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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6869—Methods for sequencing
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/06—Pyrimidine radicals
  • C07H19/073—Pyrimidine radicals with 2-deoxyribosyl as the saccharide radical
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/06—Pyrimidine radicals
  • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
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  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/14—Pyrrolo-pyrimidine radicals
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  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/16—Purine radicals
  • C07H19/173—Purine radicals with 2-deoxyribosyl as the saccharide radical
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/16—Purine radicals
  • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

• Some embodiments described herein relate to modified nucleotides or nucleosides comprising 3 '-hydroxy protecting groups and their use in polynucleotide sequencing methods. Some embodiments described herein relate to method of preparing the 3 '-hydroxy protected nucleotides or nucleosides.
• nucleic acids An example of the technologies that have improved the study of nucleic acids is the development of fabricated arrays of immobilized nucleic acids. These an-ays consist typically of a high-density matrix of polynucleotides immobilized onto a solid support material. See. e.g., Fodor et al., Trends Biotech. 12: 1 -26, 1994, which describes ways of assembling the nucleic acids using a chemically sensitized glass surface protected by a mask, but exposed at defined areas to allow attachment of suitably modified nucleotide phosphoramidites.
• Fabricated arrays can also be manufactured by the technique of "spotting" known polynucleotides onto a solid support at predetermined positions (e.g., Stimpson et al, Proc. Natl. Acad. Sci. 92: 6379-6383, 1995).
• SBS sequencing by synthesis
• a structural modification (“protecting group") is added to each labeled nucleotide that is added to the growing chain to ensure that only one nucleotide is incoiporated. After the nucleotide with the protecting group has been added, the protecting group is then removed, under reaction conditions which do not interfere with the integrity of the DNA being sequenced. The sequencing cycle can then continue with the incorporation of the next protected, labeled nucleotide.
• nucleotides and more usually nucleotide triphosphates, generally require a 3'-hydroxy protecting group so as to prevent the polymerase used to incorporate it into a polynucleotide chain from continuing to replicate once the base on the nucleotide is added.
• a 3'-hydroxy protecting group so as to prevent the polymerase used to incorporate it into a polynucleotide chain from continuing to replicate once the base on the nucleotide is added.
• the protecting group should prevent additional nucleotide molecules from being added to the polynucleotide chain whilst simultaneously being easily removable from the sugar moiety without causing damage to the polynucleotide chain.
• the modified nucleotide needs to be tolerated by the polymerase or other appropriate enzyme used to incorporate it into the polynucleotide chain.
• the ideal protecting group therefore exhibits long term stability, be efficiently incoiporated by the polymerase enzyme, cause blocking of secondary or further nucleotide incorporation and have the ability to be removed under mild conditions that do not cause damage to the polynucleotide structure, preferably under aqueous conditions.
• Some embodiments described herein relate to a modified nucleotide or nucleoside molecule comprising a purine or pyrimidine base and a ribose or deoxyribose sugar moiety having a removable 3'-hydroxy protecting group fomiing a structure -0-C(R)j T 3 covalently attached to the 3'-carbon atom, wherein
• each R 1 and R 2 is independently selected from hydrogen, optionally substituted alkyl or halogen;
• R 3 is selected from hydrogen or optionally substituted alkyl
• each R 4 and R 5 is independently selected from hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted aralkyl;
• each R 6 and R 9 is selected from hydrogen, optionally substituted alkyl or halogen; each R ⁇ R 8 , R i n and R u is independently selected from hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted aralkyl;
• n is an integer of 0 to 3;
• n is an integer of 0 to 3; provided that the total of m + n equals to 3; and p is an integer of 0 to 6; provided that
• R 1 and R 2 cannot both be halogen
• At least one R is not hydrogen.
• Some embodiments described herein relate to a method of preparing a growing polynucleotide complementary to a target single-stranded polynucleotide in a sequencing reaction, comprising incorporating a modified nucleotide molecule described herein into the growing complementary polynucleotide, wherein the incorporation of the modified nucleotide prevents the introduction of any subsequent nucleotide into the growing complementary polynucleotide.
• Some embodiments described herein relate to a method for determining the sequence of a target single-stranded polynucleotide, comprising monitoring the sequential incorporation of complementary nucleotides, wherein at least one complementary nucleotide incorporated is a modified nucleotide molecule described herein; and detecting the identity of the modified nucleotide molecule.
• the incorporation of the modified nucleotide molecule is accomplished by a terminal transferase, a terminal polymerase or a reverse transcriptase.
