate group type. The protecting group can be easily removed from the sugar moiety without damaging the polynucleotide chain. It is capable of doing so, while also preventing the addition of additional nucleotide molecules to the polynucleotide chain. It should be. Furthermore, modified nucleotides are used to incorporate them into polynucleotide chains. It must be compatible with the polymerase or another suitable enzyme used. Therefore, An ideal protecting group exhibits long-term stability and is efficiently incorporated by polymerase enzymes. This causes blockage of secondary or further nucleotide incorporation, resulting in a polynucleotide structure. It must have the ability to be removed under gentle, non-damaging conditions, preferably under aqueous conditions. It must be done.

[0007] Reversible protecting groups have been described previously. For example, Metzker et al. (Nucleic Acids Research, 22 (20): 4259-4 267, 1994) is a 2-deoxyribonucleoside 5'-triphosphate with eight 3' modifications. Synthesis and use of (3' modified dNTPs), and two DNA templates for uptake activity Test assay. WO2002 / 029003 is used to test growing DN in polymerase reaction. Sequences may include the use of allyl protecting groups to cap the 3'-OH groups on the A chain. The method is described.

[0008] Furthermore, the development of several reversible protecting groups and the deprotection of them under DNA compatibility conditions The methods have been previously reported in International Application Publication Numbers WO2004 / 014897 and WO2014 / 1395, each of which is hereby incorporated by reference in its entirety. 96 and are hereby incorporated by reference in their entirety. into this specification.

Means for Solving the Problem

[0009] (Summary) Some embodiments of the present disclosure relate to nucleotides or nucleosides containing ribose or deoxyribose having a removable 3'-OH protecting or blocking group that forms a structure covalently bonded to the 3'-carbon atom, wherein:

Chemical

[0010] Some embodiments of this disclosure have a structure covalently bonded to a 3'-carbon atom. [ka] Ribose or deoxyribo having a removable 3'-OH blocking group that forms a ribose or deoxyribo A nucleoside or nucleotide containing , wherein the formula: R 5 and R 6 Each of these is independently H, C1-C6 alkyl, and C2-C6 alkeni C2-C6 alkynyl, C1-C6 haloalkyl, C2-C8 alkoxyalkyl, (CH2) may be substituted. m -Phenyl, may be substituted-(CH2) n -(5 or 6-membered heteroaryl), may be substituted with -(CH2) k -C3~C7 Carbocyclyl, or possibly substituted-(CH2) p -(3-7 member heterozygous) (ru); -(CH2) m -,-(CH2) n -,-(CH2) k -, and -(CH2) p -of Each of them may be substituted; and Each of m, n, k, and p is independently 0, 1, 2, 3, or 4. Regarding nucleosides or nucleotides.

[0011] Some embodiments of this disclosure are 3'-OH blocked nucleotides as described herein. This relates to oligonucleotides or polynucleotides containing molecules.

[0012] Some embodiments of this disclosure describe targeting single-strand polynucleotides in sequencing reactions. In relation to methods for preparing complementary growth polynucleotides to nucleotides, the incorporation of nucleotides Complementary polynucleotides that grow the nucleotide molecules described herein, which are prevented from being introduced. This includes incorporating subsequent nucleotides into the growing complementary polynucleotide. In some embodiments, nucleotide incorporation is performed by polymerase, terminal deoxygenation This is achieved by cleotidyltransferase (TdT) or reverse transcriptase. In the embodiment, integration is achieved by polymerase (e.g., DNA polymerase). It will be done.

[0013] Some further embodiments of this disclosure include the following: Regarding methods for determining the sequence: (a) Nucleo containing a 3'-OH blocking group and the detectable label described herein The tide is a copy polynucleotide chain complementary to at least a portion of the target polynucleotide chain. To incorporate into; (b) detecting the identity of nucleotides incorporated into a copy polynucleotide chain; and (c) Labeling and 3'-OH from nucleotides incorporated into the copy polynucleotide chain Chemically removing blocking groups.

[0014] In some embodiments, the sequencing method includes (d) chemically removed labels The further method includes washing away the 3' blocking group from the copy polynucleotide chain. In some embodiments, such a cleaning step is also not incorporated into the nucleo Remove the nucleotide. In some such embodiments, the incorporated nucleotide The 3' blocking group and detectable label are removed before introducing the next complementary nucleotide. In some further embodiments, a 3' blocking group and detectable The label is removed in a single step of the chemical reaction. In some embodiments, as described herein The described continuous incorporation is at least 50 times, at least 100 times, at least 150 times It will be executed at least 200 times, or at least 250 times.

[0015] Some further embodiments of this disclosure relate to multiple nucleotides or This relates to a kit containing a nucleoside molecule and packaging materials therefor. Using nucleotides, nucleosides, oligonucleotides, or kits, biological To detect, measure, or identify a system (e.g., including its processes or components). This is possible. Exemplary examples of using nucleotides, oligonucleotides, or kits. The technologies include sequencing, expression analysis, hybridization analysis, gene analysis, and RN. A analysis, cell assay (e.g., cell binding or cell function analysis), or protein assay Sequencing (e.g., protein binding assay or protein activity assay). Automated sequencing. It can be used in automated equipment for performing specific technologies, such as single-acting devices. The SING instrument has two units that operate at different wavelengths to distinguish between different detectable labels. The above lasers may be included. [Brief explanation of the drawing]

[0016] [Figure 1] Figure 1 is a line graph showing the stability of various 3'-blocked nucleotides as a function of time in a buffer solution at 65°C. [Figure 2] Figure 2A is a line graph illustrating the percentage (%) of remaining nucleotides (starting material) as a function of time, comparing the deblocking rate of nucleotides with a 3'-AOM blocking group and nucleotides with a 3'-O-azidomethyl (-CH2N3) blocking group in solution. Figure 2B is a line graph illustrating the percentage (%) of 3'-unblocked nucleotides as a function of time, comparing the deblocking rate of 3'-blocked nucleotides with various acetal blocking groups in solution. [Figure 3] Figures 3A and 3B illustrate sequencing results using an Illumina MiniSeq® instrument with fully functionalized nucleotides (ffNs) containing a 3'-AOM blocking group in the incorporation mix. Figure 3C illustrates the sequencing error rate using fully functionalized nucleotides (ffNs) containing a 3'-AOM blocking group in the incorporation mix compared to standard ffNs containing a 3'-O-azidomethyl blocking group. [Figure 4]Figures 4A and 4B illustrate, respectively, a comparison of key sequencing metrics, including phasing, prephasing, and error rates, using fully functionalized nucleotides containing 3'-AOM and 3'-O-azidomethyl blocking groups with two different DNA polymerases (Pol812 and Pol1901). [Figure 5] Figure 5 is a line graph illustrating the sequence stability of fully functionalized nucleotides with a 3'-AOM or 3'-O-azidomethyl blocking group as a function of time in a buffer at 45°C. [Figure 6] Figure 6 is a line graph illustrating the stability of various nucleosides with 3'-blocking groups as a function of time in a buffer solution at 65°C. [Figure 7] Figure 7 is a line graph illustrating the percentage of remaining 3'-blocked nucleotides as a function of time, comparing the cleavage (deblocking) rate of the thiocarbamate 3'-blocking group dimethylthiocarbamate (DMTC) with that of the 3'-O-azidomethyl (3'-O-CH2N3) blocking group under two different conditions (Oxone® or NaIO4). [Modes for carrying out the invention]

[0017] (Detailed explanation) Embodiments of this disclosure are for sequencing applications, for example, synthesis sequencing (SB). Nucleus having a 3'-OH acetal or thiocarbamate blocking group for S) Regarding rheosides and nucleotides. These blocking groups are known in the art. Compared to other products, it offers superior stability in solution. In particular, 3'-OH block The King group improves the stability of fully functionalized nucleotides (ffNs) during synthesis. Stability in solutions during formulation, storage, and handling using the EQN instrument has also improved. Furthermore, Honmei The 3'-OH blocking group described in the details also improves data quality by reducing the amount of plastic used. Fading and low signal attenuation can be achieved, making it suitable for sequencing applications. This allows for longer readings from the source.

[0018] (definition) Unless otherwise defined, all technical and scientific terms used herein are those of the same name. It has the same meaning as commonly understood by the industry. The term "includes (inc)" "luding)" and "include", "includes" The use of other forms such as "included" is not limited. "Having," "Have," and "Has" The use of other forms such as "had" is not limited to those used herein. As shown, in the transitional phrase of the claim Even within the main body, the term "comprise(s)" The word "comprising" has an open-ended meaning. It should be interpreted as follows: That is, the above term is the phrase "having at least) or "including at least (including at l It should be interpreted as synonymous with "east." For example, when used in the context of a process. In addition, the term "comprising" means that the process is at least described. This means that it includes steps, but may include additional steps. Compounds, compositions, When used in the context of devices, the term "comprising" means The compound, composition, or device contains at least the described features or components, but further This means that additional characteristics or components may also be included.

[0019] Common organic abbreviations used herein are defined as follows: ℃ Celsius temperature dATP (deoxyadenosine triphosphate) dCTP Deoxycytidine Triphosphate dGTP (Deoxyguanosine Triphosphate) dTTP Deoxythymidine triphosphate ddNTP dideoxynucleotide triphosphate ffN: Fully functionalized nucleotides RT room temperature Sequencing by SBS synthesis SM Departure Materials

[0020] As used herein, the term "array" refers to a configuration in which different probe molecules are arranged according to their relative positions. This refers to a group of different probe molecules attached to one or more substrates, distinguishable from one another. The array consists of different probes, each positioned at a different addressable location on the substrate. It can contain molecules. Alternatively or additionally, each array contains a different probe. It may include a separate substrate having molecules, and different probe molecules on the surface to which the substrate adheres. A liquid that can be identified according to the position of the upper substrate, or according to the position of the substrate. Typical arrays located on a surface include, for example, U.S. Patent No. 6,355,431B1. As stated in U.S. Patent No. 2002 / 0102578 and PCT Publication No. WO00 / 63437 The wells shown may include, but are not limited to, arrays containing beads. For example... If so, a microfluidic device such as a fluorescence-activated cell sorter (FACS) can be used to process liquids. An exemplary format that can be used in this invention to distinguish beads within a ray is, for example, It is described in U.S. Patent No. 6,524,793. Further arrays that can be used in the present invention Examples include the U.S. 5,429,807; 5,436,327; 5,561,071; 5,583,211; 5,658,734; 5,837,858; 5,874,219; 5,919,523; 6,136,269; 6,287,768; 6,287,776; 6,288,220; 6,297,006; 6,291,193; 6,346,413; Issues 6,416,949; 6,482,591; 6,514,751 and 6,610,482 ;and WO93 / 17126;WO95 / 11995;WO95 / 35505;EP7 This includes, but is not limited to, those described in 42287 and EP799897. It will not be done.

[0021] As used herein, the term "covalently attached" "Hed)" or "covalently bonded" refers to the bond between atoms. This refers to the formation of chemical bonds characterized by the sharing of electron pairs. For example, covalent polymers. The coating is compared to bonding to the surface by other means, such as adhesion or electrostatic interaction. In contrast, it refers to a polymer coating that forms a chemical bond with the functionalized surface of the substrate. Polymers covalently bonded to a surface can also be bonded via means other than covalent bonding. This will be understood.

[0022] As used herein, the "R" group represents a substituent that can be bonded to the indicated atom. The R group may or may not be substituted. Two "R" groups are linked together. When it is stated that "together with the atoms" they form a ring or ring system, the atoms, intervening atoms Synthesizes, and this means a ring in which sets of two R-group units are enumerated. For example, the following: Substructures exist: [ka] and R 1 and R 2 It is defined as being selected from the group consisting of hydrogen and alkyl groups. R, or R 1 and R 2 along with the atoms to which they are bonded, aryl or carboxy When defined as something that forms a cyclil, R 1 and R 2 is hydrogen or alkyl The following can be selected from, or the substructure has the following structure: [ka] In the formula, A is an aryl ring or carbocyrill containing the depicted double bond.

[0023] Certain radical naming conventions, depending on the context, can refer to either monoradicals or diradicals. It should be understood that substituents can include two substituents on the rest of the molecule. When a bonding point is required, the substituent is understood to be a diradical. For example, two Substituents identified as alkyl groups requiring a bond site include -CH2- and -CH2CH2. This includes diradicals such as -CH2CH(CH3)CH2-. Other radical naming conventions This clearly indicates that the radical is a diradical such as "alkylene" or "alkenylene". vinegar.

[0024] As used herein, the terms "halogen" or "halo" refer to the radiation of elements in column 7 of the periodic table. It means any one of the linearly stable atoms, for example, fluorine, chlorine, bromine, or iodine. Electrolyte and chlorine are preferred.

[0025] As used herein, "a" and "b" are integers. a ~C b " is Al Kill, the number of carbon atoms in the alkenyl or alkynyl group, or cycloalkyl or ali This refers to the number of ring atoms in an alkyl group. That is, alkyl, alkenyl, alkynyl, cycloal Kill's ring and aryl ring can contain carbon atoms from "a" to "b". For example, the "C1-C4 alkyl" group is any alkyl group having 1 to 4 carbon atoms. That is, CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)-, and (CH3)3C-; C3-C4 cycloalkyl groups are all cyclopropyl groups having 3 to 4 carbon atoms. This refers to the pyr group, i.e., cyclopropyl and cyclobutyl. Similarly, "4-6 member hetero The "rosicryl" group is all heterocyclyl groups that have a total of 4 to 6 ring atoms, for example Azethidine, oxetane, oxazoline, pyrrolidine, piperidine, piperazine, molar This refers to foline, etc. Alkyl, alkenyl, alkynyl, cycloalkyl, or aryl compounds. If "a" and "b" are not specified for the base, then the definitions described therein are The broadest range is assumed. As used herein, the term "C1-C6" means Defined by C1, C2, C3, C4, C5, and C6, as well as any two numerical values. This includes the range of C1-C6 alkyl groups, C1, C2, C3, C4, C5 and This includes C6 alkyl, C2-C6 alkyl, C1-C3 alkyl, etc. Similarly, C2-C 6-alkenyls include C2, C3, C4, C5 and C6 alkenyls, and C2-C5 alkenyls. This includes C3-C4 alkenyls, etc. C2-C6 alkenyls are C2, C3, C4, C5 This includes C6 alkynyl, C2-C5 alkynyl, C3-C4 alkynyl, etc. 8-cycloalkyl groups each consist of 3, 4, 5, 6, 7, and 8 carbon atoms, or C3-C7. Defined by one of two numerical values, such as cycloalkyl or C5-C6 cycloalkyl. It includes a hydrocarbon ring that includes the range in which it is included.

[0026] As used herein, "alkyl" means fully saturated (i.e., double bond) This refers to a straight or branched hydrocarbon chain (that does not contain triple bonds). The alkyl group consists of 1 to 20 atoms. It may have carbon atoms (whereever it appears herein, it is always a number such as "1 to 20"). The range refers to each integer within the specified range; for example, "1 to 20 carbon atoms" is A The kill group consists of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., and up to 20 carbon atoms. It may be, but this definition also applies to the appearance of the term "alkyl" where no numerical range is specified. (to cover). Alkyl alkyl groups are also medium alkyl groups having 1 to 9 carbon atoms. Good. Alkyl alkyl groups could also be lower alkyl groups having 1 to 6 carbon atoms. Alkyl compounds may be specified as "C1-C4 alkyl" or similar designations. For example, "C1-C6 alkyl" means that the alkyl chain has 1 to 6 carbon atoms. In other words, the alkyl chain is methyl, ethyl, propyl, isopropyl, n-butyl, i This indicates that the selection is made from the group consisting of sorbyl, sec-butyl, and t-butyl. Typical alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, and isopropyl. Examples include butyl nitrate, tert-butyl nitrate, pentyl nitrate, hexyl nitrate, etc., but are not limited to these. stomach.

[0027] As used herein, “alkoxy” means that R is an alkyl group as defined above. The formula -OR refers to, for example, "C1-C9 alkoxy," and includes methoxy, ethoxy, n-p Ropoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, Examples include, but are not limited to, sec-butoxy and tert-butoxy.

[0028] As used herein, "alkenyl" refers to a linear or fractional chain containing one or more double bonds. This refers to a branched hydrocarbon chain. While an alkenyl group can have 2 to 20 carbon atoms, this definition is... It also covers the appearance of the term "alkenyl" where no numerical range is specified. Alternatively, it may be a medium alkenyl having 2 to 9 carbon atoms. The alkenyl group also It may also be a lower alkenyl having 2 to 6 carbon atoms. The alkenyl group is It may also be designated as "C2-C6 alkenil" or a similar designation. Just as an example, "C2-C6 alkenyl" means that the alkenyl chain has 2 to 6 carbon atoms, that is, The alkenyl chain consists of etenyl, propen-1-yl, propen-2-yl, propen- 3-yl, buten-1-yl, buten-2-yl, buten-3-yl, buten-4-yl , 1-methyl-propen-1-yl, 2-methyl-propen-1-yl, 1-ethyl- Ten-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl, buta-1 Selected from the group consisting of ,2,-dienyl and buta-1,2-dien-4-yl This indicates that. Typical alkenyl groups include ethenyl, propenyl, butenyl, and pentenyl. Examples include, but are not limited to, ru and hexenyl.

[0029] As used herein, "alkynyl" refers to a linear or fractional chain containing one or more triple bonds. This refers to a branched hydrocarbon chain. While an alkynyl group can have 2 to 20 carbon atoms, this definition is... It also covers the appearance of the term "alkynyl" where no numerical range is specified. Alternatively, it may be a medium-sized alkynyl having 2 to 9 carbon atoms. The alkynyl group also has, It could be a lower alkynyl having 2 to 6 carbon atoms. The alkynyl group is "C2 It may also be specified as "~C6 alkinyl" or a similar designation. Just as an example, "C2 "~C6 alkynyl" means that the alkynyl chain contains 2 to 6 carbon atoms, that is, The alkynyl chain consists of ethinyl, propyne-1-yl, propyne-2-yl, and butyne-1-yl. Selected from the group consisting of buty-3-yl, buty-4-yl, and 2-butynyl. This indicates that. Typical alkynyl groups include ethynyl, propynyl, butynyl, and pentyl. Examples include, but are not limited to, nyl and hexinyl.

[0030] As used herein, "heteroalkyl" means one or more heteroatoms, i.e. The chain skeleton contains elements other than carbon, including but not limited to nitrogen, oxygen, and sulfur. This refers to a chain or branched hydrocarbon chain. Heteroalkyl groups can have 1 to 20 carbon atoms. However, this definition also includes the appearance of the term "heteroalkyl" for which no numerical range is specified. Heteroalkyl groups are also medium heteroalkyl groups having 1 to 9 carbon atoms. It can be a heteroalkyl group. A heteroalkyl group also has a lower heteroalkyl group having 1 to 6 carbon atoms. It can be an alkyl group. Heteroalkyl groups are "C1-C6 heteroalkyl" or similar. It may be designated as a designation. A heteroalkyl group may contain one or more heteroatoms. For example only, "C4-C6 heteroalkyl" refers to a heteroalkyl chain with 4-6 carbon atoms. This indicates the presence of atoms, and furthermore, the chain's backbone contains one or more heteroatoms.

[0031] The term "aromatic" refers to a ring or ring system having a conjugated pi-electron system, and is a carbocyclic aroma. It includes both group (e.g., phenyl) and heterocyclic aromatic groups (e.g., pyridine). The term refers to monocyclic or fused polycyclic compounds (i.e., under the condition that the entire ring system is aromatic) It includes ring groups that share pairs of adjacent atoms.

[0032] As used herein, "aryl" refers to an aromatic ring or ring skeleton containing only carbon atoms. This refers to a ring system (i.e., two or more fused rings sharing two adjacent carbon atoms). In the case of a cyclic system, all rings in the system are aromatic. The aryl group has 6 to 18 carbon atoms. It may have offspring, but this definition refers to the appearance of the term "aryl" where no numerical range is specified. It also covers. In some embodiments, the aryl group has 6 to 10 carbon atoms. The aryl group is "C6~C 10 "Aryl", "C6 or C 10 "Aryl", or the same It can be designated as a designation. Examples of aryl groups include, but are not limited to, fer Examples include nyl, naphthyl, azurenyl, and anthracenyl.

[0033] "Aralkyl" or "arylalkyl" is "C 7-14 "Aralkil," The substituent is an aryl group bonded via an alkylene group, such as benzyl, 2-phenyl Examples include, but are not limited to, ethyl 3-phenylpropyl and naphthylalkyl compounds. No. In some cases, the alkylene group is a lower alkylene group (i.e., These are C1-C6 alkylene groups.

[0034] As used herein, "heteroaryl" means one or more heteroatoms, i.e., Examples include, but are not limited to, nitrogen, oxygen, and sulfur in the ring skeleton, other elements besides carbon. Aromatic rings or ring systems containing (i.e., two or more fused rings sharing two adjacent atoms) This refers to a heteroaryl ring system, where each ring in the system is aromatic. The group has 5 to 18 ring members (i.e., a ring skeleton including carbon atoms and heteroatoms). It may have the number of constituent atoms, but this definition does not specify a numerical range for the term "he". The emergence of "heteroaryl" is also covered. In some embodiments, the heteroaryl group is 5 It has a ring member number of ~10 or a ring member number of 5~7. Heteroaryl groups have a "5~7 member number" Even if designated as "terroraryl," "5-10 member heteroaryl," or a similar designation Good. Examples of heteroaryl rings include furyl, thienyl, phthalazinyl, pyrrolyl, and o Xazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl Triazolyl, thiadiazolyl, pyridinyl, pyridadinyl, pyrimidinyl, pyrazini Lu, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoi Examples include, but are not limited to, ndolyl and benzothienyl.

[0035] "Heteroaralkyl" or "heteroarylalkyl" is a group of groups with alkylates as substituents. It is a heteroaryl group bonded via an ion group. Examples include 2-thienylmethyl, 3 - Thienylmethyl, furylmethyl, thienylethyl, pyrrolylalkyl, pyridylalkyl Examples include, but are not limited to, isoxazolylalkyl and imidazolylalkyl. It is not determined. In some cases, the alkylene group is a lower alkylene group (i.e. (C1-C6 alkylene group).

[0036] As used herein, "carbocykrill" refers to a non-carbon cyclyl whose ring structure consists only of carbon atoms. This refers to an aromatic cyclic ring or ring system. If a carbocyclyl is a ring system, then two or more rings are... They may be joined together in a manner of fusion, bridging, or spiroconnection. At least one ring system Carbocyclyl may have any degree of saturation, as long as the other ring is not aromatic. Therefore, as carbocyclyls, cycloalkyl, cycloalkenyl, and cyclocyclyl are examples. Chloalkynyl is an example. The carbocyclyl group may have 3 to 20 carbon atoms. However, this definition also includes the appearance of the term "carbocykrill" for which no numerical range is specified. The carbocyclyl group is also a medium carbocyclyl having 3 to 10 carbon atoms. It may also be a carbocyclyl group having 3 to 6 carbon atoms. It is also acceptable. The carbocyclyl group is referred to as "C3-C6 carbocyclyl" or a similar designation. It may also be specified as such. Examples of carbocyclyl rings include cyclopropyl and cyclobutyl. , cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, Bisicle[2.2.2]octanyl, adamantyl, and spiro[4.4]nonanyl These are some examples, but are not limited to them.