• kits comprising a plurality of modified nucleotide or nucleoside molecule described herein, and packaging materials therefor.
• the identity of the modified nucleotide is determined by detecting the detectable label linked to the base.
• the 3 '-hydroxy protecting group and the detectable label are removed prior to introducing the next complementary nucleotide.
• the 3 '-hydroxy protecting group and the detectable label are removed in a single step of chemical reaction.
• FIG. 1A illustrates a variety of 3 -OH protecting groups.
• FIG. IB illustrated the thermal stability of various 3'-OH protecting groups.
• FIG. 2A illustrates the deprotection rate curve of three different 3'-OH protecting groups.
• FIG. 2B shows a chart of the deprotection half time of three different 3'-OH protecting groups
• FIG. 3 shows the phasing and prephasing values of various modified nucleotide with a thermally stable 3'-OH protecting group in comparison, and the standard protecting group.
• FIG. 4A shows the 2 x 400bp sequencing data of mono-F ff s-A-isomer in incorporation mix (IMX).
• FIG. 4B shows the 2*400bp sequencing data of mono-F ffNs-B-isomer in incorporatio mix ( IMX).
• One embodiment is a modified nucleotide or nucleoside comprising a 3'-OH protecting group.
• the 3'-OH protecting group is a monofluoromethyl substituted azidomethyl protecting group.
• the 3 * -OH protecting group is a C-amido substituted azidomethyl protecting group.
• Still another embodiment relates to modified nucleotides having difluoromethyl substituted azidomethyl 3'-OH protecting groups.
• the above terms are to be interpreted synonymously with the plirases "having at least” or “including at least.”
• the term “comprising” means that the process includes at least the recited steps, but may include additional steps.
• the term “comprising” means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components.
• the temi "array” refers to a population of different probe molecules that are attached to one or more substrates such that the different probe molecules can be differentiated from each other according to relative location.
• An array can include different probe molecules that are each located at a different addressable location on a substrate.
• an array can include separate substrates each bearing a different probe molecule, wherein the different probe molecules can be identified according to the locations of the substrates on a surface to which the substrates are attached or according to the locations of the substrates in a liquid.
• Exemplary arrays in which separate substrates are located on a surface include, without limitation, those including beads in wells as described, for example, in U.S. Patent No.
• covalently attached refers to the forming of a chemical bonding that is characterized by the sharing of pairs of electrons between atoms.
• a covalently attached polymer coating refers to a polymer coating that forms chemical bonds with a functionalized surface of a substrate, as compared to attachment to the surface via other means, for example, adhesion or electrostatic interaction. It will be appreciated that polymers that are attached covalently to a surface can also be bonded via means in addition to covalent attachment.
• any "R” group(s) such as, without limitation, R ⁇ R 3 , R 4 , R 5 , R f R 7 , and R 8 represent substituents that can be attached to the indicated atom.
• An R group may be substituted or unsubstituted. If two "R” groups are described as being “taken together” the R groups and the atoms they are attached to can form a cycloalkyl, aryl, heteroaryl, or heterocycle.
• R 2 and R ⁇ or R 2 , R ⁇ or R 4 , and the atom to which it is attached are indicated to be “taken together” or “joined together” it means that they are covalently bonded to one another to form a ring, an example of which is set forth below:
• alkyl refers to a straight or branched hydrocarbon chain that comprises a fully saturated (no double or triple bonds) hydrocarbon group.
• the alkyl group may have 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as “1 to 20” refers to each integer in the given range inclusive of the endpopints; e.g., " 1 to 20 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, although the present definition also covers the occurrence of the term "alkyl” where no numerical range is designated).
• the alkyl group may also be a medium, size alkyl having about 7 to about 10 carbon atoms.
• the alkyl group can also be a lower alkyl having 1 to 6 carbon atoms.
• the alkyl group of the compounds may be designated as "C 1-C4 alkyl" or similar designations.
• “C 1 -C4 alkyl” indicates that there are one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl.
• Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, and hexyls.
• the alkyl group may be substituted or unsubstitiited.
• alkenyl refers to an alkyl group that contains in the straight or branched hydrocarbon chain one or more double bonds.
• An alkenyl group may be unsubstitiited or substituted.
• alkynyl refers to an alkyl group that contains in the straight or branched hydrocarbon chain one or more triple bonds.
• An alkynyl group may be unsubstituted or substituted.