[0037] As used herein, "cycloalkyl" refers to a fully saturated carbocyclyl ring. Or it means a cyclic system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, And cyclohexyl are examples.

[0038] As used herein, "heterocyclyl" means having at least one heterocyclyl in its ring skeleton. It means a non-aromatic cyclic ring or ring system containing atoms. Heterocyclines are fused, cross-linked, and They may be joined together by spirobonding. A heterocyclyl has at least one ring system The ring may have any degree of saturation as long as the ring is not aromatic. The heteroatom is non-aromatic of the ring system. The heterocyclyl group may be present in either the aromatic ring or either of the aromatic rings. The number of ring members (i.e., the number of atoms that make up the ring skeleton, including carbon atoms and heteroatoms) It may be possible, but this definition also includes the appearance of the term "heterocycline" for which no numerical range is specified. The heterocyclyl group is also a medium heterocyclyl having 3 to 10 ring members. It is also acceptable. A heterocyclyl group is a heterocyclyl having 3 to 6 ring members. This is also acceptable. The heterocyclyl group is designated as "3-6 membered heterocyclyl" or a similar designation. This may also be done. In a preferred six-membered monocyclic heterocycline, the heteroatoms are O, N, or S. One to three are selected, and in a preferred five-membered monocyclic heterocycline, the heteroatoms are Selected from one or two heteroatoms of O, N, or S. Examples of heterocyclyl rings Examples include azepinyl, acridinyl, carbazolyl, cinolinyl, dioxolanil, and Midazolinil, imidazolidinil, morpholinil, oxyranil, oxepanil, thiepa Nyl, piperidinil, piperazinil, dioxopiperazinil, pyrrolidinil, 4-piper Donyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxynyl, 1,3-dioxanyl , 1,4-dioxynyl, 1,4-dioxanyl, 1,3-oxathianyl, 1,4- Xathiinyl, 1,4-oxathianyl, 2H-1,2-oxazinyl, trioxanyl , hexahydro-1,3,5-triazinyl, 1,3-dioxolyl, 1,3-diox Ranyl, 1,3-dithiolyl, 1,3-dithioranyl, isoxazolinil, isoxazoli Dinyl, oxazolinyl, oxazolidinyl, oxazolidinonyl, thiazolinyl, thia Zolidinyl, 1,3-oxathiolanil, indolinyl, isoindolinyl, tetrahydr Lofuran, tetrahydropyranil, tetrahydroiophenyl, tetrahydrothiopyranil Lu, tetrahydro-1,4-thiadinyl, thiamorpholinyl, dihydrobenzofuranyl, Examples include, but are not limited to, benzimidazolidinyl and tetrahydroquinoline. I can't.

[0039] The "O-carboxyl" group is defined as a C1-C6 alkyl group where R is hydrogen, as defined herein. , C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclyl, C6-C 10 Selected from aryls, 5-10 member heteroaryls, and 3-10 member heterocyclines. It refers to the "-OC(=O)R" group.

[0040] A "C-carboxyl" group is defined as a C1-C6 alkyl group where R is hydrogen, as defined herein. , C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclyl, C6-C 10 A group consisting of aryls, 5-10 membered heteroaryls, and 3-10 membered heterocyclines. It refers to the "C(=O)OR" group selected from among. Non-restrictive examples include carboxyl (that is, It contains -C(=O)OH).

[0041] A "sulfonyl" group is defined as having R as hydrogen, a C1-C6 alkyl group as defined herein, or C 2-C6 alkenyl, C2-C6 alkinyl, C3-C7 carbocyclyl, C6-C 10 Selected from aryls, 5-10 membered heteroaryls, and 3-10 membered heterocyclines. This refers to the "-SO2R" group.

[0042] The "sulfino" group refers to the "-S(=O)OH" group.

[0043] The "S-sulfonamide" group is R A and R B However, each is independently hydrogen, as specified herein. This refers to C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3- C7 Carbocyclyl, C6~C 10 Aryl, 5-10 member heteroaryl, and 3-1 Selected from 0-membered heterocyclines, "-SO2NR A R B It refers to the base.

[0044] The "N-sulfonamide" group is R A and R b However, each is independently hydrogen, as specified herein. This refers to C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3- C7 Carbocyclyl, C6~C10 Aryl, 5-10 member heteroaryl, and 3-1 Selected from 0-membered heterocyclines, "-N(R A )SO2R B It refers to the base.

[0045] The "C-amide" group is R A and R B However, each is independently hydrogen, as defined herein. , C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 calcium Bosikrill, C6~C 10 Aryl, 5-10 member heteroaryl, and 3-10 member heteroaryl Selected from rosicrill, "-C(=O)" N R A R B It refers to the base.

[0046] The "N-amide" group is R A and R B However, each is independently hydrogen, as defined herein. , C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 calcium Bosikrill, C6~C 10 Aryl, 5-10 member heteroaryl, and 3-10 member heteroaryl Selected from Rosikrill, "-N(R A )C(=O)R B It refers to the base.

[0047] The "amino" group is R A and R B However, each is independently hydrogen, as defined herein, C 1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbosyl Krill, C6~C 10 Aryl, 5-10 member heteroaryl, and 3-10 member heterozygous Selected from Krill, "-NR A R B It refers to the group. Non-restrictive examples include free amino(s In other words, it contains -NH2.

[0048] An "aminoalkyl" group refers to an amino group connected via an alkylene group.

[0049] An "alkoxyalkyl" group is a group such as "C2-C8 alkoxyalkyl". This refers to an alkoxy group bonded via a chilen group.

[0050] When used herein, substituents are defined as those in which one or more hydrogen atoms are replaced by another atom or group. It derives from a non-substituted parent base. Unless otherwise specified, the base is considered "substituted" in the following cases. The group is C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C 1-C6 heteroalkyl, C3-C7 carbocyclyl (optionally halo, C1-C6 alkyl) , C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy Substitution), C3-C7-carbocyryl-C1-C6-alkyl (optional halo, C1-C6 Alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloal (substituted with coxy), 3-10 member heterocyclyl-C1-C6 alkyl (optionally halo, C1 -C6alkyl, C1-C6alkyl, C1-C6 haloalkyl, and C1-C6 ha (substituted with alkoxy), aryl (optionally halo, C1-C6 alkyl, C1-C6 alcohol) Xyl(substituted with C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl( C1-C6) alkyl (optionally substituted with a halo, C1-C6 alkyl, C1-C6 alkoxy) (C1-C6 haloalkyl and C1-C6 haloalkoxy), 5-10 member heteroalkyl Al (optional: halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl) (substituted with C1-C6 haloalkoxy), 5-10 member heteroaryl (C1-C6 )alkyl (optionally halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 halo (substituted with alkyl and C1-C6 haloalkoxy), halo, -CN, hydroxy, C1 -C6 alkoxy, C1-C6 alkoxy (C1-C6) alkyl (i.e., ether ), aryloxy, sulfhydryl (mercapto), halo(C1-C6)alkyl (example) (-CF3), Halo(C1-C6)alkoxy (e.g., -OCF3), C1-C6 alkyl Thio, arylthio, amino, amino(C1-C6)alkyl, nitro, O-carbamyl N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amide, N-amide S-sulfonamide, N-sulfonamide, C-carboxy, O-carboxy, acyl , cyanato, isocyanato, thiocyanato, isothiocyanato, sulfinyl, sulfon From -SO3H sulfino, -OSO2C1-4 alkyl, and oxo (=O), This means that the group is substituted with one or more substituents that are selected arbitrarily. Whenever it is stated that "the group has been replaced," that group may be replaced with the substituents described above. ru.

[0051] As used herein, the term "hydroxy" refers to the -OH group.

[0052] As used herein, the term "cyano" group refers to the "CN" group.

[0053] As used herein, the term "azide" refers to the -N3 group.

[0054] As used herein, "nucleotide" refers to nitrogen-containing heterocyclic bases, sugars, and nucleotides of 1 or more. The phosphate group shown above is included. These are monomeric units of nucleic acid sequences. In RNA, sugar is ribo It is a type of DNA that does not have the hydroxyl group present in deoxyribose, that is, ribose. It is a sugar. Nitrogen-containing heterocyclic bases can be purine or pyrimidine bases. Purine bases It contains adenine (A) and guanine (G), and their modified derivatives or analogues. It contains. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U). , and their modified derivatives or analogues. The C-1 atom of deoxyribose is It binds to N-1 of the pyrimidine or N-9 of the purine.

[0055] The term "nucleoside" as used herein is structurally similar to a nucleotide, The phosphate group is missing. An example of a nucleoside analog is when the label is attached to the base and the phosphate group is missing. It is not bound to a sugar molecule. In this specification, the term "nucleoside" is used in this context. It is used in the usual sense understood by professionals. An example is ribonucleosycetes containing the ribose portion. It contains deoxyribonucleosides that include the d and deoxyribose moieties, but these Not limited. Modified pentose moieties are those in which an oxygen atom is replaced by a carbon atom and / or The nucleo is a pentose moiety in which carbon is replaced by a sulfur or oxygen atom. A "side" is a monomer that may have a substituted base and / or sugar moiety. Furthermore, nucleosides can be converted into larger DNA and / or RNA polymers and oligosaccharides. It can be incorporated into...

[0056] In this specification, the term "purine base" is used in the ordinary sense as understood by those skilled in the art. and its tautomers. Similarly, the term "pyrimidine base" is understood by those skilled in the art. The term is used herein in the usual sense and includes its tautomers. It may also be substituted. A non-restrictive list of phosphorus bases includes purines, adenines, guanines, hypoxanthines, and xanthines. Alloxanthin, 7-alkylguanine (e.g., 7-methylguanine), Theo It contains bromine, caffeine, uric acid, and isoguanine. Examples of pyrimidine bases include: Cytosine, thymine, uracil, 5,6-dihydrouracil and 5-alkylcytosine ( For example, this includes, but is not limited to, 5-methylcytosine.

[0057] When used herein, oligonucleotides or polynucleotides are used herein. Where it is stated that a nucleoside or nucleotide described herein "contains", it is stated that it "contains" the nucleoside or nucleotide described herein. The nucleoside or nucleotide is coupled with an oligonucleotide or polynucleotide. This means forming a bond. Similarly, a nucleoside or nucleotide can form an oligo nucleotides or polynucleotides are "incorporated" into oligonucleotides or When described as part of a polynucleotide, the nucleoside or nucleoside as described herein. Cleotides form covalent bonds with oligonucleotides or polynucleotides. This means that in some such embodiments, the covalent bond is an oligonucleotide. Alternatively, the 3' hydroxyl group of a polynucleotide and an oligonucleotide or polynucleotide This is a phosphodiester bond between the 3' carbon atom of the nucleotide and the 5' carbon atom of the nucleotide. It is formed between the 5' phosphate group of the nucleotide described in the specification.

[0058] As used herein, the terms "derivative" or "analog" refer to synthetic nucleotide or nucleoside derivatives having a modified base moiety and / or a modified sugar moiety. Such derivatives and analogs are discussed, for example, in Scheit, Nucleotide A nalogs (John Wiley & Son, 1980) and Uhlm an et al., Chemical Reviews 90:543-584, 1990. Phosphorodithioate, alkyl phosphonate, phosphor anilidate and phosphoramidate linkages. As used herein, the terms "derivative", "analog s" and "modified" are used interchangeably and are encompassed by the terms "nucleotide " and "nucleoside" as defined herein.

[0059] As used herein, the term "phosphate" is used in its ordinary sense as understood by those skilled in the art and includes its protonated form (e.g., ). As used herein, the terms "monophosphate", "diphosphate", and "triphosphate"

Chemical Structure

[0060] that is added to a molecule to prevent an existing group within the molecule from undergoing an unwanted chemical reaction. The terms "protecting group" and "protecting group" may be used interchangeably. As used herein, the prefix "photo" or "photo-" refers to light or electromagnetic

[0061] radiation It refers to radiation. This term includes radio waves, microwaves, infrared rays, visible and ultraviolet rays. One or more ranges commonly known as X-rays, or the gamma-ray portion of the spectrum. This may include all or part of the electromagnetic spectrum, including but not limited to the range. A portion of the spectrum is blocked by metallic areas on the surface, such as the metals described herein. It may be that. Alternatively or additionally, part of the spectrum is glass, plastic Passing through gaps in the surface, such as areas made of silica or other materials described herein. It may be such. In certain embodiments, radiation that can pass through metal can be used. Alternatively or additionally, glass, plastic, silica, or other materials as specified herein. Radiation masked by materials can be used.

[0062] As used herein, the term "fading" refers to the 3' Terminator and Incomplete removal of fluorophores, and polymers in a given sequencing cycle. This is caused by the failure of the enzyme to complete the incorporation of a portion of the DNA strand within the cluster. This refers to the SBS phenomenon. Prephasing is a nucleo without an effective 3' terminator. This is caused by the ingestion of cytoplasm. The ingestion event is triggered by a failure to complete, resulting in 1 cycle. Proceed to the next step. Fading and pre-fading allow the signal measured in a specific cycle to be transmitted. The signal strength consists of the signal from the current cycle and the noise from the preceding and succeeding cycles. As the number of clusters increases, the clusters affected by fading and prefading are affected. The proportion of the sequence increases, hindering the identification of the correct base. Prephasing occurs due to synthesis. During sequencing (SBS), unprotected or unblocked 3'- This can be caused by the presence of trace amounts of OH nucleotides. Protected The 3'-OH nucleotides that are not present may be present during the manufacturing process, or in some cases during storage and testing. It can be generated during the drug processing process. Therefore, the incidence of prephasing is The discovery of a nucleotide analog that reduces the effect is surprising, and compared to existing nucleotide analogs... It offers greater advantages in SBS applications. For example, the nucleotides provided Similar to a faster SBS cycle time, lower fading and pre-fading values, the same applies to a faster SBS cycle time, lower fading and pre-fading values. This can result in longer sequence read lengths.

[0063] (3'-Hydroxyacetal blocking group) Some embodiments of this disclosure have a structure covalently bonded to a 3'-carbon atom. [ka] Ribose or deoxyribo having a removable 3'-OH protecting or blocking group that forms a ribose or deoxyribo A nucleotide or nucleoside molecule containing -, in the formula: Each R 1a and R 1b These are independently H, C1-C6 alkyl, and C1-C6 haloalkyl C1-C6 alkoxy, C1-C6 haloalkoxy, cyano, halogen, optionally substituted. A phenyl compound, or optionally a substituted aralkyl compound; Each R 2a and R 2b These are independently H, C1-C6 alkyl, and C1-C6 haloalkyl. cyano or halogen; Alternatively, R 1a and R 2atogether with the atoms to which they are attached, optionally form a substituted 5- to 8-membered heterocyclyl group; R 3 is H, optionally substituted C2-C6 alkenyl, optionally substituted C3-C7 chloroalkenyl, optionally substituted C2-C6 alkynyl, or optionally substituted (C 1-C6 alkylene)Si(R 4 )3; and each R 4 is independently H, C1-C6 alkyl, or optionally substituted C6-C 10 ar yl; provided that when each R 1a , R 1b , R 2a , and R 2b are H, R 3 is not H , relates to a nucleotide or nucleoside molecule.

[0064] Some further embodiments of the disclosure are of the structure of formula (I):

Chemical formula

Chemical formula

Chemical formula

[0065] In some embodiments of the acetal blocking groups described herein, R 1a and R 1b at least one of is H. In some such embodiments, each R 1a and R 1b is H. In some other embodiments, R 1a and R 1b at least 1 is C1-C6 alkyl, e.g., methyl, ethyl, isopropyl or t-butyl is. In some embodiments, R 2a and R 2b each is independently H, halogen or C1-C6 alkyl. In some such embodiments, R 2a and R 2b at least one of is H or C1-C6 alkyl. In some such embodiments, each R 2a and R 2b is H. In some such embodiments each R 2a and R 2b is C1-C6 alkyl, e.g., methyl, ethyl, isopropyl or t-butyl. In one embodiment, each R 2a and R 2b is methyl [[ID=6S]]is. In some such embodiments, each R 2a and R 2b is independently It is a C1-C6 alkyl or halogen. In some such embodiments, R 2a H is R 2b These are halogens or C1-C6 alkyl groups.

[0066] In some embodiments of the acetal blocking group described herein, R 3 teeth, These are C2-C6 alkenyls that are substituted as needed. In some such embodiments... In R 3 This includes halogens, C1-C6 alkyls, C1-C6 haloalkyls, and so Optionally substituted with one or more substituents independently selected from the group consisting of these combinations These are C2-C6 alkenyls (e.g., vinyl, propenyl). Several further implementations In terms of form, R 3 teeth [ka] In some other embodiments, R 3 C2-C6 were replaced as needed. It is an alkynyl. In some such embodiments, R 3 Halogen, C1~ Independent of the group consisting of C6 alkyl, C1-C6 haloalkyl, and combinations thereof. C2-C6 alkynyl (e.g., e) optionally substituted with one or more substituents selected by the above. (Tynyl, propynyl) In one embodiment, R 3 Ethini, which is replaced as needed. Ru ( [ka] ) is. In some other embodiments, R 3 (C1~C6) are optionally replaced. Lukilen)Si(R 4)3. In some such embodiments, R 4 few At least one is C 1-4 It is alkyl. In some further embodiments, R 4 noso Each is a C1-C4 alkyl group, for example, methyl, ethyl, isopropyl, or t-butyl. In one embodiment, R 3 It is -(CH2)-SiMe3. Several alternatives In this embodiment, R 3 These are C1-C6 alkyl groups.

[0067] In some alternative embodiments, R 1a and R 2a The origin of how they are joined together Together with the offspring, it forms a heterocycline with 5 to 7 members. In some such embodiments... And, R 1a and R 2a These, along with the atoms to which they are bonded, form a six-membered heterocycline. It forms a structure. In some such embodiments, the 6-membered heterocyclyl group forms a structure [ka] It has. In some further embodiments, each R 1b , R 2b and R 3 at least One is H. In some other embodiments, each R 1b , R 2b and R 3 at least Another is C1-C6 alkyl. In one embodiment, each R 1b , R 2b and R 3 is H That is the case.

[0068] In some further embodiments, the compound of formula (I) is also by formula (Ia). Represented: [ka] In the formula, each R 2c and R 2d These are independently H and halogens (e.g., fluoro, chloro). , C1-C6 alkyl (e.g., methyl, ethyl, or isopropyl), or C1- It is a C6 haloalkyl group (e.g., -CHF2, -CH2F, or -CF3). How many? In such an embodiment, R 1a and R 1b One of them is H. In the eel method, each R 1a and R 1b In some other embodiments, R 1a and R 1b At least one of them is a C1-C6 alkyl group, e.g., methyl, ethyl, iso It is propyl or t-butyl. In some embodiments, R 2a and R 2b That These are independently H, halogens, or C1-C6 alkyl groups. In the eel method, each R 2a and R 2b H is one such embodiment. So, R 2c and R 2d Each of these is independently H, halogen, or C1-C6 aluminum It is a kill. In some such embodiments, each R 2c and R 2d C1~ C6 alkyl, for example, methyl, ethyl, isopropyl, or t-butyl. In terms of morphology, each R 2c and R 2d is methyl. In some such embodiments, Each R 2c and R 2dThese are halogens independently. In some such embodiments, R 2c H is R 2d H, halogens (fluoro, chloro) or C1-C6 alkyl These are (e.g., methyl, ethyl, isopropyl, or t-butyl). Further implementations In this state, each R 1a and R 1b H is; R 2a H is; R 2b H, Haroge It is methyl or R 2c H is; R 2d H, halogen, methyl, ethyl, It is isopropyl or t-butyl.

[0069] Non-limiting embodiments of the blocking group described herein are selected from the group consisting of the following: Includes those having a structure that: [ka] It is covalently bonded to the 3' carbon of ribose or deoxyribose.

[0070] (3'-hydroxythiocarbamate blocking group) Some additional embodiments of this disclosure involve a structure covalently bonded to a 3'-carbon atom. [ka] This includes ribose or deoxyribose having a removable 3'-OH blocking group that forms a [component]. A nucleoside or nucleotide, in which the formula: R 5 and R 6 Each of these is independently H, C1-C6 alkyl, and C2-C6 alkeni C2-C6 alkynyl, C1-C6 haloalkyl, C2-C8 alkoxyalkyl, Optional substitution - (CH2) m-Phenyl, may be substituted-(CH2) n -(5 also (6-membered heteroaryl), may be substituted -(CH2) k -C3-C7 Karbosik Lil, or possibly substituted - (CH2) p -(3-7 member heterocyclyl) ; Alternatively, R 5 and R 6 Even if they are substituted along with the atoms to which they are bonded Forms good 5-7 member heterocyclines; -(CH2) m -,-(CH2) n -,-(CH2) k -, and -(CH2) p -of Each of them may be substituted; and Each of m, n, k, and p is independently 0, 1, 2, 3, or 4. Regarding nucleosides or nucleotides.

[0071] Some additional embodiments relate to compounds of formula (II): [ka] In the formula, R' is H, monophosphate, diphosphate, triphosphate, thiophosphate, phosphate ester analogs. -O- bonded to a reactive phosphorus-containing group, or -O- protected by a protecting group; R '' is H or OH; B is a nucleic acid base; R 5 and R 6 Each of them is defined above In some further embodiments, B is [ka] In some further embodiments, nucleic acid bases are optionally linked via a linker. Then, it is covalently bound to a detectable label (e.g., a fluorescent dye), for example, B is [ka] In some such embodiments, R' is triphosphate. In such embodiments, R'' is H.

[0072] In some embodiments of the thiocarbamate blocking group described herein, R 5 oh Call R 6 At least one of them is H. In some such embodiments, each R 5 Oh biR 6 is H. In some such embodiments, R 5 H is R 6 is C1~ C6 alkyl groups, such as methyl, ethyl, isopropyl, or t-butyl. In some such embodiments, R 5 H is R 6 C2~C6 alkenyl ( For example, vinyl or allyl) or C2-C6 alkynyl (for example, ethynyl or p) In some such embodiments, R 5 H is R 2 teeth, Optionally substituted -(CH2) m -phenyl, optionally substituted-(CH2) n -(5 (or 6-membered heteroaryl), optionally substituted -(CH2) k -C3~C7 Carbocycline Lu, or replace as needed - (CH2) p -(3-7 member heterocyclyl). Several In a further embodiment, the C3-C7 carbocykyl group is a C3-C7 cycloalkyl group. It may be a C3-C7 cycloalkenyl. The 3- to 7-membered heterocyclyl group is ring The structure may contain zero or one double bond. In further embodiments, R 5 It is H R 6 is an optionally substituted -(CH2) m -phenyl, optionally substituted-(CH2 ) n -6-membered heteroaryl, optionally substituted-(CH2) k -C5 or C6 carboshi krill, or optionally substituted -(CH2) p -(5 or 6-membered heterocyclyl). In some embodiments, m, n, k, or p are 0. In other embodiments, m, n , k or p is 1 or 2. In some other embodiments, R 5 and R 6 few At least one of them is a C1-C6 alkyl group. For example, methyl, ethyl, isopropyl, or is t-butyl. In some further embodiments, R 5 and R 6 Both are C1~ It is a C6 alkyl group. In one embodiment, R 5 and R 6 Both are methyl.

[0073] In some alternative embodiments, R 5 and R 6 The atoms to which they are bonded and Together, they form 5 to 7-membered heterocyclines with optional substitutions. In one embodiment, R 5 and R 6 They, along with the atoms to which they are bonded, as needed It forms a substituted piperidinyl.

[0074] Non-limiting embodiments of the 3'-O-thiocarbamate blocking group described herein are , including those having a structure selected from the following group: [ka] It is covalently bonded to the 3' carbon of ribose or deoxyribose.

[0075] Additional embodiments of this disclosure include nucleosides or nucleotides as described herein. This relates to oligonucleotides or polynucleotides.

[0076] In any of the embodiments of the blocking group described herein, the group is optionally substituted When it is written as "reta," it can be either a non-substitution or a substitution.

[0077] The nucleotides or nucleosolecules having a 3' hydroxy-blocking group described herein. In any embodiment of the side, the nucleoside or nucleotide may be a linker. —can be covalently bonded to a detectable label (e.g., a fluorophore) via —. The linker is , may be severable or inseverable. In some such embodiments, detection Possible labels (e.g., fluorophores) can be cleaved through linkers to the nucleoside. Or it is covalently bonded to the nucleic acid base of a nucleotide. In some other embodiments, detectable A cleavable label (e.g., a fluorophore) can be used to cleave nucleosides via a linker. It is covalently bonded to the 3' oxygen of the nucleotide. In some further embodiments, Such a severable linker consists of an azide portion or a disulfide portion, and an acetal portion. , or may include a thiocarbamate moiety. In some embodiments, 3' hydroxy The blocking groups and cleavable linkers (and attached labels) are the same or substantially the same. They can be removed under the same chemical reaction conditions, for example, blocking groups and detectable labels. , can be removed in a single chemical reaction. In other embodiments, blocking groups and detection Any possible signs are removed in two separate steps.

[0078] In some embodiments, the nucleotides or nucleosides described herein It contains 2'-deoxyribose. In some further aspects, 2'-deoxyribose The sugar ring contains one, two, or three phosphate groups at the 5' position. In one embodiment, the nucleotide described herein is a nucleotide triphosphate.

[0079] In some embodiments, the 3' block nucleotide or nucleotide described herein The rheoside is protected by the same standard 3'-OH blocking group as disclosed in the prior art. Compared to creotides or nucleosides, the solution or sequencing applicator during storage It provides excellent stability in handling reagents during use. For example, 3'-O-azide Methyl protecting group. For example, acetal or thiocarbamate blots disclosed herein. The King group is simultaneously protected with azidomethyl under the same conditions for the same period of time and 3'-OH Compared to at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, and 70%. 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 7 00%, 800%, 900%, 1000%, 1500%, 2000%, 2500%, and This can provide 3000% improved stability, thereby reducing the prephase value and sequence The reading length of the signal becomes longer. In some embodiments, stability is determined by ambient temperature or ambient temperature. It is measured at a temperature lower than the temperature (e.g., 4-10°C). In other embodiments, stability is Measurements are taken at high temperatures such as 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C. In such embodiments, stability is maintained in a basic pH environment, for example, pH 9.0, 9.2. It is measured in solutions of 9.4, 9.6, 9.8, or 10.0. Some such actual In terms of application form, stability is determined by polymerase (e.g., DNA polymerase) and terminal deoxygenation. Regardless of the presence or absence of enzymes such as synucleotidyltransferase or reverse transcriptase It is measured without being measured.

[0080] In some embodiments, the 3' block nucleotide or nucleotide described herein The rheoside is protected by the same nucleus as the standard 3'-OH blocking group disclosed in the prior art. Chemical cleavage for sequencing applications, compared to rheosides or nucleosides. It provides excellent deblocking rates in solution during the step. For example, 3'-O-azide Methyl protecting group. For example, acetal or thiocarbamate blots disclosed herein. The King group can be deblocked using a standard deblocking reagent (such as tris(hydroxypropyl)phosphine). Compared to the azidomethyl-protected 3'-OH used, at least 5%, 10%, 20%, 30% %, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000 It may be possible to provide a deblocking rate that is improved by %, 1500%, or 2000%, thereby Reduce the overall duration of the densification cycle. In some embodiments, the deblockization rate It is measured at ambient temperature or a temperature lower than ambient temperature (e.g., 4-10°C). In terms of application method, the deblockization rate is 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C. It is measured at high temperatures such as °C. In some such embodiments, the deblockization rate is measured by salt Basic pH environment, e.g., pH 9.0, 9.2, 9.4, 9.6, 9.8 or 10.0 It is measured in solution. In some such embodiments, a deblocking reagent and a substrate (sand The molar ratio of 3'-block nucleosides (or nucleotides) is approximately 10:1, and approximately 5: 1. Approximately 2:1, or approximately 1:1.

[0081] In some embodiments, a palladium deblocking reagent (e.g., Pd(0)) is used. , remove the 3'-acetal blocking group (e.g., AOM blocking group). Pd is Both oxygen atoms of the AOM group can form chelate complexes. This allows for the removal of deblocking reagents located very close to the functional groups, thereby increasing the deblocking rate. It can be made faster.

[0082] (Deprotection of 3'-OH blocking group) The 3'-acetal blocking groups described herein can be removed under various chemical conditions. It can be cut. Acetal blocking group containing vinyl or alkenyl moiety [ka] In this case, non-restrictive cleavage conditions include phosphine ligands, such as tris(hydroxymethyl Pd(OAc)2 or allyl Pd(II) chloride dimers, etc., in the presence of Pd( II) Contains complexes. Phosphine (THMP) or tris(hydroxypropyl )Phosphine (THP or THPP). It contains an alkynyl group (e.g., ethynyl). These blocking groups are also phosphine ligands (e.g., THP) In the presence of THMP, Pd(II) complexes (e.g., Pd(OAc)2 or allyl) It can be removed by Pd(II) chloride dimer.

[0083] (Palladium cutting reagent) In some embodiments, the acetal blocking group described herein is palladium It can be cleaved by a catalyst. In some such embodiments, the Pd catalyst is water-soluble. In some such embodiments, Pd(0) complexes (e.g., Tris(3,3)) are used. ',3''-Phosphine dinetris (benzenesulfonate)palladium (0) non-sodium It is a nonhydrated salt. In some cases, the Pd(0) complex is an alkene, alcohol, or From the reduction of Pd(II) complexes with reagents such as mine, phosphine, and metal hydrides, in situ production Suitable palladium sources include Na2PdCl4, Pd(CH3CN)2Cl2, (PdCl(C3H5))2, [Pd(C3H5)(THP)]Cl, [Pd(C3H5) )(THP)2]Cl, Pd(OAc)2, Pd(Ph3)4, Pd(dba)2, Pd (Acac)2, PdCl2(COD), in one such embodiment, the Pd(0) complex is , generated in situ from Na2PdCl4. In another embodiment, the palladium source is ant It is a lupalladium(II) chloride dimer [(PdCl(C3H5))2]. How many? In that embodiment, the Pd(0) complex is mixed with the Pd(II) complex with phosphine. Therefore, it is produced in aqueous solution. Suitable phosphines include tris(hydroxypropyl) phosphate. Sphin (THP), Tris (hydroxymethyl), Phosphine (THMP), 1,3, 5-Triaza-7-phosphaadamantane (PTA), bis(p-sulfonatophenyl) Phenylphosphine dihydrate potassium salt, tris(carboxyethyl)phosphine (TC EP), and triphenylphosphine-3,3',3'-trisulfonate trisodium. It contains water-soluble phosphines such as salts.

[0084] In some embodiments, Pd(0) is a Pd(II) complex [(PdCl(C3H5) )2] is prepared by mixing it in situ with THP. Pd(II) complex and THP The molar ratios are approximately 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and Or it may be 1:10. In some further embodiments, ascorbin Add one or more reducing agents, such as an acid or its salt (e.g., sodium ascorbate). This is possible. In some embodiments, the cleavage mixture is a primary amine, a secondary amine. , tertiary amines, carbonates, phosphates, or borates, or combinations thereof, etc. It may include additional buffering agents. In some further embodiments, the buffering agent is ethanol Alamine (EA), Tris(hydroxymethyl)aminomethane (Tris), Glycine, Sodium carbonate, sodium phosphate, sodium borate, 2-dimethylaminomethanol (DMEA), 2-diethylaminomethanol (DEEA), N,N,N',N'-Tet Methylethylenediamine (TEMED), or N,N,N',N'-tetraethyl Includes ethylenediamine (TEEDA) or a combination thereof. In one embodiment, buffered The drug is DEEA. In another embodiment, the buffering agent is a carbonate, phosphate, or boric acid. It contains one or more inorganic salts, such as salts or combinations thereof. In one embodiment, inorganic Salt is a sodium salt.

[0085] Alternatively, the alkynyl moiety containing the blocking group is present in the presence of (NH4)2MoS4 It can also be cleaved. Other non-limiting cleavage conditions for the alkynyl moiety include the THPTA ligand ( Cu(II) complex with ris(3-hydroxypropyltriazolylmethyl)amine, It contains ascorbates. It contains a six-membered heterocyclic compound (e.g., tetrahydropyran). Non-restrictive cleavage conditions for locking groups include cyclodextrin or Ln(OTf)3( It contains lanthanide triflate. Blocking groups containing alkylsilane groups (for example) Non-limiting cleavage conditions for -CH2SiMe3 include LiBF4 (lithium tetrafluoroborate). It contains other acetal blocks such as -O(CH2)O-C1~C6 alkyl. The lig group can be removed by LiBF4 or Bi(OTf)3 (bismuth triflate). The following are non-limiting, exemplary conditions for cleaving the various blocking groups described. This is shown in Scheme 1. Scheme 1.3' - Example of deblocking conditions [ka]

[0086] The 3'-O-thiocarbamate blocking group described herein is available under various chemical conditions. The thiocarbamate blocking groups described herein can be removed or cut off. Non-limiting exemplary conditions for cutting include NaIO4 and Oxone (registered trademark). It contains potassium peroxymonosulfate.

[0087] Furthermore, the azide group of -CH2N3 can be converted to an amino group by phosphine. The azide group of -CH2N3 makes such molecules thiols, especially dithiothreitols. It can be converted to an amino group by contact with a water-soluble thiol such as DTT. In one embodiment, the phosphine is THP.

[0088] (Compatibility with linearization) To maximize the throughput of nucleic acid sequencing reactions, multiple template molecules The advantage is that sequencing can be performed in parallel. Parallel processing of multiple templates is advantageous. This can be achieved using nucleic acid array technology. These arrays are typically fixed on a solid support material. It consists of a high-density matrix of ionized polynucleotides.

[0089] Both WO98 / 44151 and WO00 / 18957 are multiple identical fixed points Clusters formed from renucleotide chains and multiple identical immobilized polynucleotide chains. - Or, to form an array consisting of "colonies", the amplification product is immobilized on a solid support. This document describes a nucleic acid amplification method that enables the amplification of multiple identical immobilized complementary strands. This type of array is referred to herein as a "clustered array." Nucleic acid molecules present in DNA colonies on a clustered array prepared according to the method are, for example, For example, as described in WO98 / 44152, a tensile We can provide plates. WO98 / 44151 and WO00 / 18957 The products of the solid-phase amplification reactions described are immobilized polynucleotide chains and immobilized complements. This is a so-called "bridged" structure formed by the annealing of a pair of chains, where both chains are 5' Strong edge support. Provides a more suitable template for nucleic acid sequencing. To do this, in order to generate a template that is at least partially single-stranded, "bridge" It is preferable to remove substantially all or at least part of one of the immobilizing chains in the structure. Therefore, the single-stranded template portion is transferred to the sequencing primer. It can be used for hybridization. "Cross-linking" immobilization of one strand within a double-stranded nucleic acid structure. The process of removing all or part of a chain is called "linearization." Linearization involves enzymatic processes. There are various methods, including but not limited to cleavage, photochemical cleavage, or chemical cleavage. A non-limiting example of the linearization method is PCT publication number WO2007 / 010251, U.S. Patent. Publication number 2009 / 0088327, US Patent Publication number 2009 / 0118128, These are disclosed in U.S. Patent Application No. 62 / 671,816, and are incorporated by reference throughout. Born.

[0090] In some embodiments, for deprotection or removal of the 3'-OH blocking group The conditions are also compatible with the linearization process. In some further embodiments, The protective conditions involve the use of Pd complexes and phosphines, such as Pd(OAc)2 and THP. It is compatible with chemical linearization processes that include. In some embodiments, the Pd complex is This is a Pd(II) complex that generates Pd(0) in situ in the presence of phosphine.

[0091] Unless otherwise specified, references to nucleotides also apply to nucleosides. That is the intention.

[0092] (labeled nucleotides) According to one aspect of this disclosure, the described 3'-OH blocked nucleotides are also detected Including possible labels, such nucleotides are called labeled nucleotides. For example, fluorescent dyes are attracted by various means including hydrophobic attraction, ionic attraction, and covalent bonding. They can be bonded via an optional linker. In some aspects, dyes It is conjugated to the substrate by covalent bonds. More specifically, covalent bonds are linked by linkers - It depends on the group. In some cases, such labeled nucleotides are also called "modified nucleotides". I was called.

[0093] Labeled nucleotides are used in PCR amplification, isothermal amplification, solid-phase amplification, and polynucleation, as non-limiting examples. Cleotide sequencing (e.g., solid-phase sequencing), nick translation It is useful for labeling polynucleotides formed by enzymatic synthesis in reactions, etc. be.

[0094] In some embodiments, the dye is an oligonucleotide via a nucleotide base. Alternatively, they can be covalently bonded to nucleotides. For example, labeled nucleotides or oligonucleotides. Otide is located at the C5 position of the pyrimidine base or 7-deazaprine salt via the linker moiety. It may have a label attached to the base at position C7.

[0095] Unless otherwise specified, references to nucleotides are also applicable to nucleosides. This application is also described further with reference to DNA, but unless otherwise specified, the description is R It is also applicable to NA, PNA, and other nucleic acids.

[0096] (Linker) In some embodiments described herein, the nucleotides or nucleotides described herein The purine or pyrimidine base of the reoside molecule can bind to the above-mentioned detectable label. Yes, it is possible. In some such embodiments, the linker used is cuttable. Using a disconnectable linker ensures that the label can be reliably removed after detection if necessary, and then... Interference signals from the incorporated labeled nucleotide or nucleoside can be avoided. In this embodiment, the severable linker is an azide portion, -O-C2~C6 alkenyl Part (e.g., -O-allyl), disulfide part, acetal part (as described herein) Contains the same or similar 3'-acetal blocking group, or thiocarbamate. A portion (same as or similar to the 3'-acetal blocking group described herein).

[0097] In some other embodiments, the linker used is non-cuttable. In each case where a nucleotide is incorporated, there is no need to incorporate another nucleotide afterward. Therefore, there is no need to remove the label from the nucleotide.

[0098] Cuttable linkers are known in the art, and by applying conventional chemistry, linkers can be made It can be bound to nucleotide bases and labels. The linker can be an acid, base, or nucleophile. This includes exposure to electrophiles, radicals, metals, reducing or oxidizing agents, light, temperature, enzymes, etc. It can be cleaved by any suitable method. The 3'-O-blocking bond is cleaved. It is used for this purpose. Suitable linkers include Greene & Wuts, Prote active Groups in Organic Synthesis, John It can be adapted from the standard chemical protecting groups disclosed by Wiley & Sons. Yes, it is possible. A more suitable cleavable linker used in solid-phase synthesis is the Guillier et al. (Chem. Rev. 100:2092-2157, 2000) It will be disclosed.

[0099] If a detectable label is attached to the base, Watson-Crick base pairing still occurs. As long as it is feasible, the linker can attach to any position on the nucleotide base. In the context of purine bases, the linker is at position 7 of the purine or preferred deazapurine analogue. via 8-modified purines, via N-6 modified adenosine or N-2 modified guanine It is preferable that they are bound together. In the case of pyrimidines, the binding is to cytosine, thymidine, or u Preferably via the 5-position of racil and the N-4 position of cytosine.

[0100] In some embodiments, the linker may include a spacer unit. The label is nucleotide so as not to interfere with the interaction between the oside and the enzyme, such as polymerase. As long as you maintain a sufficient distance from Ochid, the length of the linker is not important.

[0101] In some embodiments, the linker consists of functional groups similar to the 3'-OH protecting group. This allows for the removal of both the label and the protecting group in a single process. The processes of protection and deprotection become more efficient.

[0102] The use of the term "cuttable linker" implies that the entire linker needs to be removed. It does not have a taste. The cut area is where part of the linker is cut, and the pigment and / or substrate part It can be positioned on the linker to ensure it remains connected for a while. Possible linkers, as non-limiting examples, include electrophilically cleavable linkers and nucleophilically cleavable linkers. Possible linker, light-cuttable linker, under reductive conditions (e.g., disulfide or a (Zide-containing linker), can be cut under oxidative conditions, via the use of a safety catch linker. It may be cut by cutting, and may also be cut by an exclusion mechanism. By using a linker as needed to bind the dye compound to the substrate portion, This allows the label to be removed after detection, and the interference signal in the downstream step can be reversed. It can be avoided.

[0103] Useful linker groups are found in PCT Publication No. 2004 / 018493 (see reference). Examples of such may be found in (and incorporated herein), including transition metals and at least Using a water-soluble phosphine or water-soluble transition metal catalyst formed from a partially water-soluble ligand A linker that can be cut using a linker is an example. In aqueous solution, the latter is at least partially aqueous soluble. It forms transition metal complexes, such as Pd(II) complexes and THP. Using a possible linker, the bases of the nucleotides are labeled with dyes as described herein. It can connect to

[0104] Special linkers include those that include the following part of the formula, such as the PCT Public Number International Public What is disclosed in Patent Publication No. 2004 / 018493 (which is incorporated herein by reference) Includes: [ka] (In the formula, X is selected from groups including O, S, NH and NQ, and Q is C 1-10 Replace Y is an unsubstituted alkyl group, and Y is selected from the group including O, S, NH, and N (allyl). Therefore, T is hydrogen or C1-C 10 A substituted or unsubstituted alkyl group, and * is a part (This indicates where the part is attached to the rest of the nucleotide or nucleoside.) In some embodiments, the linker converts the bases of the nucleotides, for example, to the dyeing described herein. Connects to signs such as "combined objects".

[0105] An additional example of a linker is one that includes the following part of the formula, U.S. Publication No. 20 Includes the information disclosed in 16 / 0040225 (which is incorporated herein by reference). ru: [ka] The linker portion shown herein is a linker between the nucleotide / nucleoside and the label. - May include the entire structure or parts thereof.

[0106] An example of adding a linker ("L") includes part of the formula: [ka] Here, B is a nucleic acid base; Z is -N3 (azide), -O-C1~C6 alkyl, -O -C2~C6 alkenyl, or -O-C2~C6 alkinyl; F1 is an additional linker Includes a fluorescent label which may include a structure. Those skilled in the art can use the functional group (e.g., carboxyl) of the label. By reacting with the functional group of the linker (e.g., amino), the label is covalently bonded to the linker. I understand that they are in agreement.

[0107] In a special embodiment, the linker between the fluorescent dye (fluorophore) and the guanine base The length can be changed, for example, by introducing polyethylene glycol spacer groups. This allows for the binding of guanine bases to other linkages known in the art. It increases fluorescence intensity compared to the same fluorophore. Exemplary linker and it These characteristics are described herein by reference in PCT Publication No. 2007020457 (PCT Publication No. 2007020457). (As shown in the diagram) Linker design, especially the increase in their length, allows for the integration of DNA etc. When incorporated into a polynucleotide, it binds to the guanine base of the guanosine nucleotide. The brightness of the fluorophore can be improved. Therefore, the dye contains guanine. When used in analytical methods that require the detection of fluorescent dye labels bound to nucleotides, phosphorus Carr, as described in International Publication No. 2007 / 020457, formula -((CH2) 20) n - A spacer base including one in which n is an integer between 2 and 50 in the formula , it is advantageous.

[0108] Nucleosides and nucleotides can be labeled with a site on a sugar or nucleic acid base. As is well known in the field, a "nucleotide" consists of a nitrogenous base, a sugar, and one or more phosphate groups. Yes. In RNA, the sugar is ribose, and in DNA, it is deoxyribose, i.e., ribose. It is a sugar that lacks the hydroxyl group found in Bose. Nitrogen bases are purines or pyrimidines. It is a derivative. Purines are adenine (A) and guanine (G), and pyrimidines are These are cytosine (C) and thymine (T), or uracil (U) in the context of RNA. The C-1 atom of deoxyribose binds to the N-1 atom of a pyrimidine or the N-9 atom of a purine. Nucleotides are also phosphate esters of nucleosides, attached to the C-3 or C-5 of sugars. Esterification occurs at the bonded hydroxyl group. Nucleotides are usually mononucleotides, dinucleotides, or trinucleotides. It is acid.

[0109] Nucleosides are structurally similar to nucleotides, but they lack a phosphate group. Examples of nucleoside analogs include those in which the label is attached to a base and a phosphate group is attached to a sugar molecule. It is not something that exists.

[0110] Bases are usually called purines or pyrimidines, but those skilled in the art will call them nucleotides or nuclei. Derivatives that do not alter the ability of rheoside to undergo Watson-Crick base pairing You will understand that derivatives and analogues are available. "Derivatives" or "analogs" are... The structure is the same as or very similar to that of the parent compound, but is different, for example. Alternatively, derivative nucleotides or nucleosides, such as additional side chains, can be attached to another molecule. This refers to a compound or molecule that has chemical or physical modifications that enable it to do so. For example, the base may be a deazaprine. In a special embodiment, the derivative is Wat It should be possible to receive son-Crick pairing. "Derivatives" and "Analogs" also have, for example, modified base moieties and / or modified sugar moieties. This includes synthetic nucleotides or nucleoside derivatives. Such derivatives and analogs For example, Scheit, Nucleotide analogs (John Wile Y & Son, 1980) and Uhlman et al., Chemical Rev. This is discussed in iews 90:543-584, 1990. Nucleotide analogs are... Phosphothioates, phosphorodithioates, alkylphosphonates, phosphoranidine It may contain modified phosphodiester bonds, including dates and phosphoamide date bonds.