• cycloalkyl refers to a completely saturated (no double or triple bonds) mono- or multi- cyclic hydrocarbon ring system. When composed of two or more rings, the rings may be joined together in a fused fashion. Cycloalkyl groups can contain 3 to 10 atoms in the ring(s). In some embodiments, cycloalkyl groups can contain 3 to 8 atoms in the ring(s). A cycloalkyl group may be unsubstituted or substituted.
• Typical cycloalkyl groups include, but are in no way limited to, cyclopropyl, cyc!obutyl, cyclopentyl, cyclohexyl, cyclohcptyl, and cyclooctyl.
• aryl refers to a carbocyclic (all carbon) monocyclic or multicyclic aromatic ring system (including, e.g., fused, bridged, or spiro ring systems where two carbocyclic rings share a chemical bond, e.g., one or more aryl rings with one or more aryl or non- aryl rings) that has a fully delocalized pi-electron system throughout at least one of the rings.
• the number of carbon, atoms in an aryl group can vary.
• the aryl group can be a Q-C aryl group, a Cfi-C-w aryl group, or a C F) aryl group.
• aryl groups include, but are not limited to, benzene, naphthalene, and azulene.
• An aryl group may be substituted or unsubstituted.
• heterocyclyl refers to ring systems including at least one lieteroatom (e.g., O, N, S). Such systems can be unsaturated, can include some unsaturation, or can contain some aromatic portion, or be all aromatic. A heterocyclyl group may be unsubstituted or substituted.
• heteroaryl refers to a monocyclic or multicyclic aromatic ring system (a ring system having a least one ring with a fully delocalized pi-electron system) that contain(s) one or more hcteroatoms, that is, an element other than carbon, including but not limited to, nitrogen, oxygen, and sulfur, and at least one aromatic ring.
• the number of atoms in the ring(s) of a heteroaryl group can vary.
• a heteroaryl group can contain 4 to 14 atoms in the ring(s), 5 to 10 atoms in the ring(s) or 5 to 6 atoms in the ring(s).
• heteroaryl includes fused ring systems where two rings, such as at least one aryl ring and at least one heteroaryl ring, or at least two heteroaryl rings, share at least one chemical bond.
• heteroaryl rings include, but are not limited to, furan, furazan, thiophene, benzothiophene, phthalazine, pyrrole, oxazole, benzoxazole, 1 ,2,3-oxadiazole, 1 ,2,4-oxadiazole, thiazole, 1 ,2,3-thiadiazole, 1 ,2,4-thiadiazole, benzothiazole.
• a heteroaryl group may be substituted or unsubstituted.
• heteroalicyclic or “heteroalicyclyl” refers to three-, four-, five-, six-, seven-, eight-, nine-, ten-, up to 18-membered monocyclic, bicyclic, and tricyclic ring system wherein carbon atoms together with from 1 to 5 heteroatoms constitute said ring system.
• a heterocycle may optionally contain one or more unsaturated bonds situated in such a way, however, that a fully delocalized pi-electron system does not occur throughout all the rings.
• the heteroatoms are independently selected from oxygen, sulfur, and nitrogen.
• a heterocycle may further contain one or more carbonyl or thiocarbonyl functionalities, so as to make the definition include oxo- systems and thio-systems such as lactams, lactones, cyclic imides, cyclic thioimides, and cyclic carbamates. When composed of two or more rings, the rings may be joined together in a fused fashion. Additionally, any nitrogens in a heteroalicyclic may be quaternized. Heteroalicyclyl or heteroalicyclic groups may be unsubstituted or substituted.
• heteroalicyclic or “heteroalicyclyl ' " groups include but are not limited to, 1 ,3-dioxin, 1 ,3-dioxane, 1 ,4-dioxane, 1 ,2- dioxolane.
• benzo- fused analogs e.g., benzimidazolidinone, tetrahydroquino!ine, 3 ,4-methy!enedioxyphenyl.
• aralkyl and aryl(alkyl) refer to an aryl group connected, as a substituent, via a lower alkylene group.
• the lower alkylene and aryl group of an aralkyl may be substituted or unsubstituted. Examples include but are not limited to benzyl, 2-phenylalkyl, 3- phenylalkyl, and naphthylalkyl.
• heteroarylkyf and “heteroaryl(alkyl)” refer to a heteroaiyl group connected, as a substituent, via a lower alkylene group.
• the lower alkylene and heteroaiyl group of heteroaralkyl may be substituted or unsubstituted. Examples include but are not limited to 2- thienylalkyl, 3 -thienylalkyi, fury!alkyl, thienylalkyi, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyi, and imidazolylalkyh and their benzo- fused analogs.