[0111] Dyes can be attached to any position on a nucleotide base, for example, via a linker. This is possible. In a special embodiment, Watson-Crick base pairing is obtained similar to the It can still be performed on the body. Specific nucleic acid base labeling sites include pyrim Examples include the C5 position of the din base or the C7 position of the 7-deazapurine base. A linker group can be used to covalently bond the dye to a nucleoside or nucleotide.

[0112] In a particular embodiment, the labeled nucleoside or nucleotide is enzymatically incorporated Rarely, it may be enzymatically elongable. Therefore, the linker portion is used by the nucleic acid replication enzyme. To avoid significantly interfering with the overall binding and recognition of nucleotides by nucleotids, The linker can be long enough to connect the compound. Therefore, the linker is a spacer. It may also include knitting. The spacer is, for example, a cut or marking. Release the base.

[0113] The nucleosides or nucleotides labeled with the dyes described herein have the following formula: You may: [ka] In the formula, Dye is a dye compound; B is, for example, uracil, thymine, cytosine, adenocarcinoma. Nucleic acid bases such as nin and guanine; L is an optional base that may or may not be present. It is a linker group; R' is H, monophosphate, diphosphate, triphosphate, thiophosphate, phosphate e Protected by a ster analog, an -O- bonded to a reactive phosphorus-containing group, or a blocking group. It may be -O-; R''' is H, OH, phosphoramidite, or as specified herein The 3'-OH blocking group described is R'', where R'' is H or OH. If it is a sforamidite, R' is a hydroxy protecting group that can be cleaved with acid, and autocombination This enables subsequent monomer coupling under specific conditions.

[0114] In a particular embodiment, the linker (between the dye and the nucleotide) and the blocking group are both Both exist and are separate parts. In a particular embodiment, the linker and the blocking Both groups can be cleaved under substantially similar conditions. Therefore, the dye compound and the group Since only one treatment is required to remove both locking groups, deprotection and deprotection are necessary. The blocking process may be more efficient. However, in some embodiments, The linker and blocking groups do not need to be cleavable under the same conditions, instead They can be cut individually under different conditions.

[0115] This disclosure also includes polynucleotides incorporating dye compounds. Nucleotides are deoxyribonucleotides or ribonucleotides linked by phosphodiester bonds. It may be DNA or RNA, each composed of a bonucleotide. nucleotides are naturally occurring nucleotides, and non-natural nucleotides other than the labeled nucleotides described herein. Nucleotides present (or modified) in this specification, or any combination thereof, Combined with at least one modified nucleotide (e.g., labeled with a dye compound) as described above. The polynucleotides disclosed herein may also include non-natural skeletal linkages and / or Non-nucleotide chemical modifications may be included. Ribonucleotide and at least one label Chimeric structures consisting of a mixture of deoxyribonucleotides including nucleotides are also being considered. It will be done.

[0116] Examples of non-exclusive labeled nucleotides described herein include: reru: [ka] In the formula, L represents the linker, and R represents the sugar residues mentioned above, or 1, 2, or 3 at the 5' position. This represents a sugar residue substituted with phosphate.

[0117] In some embodiments, non-limiting exemplary fluorescent dye conjugates are shown below. reru: [ka] In the formula, PG represents the 3'-hydroxy blocking group as described herein. In any embodiment of the labeled nucleotide, the nucleotide is a nucleotide triphosphate. ru.

[0118] (kit) This disclosure also relates to one or more 3' block nucleosides and / or the information described herein. Nucleotides, for example, the 3' block nucleoty of formula (I), (Ia), or (II) We provide kits that include a [product name]. Such kits generally include at least one further [product name]. Along with the minutes, at least one 3'-blocked nucleotide or nucleus labeled with a dye is detected. It will contain osides. Further components may be as described herein or in the following examples. The kit of this disclosure may be one or more components as specified in the section. Some non-restrictive examples of components that can be combined with the following are listed below.

[0119] In certain embodiments, the kit includes at least one labeled 3'-blocked nucleotide. or nucleosides, together with labeled or unlabeled nucleotides or nucleosides. This is possible. For example, dye-labeled nucleotides can be unlabeled or natural nucleotides. , and / or in combination with fluorescently labeled nucleotides or any combination thereof It can be supplied. The combination of nucleotides is a separate individual component (e.g., container or chip (One nucleotide type per tube) or a mixture of nucleotides (e.g., in the same container) It is provided as (or as two or more nucleotides mixed in a tube).

[0120] The kit contains multiple, particularly two or three, or more specifically, dye compounds labeled with a dye compound. If it contains four 3'-blocked nucleotides, different nucleotides will have different dye compounds. They may be labeled with substances, or one may be dark in color without a pigment compound. Different nucleos If cydides are labeled with different dye compounds, those dye compounds can be spectrally distinguished. The kit's distinguishing feature is its ability to produce fluorescent dyes. When used herein, "Spec" refers to the fluorescent dyes. The term "fluorescently distinguishable fluorescent dye" is used when there are two or more such dyes. In addition, a fluorescence detection device (for example, a commercially available capillary-based DNA sequencing platform) This refers to a fluorescent dye that emits fluorescence energy at wavelengths that can be identified by a specific type of pigment. It is present in the sample. Two nucleotides labeled with a fluorescent dye compound are provided in the form of a kit. When provided, spectrally distinguishable fluorescent dyes are subjected to the same laser, for example. A key feature of some embodiments is that they can be excited at the same wavelength. Labeled with a fluorescent dye compound. The four 3' block nucleotides (A, C, T, and G) are provided in kit form. In this case, both spectrally distinguishable fluorescent dyes are excited at one wavelength, and the other These two spectrally distinguishable dyes can both be excited at different wavelengths. Specific excitation The wavelengths are 488 nm and 532 nm.

[0121] In one embodiment, the kit includes a first 3' block nucleotide labeled with a first dye. and a second nucleotide labeled with a second dye, the dye having at least 10 nm , in particular, there is a difference in maximum absorbance from 20 nm to 50 nm. More specifically, two dyes The compound has a Stokes shift of 15-40 nm, where "Stokes shift" is defined as follows: This is the distance between the peak absorption wavelength and the peak emission wavelength.

[0122] In an alternative embodiment, the kit of this disclosure is one in which the same base is labeled with two or more different dyes. It may contain a 3' block nucleotide. The first nucleotide (for example, the 3' block (Cut T nucleotide triphosphate or 3'-blocked G nucleotide triphosphate) , can be labeled with a first dye. A second nucleotide (e.g., 3' block C nucleoty (Phosphoric acid) is a second dye that is spectrally different from the first dye, for example, at 600 nm It can be labeled with a "green" dye that absorbs less than 50, and a "blue" dye that absorbs less than 50. 0 nm, for example 400 nm to 500 nm, especially 450 nm to 460 nm). The third nucleo Tides (e.g., 3' block A nucleotide triphosphate) are a mixture of the first and second dyes. The substance, or the first, second and third pigments and the fourth nucleotide (e.g., 3' block G) ) may be labeled as a mixture of nucleotide triphosphates or 3' block T nucleotides. (Diphosphate) may be "dark" and may not have a label. For example, nucleo Chids 1-4 may be labeled as "blue," "green," "blue / green," and dark. To further simplify, two dyes excited by a single laser using four nucleotides. They can be labeled with "Blue 1" and "Blue 2". Therefore, the labeling of nucleotides 1-4 is done with "Blue 1" and "Blue 2". "Blue 1 / Blue 2", and Darkness.

[0123] In certain embodiments, the kit includes four labeled 3' block nucleotides (e.g., It may include A, C, T, and G nucleotides, where each type of nucleotide is the same 3' block. It includes a group and a fluorescent label, and each fluorescent label is separate. Maximum fluorescence and each fluorescent The label can be distinguished from the other three labels. The kit contains two or more fluorescent labels that have similar maximum absorbance. It could be something that has degrees but has different Stokes shifts. In the application form, one type of nucleotide is not labeled.

[0124] This specification relates to a configuration having different nucleotides labeled with different dye compounds. Although the kit is illustrated, the kit may contain the same dye compound as kits 2, 3, 4 or the same It will be understood that the above different nucleotides may be included. The kit also includes enzymes and buffers suitable for the enzyme's action. In one embodiment, the enzyme is polymerase, terminal deoxynucleotidyl transfer It is a polymerase, or reverse transcriptase. In certain embodiments, the enzyme is DNA polymerase. DNA polymerases such as 812 (Pol 812) or 1901 (Pol 1901) It is a DNA polymerase. Pol 812 and Pol 1901 polymerases The no-acid sequence is, for example, in U.S. Patent Application No. 16 / 670 filed on October 31, 2019. As described in patent no. 876 and 16 / 703,569 filed on December 4, 2019 It is incorporated herein by reference.

[0125] Other components included in such kits include buffer solutions, etc. D, and any other nucleotide components including mixtures of different nucleotides, should be used before use. The kit may be provided in a concentrated form that is diluted. In such embodiments, a suitable dilution buffer is used. Liquids may also be included. Here again, one or more of the components specified by the method described herein may be included. It can be included in the disclosure kit.

[0126] (Sequencing method) The labeled nucleotides or nucleosides in this disclosure are nucleotides or nucleosides. It can be used in any analytical method, including methods that involve detecting fluorescent labels attached to the material. In this context, the term "integrated into polynucleotides" means that the 5' phosphate is phosphorus The 3' hydroxy-OH group of the second (modified or unmodified) nucleotide is linked by an acid diester bond. This means that it is bound to and itself forms part of a long polynucleotide chain. It is possible that the 3' end of the nucleotides described herein may be further modified or unmodified. (Decoration) Whether or not a phosphodiester bond is attached to the 5' phosphate of a nucleotide This may also apply. Therefore, in one non-limiting embodiment, this disclosure may (a) apply to the (b) Incorporating at least one nucleotide into the polynucleotide This includes detecting the nucleotides incorporated into polynucleotides. A method for detecting nucleotides is provided. Fluorescent signals from dye compounds bound to the nucleotides are provided. By detecting the nucleotide, it is inserted into the polynucleotide.

[0127] This method involves incorporating one or more nucleotides into a polynucleotide according to the present disclosure. Step (a), and detection of one or more nucleotides incorporated into the polynucleotide The detection step (b) may include detecting otides or quantifying their fluorescence. To measure accurately.

[0128] Some embodiments of this application relate to sequencing methods including: (a) Incorporating at least one labeled nucleotide described herein into a polynucleotide. (b) To detect a fluorescent signal from a new fluorescent dye attached to the nucleotide. This method detects labeled nucleotides incorporated into polynucleotides.

[0129] Some embodiments of this disclosure include determining the sequence of a target single-stranded polynucleotide, including the following: Regarding the method of determination: (a) Nucleo containing a 3'-OH blocking group and the detectable label described herein The tide is a copy polynucleotide chain complementary to at least a portion of the target polynucleotide chain. To incorporate into; (b) To detect the identity of nucleotides incorporated into a copy polynucleotide chain; and (c) Labeling and 3'-OH from nucleotides incorporated into the copy polynucleotide chain Chemically removing blocking groups.

[0130] In some embodiments, the sequencing method includes (d) chemically removed labels The further method includes washing away the 3' blocking group from the copy polynucleotide chain. In some such embodiments, the 3' blocking group and the detectable label are It is removed before introducing the next complementary nucleotide. In some further embodiments, The 3' blocking group and detectable label are removed in a single step of the chemical reaction. In some embodiments, the cleaning step (d) is also not incorporated into the nucleo Removes chlorine. In some further embodiments, the palladium scavenger is Furthermore, it is used in the washing process after the chemical cleavage of the label and 3' blocking groups.

[0131] In some embodiments, steps (a) to (d) are performed on a template polynucleotide. This process is repeated until the arrangement of the cytoplasmic chain segments is determined. In some such embodiments, Steps (a) to (d) are performed at least 50 times, at least 75 times, and at least 100 times. times, at least 150 times, at least 200 times, at least 250 times, or at least It is repeated 300 times.

[0132] In some embodiments, the label and the 3'-blocking group are formed by two separate chemical reactions. It is removed. In some such embodiments, it is incorporated into the copy polynucleotide chain. Removing the label from the embedded nucleotide is possible. - This involves contacting the chain with a first cleavage solution. In some such embodiments, the The cleavage solution of 1 contains a phosphine such as a trialkylphosphine. Non-specific examples of tris(hydroxypropyl)phosphine (THP), tris- (2-carboxyethyl)phosphine (TCEP), tris(hydroxymethyl)phosphine Contains phosphate (THMP) or tris(hydroxyethyl)phosphine (THEP) In one embodiment, the first cutting solution contains THP. In some such embodiments... In this case, the nucleotides incorporated into the copy polynucleotide chain have 3' blocking groups Removing the incorporated nucleotides involves contacting the copy strand containing the nucleotides with the second cleavage solution. This includes the following. In some such embodiments, the second cleavage solution is palladium ( The catalyst includes a Pd(0) catalyst. In some further embodiments, the Pd catalyst is a Pd(0) catalyst. In some such embodiments, Pd(0) is a Pd(II) complex [( It is prepared by mixing PdCl(C3H5)2) in situ with THP. The molar ratios of d(II) complex to THP are approximately 1:2, 1:3, 1:4, 1:5, 1:6, 1: The ratio may be 7, 1:8, 1:9, or 1:10. In one embodiment, Pd:TH The molar ratio of P is 1:5. In some further embodiments, ascorbic acid and Adding one or more reducing agents, such as a salt of that (e.g., sodium ascorbate), is possible. Yes, it is possible. In some embodiments, the second cleavage solution is a primary amine, a secondary amine , tertiary amines, carbonates, phosphates, or borates, or combinations thereof, etc. It may contain one or more buffering reagents. In some further embodiments, the buffering reagent is ethanol. Nolamine (EA), Tris(hydroxymethyl)aminomethane (Tris), Glycine Sodium carbonate, sodium phosphate, sodium borate, 2-dimethylaminomethano (DMEA), 2-diethylaminomethanol (DEEA), N,N,N',N'-Te Tramethylethylenediamine (TEMED), or N,N,N',N'-tetraethyl Includes ethylenediamine (TEEDA) or a combination thereof. In one embodiment, buffer The reagent is DEEA. In another embodiment, the buffering agent is a carbonate, phosphate, or boron It comprises one or more inorganic salts, such as salts or combinations thereof. In one embodiment, The inorganic salt is a sodium salt. In some other embodiments, the second cleavage solution is Na Includes IO4 or Oxone®. In some further embodiments, 3 The block nucleotide contains an AOM group, and the second cleavage solution is palladium (Pd) catalyzed. The medium and one or more buffering reagents described herein (e.g., tertiary amines such as DEEA) It contains and has a pH of approximately 9.0 to approximately 10.0 (for example, 9.6 or 9.8).

[0133] In some alternative embodiments, the label and the 3'-OH blocking group are removed in a single chemical reaction. It is removed. In some such embodiments, the label is the same as the 3' blocking group. The nucleotide is bound via a cleavage linker containing a portion, for example, the linker and the 3' Both blocking groups are acetal moieties as described herein. [ka] or thiocarbamate portion [ka] It may include. In some such embodiments, a single chemical reaction is the Pd reaction described above. This is carried out in a cleavage solution containing a fertilizer.

[0134] In some further embodiments, the nucleo used in the integration step (a) Tides are fully functionalized A, C, T, and G nucleotide triphosphates, respectively , including the 3' blocking group described herein. In some such embodiments, The nucleotides herein are protected with a standard 3'-O-azidomethyl blocking group. Compared to the same nucleotide, it exhibits superior stability in solution during sequencing. Provided, for example, acetal or thiocarbamate blocking as disclosed herein. The group is less than 3'-OH protected with azidomethyl under the same conditions and for the same period. 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000%, 2500%, or 3000% improvement This can provide stability, thereby reducing the prephase value and the sequence read length. The duration increases. In some embodiments, stability is maintained at ambient temperature or below ambient temperature. (For example, measured at 4-10°C). In other embodiments, stability is measured at 40°C, 45°C, Measurements are taken at high temperatures such as 50°C, 55°C, 60°C, or 65°C. Several such implementations In terms of form, stability is maintained in basic pH environments, for example, pH 9.0, 9.2, 9.4, 9.6. It is measured in a solution of 9.8 or 10.0. In some further embodiments, The prephasizing value using 3' block nucleotides as described in the specification is SBS = 50 After running 100 or 150 cycles or more, approximately 0.25, 0.24, 0.23, 0 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0 It is less than 0.06 or 0.05. In some further embodiments, the 3' block Phase values ​​with nucleotides are obtained by performing SBS for 50, 100, or 150 cycles or more. After the operation, the values ​​were approximately 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, and 0.19. , 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11 It is less than 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05. In this embodiment, each ffN includes a 3'-AOM group.

[0135] In some embodiments, the 3' block nucleotides described herein are standard Compared to the same nucleotide protected with a 3'-O-azidomethyl blocking group, Provides excellent deblocking rates in solution during the chemical cleavage step of the sizing process. For example, acetals (e.g., AOM) or thiocarbamates disclosed herein Blocking groups are present in amounts of at least 5%, 10%, 20%, 30%, 40%, 50%, and 60%. 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 50 0%, 600%, 700%, 800%, 900%, 1000%, 1500%, or 20 00% shows improved deblocking rate compared to 3'-OH protected with azidomethyl. Use a standard deblocking reagent (such as tris(hydroxypropyl)phosphine). This reduces the overall time of the sequence cycle. In some embodiments, each The nucleotide deblocking times are approximately 5%, 10%, 20%, 30%, 40%, and 50%. or shortened by 60%. For example, the desorption of 3'-AOM and 3'-O-azidomethyl. The blocking time is approximately 4-5 seconds and approximately 9-10 seconds, respectively, under specific chemical reaction conditions. In some embodiments, the half-life of the AOM blocking group (t 1 / 2 ) is azidomethyl It is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times faster than the blocking group. In such an embodiment, the t of AOM 1 / 2 The time is approximately 1 minute, and the t of azidomethyl 1 / 2 is approximately 11 minutes. In some embodiments, the deblocking rate is the ambient temperature or ambient temperature. Measurements are taken at temperatures lower than the actual temperature (e.g., 4-10°C). In other embodiments, deblocking is performed. The concentration is measured at high temperatures such as 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C. In some such embodiments, the deblockization rate is determined in a basic pH environment, for example, Measurements are taken in solutions with pH 9.0, 9.2, 9.4, 9.6, 9.8, or 10.0. In some such embodiments, a deblocking reagent and a substrate (i.e., 3' block) The molar ratios of the nucleosides (or nucleotides) are approximately 10:1, 5:1, and 2:1. , approximately 1:1, approximately 1:2, approximately 1:5, or approximately 1:10. In one embodiment, each ffN is Includes the 3'-AOM group.

[0136] In any embodiment of the method described herein, the labeled nucleotide is a nucleotide. It is othide triphosphate. In any embodiment of the method described herein, the target polynucleotide The rheotide chain is attached to a solid support such as a flow cell.

[0137] In one embodiment, at least one nucleotide is involved in the action of the polymerase enzyme. This is incorporated into the polynucleotide in the synthesis step. Several such embodiments In this context, the polymerase is DNA polymerase Pol 812 or Pol 1901. It is possible. However, there are other ways of joining nucleotides to polynucleotides, for example If, chemical oligonucleotide synthesis or unlabeled oligonucleotides You can use ligation to Ochido. Therefore, the use of "integrate" The term, when used in relation to nucleotides and polynucleotides, refers to chemical methods and This can include polynucleotide synthesis by enzymatic methods.

[0138] In certain embodiments, the synthesis step is performed, and the labeled 3'-blocked nucleus of the Disclosure The template polynucleotide chain is incubated with a reaction mixture containing ocide. It may optionally include the following: a template polynucleotide chain annealed to a polynucleotide. Phosphodiester bonds between the free 3'-OH group on the nucleotide chain and the 5'-phosphate group on the nucleotide. It is also possible to provide polymerases under conditions that enable the formation of the polymer. Therefore, synthesis The step is directed by complementary base pairing of nucleotides with respect to the template strand. This may include the formation of polynucleotide chains.

[0139] In all embodiments of this method, the labeled nucleotide is incorporated into the polynucleotide While the dove chain is annealed to the template chain, or after the denaturation process in which the two chains are separated A detection process can be carried out. Further steps between the synthesis process and the detection process, for example This may include chemical or enzymatic reaction steps or purification steps. In particular, labeled nucleotides The target chain incorporating the drug is isolated or purified, further processed, or used for subsequent analysis. It can be used. For example, in the synthesis step, the nucleotides described herein can be used. The labeled target polynucleotide is then used as a labeled probe or primer. In other embodiments, the products of the synthesis steps described herein may be further reacted. They may be subjected to a step, and if necessary, the products of these subsequent steps may be purified or They may be isolated.

[0140] The conditions suitable for the synthesis steps are well known to those familiar with standard molecular biology techniques. In one embodiment, the synthesis step involves a nucleotide comprising the nucleotides described herein. Similar to standard primer extension reactions using rheotide precursors, appropriate polymerase enzymes In the presence of the base material, a complementary extension target chain can be formed to the template chain. Other implementations In this state, the synthesis process itself forms part of the amplification reaction, and the target and template polynucleotide chains It generates a labeled double-strand amplification product consisting of an annealed complementary strand derived from a copy. Other examples Typical synthesis steps include nic translation, chain substitution polymerization, and random priming. This includes NA labeling, etc. Polymerase enzymes particularly useful in the synthesis steps are described herein. It can catalyze the incorporation of nucleotides. Various natural or modified polymerases For example, thermally stable polymerases can be used under thermal cycling conditions. It can be used in synthetic reactions carried out by [unspecified method], but isothermal primer extension reactions require thermal stability Polymerase may be undesirable. The nucleotides can be incorporated according to this disclosure. For suitable heat-stable polymerases, use WO2005 / 024010 or WO06 / 1 This includes items described in 20433, which are incorporated herein by reference. In synthesis reactions carried out at low temperatures such as 37°C, polymerase enzymes are not necessarily heat-stable. It does not need to be a sexual polymerase. Therefore, the selection of polymerase depends on the reaction temperature and pH. , chain displacement activity, and favorites.

[0141] In certain non-limiting embodiments, this disclosure relates to nucleic acid sequencing, resequencing, Whole genome sequencing, single nucleotide polymorphism scoring methods, and integration into polynucleotides Other applications, including detection of labeled nucleotides or nucleosides as described herein, if present. It includes the benefits of using polynucleotides labeled with nucleotides containing fluorescent dyes. The various other applications that result include labeling nucleotides or nucleotides with the dyes described herein. Creosides can be used.

[0142] In certain embodiments, the present disclosure relates to the synthesis of labeled nucleotides by the present disclosure. Provides use in nucleotide sequencing (SBS) reactions. Synthetic sequencing Processing typically involves using polymerase or ligase to process the growing polynucleotides. Add one or more nucleotides or oligonucleotides to the chain sequentially from 5' to 3'. In addition, it forms an elongated polynucleotide chain complementary to the template nucleic acid. The identity of the bases present in one or more of the added nucleotides is detected or This can be determined in the "imaging" step. The identity of the added base is determined by each nucleus. This can be determined after the Otid incorporation step. Next, the template sequence This can be inferred using the conventional Watson-Crick base pairing rules. Single salt The use of labeled nucleotides as described herein to determine the identity of a group is, for example, single This may be useful in scoring nucleotide polymorphisms, such as single base The extension reaction is within the scope of this disclosure.

[0143] In one embodiment of the present disclosure, the sequence of the template polynucleotide is incorporated The sequencing template is obtained by detecting the fluorescent label attached to the polynucleotide. One or more 3'-blocked nucleotides described herein to the nascent chain complementary to the topolynucleotide. Determined by detecting the incorporation of creotides. Nucleotides. Template Polynucleotide sequencing requires appropriate primers (or as part of a hairpin). Can be primed with a hairpin construct (prepared with primer), 3' The nascent chain is gradually extended by the addition of nucleotides to the ends. Polymerase-catalyzed reaction The amount of primer in the response.

[0144] In certain embodiments, different nucleotide triphosphates (A, T, G, and C) It can be labeled with a unique fluorophore to prevent uncontrolled polymerization at the 3' position. It also includes a blocking group. Alternatively, one of the four nucleotides may be unlabeled (dark colored). It is possible. The polymerase enzyme adds a nascent chain complementary to the template polynucleotide. The creotide is incorporated, and the blocking group prevents further incorporation of nucleotides. All unincorporated nucleotides can be washed away, and each incorporated nucleotide The fluorescence signal from is obtained using laser excitation and an appropriate emission filter for charge coupling elements. It can be optically "read" by appropriate means. Next, 3'-Blocking The nucleotide group and the fluorescent dye compound are removed simultaneously or sequentially (deprotected) to obtain further nucleotides. The nascent strand can be exposed for incorporation. Typically, incorporated nucleotides The identity is determined after each integration step, but this is not strictly required. As such, U.S. Patent No. 5,302,509 (as incorporated herein by reference) The document discloses a method for arranging polynucleotides immobilized on a solid support.

[0145] This method, as illustrated above, uses fluorescently labeled 3'-block nucleotides A and G C and T are added to the immobilized polynucleotide in the presence of DNA polymerase. This utilizes the process of incorporating the target polynucleotide into the growth chain. The polymerase uses a salt complementary to the target polynucleotide. Although it incorporates a group, further addition is prevented by a 3'-blocking group. Next, the label of the incorporated nucleotide is determined, and the protecting group is removed by chemical cleavage. Furthermore, polymerization can be carried out. In sequencing reactions by synthesis, The nucleic acid template to be sequenced is any polynucleotide of which sequencing is desired. It is possible. Nucleic acid templates for sequencing reactions are typically used for sequencing. It functions as a primer or starting point for further nucleotide addition in the reaction. It contains a double-stranded region having a free 3'-OH group. The region overhangs this free 3'-OH group on the complementary chain. Sequencing The overhang region of the template may be single-stranded, but the sequencing of the template The presence of "nicks" on the chain complementary to the plate chain allows for the free 3' of the initiation. -It may be a double helix, provided it provides an OH group. Sequence reaction. Such implementation In this state, sequencing can proceed by chain substitution. In certain embodiments, free 3 Primers having an OH group are used to create a single-stranded region of the template being sequenced. It can be added as a separate component to be absorbed (e.g., a short oligonucleotide). Alternatively, the primer and template strands to be sequenced are, for example, hairpins. A portion of a partially self-complementary nucleic acid chain that can form an intramolecular double helix structure such as a loop structure. Each can be formed. Hairpin polynucleotides and they on a solid support. The method of attachment is described in PCT publication numbers WO01 / 57248 and WO2005 / 0473. Disclosed in 01, which are incorporated herein by reference. Nucleotides are, The nucleotides are successively added to the growing primer, synthesizing a polynucleotide chain in the 5' to 3' direction. The properties of the added bases are determined, in particular, after each nucleotide addition, but not necessarily. It is not necessary to do so, so we provide the sequence information of the nucleic acid template. Nucleotides are converted into nucleic acids through the formation of phosphodiester bonds with the 5' phosphate group of rheotide. By binding to the free 3'-OH group of the chain, the nucleotide becomes a nucleic acid chain (or polynucleotide). It will be incorporated into Ochido.

[0146] The nucleic acid template to be sequenced is DNA or RNA, or deoxynucleate It could even be a hybrid molecule consisting of rheosides and ribonucleotides. The rate is natural and does not interfere with the copying of the template in the sequencing reaction. It may also contain / or non-natural nucleotides and natural or non-natural skeletal linkages.

[0147] In certain embodiments, the nucleic acid template to be sequenced is known in the art. It can be bonded to a solid support by any suitable bonding method, for example, covalent bonding. Specific implementation In terms of morphology, the template polynucleotide is supported by a solid support (e.g., silica-based). It can be directly attached to the support. However, in other embodiments of the present disclosure, a solid support The surface of the body allows for direct covalent bonding of template polynucleotides, or Template polymers through a hydrogel or polyelectrolyte multilayer which may themselves be non-conforming. It can be modified in some way to immobilize the creotide. - Covalently bonded to a solid support. They are doing it.

[0148] (Embodiments and alternatives to sequencing by synthesis) Some embodiments include pyrosequencing technology. Pyrosequencing is, The release of inorganic pyrophosphate (PPi) when certain nucleotides are incorporated into the nascent chain is detected. (Ronaghi, M., Karamohamed, S., Pettersson) , B., Uhlen, M. and Nyren, P. (1996) “Real-pylori Time-dependent DNA sequencing using phosphate release detection. "Analytical Bio chemistry 242(1), 84-9; Ronaghi, M. (2001) Pyrosequencing sheds light on DNA sequencing. "GenomeRes." 11(1), 3-11; Ronaghi, M., Uhlen, M. and Nyren P. (1998) "Real-time pyrophosphate-based sequencing method" Science CE 281(5375), 363; U.S. Patent No. 6,210,891; No. 6,258, Disclosures No. 568 and No. 6,274,320. These are, in their entirety, included in this specification by reference. (It is incorporated into the book). In pyrosequencing, the released PPi is ATP sulfa It can be detected and produced by being immediately converted to adenosine triphosphate (ATP) by ze. The ATP level is detected via photons produced by luciferase. The nucleic acids can be attached to features within the array, and the array can then be attached to the features of the array. To capture the chemiluminescent signal generated as Otid is incorporated, imaging is performed. This can be done. After treating the array with a specific nucleotide type (A, T, C, G, etc.), the image An image can be obtained. The image obtained after adding each nucleotide type will show which features in the array It differs in whether they are detected. These differences in the images are due to the sequence of features on the array. This reflects the differences in content. However, the relative position of each feature is within the image. Unchanged. Images may be stored, processed, and analyzed using the methods described herein. This can be done. For example, after processing the array with each different nucleotide type, The resulting images are used for different detection channels in a reversible terminator-based sequencing method. Images obtained from Nell can be processed in the same manner as illustrated herein. ru.

[0149] In another exemplary type of SBS, cycle sequencing is, for example, WO04 / 0 As described in U.S. Patent Nos. 18497 and 7,057,026, for example, Stepwise addition of reversible terminator nucleotides containing discontinuable or photobleachable dye labels. This is achieved by adding. Its disclosure is incorporated herein by reference. This approach These products are commercialized by Solexa (now Illuminati), and each is based on the original. Also described in WO91 / 06678 and WO07 / 123,744 incorporated into the specification. It is. Both ends can be reversed, and the fluorescent label is cut off. The availability of the terminal is due to the efficient cyclic reversible termination (CRT) sequence. It facilitates the process. Polymerase also efficiently incorporates these modified nucleotides. And from there, we can collaborate on the design to extend further.

[0150] Preferably, in an embodiment of reversible terminator-based sequencing, the sign It does not substantially inhibit elongation under SBS reaction conditions. However, the detection label is, for example, For example, they can be removed by cutting or decomposition. Labeling the characteristics of the sequenced nucleic acids. After integration, images can be captured. In a particular embodiment, each cycle is , including simultaneous delivery of four different nucleotide types to the array, each nucleotide type These have spectrally different labels. Next, we select one of the four different labels. Four images can be obtained using different detection channels. Alternatively, different nuclei Otidotypes can be added sequentially, and an image of the array can be obtained between each addition step. This is possible. In such embodiments, each image contains a specific type of nucleotide. It will show the characteristics of the incorporated nucleic acid. Because the sequence content of each function is different, Each image may or may not have various functions. However, the relative position of the features is in the image. The image remains unchanged. Images obtained from this reversible terminator-SBS method are... It can be stored, processed, and analyzed as described in the specification. Image capture Following step A, the label can be removed, and the subsequent addition and detection of nucleotides can be performed. The reversible terminator portion can be removed for a given cycle. After detection, removing the label before the next cycle will reduce the background signal and the cyanotype. It has the advantage of reducing crosstalk between cells. Examples of useful labels and removal methods are provided below. See below.

[0151] Some embodiments use four different nucleotides with fewer than four different labels. Detection can be used. For example, SBS has US public disclosure number 2013 / 00792 Issue 32. As a first example, nucleotide type pairs can be detected at the same wavelength, but pairs Based on the difference in strength between one member and the other member, or one member of the pair They are distinguished based on changes to (e.g., via chemical modification, photochemical modification, or physical modification) ), the apparent signal is shown or hidden compared to the signals detected by other members of the pair. As a second example, three of the four different nucleotide types are under certain conditions. Although detectable below, the fourth nucleotide type is a detectable label under those conditions. Either not detected, or minimally detected under these conditions (e.g., by background fluorescence). (Minimal detection, etc.). The incorporation of the first three nucleotide types into nucleic acids is their... Based on the presence of each signal, it can be determined that the fourth nucleotide in the nucleic acid The inclusion of the IP can be determined based on the absence or minimal detection of any signal. Yes, it is possible. As a third example, one nucleotide type can be detected by two different channels. It can include a label, but other nucleotide types can be detected by one or fewer channels. The three exemplary configurations mentioned above are not considered mutually exclusive and can be combined in various ways. They can be used in combination. An exemplary embodiment combining all three examples is shown below. The first nucleotide type detected in channel 1 (for example, by the first excitation wavelength) When excited, dATP (which has a label detected in the first channel) is detected in the second channel The second nucleotide type detected by the meter (for example, when excited by the second excitation wavelength) (dCTP) having a label detected in the second channel, both the first and second channels The third nucleotide type detected by (for example, by the first and / or second excitation wavelengths) A dTT having at least one label that is detected in both channels when excited. P, and any other markers that are not detected or minimally detected in either channel. This is a fluorescence-based SBS method that uses a fourth nucleotide type (e.g., label). .

[0152] Furthermore, as stated in the incorporated document of U.S. Public Publication No. 2013 / 0079232 As such, sequence data can be acquired using a single channel. In the one-dic sequencing approach, the first nucleotide type is labeled. However, the label is removed after the first image is generated, and the second nucleotide type is the most Labeling occurs only after the first image is generated. The third nucleotide type is the same as the first. The label is retained in both the first and second images, and the fourth nucleotide type is in both images. It remains unlabeled.

[0153] Some embodiments can utilize sequencing by ligation technology. Yes, such technology utilizes DNA ligase to incorporate oligonucleotides. to identify the incorporation of such oligonucleotides. Oligonucleotides are typical This involves the identity of a specific nucleotide in the sequence in which the oligonucleotide hybridizes. It has different correlated labels. As with other SBS methods, the labeled sequencing reagent is used. After processing a series of nucleic acid functions, an image can be obtained. Each image has a specific type of label. The features of the embedded nucleic acids are displayed. Because the sequence content of each feature is different, Different images may or may not have different features, but the relative positions of the features change within the image. Images obtained from ligation-based sequencing methods are described herein. It can be stored, processed, and analyzed as described herein. Exemplary SBS systems and methods that can be used in conjunction with the system are U.S. Patent As described in permits No. 6,969,488, 6,172,218, and 6,306,597 (These disclosures are incorporated herein by reference in their entirety.)

[0154] Some embodiments can utilize nanopore sequencing. r, DW & Akeson, M. "Nanopores and Nucleic Acids: Prospects for Ultrafast Sequencing". "Analysis of nucleic acids by nanopore analysis," Acc. Chem. Res. 35:817-825( 2002); Li, J., M. Gershow, D. Stein, E. Brandin , and JA Golovchenko, "DNA Molecules and Constituent Solid-State Nanopore Microscopy" Nat. Mater. 2:611-615 (2003), its disclosure is by reference in its entirety. (The body is incorporated herein). In such embodiments, the target nucleic acid passes through the nanopore. Nanopores may be synthetic pores or biological membrane proteins such as α-hemolysin. When the target nucleic acid passes through the nanopore, the change in the pore's electrical conductance is measured. This allows for the identification of each base pair. (US Patent No. 7,001,792; So ni, GV & Meller, “A. Ultrafast DNA sequencing using solid nanopores Progress toward [a certain goal]. Clin. Chem. 53, 1996-2001 (2007); Healy, K. "Nanopore-based. Single-molecule DNA analysis." "Nanomed. 2, 459-481(2007); Cockroft, S.L., Chu, J., Amorin M. & Ghadiri, MR. “Single-molecule nanopore devices with single nucleotides and DNA Resolution for detecting polymerase activity. J. Am. Chem. Soc. 130, 818-8 20 (2008), its disclosure is incorporated herein in its entirety by reference. Nano The data obtained from pore sequencing will be stored and processed as described herein. , and can be analyzed. In particular, the data can be used to analyze optical images as described herein. It can be treated as an image according to the example processing of other images.

[0155] Several other embodiments of the sequencing method are described in U.S. Patent No. 9,222,132. The nanoball sequencing technology described herein, etc. This includes the use of 3' block nucleotides, the disclosure of which is incorporated by reference. Through the RCA (Ring Circle Amplification) process, numerous individual DNA nanoballs are generated. It is possible that this will happen. Next, the nanoball mixture associates a single nanoball with each position. It is distributed onto a patterned slide surface that includes features that enable it. During ball formation, DNA is fragmented and ligated into the first sequence of the four adapter sequences. The template is amplified, cyclized, and cleaved with type II endonuclease. A second set of adapters is added, followed by amplification, ringing, and cutting. This process is repeated for the remaining two adapters. The final product is... A circular template with four adapters separated by a template sequence. The library molecules undergo a rolling circle amplification step, and DNA nanoballs and It generates a large amount of concatemer called [name of concatemer], which deposits in the flow cell. Goodwin et al. al., “Coming of age: ten years of next-g generation sequencing technologies,” Nat Rev Genet. 2016;17(6):333-51.

[0156] Some embodiments include real-time monitoring of DNA polymerase activity. The law can be used. Nucleotide incorporation is, for example, U.S. Law 7,329 Nos. 492 and 7,211,414 (both incorporated herein by reference) As described in (included), fluorophore-containing polymerase and γ-phosphate labeling Detection is performed via fluorescence resonance energy transfer (FRET) interactions between nucleotides. This is possible. Alternatively, nucleotide incorporation is, for example, U.S. Patent No. 7,315,019. As described in (incorporated herein by reference), detection in a zero-mode waveguide It can be issued. For example, U.S. Patent No. 7,405,281 and U.S. Publication No. 2 As described in Issues 008 / 0108082 (both of which are incorporated herein by reference): As described, fluorescent nucleotide analogs and engineered polymerases are used. The lighting is designed so that the uptake of fluorescently labeled nucleotides can be observed with low background. It is possible to limit the volume around the polymerase attached to the surface to a zeptolite scale. It is possible (Levene, MJ et al. "Zero-mode wave") guides for single-molecule analysis at h Science 299, 682-68 6 (2003); Lundquist, PM et al. lel confocal detection of single molecule es in real time.” Opt. Lett. 33, 1026-10 28 (2008); Korlach, J. et al. aluminum passivation for targeted immob ilization of single DNA polymerase molec ules in zero-mode waveguide nano structure res.” Proc. Natl. Acad. Sci. USA 105, 11 76-1181 (2008), its disclosure is incorporated in its entirety herein by reference. .) Images obtained by such methods may be stored, processed, or otherwise preserved as described herein. It can be called and analyzed.

[0157] In some embodiments of SBS, the nucleotide is released once it is incorporated into the elongation product. This includes proton detection. For example, sequencing based on the detection of emitted protons, Ion Torrent (Guilford, CT, Life Technologie) Electrical detectors and related technologies commercially available from a subsidiary of s, or U.S. Publication No. 20 09 / 0026082;2009 / 0127589;2010 / 0137143;and In publication No. 2010 / 0282617 (all of which are incorporated herein by reference) The sequencing methods and systems described can be used. The method described herein for amplifying target nucleic acids using proton detection It can be easily applied to substrates used for this purpose. More specifically, as described herein. Using this method, a clonal population of amplicons used to detect protons is generated. It is possible.

[0158] The above SBS method uses a multiplexer so that multiple different target nucleic acids are manipulated simultaneously. It can be performed advantageously in a set. In certain embodiments, different target nucleic acids have common reactions. It can be processed inside the vessel or on the surface of a specific substrate. This allows for sequencing experiments. Convenient drug delivery, removal of unreacted reagents, and detection of uptake events are all possible in a multi-faceted manner. In embodiments using surface-bound target nucleic acids, the target nucleic acid is array format This is possible. In array format, target nucleic acids are usually represented in a spatially distinguishable manner. It can bind to surfaces. Target nucleic acids can be directly covalently bonded, or attached to beads or other particles. , or binding by binding to polymerase or other molecules attached to the surface. This is possible. The array contains a single copy (also called a feature) of the target nucleic acid at each site. This can occur, or multiple copies with the same sequence can exist in each site or feature. Multiple copies are bridged or emulsion, as will be explained in more detail below. It can be generated by amplification methods such as PCR.

[0159] The methods described herein include, for example, at least about 10 features / cm 2 , 100 special Features / cm 2 500 features / cm 2 1000 features / cm 2 , 5000 features / cm 2 , 10,000 functions / cm 2 50,000 functions / cm 2 , 100,000 functions / cm 2 1,000,000 functions / cm 2 5,000,000 functions / cm 2 The above Arrays with characteristics of various densities can be used.

[0160] The advantage of the methods described herein is that they enable the rapid and efficient detection of multiple target nucleic acids in parallel. The disclosure is to provide by doing so. Therefore, this disclosure applies to the art, such as those exemplified above. We propose an integrated system that can prepare and detect nucleic acids using technologies known to be used. Provides amplification reagents and / or sequencing. A fluid component capable of delivering reagents to one or more immobilized DNA fragments. The system may include components such as pumps, valves, reservoirs, and fluid lines. This includes. The flow cell consists of and / or uses an integrated system for detecting target nucleic acids. It can be used. An example flow cell is, for example, U.S. Publication No. 2010 / 011 These are listed in serial number 1768 and U.S. serial number 13 / 273,666. Each of these is incorporated herein by reference, as exemplified by the flow cell. One or more fluid components of the integrated system are used in amplification and detection methods. This is possible. Taking nucleic acid sequencing as an example, the integrated system One or more fluid components are amplified according to the amplification method described herein, and as exemplified above. It can be used for delivering sequencing reagents in sequencing methods. Furthermore, the integrated system has separate fluid systems for performing amplification and detection methods. It can include a system for creating amplified nucleic acids and determining the sequence of nucleic acids. Examples of integrated sequencing systems include the MiSeq (trademark) platform (Il Lumina, Inc. (San Diego, California) and U.S. Serial Number 1 Examples include the devices described in issue 3 / 273,666, which are incorporated herein by reference. .

[0161] Arrays in which polynucleotides are directly attached to a silica-based support are, for example, WO00 Disclosed in / 06770 (incorporated herein by reference), The nucleotide consists of a pendant epoxide group on the glass and an internal amino acid on the polynucleotide. Furthermore, polynucleotides are, for example, WO2005 / 047301 (see reference). As described in the specification, a sulfur-based nucleophile and a solid support The reaction allows it to be bonded to a solid support. Another example of a polynucleotide is a template polynucleotide, for example, WO00 / 31148, WO01 / 01143, WO02 / 12566, WO03 / 014392 See U.S. Patent No. 6,465,178 and WO00 / 53812. These are each incorporated herein by reference.

[0162] Certain surfaces to which template polynucleotides can be immobilized include polyacrylamide. It is a hydrogel. Polyacrylamide hydrogel is the reference cited above and WO2 It is described in 005 / 065814 and incorporated herein by reference. For certain hydrogels, see WO2005 / 065814 and USPub. No. 20 14 / 0079923. In one embodiment, the hydrogel is PAZAM(poly(N-(5-a It is (didoacetamidylpentyl)acrylamide-co-acrylamide).

[0163] DNA template molecules are described, for example, in U.S. Patent No. 5,253,003. It can adhere to beads or fine particles. (US Patent No. 6,172,218) No. (as incorporated herein by reference). Adhesion to beads or fine particles is due to sequencing Useful for sculpting applications. The bead library contains beads with different DNA sequences. It can be prepared to include. Examples of libraries and how to create them are in Nature. This is described in 437, 376-380 (2005). Science, 309, 5741, 1728-1732 (2005), these refer to the details of this specification, respectively. To be incorporated into the book. An array of such beads using the nucleotides described herein. The sequencing is within the scope of this disclosure.

[0164] The sequenced templates form part of an "array" on a solid support. This is possible, and in that case, the array can take any convenient form. Therefore, this disclosure The method includes any single-molecule array, clustered array, and bead array. Applicable to high-density arrays of this type. The labeled nucleotides of this disclosure are placed on a solid support. This includes, but is not limited to, those formed by the immobilization of nucleic acid molecules, and is essentially flexible. It can be used to sequence templates on an array of a specific type.

[0165] However, the labeled nucleotides of this disclosure are in the context of sequencing clustered arrays. This is particularly advantageous in clustered arrays. In clustered arrays, different regions (sites) on the array are advantageous. (Often called a feature) Multiple polynucleotide template molecules It constitutes. Generally, multiple polynucleotide molecules are individually decomposed by optical means. It is not possible to detect them individually, but rather they are detected as a group. Depending on how the array is formed, on the array Each site is made up of multiple copies of a single, distinct polynucleotide molecule (for example, that site is specific). (Homogeneous for single-stranded or double-stranded nucleic acid species) or containing a small number of copies There is a match. This is a combination of different polynucleotide molecules (for example, multiple copies of two different nucleic acid species). ). Clustered arrays of nucleic acid molecules are a technique commonly known in this field. It can be generated using the following: For example, WO98 / 44151 and WO00 / 18957( Each of these (as incorporated herein) forms an array consisting of clusters A nucleic acid amplification method in which both the template and the amplification product remain immobilized on a solid support. It describes or immobilized nucleic acid molecules as "colonies". Prepare according to these methods Nucleic acid molecules present on the prepared clustered array are labeled with the dye compound of this disclosure. This is a suitable template for sequencing using nucleotides.