• alkoxy refers to the formula -OR wherein R is an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl or a cycloalkynyl is defined as above.
• R is an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl or a cycloalkynyl is defined as above.
• a non- limiting list of alkoxys is methoxy, ethoxy, n-propoxy, 1 -methyl ethoxy (isopropoxy), n-butoxy, iso- butoxy, sec-butoxy, and tert-butoxy.
• An alkoxy may be substituted or unsubstituted.
• a C-amido may be substituted or unsubstituted.
• An N-amido may be substituted or unsubstituted.
• halogen atom means any one of the radio-stable atoms of column 7 of the Periodic Table of the Elements, such as, fluorine, chlorine, bromine, and iodine.
• amine refers to a -NH 2 group wherein one or more hydrogen can be optionally substituted by a R group.
• R can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or (heteroal icyclyl )al kyl .
• aldehyde refers to a -R -C(0)H group, wherein R c can be absent or independently selected from alkylene, alkenyl ene. alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, arylene, heteroaiylene, heteroal icyclylene, aralky!ene, or (heteroa!icyclyl)alkylene.
• hydroxy refers to a -OH group.
• cyano refers to a "-CN” group.
• thiol refers to a -SH group.
• carboxylic acid refers to ⁇ C(0)OH.
• thiocyanate refers to -S-C ⁇ N group.
• oxo-amlne refers to -0-NH 2 group, wherein one or more hydrogen of the -N3 ⁇ 4 can be optionally substituted by a R group.
• R can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryi, heteroalicyclyl, aralkyl, or ( eteroalicyclyl)alkyl. .
• a "nucleotide” includes a nitrogen containing heterocyclic base, a sugar, and one or more phosphate groups. They are monomenc units of a nucleic acid sequence.
• the sugar is a ribose, and in DNA a deoxyribose, i.e. a sugar lacking a hydroxyl group that is present in ribose.
• the nitrogen containing heterocyclic base can be purine or pyrimidine base.
• Purine bases include adenine (A) and guanine (G), and modified derivatives or analogs thereof.
• Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and modified derivatives or analogs thereof.
• C cytosine
• T thymine
• U uracil
• the C- l atom of deoxyribose is bonded to N- l of a pyrimidine or N-9 of a purine.
• nucleoside is structurally similar to a nucleotide, but is missing the phosphate moieties.
• An example of a nucleoside analogue would be one in which the label is linked to the base and there is no phosphate group attached to the sugar molecule.
• nucleoside is used herein in its ordinary sense as understood by those skilled in the art. Examples include, but are not limited to, a ribonucleoside comprising a ribose moiety and a deoxyribonucleoside comprising a deoxyribose moiety.
• a modified pentose moiety is a pentose moiety in which an oxygen atom has been replaced with a carbon and/or a carbon has been replaced with a sulfur or an oxygen atom.
• a "nucleoside” is a monomer that can have a substituted base and/or sugar moiety. Additionally, a nucleoside can be incorporated into larger DNA and/or RNA polymers and oligomers.
• purine base is used herein in its ordinary sense as understood by those skilled in the art, and includes its tautomers.
• pyrimidine base is used herein in its ordinary sense as understood by those skilled in the art, and includes its tautomers.
• a non- limiting list of optionally substituted purine-bases includes purine, adenine, guanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (e.g. 7-methylguanine), theobromine, caffeine, uric acid and isoguanine.
• pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil and 5-alkylcytosine (e.g., 5-methylcytosine).
• “derivative” or “analogue” means a synthetic nucleotide or nucleoside derivative having modified base moieties and/or modified sugar moieties. Such derivatives and analogs are discussed in, e.g., Scheit, Nucleotide Analogs (John Wiley & Son, 1980) and Uhlman et al, Chemical Reviews 90:543-584, 1990. Nucleotide analogs can also comprise modified phosphodiester linkages, including phosphorothioate, phosphorodithioate, a!kyl- phosphonate, phosphoranilidate and phosphoramidate linkages. "Derivative”, “analog” and “modified” as used herein, may be used interchangeably, and are encompassed by the terms “nucleotide” and “nucleoside” defined herein.
• phosphate is used in its ordinary sense as understood by those skilled in the art, and includes its protonated forms (for example, ).
• diphosphate diphosphate
• triphosphate are used in their ordinary sense as understood by those skilled in the art, and include protonated forms.
• protecting group and “protecting groups” as used herein refer to any atom or group of atoms that is added to a molecule in order to prevent existing groups in the molecule from undergoing unwanted chemical reactions. Sometimes, “protecting group” and “blocking group” can be used interchangeably.