[0166] The labeled nucleotides of this disclosure can also be used for sequencing templates on single-molecule arrays. Useful. The terms “single molecule array” or “SMA” as used herein refer to This refers to a collection of polynucleotide molecules dispersed (or arranged) on a solid support, and individual poly The spacing between nucleotides and all other populations separates individual polynucleotide molecules individually. It is possible to do so. Therefore, target nucleic acid molecules immobilized on the surface of a solid support can be In some embodiments, it can be disassembled by optical means. One or more different signals, each representing a single polynucleotide, are used in a specific way. This means it occurs within the disassemblable region of the imaging device.

[0167] The spacing between adjacent polynucleotide molecules on the array is at least 100 nm, more details For at least 250nm, and for even finer detail at least 300nm, and for even finer detail... This allows for the detection of single molecules at least 350 nm in size. The molecules can be individually decomposed and detected as single-molecule fluorescence points, and the fluorescence points of the single molecules The fluorescence from this source also exhibits a single-step photobleaching.

[0168] In this specification, the terms “individual breakdown” and “individual breakdown” are used to describe the visualization process. This specifies that one molecule on the array can be distinguished from its neighboring molecules. The separation between individual molecules shown above is partly due to specific techniques used to break down individual molecules. Determined by the technique. General characteristics of single-molecule arrays are described in the published application WO00 / 0 This will be understood by referring to 6770 and WO01 / 57248. These are each incorporated herein by reference. One use of the nucleotides of this disclosure is While sequencing is performed through synthetic reactions, the usefulness of nucleotides is not limited to such methods. No, it doesn't. In fact, nucleotides attach to nucleotides incorporated into polynucleotides. It can be advantageously used in any sequencing method that requires the detection of a fluorescent label. ru.

[0169] In particular, the labeled nucleotides of this disclosure are used in automated fluorescence sequencing protocols, especially Sanga - and the collaborator's fluorescent dye terminator cycle based on chain termination sequencing method This method can be used in cyclic sequencing. Such methods typically involve enzymes and cyclic sequencing. Using quenching, fluorescently labeled dideoxynucleotides are used in primer extension screening. Incorporate it into the sequencing reaction. This is the so-called Sanger sequencing method and related protocols. Sanger type is randomized chain termination by labeled dideoxynucleotides. Use the stop function.

[0170] Therefore, this disclosure relates to dideoxyxine which lacks hydroxyl groups at both the 3' and 2' positions. This also includes labeled nucleotides that are creotides, and such dideoxynucleotides are It is suitable for use in methods such as the Ninger sequencing method.

[0171] The labeled nucleotides of this disclosure incorporating a 3'-blocking group are subject to Sanger and related processes. It may also be useful in linked protocols because it uses dideoxynucleotides. The same effect achieved by having a nucleotide with a 3'-OH blocking group This can be achieved by using : both of which involve the subsequent nucleotide To prevent contamination. When the nucleotides provided in this disclosure are used in Sanger sequencing. A nucleotide-bound dye compound or a detectable label is cleaved via a cleavable linker. It will be understood that there is no need to bind the labeled nucleotide of this disclosure. Each example is included. Since there is no need to incorporate the nucleotide later, the label is removed from the nucleotide. There is no need to leave.

[0172] In any embodiment of the method described herein, in a sequencing application The nucleotides used are 3' block nucleotides as described herein, for example, formula ( It is a nucleotide of (I), (Ia), or (II). In any embodiment, 3' Block nucleotides are nucleotide triphosphates. [Examples]

[0173] Additional embodiments are provided below, which are not intended to limit the scope of the claims. Further details will be disclosed later.

[0174] (Example 1. Preparation of 3'-acetal block nucleosides) In this example, various 3'-acetal protected T nucleosides are prepared according to Scheme 2. Ta. [ka]

[0175] Preparation of T1: In a nitrogen-purged 100 mL flask that has been dried in an oven, add 5-iodine -2'-deoxyuridine (5.0 g, 14.12 mmol) was added. This was then added to 30 mL. After evaporating the pyridine three times, the mixture was placed under nitrogen. Anhydrous pyridine (25 mL) was added. The reactants were stirred at room temperature until a homogeneous solution was obtained (approximately 15 minutes). The mixture was then placed in an ice bath. Cool to 0°C, then add tert-butyldiphenylsilyl chloride (4.04 mL, 15.5 mmol). l) was added slowly by drop while vigorously stirring (for about 1 hour). All SMs were TL The reaction was maintained at 0°C for 8 hours until it was consumed by C. Saturated ammonium chloride aqueous solution. (Approximately 15 mL) was added and the reaction mixture was warmed to room temperature. The mixture was then mixed with ethyl acetate (100 mL). The solution was diluted and washed with saturated ammonium chloride aqueous solution (200 mL). The organic layer was separated, and the aqueous layer was separated. The mixture was extracted with ethyl acetate (4 x 50 mL). The organic layers were combined and dried, then (MgSO4) was used. After concentration under vacuum and removal of residual solvent under high vacuum, approximately 8 g of clear yellow oil was obtained. Crude Product T1 was obtained by flash column chromatography on silica, revealing a white crystalline structure. It was purified as a solid. The yield was 6.94 g (83%). LC-MS (electrosprung mass) (Ray Negative) 591.08 [MH]

[0176] Preparation of T2: In an oven-dried, nitrogen-purged brown 500 mL three-necked flask, prepare T 1 (6.23g, 10.5 mmol), copper(I) iodide (200mg, 1.05 mmol) ) and bis(triphenylphosphine) were added. Palladium(II) dichloride under nitrogen. (369 mg, 0.526 mmol). Protect the flask from light and store in anhydrous, degassed DMF. (200 mL) was added to this solution. 2,2,2-trifluoro-N-prop-2- Add nyl-acetamide (4.74 g, 31.6 mmol), followed by degassed triethyl acetate. Luamine (2.92 mL, 21.0 mmol) was added. The reaction mixture was incubated under nitrogen at room temperature for 6 hours. After stirring, no further starting materials were observed by TLC analysis. Volatile substances were removed under vacuum. The residue was removed in approximately 15 minutes, and the DMF was removed under high vacuum for approximately 1 hour, resulting in a brown residue. Dissolve this in ethyl acetate (200 mL) and prepare 0.1 MEDTA aqueous solution (2 x 200 mL) Extraction was performed. The aqueous layer was combined and further extracted with ethyl acetate (200 mL). The organic phase was combined. Then, dry (MgSO4), remove volatile substances under vacuum (~30 minutes), and further under high vacuum. Dry it (for about 1 hour) to obtain about 8 g of crude brown / yellow oil. Flush the mixture with silica gel. The substance was purified as an off-white solid by sugar column chromatography. Yield: 6.0 g (85%). LC-MS (electrospray negative) 614.19 [MH].

[0177] Preparation of T3: Start under nitrogen conditions with nucleoside T2 (2.0 g, 3.25 mmol) - In a 100 mL flask, add anhydrous DMSO (6.9 ml, 97.5 ml) to a dry nitrogen-purged flask. Add ol) all at once at room temperature and stir until a homogeneous solution is formed. Acetic acid (11. 1 mL, 195 mmol), followed by acetic anhydride (15.1 mL, 162.09 mmol) Both were added dropwise (for about 5 minutes each). The mixture was heated to 50°C and TLC (alkyl ethyl acetate / petroleum). Stir with ether (3:2) until the starting nucleoside is completely consumed (about 5 hours). Next, the reactants were concentrated to half their volume and cooled to approximately 0.5°C in an ice bath. ) N Slowly add aHCO3 (aq, sat.) (45 mL), and stir further. Post-treatment was started until no more bubbles were observed (~15 minutes). The solution was warmed to room temperature, and then Water was extracted with toluene (3 x 100 mL). The combined organic layer was dried with MgSO4. The mixture was filtered, and volatile substances were evaporated under reduced pressure and then under high vacuum. The crude product T3 was then processed off-white. The solid was purified by flash chromatography using silica gel. Yield: 1 0.79g (82%). LC-MS (Electrospray Negative) 674.20[M- H] - .

[0178] Preparation of T4: Starting nucleoside T3 (1.5 mL) in anhydrous CH2Cl2 under N2. To a solution of 79g, 2.649 mmol, add cyclohexene (1.34 mL, 13.2 mm (ol) was added. The mixture was cooled to 0°C in an ice bath, and distilled sulfuryl chloride (322 μL, 3. 97 mmol) was slowly added dropwise under N2 conditions (approximately 20 minutes), and the mixture was stirred at that temperature for 20 minutes. Furthermore, TLC (SiO: petroleum ether) = 3:2v / v) is complete when the initial nucleoside is complete. This indicates that it was consumed. Next, as shown in Scheme 3, the newly distilled corresponding The chloride intermediate was quenched by directly adding unsaturated alcohol (5 equivalents) dropwise. The liquid was stirred at room temperature for 2 hours, followed by evaporation of volatile substances under reduced pressure. The oily residue was EtO2. The mixture was divided into Ac:brine (3:2) (125 mL). The organic layer was separated, and the aqueous mixture was further separated. Extraction was performed using ethyl acetate (2 x 50 mL). The combined organic extracts were dried with MgSO4 and filtered. The volatile substances were evaporated under reduced pressure. The oily residue was treated with Â:brine (3:2). The mixture was divided into (125 mL). The organic layer was separated, and the aqueous solution was further divided into toluene (2 × 50 mL). The organic extracts were extracted using ) and dried with MgSO4, filtered, and volatile substances were removed under reduced pressure. It was evaporated. The crude product T4 was purified by flash chromatography using silica gel. The final product was obtained as a yellow oil. Yield: 1.20 g (69%) by AOM. PrO For M, the dose is 1.29g (71%). For DPrOM, the dose is 1.34g (71%).

[0179] 3'-AOM: Yellow oil. LC-MS (Electrospray Negative) [MH] ]684.24.

[0180] 3'-PrOM: Yellow oil. LC-MS (Electrospray Negative) [M- H]682.22.

[0181] 3'-DPrOM: Yellow oil. LC-MS (Electrospray Negative) [M -H]710.25. [ka] Scheme 3.

[0182] Preparation of T5: Starting material T4 (1.04 g, 1.51 g) in a 50 mL round-bottom flask under nitrogen. 6 mmol) was added to anhydrous THF (9 mL) at room temperature. Next, TBAF (1. Add 0M, 1.7mL, and 1.70 mmol drops, and all SM is consumed by TLC. The solution was stirred until it was completely dissolved (about 2 hours). During the reaction, the solution turned orange. The chemical substances were removed under vacuum to obtain an orange residue, which was then dissolved in methoxy (100 mL). It was dissolved and separated with NaHCO3 (saturated aqueous solution) (60 mL). The two layers were separated, and the aqueous layer was Extracted with HCl (60 mL). The organic layers were combined, dried (with MgSO4), filtered, The crude product was evaporated to obtain a yellow oil. The crude product was then flash-chromized with silica gel. Refined by tography, a clear yellow oil was obtained. Yield: 637 mg (94) AOM %). For PrOM, 526 mg (78%). For DPrOM, 617 mg (86%) ).

[0183] 3'-AOM: Transparent yellow oil. LC-MS (Electrospray Negative) [ MH] 446.12.

[0184] 3'-PrOM: Clear yellow oil (526 mg 78%). LC-MS (Electrospray) (Ray Negative): [MH] 444.10.

[0185] 3'-DPrOM: Transparent yellow oil (617 mg 86%). LC-MS (Electroscopy) Play Negative): [MH] 472.13.

[0186] Furthermore, two additional 3'-block T nucleosides (3'-eAOMT and 3'-i AOMT was prepared using the same method as described above. 3'-iAOMT:LC-MS(ES): (Negative ions) m / z 325.5 (MH) + ), (positive ions) 327.3 (M+H + ). 3 '-eAOMT:LC-MS(ES):(cation) m / z341.3(M+1H + ). [ka]

[0187] (Example 2.3'-OH blocking group stability test) In this example, the stability test of the 5'-mP 3'-AOM T nucleotide was performed using the standard 5 Run side-by-side in an incorporation buffer containing '-mP3'-O-azidomethyl T nucleotide. Ta. [ka]

[0188] (Formulation of buffer solutions) 100 mM ethanolamine buffer (pH 9.8), 100 mM NaCl, and 0.1 mM each of the 5'-monophosphate 3'-protective T nucleotide in a 2.5 mM EDTA solution 1 mL was incubated in a heating block at 65°C for 2 weeks. At the time of setting, 4 Take a 0 μL aliquot and analyze it by HPLC to determine the remaining blocked nucleotides. The proportion of unblocked nucleotides and the final formation of unblocked nucleotides were determined.

[0189] AOM, PrOM, DPrOM acetal protecting groups and standard azidomethyl blocking Figure 1 shows the results of stability tests on 5'-monophosphate 3' protected nucleotides containing the group. 3' block nucleotide monophosphates, including AOM, PrOM, and DPrOM, were observed. The blocking group improved the reduction in the deblocking rate in solution by 30 to 50 times or more. This experiment shows that the corresponding fully functionalized nucleotide (ffN) is sequenced by a sequencing device. This simulates how it works when stored in the built-in mix on the chair's cartridge. To imitate. The stability improvements provided by these acetal protecting groups are also due to the sequencing This will lead to a lower pre-phasing rate during execution. Finally, it will To improve the shelf life of mixed reagents.

[0190] (Example 3.3' - AOM deblocking test) In this example, we have 5'-mP3'-AOM T and the standard 5'-mP3'-O-azid Deblocking tests of methyl T nucleotides provide solutions specific to each block group. The tests were performed individually. The conditions were designed to mimic Illumina's standard deblocking reagents as closely as possible. It was formulated to imitate and follow the same methodology. Activated deblocking reagents, buffers, nucleos While the sid concentration remained constant across all tests, the identity of each component was unique. Thus, the differences in rates observed between individual deblocking chemicals are due to differences in formulation concentrations. It is not due to [unclear / unclear]. [ka]

[0191] (Standard azidomethyl deblocking conditions) Nucleotide: 5'-monophosphate 3'-O-azidomethyl T. Active deblocking reagent : Tris(hydroxypropyl)phosphine (THP) (1M in 18mΩ water). (Op Additive: Sodium ascorbate (0.1 mM in 18 mΩ water) Final concentration = 1 ml M. Buffer solution: Ethanolamine pH 9.8 (2M in 18mΩ water). Quenching reagent: H2O2.

[0192] (AOM deblocking conditions) Nucleotide: 5'-monophosphate 3'-O-azidomethyl T. Stock of 3'-AOMT The solution was placed in a glass vial under nitrogen and heated in 100 mM ethanolamine buffer (pH 9 .8) Diluted to 0.1 mM. Stock solution of sodium ascorbate additive was used as the final solution. The solution was added to a concentration of 0.1 mM, and the solution was stirred for 5 minutes. To start the assay, In addition, deblocking reagent (Pd / THP=1 / 5; sodium ascorbate; ethanol Min was added to a stirred solution at room temperature to a final concentration of 1 mMTHP. At the specified time, 40 Take a μL aliquot and mix with EDTA / H2O2 (0.025:0.075M) in a 1:3 ratio. Quenched with 6 μL of the mixture. HPLC analysis showed an initial nucleoside peak, followed by a 3'-OH peak. The area of ​​the nucleotide peaks and other nucleotide peaks shown in the HPLC chromatogram. This was performed by measurement. No other nucleotide-based byproducts were observed. .

[0193] The comparison results are shown in Figure 2A. AOM is different from the standard azidomethyl blocking group. It was observed that this provided a tenfold improvement in the non-blocking rate in solution. The experiment investigates how the corresponding ffN behaves in the sequence during the deblocking step. It serves the purpose of mimicking. A significant improvement in deblocking speed allows certain Illuminati Instead of the 10-20 second incubation time typically used on the Kens platform This allows for a flush-through deblocking step. As a result, deblocking is achieved. The conversion rate has a significant impact on the synthesis-based sequencing (SBS) cycle time.

[0194] Under similar experimental conditions, the deblocking aggregates of 3'-eAOM T and 3'-iAOM T were subjected to the same experimental conditions. Used in (i). As a single change, to observe a clear difference in the deblockization rate, Pd The catalyst-to-substrate ratio was reduced to 5:1. 3'-AOM T was used as a reference, and the results are shown in figure. 2B These results show that the deblocking speed of eAOM and iAOM is specific to this particular This shows that the Pd-catalyzed deblocking reagent at this concentration is 2-3 times slower than AOM. The difference in deblocking rates between the substituted and unsubstituted versions of the M blocking group is P It can be expected that the value will decrease as the ratio of catalyst to substrate increases.

[0195] (Example 4. Optimization of palladium-cleaved mixture for sequencing) The Pd / THP catalyst used in the deblockization reaction described in Example 2 is highly sensitive to air. It felt like it was oxidizing. When exposed to air, it showed a substantial loss of activity. In this example, oxidation We developed a stress assay to evaluate the air sensitivity of various formulations of palladium cleavage mixtures. did.

[0196] Dispense 0.5 mL of Pd Cleave Mix into a 5 mL glass vial and leave at room temperature for 3 hours. It was left open to the atmosphere. The residual activity of the oxidized cleavage mixture was 3'-A as follows: The cleavage of OM T was evaluated by measuring the stock solution of 3'-AOM T. It was diluted to 0.1 mM with 100 mM cleavage mixing buffer. Sodium ascorbate Add the stock solution to a final concentration of 1 mM, then add the oxidative cleavage mixture. The final dilution was 1 / 20. After 1 hour, 40 μL of the solution was mixed with 10 μL of EDTA / H2O2(0 The sample was immediately quenched with a 1:1 mixture of 0.25 M and 0.25 M, and analyzed by HPLC. In the experiment, various buffer reagents were screened, including primary amines (ethanol). (Sodium amines, trisulfone, glycine, etc.). Tertiary amines (2-dimethylaminomethanol(( DMEA), 2-diethylaminomethanol (DEEA), N,N,N',N'-tetra Methylethylenediamine (TEMED) or N,N,N',N'-tetraethylethylene Diamines (TEEDA, etc.); and various inorganic salts (borates, carbonates, phosphates, etc.) Inorganic buffering reagents (sodium borate, sodium carbonate, sodium phosphate, etc.) are the best. It was observed that the palladium complex maintained high % activity while providing air stability. Furthermore, Compared to primary amines, tertiary amines also showed significantly improved stability in the Pd cleavage mixture. .

[0197] Based on these findings, two palladium cleavage mixtures were prepared. In the first example, 2 Use a stock solution of 50 mM borate buffer (pH 9.6, 20 mL) and water (14 After dilution (mL), THP (1M in 100mM Tris, pH 9, 5mL, 5.0mm (ol) and allyl palladium(II) chloride dimer (183 mg, 0.5 mmol). After vigorously stirring the mixture at room temperature for several minutes, add 1M sodium ascorbate aqueous solution. (0.5 mL, 0.5 mmol), 5 M NaCl aq. (10 mL) and 10% v / vTween20 (0.5 mL). In the second example, 2 M DEEA buffer aqueous solution. Stock solution of the liquid (pH 9.6, 0.6 mL) is diluted with water (7.6 mL), then TH P (1 M in 100 mM Tris, pH 9, 1.2 mL, 1.2 mmol) and solid chloride A stock solution of allylpalladium(II) dimer(II) was added. 43.9 mg, (0.12 mmol). After vigorously stirring the mixture at room temperature for several minutes, add 1M sodium ascorbate. A solution of 5 M NaCl was added (0.12 mL, 0.12 mmol). (2.4 mL) and 10% v / vTween20 (0.12 mL).

[0198] (Example 5. Preparation of fully functionalized nucleotides and their use in sequencing applications) ) In this example, various fully functionalized nucleotides containing a 3'-AOM blocking group The preparation of ffN will be explained in detail. These ffNs are Illumina MiniSe It is also used for sequencing by synthesis applications on the q(registered trademark) platform. It was done. Synthesis of scheme 4.3'-AOM-ffC-LN3-SO7181 [ka]

[0199] Synthesis of intermediate AOM C2: Nucleoside C1 (0.5g, 0.64 mmol) is converted to N2 Dissolve in anhydrous DCM (12 mL) and cool the mixture to 0°C. Cyclohexene (0. Add 32 mL, 3.21 mmol) followed by SO2Cl2 (1.0 M, 1.2 mL in DCM). 7 mL (1.27 mmol) was added dropwise. The reaction mixture was quickly transferred to a rotary evaporator. Then, before removing all volatile substances under reduced pressure, add an additional cyclohexene (0.32 mL) (3.21 mmol) was added. The solid residue was further dried under high vacuum for 10 minutes, The mixture was dissolved in anhydrous DCM (5 mL) under N2 conditions. The mixture was cooled to 0°C and ice-cold allyl alcohol. 5 mL of ru (amount) was added dropwise. The reaction mixture was stirred at 0°C for 2 hours, then sat (amount) was added to quench it. The following was done: NaHCO3 aqueous solution (50 mL) and DCM (30 mL). The two phases were separated. The aqueous layer was extracted with toluene (2 x 50 mL). The organic layers were combined and dried over MgSO4. The mixture was filtered, and volatile substances were evaporated under reduced pressure. The crude product was then treated with acetone / petroleum ether. AOM C2 is purified by flash chromatography using silica gel. Obtained as a white solid (264 mg, 52% yield). LC-MS (electrospray negative) Tib): [MH]787, [M+Cl]823.

[0200] Synthesis of intermediate AOM C3: AOM C2 (246 mg, 0.31 mmol) under N2 Dissolve in anhydrous THF (9.5 mL) and cool the mixture to 0°C. Acetic acid (0.054 mL) Add 0.94 mmol) and then TBAF (1.0 M in THF, 5 wt. % water, 0. 99 mL (0.94 mmol) was added dropwise. After stirring the reaction mixture at 0°C for 5 hours, EtOA was added. Diluted with c (20 mL), then poured into a 0.05 M HCl aqueous solution (20 mL). The layers were separated, and the aqueous layer was extracted with SiO2 (2 × 20 mL). The organic layers were combined and MgS The product was dried with O4, filtered, and volatile substances were evaporated under reduced pressure. The crude product was divided into DCM / EtO Purified by flash chromatography on silica gel using Ac, AOM C3 was obtained as a yellowish solid (114 mg, 66% yield). LC-MS (Electron Trospray Negative): [MH]549, [M+H2O-H]567, [M+Cl ]585, (Electrospray positive): [M+H]551, [M+H2O+H] 569.

[0201] Synthesis of intermediate AOMC4: AOM C3 (0.114g, 0.21 mmol), newly Activated 4 Å molecular sieve, proton sponge (0.066 g, 0.31 mmol) l) Place a magnetic stirrer under N2 and add anhydrous trimethyl phosphate (1.0 mL) The reaction mixture was cooled to -10°C, and freshly distilled POCl3 (23 μL, 0.25) was added. A molecule was added dropwise. The reaction mixture was stirred at -10°C for 1 hour. Bis-tri-n-butyl Solution of pyrophosphate as monium salt (0.5 M in DMF, 1.7 mL, 0.85 ml) Premix (moles) and anhydrous tri-n-butylamine (0.41 mL, 1.74 mmol). Then, it was added to the ice-collapsed activated solution. Some of the nucleoside solution. The mixture was stirred vigorously at room temperature for 5 minutes. The reaction mixture was then placed in another flask containing a vigorously stirred solution of 2M TEAB aqueous solution. Pour into the flask (~10 mL). Rinse the reaction flask with a small amount of H2O and add the washing solution in 2 M TE. It was added to solution AB. Next, the combined mixture was stirred at room temperature for 4 hours, and then the solvent was reduced under reduced pressure. It was evaporated. The residue was dissolved in an NH3 aqueous solution (35%, approximately 10 mL) and left overnight at room temperature. The mixture was stirred. The reaction mixture was concentrated under vacuum and flash-chromatped using DEAE-Sephadex. The product was purified by tography. The product was further purified by preparative HPLC to obtain pure AO. M C4 (62 μmol, 30% yield, measured by UV-Vis spectroscopy, λ) max =29 4nm, ε=8600M -1 cm -1 ) was obtained. LC-MS (Electrospray Negative) Tib): [MH]589.

[0202] Synthesis of 3'-AOM-ffC-LN3-SO7181: LN3-SO7181(0.0 205 mmol) was dissolved in anhydrous DMA (4 mL) under N2 conditions. N,N-diisopropyl Ethylamine (28.6 μL, 0.164 mmol) was added, followed by TSTU (in DMA). (0.1 M, 234 μL, 0.0234 mmol) was added. The reaction mixture was incubated under N2 at room temperature for 1 hour. The mixture was stirred. During this time, an aqueous solution of AOM C4 (0.0101 mmol) was evaporated under reduced pressure. It was allowed to dry and resuspended in 0.1 M TEAB aqueous solution (400 μL), and then LN3-SO It is added to solution 7181. The reactants are stirred at room temperature for 17.5 hours, then 0.1 M TEA is added. Quenched with aqueous solution B (4 mL). The crude product was diluted with DEAE-Sephadex. The product was purified by sch chromatography. The product was further purified by preparative HPLC. Then, pure 3'-AOM-ffC-LN3-SO7181 (6.81 mol, 67% yield) Determined by UV-Vis spectroscopy, λ max =644nm, ε=2,000,000M -1 c m -1 ). Obtained: LC-MS (Electrospray Negative): [MH]1561 [M-2H] 781, [M-3H] 520. Synthesis of Scheme 5.3'-AOM-ffA [ka] JPEG0007833596000037.jpg33115

[0203] Synthesis of intermediate AOM A2: Nucleoside A1 (716 mg, 0.95 mmol) is converted to N Dissolve in 10 mL of anhydrous dichloromethane under 2 atmospheres, then dissolve in cyclohexene (481 μL, 4 Add 0.75 mmol of sulfuryl chloride (distilled, 92 μg). The solution was cooled to approximately -15°C. Add L (1.14 mmol) dropwise and stir the reaction mixture for 20 minutes. All starting materials were consumed. Afterward, the excess portion of cyclohexene (481 μL, 4.75 mmol) was added and the reaction proceeded. The material was evaporated to dryness under reduced pressure. The residue was quickly purged with nitrogen, and then allyl alcohol ( 5 mL (approximately 100 mmol) was added while stirring at 0°C. The reaction mixture was stirred at 0°C for 1 hour. Next, the mixture was quenched with 50 mL of saturated NaHCO3 aqueous solution. The mixture was then mixed with 2 × 100 mL of vinegar. Extracted with ethyl acid. The pooled organic phase was diluted with 100 mL of water and 100 mL of brine. The material was washed, then dried with MgSO4, filtered, and evaporated to dryness. The residue was then converted to petroleum ether. Purified using flash chromatography on silica gel with / IgG 60% yield (435 mg, 0.57 mmol). LC-MS (ES and CI): ( (Cation) m / z 763 (M+H+); (Anion) m / z 761 (M-H+).

[0204] Synthesis of intermediate AOM A3: Nucleoside AOM A2 (476 mg, 0.62 mmol) l) Dissolve in dry THF (5 mL) under an N2 atmosphere, then THF (750 μL, 0.7 A 1.0 M TBAF solution in 5 mmol was added. The solution was stirred at room temperature for 1.5 hours. The solution was diluted with 50 mL of acetoxide, and then washed with 100 mL of NaH2PO4sat. The solution was purified (pH=3), using 100 mL of brine. The organic phase was dried with MgSO4 and filtered. The mixture was allowed to dry by evaporation. The residue was then frothed in silica gel using à / MeOH. Purified by sch chromatography. 90% yield (292 mg, 0.55 mg). (moles). LC-MS (ES and CI): (cations) m / z 525 (M + H +); (cations) Ion) m / z523 (M-H+).

[0205] Synthesis of intermediate AOM A4: Nucleoside AOM A3 (285 mg, 0.544 mm The solution (ol) was dried under reduced pressure on P2O5 for 18 hours. Anhydrous triethyl phosphate (2 mL) Then, several newly activated 4 Å molecular sieves were added to it under nitrogen, and then the reaction was reversed. The test flask was cooled to 0°C in an ice bath. Freshly distilled POCl3 (61 μL, 0.65 Administer mmol) as drops, followed by ProtonSponge(registered trademark) (175 mg, 0. 816 mmol) was added. After the addition, the reaction mixture was stirred further at 0°C for 15 minutes. Next, Bis-tri-n-butylammonium salt in water DMF (5.4 mL, 2.72 mmol) Quickly add a 0.5 M solution of pyrophosphate as the base, followed immediately by tri-n-butylamine. (540 μL, 2.3 mmol) was added. The reaction mixture was kept in an ice bath for another 10 minutes. Next, pour it into a 1M triethylammonium bicarbonate aqueous solution (TEAB, 20 mL). The mixture was quenched and stirred at room temperature for 4 hours. All solvent was evaporated under reduced pressure. Add 20 mL of a 35% aqueous solution of ammonia to the residue and let the mixture sit at room temperature for at least 5 minutes. The mixture was stirred for a certain amount of time. Next, the solvent was evaporated under reduced pressure. The crude product was first prepared by adding DEAE-SEF. Purified by ion exchange chromatography using Adex A25 (100g). Lamb was eluted with a gradient of aqueous triethylammonium bicarbonate. The fraction containing triphosphate was The mixture was pooled and the solvent was evaporated to dryness under reduced pressure. The crude material was then processed using YMC-Pack-Pro C1. Preparative scale H using 8 columns and eluting with 0.1M TEAB and acetonitrile. Further purification was performed by PLC. Compound AOM A4 was obtained as a triethylammonium salt. Obtained in 56% yield (306 μmol). LC-MS (ES and CI): (Antidote) (ON) m / z 612 (M-H+); (Cation) m / z 614 (M+H+), 715 (M +Et3NH+).

[0206] General procedure for ffA synthesis: Add dye linker (0.020 mmol) to 2 mL of anhydrous N, Dissolved in N'-dimethylacetamide (DMA). N,N-diisopropylethylamide Add 28.4 μL, 0.163 mmol of N, followed by N, N, N', N'-tetramethyl Chil-O-(N-succinimidyl)uronium tetrafluoroborate in 0.1 M anhydrous solution The DMA solution (TSTU, 232 μL) was added. (0.023 mmol). The reaction products were then added. The mixture was stirred under nitrogen at room temperature for 1 hour. During this time, triphosphate AOM A4 (0.01 mmol) The aqueous solution is evaporated to dryness under reduced pressure, and 200 μL of 0.1 M triethylammonium bicarbonate is added. The activated dye linker solution was resuspended in an aqueous solution of TEAB. The activated dye linker solution was added to triphosphate and reacted. The mixture was stirred at room temperature for 18 hours. The crude product was first mixed with DEAE-Sephadex A25 ( Purified by ion-exchange chromatography (25g). The fraction containing triphosphate was selected. The mixture was then evaporated under reduced pressure to dryness. The crude material was then processed using YMC-Pack-ProC18. Further purification was performed by preparative scale RP-HPLC using ram. 3'-AOM-f fA-LN3-NR7180A: 38% yield (3.8 μmol). LC-MS(ES) :(Anion) m / z1459(M-H+), 729(M-2H+), 486(M-3H +) 3'-AOM-ffA-LN3-BL-NR 5 50S0: 37% yield (3.7μ (mol). LC-MS(ES): (Anion) m / z 1771 (M-H+), 885 (M -2H+), 589(M-3H+). 3'-AOMffA-LN3-BL-NR 6 50C 5:51% yield (51 μmol). LC-MS(ES): (Anion) m / z 1917 (M-H+), 958(M-2H+), 645(M-3H+). Synthesis of Scheme 6.3'-AOM-pppG [ka]

[0207] Synthesis of intermediate AOMG4: Known nucleoside dG3 (100 mg, 0.143 mmol) l) Dissolve in 10 mL of anhydrous dichloromethane under an N2 atmosphere, and cyclohexene (72 μL) (L, 0.714 mmol) was added and the solution was cooled to -12°C. Sulfuryl chloride (distillation, Add (1M in DCM, 171 μL, 0.171 mmol) dropwise and stir the reaction mixture for 10 minutes. The excess cyclohexene (72 μL, 0.714 mmol) was added, and the reaction mixture was prepared. The mixture was stirred at -12°C for 30 minutes. The reaction mixture was evaporated to dryness under reduced pressure, and the residue was purged with nitrogen. Stir ice-cold neat allyl alcohol (distilled, 0.8 mL, 12 mmol) at -12°C. It was added while stirring. The reaction mixture was stirred at -12°C for 60 minutes, then 2 mL of saturated aqueous solution was added. I did it. NaHCO3. Separate the mixture with ethyl acetate (2 mL), and the aqueous layer with ethyl acetate. Extraction was performed. The combined organic phase was washed with 4 mL of water and 4 mL of brine, and then with MgSO4. The oil was dried, filtered, and evaporated to obtain crude oil. The residue was then flash-chromatographed using silica gel. Purified with a filtration system, AOMG4 was obtained as a clear oily substance. 36% yield (50.9 mg). (0.072 mmol). LC-MS (ES and CI): (cation) m / z 710[ M+H]+; (anion) m / z708[MH]-.

[0208] Synthesis of intermediate AOM G5: Nucleoside AOM-G4 (111 mg, 0.156 mm) (ol) was dissolved in dry THF (5 mL) under an N2 atmosphere. Acetic acid (27 μL, 0.468 Add mmol) followed by 1.0 M TBAF THF solution (296 μL, 0.296 ml), then 1.0 M TBAF THF solution (296 μL, 0.296 ml). (mol) was added. The solution was stirred at room temperature for 5 hours. The solution was diluted with 10 mL of HCl. The sample was washed with 10 mL of 0.05 M aqueous solution. HCl and organic matter were separated. The aqueous phase was treated with acetate. Extraction was performed using chill extraction. The combined organic phase was dried with MgSO4, filtered, and evaporated to dryness. Residue The solution was purified by silica gel flash chromatography, and AOMG5 was obtained as a white solid. Obtained. 44% yield (32.4 mg, 0.068 mmol). LC-MS (ES and C I): (Cation) m / z 472[M+H]+; (Anion) m / z 470[MH]- .

[0209] Synthesis of 3'-AOM-pppG: A newly activated 4Å molecular sieve containing Nucleoside AOM-G5 (79 mg, 0.168 mmol) was measured under reduced pressure on P2O5. Dry for 18 hours. Proton sponge (registered trademark) (175 mg, 0.816 mmol) ) and anhydrous triethyl phosphate (0.8 mL) were added under nitrogen, and the mixture was stirred at room temperature for 1 hour. The reaction flask was cooled to 0°C in an ice bath, and freshly distilled POCl3 (19 μL, 0.20 2 mmol) was added dropwise, and the reaction mixture was stirred at 0°C for 15 minutes. Next, the bis- in anhydrous DMF was added. Pyrroline as tri-n-butylammonium salt (1.68 mL, 0.84 mmol) Quickly add the 0.5 M solution of acid, and immediately add tri-n-butylamine (168 μL, 0.70 (5 mmol) was added. Remove the reaction mixture from the ice / water bath and stir vigorously for 5 minutes, then... By pouring it into a 1M triethylammonium bicarbonate aqueous solution (TEAB, 6 mL), the quenching The mixture was heated and stirred at room temperature for 18 hours. All solvent was evaporated under reduced pressure. The residue was reduced to 35%. It was dissolved in an aqueous ammonia solution (10 mL) and stirred at room temperature for at least 5 hours. Next, the solvent The solution was evaporated under reduced pressure and then co-evaporated with water. The crude product was first treated with DEAE-Sepha Purified by ion exchange chromatography using a Dex A25 (50g) column. It was eluted with a linear gradient of aqueous triethylammonium. The fraction containing triphosphate was collected. The solvent was evaporated to dryness under reduced pressure. The crude material was analyzed using a YMC-Pack-ProC18 column. Further purification was performed by preparative scale HPLC. 3'-AOM-pppG is trie It was obtained as ammonium thyl salt. 24% yield (39.7 μmol). LC-MS ( ES and CI): (Anion) m / z 576 [MH]-; (Cation) m / z 578 [M+H]+.

[0210] 3'-AOM-ffT-LN3-NR 5 50S0 is 3'-AOMffA and ffC It was synthesized using the same method as described in the preparation section. Scheme 7.3'-AOM-ffT-LN3'-NR 5 Synthesis of 50S0 [ka]

[0211] Synthesis of intermediate T1: 5-iodo-2'-deoxyuridine (3g, 8.4 mmol) Dissolve palladium(II) acetate (1.6 g, 7.14 mmol) in degassed DMF. Next, it was dissolved in N-allyltrifluoroacetamide (6.4 mL, 42 mmol). ) was added. The solution was placed under vacuum, then purged three times with nitrogen, and then degassed triethylene Luamine (2.3 mL, 16.8 mmol) was added. The solution was heated at 80°C for 2 hours. The black mixture was cooled to room temperature and then diluted with 50 mL of methanol. Approximately 0.5 g of active Carbon was added, the solution was filtered through Celite, and then evaporated under reduced pressure to obtain a thick, brownish oil. This crude product is subjected to chromatography using silica gel with ¼ / MeOH. Purified by: (2.27g, 5.99 mmol). LC-MS (ES and CI): (Anion) m / z 378 (M-H+).

[0212] Synthesis of intermediate T2: 5-[3-(2,2,2-trifluoroacetamide)-allyl] -2'-deoxyuridine (T1) (2.55 g, 6.72 mmol) dissolved in dry DMF Soaked. Add imidazole (1.37g, 20.1 mmol), followed by 4-(dimethyl Amino)pyridine (410 mg, 3.36 mmol) was added. The reaction mixture was cooled to 0°C. Next, tert-butyl(chloro)diphenylsilane (1.92 mL, 7.39 mmol) The mixture was slowly added in three separate additions at 30-minute intervals. The reaction mixture was stirred at 0°C for 6 hours. Next, The solvent is evaporated, the residue is resuspended in 200 mL of acetylene, and a 2 × 200 mL aqueous solution is prepared. Washed with saturated NaHCO3 and 200 mL of water, then 100 mL of brine. The organic phase The product was dried with MgSO4, filtered, and evaporated to dryness. The crude product was treated with DCM / Âi. Purified by flash chromatography in silica. 68% yield (2.8 (0.6g, 4.54 mmol). LC-MS (ES and CI): (cation) m / z 61 8(M+H+); (anion) m / z 616(M-H+).

[0213] Synthesis of intermediate T3: 5'-O-(tert-butyldiphenylsilyl)-5-[3-( 2,2,2-trifluoroacetamide)-allyl]-2'-deoxyuridine(T2) (2.8g, 4.53 mmol) dissolved in 10 mL of anhydrous DMSO (136 mmol) Next, glacial acetic acid (16 mL, 272 mmol) and acetic anhydride (16 mL, 158 mmol) are added. (L) was added. The reaction mixture was heated at 50°C for 6 hours, and then quenched with 200 mL of aqueous solution. Saturated NaHCO3. After the foaming of the solution stops, extract with 2 x 150 mL of toluene. The organic phase was pooled and washed with 2 x 200 mL of aqueous solution. Saturated NaHCO3, 200 mL of water and 100 mL of brine. Dry the organic phase with MgSO4, filter, and evaporate to dryness. The crude product was then flash-chromatographed in silica using DCM / .'' Purified by Fee. 77% yield (2.375g, 3.51 mmol). LC-M S (ES and CI): (Cation) m / z 678 (M + H +); (Anion) m / z 6 76 (M-H+).

[0214] Synthesis of intermediate T4: 5'-O-(tert-butyldiphenylsilyl)-3'-O- Tylthiomethyl-5-[3-(2,2,2-trifluoroacetamide)-allyl]-2 - Deoxyuridine (T3) (310 mg, 0.45 mmol) under an N2 atmosphere for 5 minutes. Dissolve cyclohexene (228 μL, 2.25 mmol) in L of anhydrous dichloromethane. In addition, the solution was cooled to approximately -15°C. Sulfuryl chloride (distilled, 55 μL, 0.675 mm) Add ol) dropwise and stir the reaction mixture for 20 minutes. After all the starting materials were consumed, cyclo Add the excess hexene (228 μL, 2.25 mmol) and evaporate the reactants under reduced pressure. It was allowed to dry completely. The residue was quickly purged with nitrogen, and then ice-cold allyl alcohol (2.5 mL) was added. The mixture was added while stirring at 0°C. The reaction mixture was stirred at 0°C for 35 minutes, and then 25 mL of saturated aqueous solution was added. Quenched with the solution. Next, further dilute NaHCO3 with 100 mL of saturated aqueous solution. aHCO3. The mixture was extracted with 2 × 50 mL of ethyl acetate. The pooled organic phase was extracted with MgS The residue was dried with O4, filtered, and evaporated to dryness. The residue was then processed using DCM / Â. Purified by flash chromatography in Kagel. 69% yield (214 mg) (0.311 mmol). LC-MS (ES and CI): (cation) m / z 688( M+H+); (anion) m / z 686(M-H+).

[0215] Synthesis of intermediate T5: 5'-O-(tert-butyldiphenylsilyl)-3'-O- Ryloxymethyl-5-[3-(2,2,2-trifluoroacetamide)-allyl]- 2'-Deoxyuridine (T4) (210 mg, 0.305 mmol) under an N2 atmosphere Dissolved in dried THF (3 mL). Solution of 1.0 MTBAF in THF (367 μL, 0 (0.367 mmol) was added. The solution was stirred at room temperature for 3 hours. 50 mL of EtOA was added to the solution. Diluted with c, then washed with 50 mL of NaH2PO4sat (pH=3), and Use 50 mL of water. The organic phase was dried with MgSO4, filtered, and evaporated to dryness. Residual The material was subjected to flash chromatography using silica gel with DCM / alkyl. It was purified using LC-MS (ESO). 95% yield (130 mg, 0.289 mmol). (Called CI): (Anion) m / z 448 (M-H+), 484 (M+Cl-).

[0216] Synthesis of intermediate T6: 3'-O-allyloxymethyl-5-[3-(2,2,2-triph Luoroacetamide)-allyl]-2'-deoxyuridine (T5) (120 mg, 0. 267 mmol was dried under reduced pressure. It was then dried in P2O5 for 18 hours. Anhydrous triethyl phosphate. (1 mL) and several newly activated 4 Å molecular sieves were added to it under nitrogen. Next, the reaction flask was cooled to 0°C. Freshly distilled POCl3 (30 μL, 0.3 Add 2 mmol) dropwise, followed by ProtonSponge (registered trademark) (85 mg, 0. 40 mmol was added. After the addition, the reaction mixture was stirred further at 0°C for 15 minutes. Next, anhydrous Bis-tri-n-butylammonium salt in DMF (2.7 mL, 1.33 mmol) and Then quickly add a 0.5 M solution of pyrophosphate, followed immediately by tri-n-butylamine ( 270 μL (1.2 mmol) was added. The reaction mixture was kept in an ice bath for another 10 minutes, and then... Then pour it into a 1M triethylammonium bicarbonate aqueous solution (TEAB, 10 mL). Therefore, the mixture was quenched and stirred at room temperature for 4 hours. All solvent was evaporated under reduced pressure. Add 10 mL of a 35% aqueous solution of ammonia to the residue and stir the mixture at room temperature for 18 hours. Next, the solvent was evaporated under reduced pressure, and the residue was resuspended in 10 mL of 0.1 MTEAB. The solution was filtered. The filtrate was first subjected to ion exchange using DEAE-Sephadex A25 (100g). Purified by chromatography. The column was treated with aqueous triethylammonium bicarbonate (T Elution was performed using EAB. The fraction containing triphosphate was pooled, and the solvent was evaporated to dryness under reduced pressure. The crude material was subjected to preparative scale HPLC using a YMC-Pack-ProC18 column. It was then further purified. Compound T6 was obtained as the triethylammonium salt. 33% yield. (89 μmol). LC-MS (ES and CI): (Anion) m / z 592 (MH) +), 295 (M-2H+).

[0217] 3'-AOM-ffT-LN3'-NR 5 Synthesis of 50S0: Dried known compound LN 3-NR 5 50S0 (0.015 mmol) was dissolved in anhydrous DMA (2 mL) under N2 conditions. Add N,N-diisopropylethylamine (17 μL, 0.1 mmol), followed by T STU (0.1 M in DMA, 180 μL, 0.018 mmol) was added. The reaction mixture was then converted to N2 The mixture was stirred at room temperature for 1 hour. During this time, an aqueous solution of T6 (0.01 mmol) was steamed under reduced pressure. It was dried and resuspended in 0.1 M TEAB aqueous solution (200 μL), and then LN3-NR 5 It is added to the 50S0 solution. The reaction mixture is stirred at room temperature for 18 hours, then 0.1 MTEAB water is added. Quenched with solution (4 mL). The crude product was flushed with DEAE-Sephadex. The product was purified by chromatography. The product was further purified by preparative HPLC. Pure 3'-AOM-ffT-LN3'-NR 5 50S0 was obtained. Yield of 67% (41. mol, measured by UV-Vis spectroscopy, λ max =550nm, ε=125000M-1c m-1). LC-MS(ES): (Anion) m / z 1521 (M-H+), 761 (M -2H+), 507(M-3H+).

[0218] (Sequencing experiment using synthesis) Next, we used an Illumina MiniSeq® instrument to perform sequencing to determine ffN. It stopped. All standard cities except for the new built-in mix which includes these ffNs. Commercially available reagents were used. A standard 2x150 recipe was used. Sequencing was performed using standard synthesis. In addition to the SBS protocol, a 5-second interval in the solution of the palladium cleavage mixture is performed. Add incubation (Pd:THP=1 / 5 in DEEA as described in Example 4), 3 '-AOM has been unblocked.

[0219] In the initial experiment, the following ffN was used in the embedded mix: 3'-AOM-ffT- LN3-NR 5 50S0, 3'-AOM-ffA-LN3-BL-NR 5 50S0, 3' -AOM-ffA-LN3-BL-NR 6 50C5, 3'-AOM-ffA-LN3-N R7180A, 3'-AOM-ffC-LN3-SO7181, and 3'-AOM-p ppG (Dark G). The sequencing results for Read 1 are summarized below.

[0220] [Table 1]

[0221] In the second experiment, unlabeled 3'-AOM was prepared in the same way as the preparation of 3'-AOM-pppG described above. -pppT was synthesized (LC-MS(ES): (negative ion) m / z 551 (M-H+)) Commercially available Green ffG-LN3-PEG12-ATTO532 (Illumina 4-channel) Used in the system in the presence of the sequencer, as described in the first experiment above. The same ffA and ffC were used. The results are summarized below. Fading value and pref Significant improvement was observed in the aging value, and no signal attenuation was observed (Figure 3A). Furthermore, The error rates for both read 1 and read 2 also decreased.

[0222] [Table 2]

[0223] In another experiment, 3'-AOM-ffT-LN3'-NR 5 50S0, 3'-AOM-f fA-LN3-BL-NR 5 50S0, 3'-AOM-ffA-LN3-BL-NR 6 5 Embedded mix NR7180A, 3', including 0C5, 3'-AOM-ffA-LN3- -AOM-ffC-LN3-SO7181, and 3'-AOM-pppG (dark G) We used a palladium catalyst (Pd / THP=1:10; explained in Example 4), similar to previous executions. Incubation for 5 seconds with the cleavage mixture containing the specified 100 mM DEEA is standard. Added to the SBS cycle. Standard MiniSeq® DNA polymerase was used. Although this was used, the acquisition time was twice as long. No signal attenuation phenotype was observed (Figure 3). B). Furthermore, these sequencing results show that f has a standard azidomethyl blocking group. This was compared to a commercially available MiniSeq® run (mean 3; N=3) using fN. The error rates were observed to be almost the same (Figure 3C). The sequencing results are as follows: To summarize:

[0224] [Table 3]

[0225] Furthermore, the main sequencing metrics of ffN with a 3'-AOM blocking group are as follows: Standard MiniSeq® commercial kit containing DNA polymerase Pol 812 The results of the comparison with the one produced by 3'-AOM-ffN are shown in Figure 4A. Due to improved qualitative analysis, very low pre-phasing was observed. However, twice the amount of uptake was observed. Even when using the time interval, the phase was still rising.

[0226] In yet another experiment, a commercially available MiniSeq® kit (Pol 812) Instead of using DNA polymerase, use a different DNA polymerase (Pol 1901). Pol 1901, instead of twice the acquisition time mentioned above, uses the standard 1 in the sequence. This allowed for double the incorporation time. Furthermore, incubation with Pd-cleaved mixtures was standard This was reduced by half compared to the standard run. This reduces the overall SBS chemistry cycle. We saved 10% of the time. Sequencing metrics were significantly improved, and 3'-O-A The values ​​exceeded those obtained from standard commercially available kits containing a didomethyl blocking group (Figure 4B). ).

[0227] (3'-blocking group stability test in sequencing) To demonstrate the improved stability of ffN by 3'-AOM, 3'-O-azidomethyl It was compared side-by-side with a standard MiniSeq(registered trademark) ffN that has a base. Two sets of ffNs. This is a standard incorporation mixture formulation that excludes only DNA polymerase, and the ink can be stored at 45°C for several days. Revised. At each point in time, just before loading new poly into MiniSeq(registered trademark) Melase was added to complete the incorporation mix. The aforementioned sequencing conditions were used. Prephase % is a direct indicator of the percentage of 3'OH-ffN present in the mixture. Therefore, it directly correlates with the stability of the 3' block group. Rephase values ​​were recorded and plotted (Figure 5). At 45°C, 3'-A containing ffN was observed. OM appears to be six times more stable than standard ffN containing a 3'-O-azidomethyl group. This was observed. Sequencing metrics were observed during the stability assay in solution. The observed trend was also confirmed, and the -3'-AOM block is more significant than the 3'-O-azidomethyl group. It remained stable.

[0228] (Example 6. Preparation of 3'-O-thiocarbamate block nucleosides) In this example, T nucleosides protected with various 3'-O-thiocarbamates are skimmed. Prepared according to M8. Scheme 8. Synthesis of 3'-O-dimethylthiocarbamate T nucleoside [ka]

[0229] Preparation of T-7: Put 5'-O- into a nitrogen-purged 100 mL flask that has been dried in an oven. (4,4'-dimethoxytrityl)thymidine (1.0 g, 1.836 mmol) was added. This was co-evaporated with anhydrous DMF (3 x 20 mL) and placed under nitrogen. Anhydrous DCM (9. Add 2 mL of 4-dimethylaminopyridine (224 mg, 0.184 mmol) to the solution. The mixture was stirred at room temperature until a homogeneous solution was formed. Next, 1,1'-thiocarbonyldiimi Quickly add dazole (360 mg, 2.02 mmol) to the nitrogen stream, reseal the reaction mixture, The reaction mixture was stirred at room temperature for 2 hours until all the starting materials were consumed. Filter through a filter and wash the filter cake with dimethyl sulfate (10 mL). Volatile substances The quality was removed under vacuum, and the crude residue was used without further purification.

[0230] Preparation of T-8: Compound T-7 from the previous step was used immediately after being dried in a vacuum. The residue was placed in a 25 mL round-bottom flush under nitrogen, and dimethylamine (2 in THF) was removed. Add M (7.3 mL, 14.6 mmol) and consume all starting material according to TLC. The reaction mixture was stirred for 2 hours until it was clear. All volatile substances were removed under vacuum, and a clear crude residue was obtained. This is formed and purified by silica gel flash column chromatography. T-8 was obtained as a white solid. Yield: 1.15 g (99%). LC-MS (electronography) (Spray negative) 630.23 [MH].

[0231] Preparation of T-9: Starting nucleoside T-8 (320 mg, 0.504 mmol) is subjected to air. It was dissolved in the smallest amount of acetonitrile in the 50 mL round-bottom flask. AcOH / H2O 5: Add 1 (12.5 mL:2.5 mL) of solution all at once, and continue until all the starting material is consumed. The reactants were stirred at room temperature for 2-4 hours. Under vacuum, all volatile substances were evaporated. When the residue is co-evaporated with toluene (2 x 60 mL), the crude product is an off-white solid. It is obtained as follows. The crude product is purified by flash column chromatography and T- 9 was obtained as a white solid. Yield: 123 mg (74%). LC-MS (electrospray) (Negative) [MH] 328.10.

[0232] Following a similar synthesis procedure using the corresponding MeNH2 or NH3, the other two thio Nucleosides with carbamate protecting groups were also prepared. The general reaction scheme is shown below. . [ka]

[0233] (Stability test of 3'-O-thiocarbamate blocking group) Stability testing of 5'-mP3'-DMTC T nucleotides is performed using standard 5'-mP3' The procedure was performed side-by-side in an incorporation buffer containing -O-azidomethyl T nucleotide. [ka]

[0234] In both 5'-mP 3'-DMTC T and 5'-mP 3'-O-azidomethyl T, The final solution volume was 1 mL, and the final concentration of the corresponding nucleotides was 0.1 mM for both. Other components of the buffer solution include ethanolamine (EA), ethanolamine HCl, and N. It contains aCl (100 mM) and EDTA (2.5 mM). The concentrations of the buffer are as follows: That's correct. 0.5 MEA buffer, 0.5 M NaCl, 0.01 M EDTA.

[0235] (Stability testing method) Dissolve 200 μL of 10x buffer in a 1.7 mL polypropylene snap-lock microtube. In addition to the above, it was diluted with an appropriate amount of 18 mΩ water. Next, the corresponding nucleoside was added, and the above was added. Seal the bottle, invert it, gently agitate, or pump it with a micropipette. Mixed further. Take a 40 μL aliquot and analyze by HPLC to determine the starting value (or t It was set to function as (=0). Next, the vial was placed in a preheated heating mantle set to 65°C. Place in a container, cover with a thick layer of aluminum foil, and heat for one month. Take 40 μL aliquots regularly. Samples are collected (once a day during the first week, and once every two days from the second to fourth week), and analyzed by HPLC. The proportion of starting material and the proportion of unblocked (3'-OH) nucleotides in the sample It was decided. By measuring the area of ​​the nucleotide peak and the 3'OH peak, HPLC An analysis was conducted. These values ​​are displayed in a graph, showing the intake buffer between samples. The percentage of unblocked nucleotides used to compare stability. This was used to calculate the 3'-O-azidomethyl blocking group at 65°C. Figure 6 shows the 3'-O-azidomethyl blocking group. Three different thiocarbamate 3' blocking agents for nucleotides blocked by The results of the nucleotide stability comparison are shown. 3'-OC(=S) N H2 or 3'- OC(=S) N The nucleotide containing HCH3 is protected with a standard 3'-O-azidomethyl group. Although less stable than the nucleotides that were previously used, nucleotides containing 3'-DMTC have been improved. It was observed that stability was maintained during the 9-day test period. Therefore, DMTC is a standard A It showed superior stability compared to the didomethyl blocking group.

[0236] (Example 7. 3'-O-thiocarbamate blocking group deblocking test) In this example, 5'-mP 3'-DMTC T and standard 5'-mP 3'-O-A Deblocking or deblocking tests of didomethyl T nucleotides, each blocking The solutions were performed individually based on the specific underlying conditions. It was formulated to mimic the removal reagent as closely as possible and to follow the same methodology. Activated deblocking reagent, The concentrations of buffer and nucleoside are kept the same in all tests, but the concentrations of each component are the same. The uniformity was unique. Thus, the difference in rates observed between individual deblocking chemicals This is not due to differences in the concentration of the formulation. [ka]

[0237] (General methodology for block removal testing) Each reaction component is individually formulated as a concentrated stock in 18 mΩ of water and stored appropriately. The aliquots were combined in the following specific order: pre-formulated deblocking reagents. The reaction was initiated by the addition of [substance name]. Final concentration: nucleoside (0.1 mM), activated deblocking. Reagent (1 mM), additive (specific to the deblocking reagent), buffer (100 mM). Final volume Volume: 2000μL.

[0238] In a 3 mL glass vial, add the pre-prescribed buffer, followed by the pre-prescribed additive. The solution was added. This was diluted with the correct amount of 18 mΩ water and stirred for 10 minutes. Next, Nuku An aliquot of the rheotide solution was added and stirred for 5 minutes. Next, a 40 μL aliquot was taken. Then, add the quenching reagent and analyze the reference (or t=0 min) peak by HPLC. Next, the deblocking reagent was added to the stirred solution all at once, and timing was started. Then, take a 40 μL aliquot and immediately quench it with an appropriate quenching reagent, and HP LC analysis was performed to determine the unblocked nucleotides that occurred at these specified points in time. The quantity was determined. The results were plotted in a graph to compare the efficiency and effectiveness of unblocking. It is used for [purpose].

[0239] (DMTC deblocking) Nucleotides: 5'-mP 3'-DMTC T. Active deblocking reagent: NaI O4 (0.1M in 18mΩ water) or Oxone (registered trademark) (0.1M in 18mΩ water) M). Additives: None. Buffer for NaIO4: pH 6.75 phosphate buffer (18m Ω (1M in water). Buffer for Oxone(registered trademark): pH 8.65 phosphate buffer ( (18 mΩ in water, 1 M). Rapid cooling reagent: sodium thiosulfate. 3'-O-azidomethyl deblocking agent. The fermentation conditions are the same as those described in Example 3.

[0240] HPLC analysis involves nucleoside peaks, 3'-OH peaks, and HPLC chromatography. This was performed by measuring the area of ​​other nucleotide peaks displayed in the lamb. These values ​​are used to determine the parsing of the start nucleotide and the unblocked nucleotide. Calculate the duration and display it in a graph to show the unblocking rate, efficiency, and effectiveness between samples. The efficiency was compared. The comparison results are shown in Figure 7. Deblocking of DMTC by NaIO4 is efficient. It was observed that this was not the case. However, when DMTC was cut with Oxone(registered trademark) In combination, the proportion of remaining starting material containing nucleotides with DMTC blocking groups is significantly reduced. In summary, DMTC has a standard azidomethyl blocking group deblocking rate. It exhibits a superior deblocking rate (using Oxone®).

Claims

1. A removable 3'-OH blocking group covalently bonded to a 3'-carbon atom 【Chemistry 1】 A nucleotide comprising ribose or deoxyribose having a detectable label, which is covalently bonded to the ribose.

2. The nucleotide according to claim 1, comprising 2'-deoxyribose.

3. The nucleotide according to claim 1 or claim 2, comprising a triphosphate group at the 5' position of the ribose or deoxyribose.

4. The nucleotide according to any one of claims 1 to 3, covalently bonded to a detectable label via a cleavable linker.

5. The nucleotide according to claim 4, wherein the detectable label is covalently bonded to the nucleic acid base of the nucleotide via a cleavable linker.

6. The nucleotide according to claim 5, wherein the cleavable linker comprises an azide moiety, an -O-allyl moiety, a disulfide moiety, an acetal moiety, or a thiocarbamate moiety.

7. The severable linker is a portion selected from the group consisting of: 【Chemistry 2】 (In the formula, X is selected from the group consisting of O, S, NH, and NQ; Q is a C1-C10 substituted or unsubstituted alkyl group; Y is selected from the group consisting of O, S, NH, and N (allyl); T is hydrogen or a C1-C10 substituted or unsubstituted alkyl group; and * indicates the location where the said part is attached to the rest of the nucleotide.) The nucleotide according to claim 5, comprising:

8. The severable linker is as follows: 【Transformation 3】 (In the formula, B is a nucleic acid base; Z is a -N3 (azide), -O-C1-C6 alkyl, -O-C2-C6 alkenyl, or -O-C2-C6 alkynyl; F1 includes a fluorescent label which may include an additional linker structure.) A nucleotide according to claim 5, selected from the group consisting of the following.

9. The nucleotide according to any one of claims 5 to 8, wherein the 3'-OH blocking group and the cleavable linker are removed under the same chemical reaction conditions.

10. The following: 【Chemistry 4】 A nucleotide according to any one of claims 1 to 3, comprising a nucleic acid base selected from the group consisting of the following.

11. The following: 【Transformation 5】 (In the formula, L is the linker and Dye is the fluorescent dye.) A nucleotide according to any one of claims 1 to 9, comprising a nucleic acid base selected from the group consisting of the following.

12. The following: 【Transformation 6】 (In the formula, L is a linker, Dye is a fluorescent dye, and R is a removable 3'-OH blocking group covalently bonded to the 3'-carbon atom) 【Transformation 7】 (The ribose or deoxyribose having the above properties.) A nucleotide according to any one of claims 1 to 9, having a structure selected from the group consisting of the following.

13. The nucleotide according to any one of claims 1 to 12, wherein the removable 3'-OH blocking group simultaneously imparts at least 5% improved stability compared to a 3'-OH protected with azidomethyl under the same conditions for the same period.

14. An oligonucleotide incorporating a nucleotide according to any one of claims 1 to 13, wherein a phosphodiester bond is formed between the 3'-carbon atom of the oligonucleotide and the 5'-carbon atom of the nucleotide.

15. The oligonucleotide according to claim 14, which is immobilized on the surface of an array.

16. A method for preparing a growth polynucleotide complementary to a target single-stranded polynucleotide in a sequencing reaction, comprising incorporating a nucleotide according to any one of claims 1 to 13 into a growth complementary polynucleotide, wherein the incorporation of the nucleotide prevents the subsequent introduction of any nucleotides into the complementary growth polynucleotide.

17. The method according to claim 16, comprising incorporating the nucleotide according to any one of claims 3 to 13 into the complementary growth polynucleotide.

18. The method according to claim 16 or 17, wherein the incorporation of nucleotides is achieved by polymerase.

19. A method for determining the sequence of a target single-stranded polynucleotide, comprising: (a) Incorporating the nucleotide according to any one of claims 4 to 13 into a copy polynucleotide chain complementary to at least a portion of the target single-stranded polynucleotide; (b) detecting the identity of the nucleotides incorporated into the copy polynucleotide chain; and (c) Chemically remove a detectable label and a removable 3'-OH blocking group from the nucleotides incorporated into the copy polynucleotide chain according to any one of claims 4 to 13.

20. The method according to claim 19, wherein the nucleotide incorporated in step (a) is a nucleotide triphosphate.

21. (d) The method according to claim 19 or 20, further comprising washing off the chemically removed detectable label and the removable 3'-OH blocking group from the copy polynucleotide chain.

22. The method according to claim 21, wherein a palladium scavenger is used in step (d).

23. The method according to claim 21 or claim 22, further comprising repeating steps (a) to (d) until the sequence of the portion of the template polynucleotide chain is determined.

24. The method according to any one of claims 21 to 23, wherein steps (a) to (d) are repeated at least 50 times.

25. The method according to any one of claims 19 to 24, wherein a detectable label and a removable 3'-OH blocking group derived from a nucleotide incorporated in the copy polynucleotide chain is removed in a single chemical reaction.

26. The method of claim 25, wherein step (c) comprises contacting the incorporated nucleotide with a cleavage solution containing a palladium catalyst.

27. ​​The method according to any one of claims 19 to 24, wherein a detectable label and a removable 3'-OH blocking group derived from a nucleotide incorporated in the copy polynucleotide chain are removed in two separate chemical reactions.

28. The method of claim 27, wherein step (c) comprises contacting the incorporated nucleotide with a cleavage solution comprising a phosphine and a palladium catalyst.

29. The method according to claim 28, wherein the phosphine is tris(hydroxymethyl)phosphine, tris(hydroxyethyl)phosphine, or tris(hydroxypropyl)phosphine.

30. The method according to any one of claims 26, 28, or 29, wherein the cleavage solution containing the palladium catalyst further comprises one or more buffering reagents selected from the group consisting of primary amines, secondary amines, tertiary amines, carbonates, phosphates, and borates, and combinations thereof.

31. The method according to claim 30, wherein the buffer reagent is selected from the group consisting of ethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris), glycine, carbonate, phosphate, borate, 2-dimethylaminoethanol (DMAA), 2-diethylaminoethanol (DEEA), N,N,N',N'-tetramethylethylenediamine (TEMED), and N,N,N',N'-tetraethylenediamine (TEEDA), and combinations thereof.

32. The method according to any one of claims 26 or 28-31, wherein the cutting solution further comprises ascorbic acid or a salt thereof.

33. The method according to any one of claims 19 to 32, wherein the method is carried out in multiple formats.

34. The method according to any one of claims 19 to 33, wherein the method is carried out in an array format using surface-bound target nucleic acids, and the target nucleic acids are bound to the surface in a spatially distinguishable manner.

35. The method according to claim 34, wherein the array includes a plurality of copies, each having the same sequence of target nucleic acid at a different position.

36. The method according to claim 35, wherein a plurality of copies of the target nucleic acid are formed by bridge amplification.

37. The method according to claim 35, wherein a plurality of copies of the target nucleic acid are present in the concatemer.

38. The use according to any one of claims 34 to 37, wherein the array has a concentration of at least 10 features / cm².

39. The method according to any one of claims 19 to 38, wherein the method uses a first nucleotide type detected in a first channel, a second nucleotide type detected in a second channel, a third nucleotide type detected in both the first and second channels, and a fourth nucleotide type lacking a label that is not detected or is minimally detected in either channel.

40. The method according to any one of claims 19 to 38, wherein the method uses four distinct nucleotide types, and each nucleotide type has a spectrally different label.

41. The method according to any one of claims 24 to 40, wherein the pre-phasing value is less than 0.25 after exceeding 50 cycles.

42. The method according to any one of claims 24 to 41, wherein the fading value is less than 0.25 after exceeding 50 cycles.

43. The method according to any one of claims 19 to 42, wherein the incorporation of the nucleotide is achieved by DNA polymerase.

44. A kit comprising one or more nucleotides corresponding to the nucleotides described in any one of claims 1 to 13.

45. The following structure in which at least one nucleotide is covalently bonded to the 3'-carbon atom of 2'-deoxyribose: 【Transformation 8】 The kit according to claim 44, wherein the nucleotide 5'-triphosphate has a 3'-OH blocking group.

46. The kit according to claim 44 or 45, further comprising an enzyme and a buffer suitable for the operation of the enzyme.

47. The kit according to claim 46, wherein the enzyme is a polymerase.

48. The kit according to claim 47, wherein the polymerase is DNA polymerase.

49. The kit according to any one of claims 44 to 48, comprising at least one nucleotide labeled with a detectable label according to any one of claims 4 to 13.

50. A kit according to any one of claims 44 to 48, comprising two, three, or four types of nucleotides labeled with a detectable label according to any one of claims 4 to 13.

51. The kit according to claim 50, further comprising a dark nucleotide that does not have a detectable label.

52. The kit according to claim 50 or 51, wherein the different types of labeled nucleotides are labeled with spectrally distinguishable dye compounds.

53. The kit according to any one of claims 50 to 52, wherein at least one type of nucleotide is labeled with two or more different dyes in the nucleic acid base.

54. The kit according to any one of claims 50 to 53, wherein a first type of nucleotide is labeled with a first dye, a second type of nucleotide is labeled with a second dye different from the first dye, a third type of nucleotide is labeled as a mixture of the first and second dyes, or as a mixture of the first, second and third dyes, and a fourth nucleotide is unlabeled.

55. The kit according to claim 50, comprising four types of nucleotides labeled with detectable labels, wherein the nucleotides labeled with detectable labels are A, C, T, and G, each type of nucleotide has a fluorescent label having a different fluorescence maximum value, and each of the fluorescent labels is distinguishable from the other three fluorescent labels.

56. The kit according to any one of claims 44 to 55, further comprising tris(hydroxypropyl)phosphines, a palladium (Pd) catalyst, ascorbic acid or salts thereof, and a buffer selected from the group consisting of ethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris), glycine, carbonate, phosphate, borate, 2-dimethylaminoethanol (DMAA), 2-diethylaminoethanol (DEEA), N,N,N',N'-tetramethylethylenediamine (TEMED), and N,N,N',N'-tetraethylenediamine (TEEDA), and combinations thereof.

57. The kit according to any one of claims 44 to 56, further comprising a washing solution containing a palladium scavenger.

58. A kit according to any one of claims 44 to 57 for use in multiple sequencing by synthesis.

59. Use of a nucleotide according to any one of claims 1 to 13 in synthetic multiple sequencing.

60. The use according to claim 59, wherein the sequence determination step is repeated at least 50 times.

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