• the prefi es "photo” or "photo-" mean relating to light or electromagnetic radiation.
• the ter can encompass all or part of the electromagnetic spectrum including, but not limited to, one or more o the ranges commonly known as the radio, microwave, infrared, visible, ultraviolet. X-ray or gamma ray parts of the spectrum.
• the part of the spectrum can be one that is blocked by a metal region of a surface such as those metals set forth herein.
• the part of the spectrum can be one that passes through an interstitial region of a surface such as a region made of glass, plastic, silica, or other material set forth herein.
• radiation can be used that is capable of passing through a metal.
• radiation can be used that is masked by glass, plastic, silica, or other material set forth herein.
• phasing refers to phenomena in SBS that is caused by incomplete removal of the 3' terminators and fluorophores, and failure to complete the incorporation of a portion of DNA strands within clusters by polymerases at a given sequencing cycle.
• Pre- phasing is caused by the incorporation of nucleotides without effective 3' terminators and the incorporation event goes 1 cycle ahead. Phasing and pre-phasing cause the extracted intensities for a specific cycle to consist of the signal of the current cycle as well as noise from the preceding and following cycles. As the number of cycles increases, the fraction of sequences per cluster affected by phasing increases, hampering the identification of the correct base.
• Pre-phasing can be caused by the presence of a trace amount of unprotected or unblocked 3'-OH nucleotides during sequencing by synthesis (SBS).
• SBS sequencing by synthesis
• the unprotected 3'-OH nucleotides could be generated during the manufacturing processes or possibly during the storage and reagent handling processes.
• nucleotide analogues which decrease the incidence of pre-phasing is surprising and provides a great advantage in SBS applications over existing nucleotide analogues.
• the nucleotide analogues provided can result in faster SBS cycle time, lower phasing and pre-phasing values, and longer sequencing read length.
• one of R is hydrogen and the other R is -C(R ) m (R ) endeavour.
• -C(R' ) M (R 2 ) N is selected from -CHF 2 , -CH 2 F, -CHC1 2 or -CH 2 CI.
• -C(R L ) M (R 2 ) N is -CHF 2 .
• -C(R') m (R 2 ) n is -CH 2 F.
• R 3 is hydrogen.
• one of R is hydrogen and the other R is - C(R 6 ) 2 0(CH 2 ) p NR 7 R 8 .
• both R are hydrogen.
• both R 7 and R 8 are hydrogen.
• p is 0. In some other such embodiment, p is 6.
• both R are hydrogen.
• both R 10 and R 1 1 are hydrogen.
• R 10 is hydrogen and R 1 1 is a substituted alkyl.
• R 1 1 is an amino substituted alkyl.
• the 3' -OH protecting group is removed in a deprotecting reaction with a phosphine.
• the azido group in -C(R) 2 N3 can be converted to an amino group by contacting the modified nucleotide or nucleoside molecules with a phosphine.
• the azido group in -C( ) 2 N3 may be converted to an amino group by contacting such molecules with the thiols, in particular water-soluble thiols such as dithiothreitol (DTT).
• the phosphine is tris(hydroxymethyl)phosphinc (THP). Unless indicated otherwise, the reference to nucleotides is also intended to be applicable to nucleosides.
• Detection can be carried out by any suitable method, including fluorescence spectroscopy or by other optical means.
• the preferred label is a fluorophore, which, after absorption of energy, emits radiation at a defined wavelength.
• Many suitable fluorescent labels are known. For example, Welch et al. (Chem. Eur. J. 5(3):951 -960, 1999) discloses dansyl-fonctionalised fluorescent moieties that can be used in the present invention. Zhu et al. (Cytometry! 28:206-21 1 , 1997) describes the use of the fluorescent labels Cy3 and CyS, which can also be used in the present invention.
• Labels suitable for use are also disclosed in Prober et al. (Science 238:336-341 , 1987); Connell et al. (BioTechmgues 5(4):342-384, 1987), Ansorge et al. (Nucl Acids Res. 15(1 1):4593- 4602, 1987) and Smith et al. (Nature 321 :674, 1986).
• Other commercially available fluorescent labels include, but are not limited to, fluorescein, rhodamine (including TMR, texas red and Rox), alexa, bodipy, acridine, coumarin, pyrene, benzanthracene and the cyanins.
• Multi-fluor dendrimeric systems J. Am. Chem. Soc. 123:8101 -8108, 2001
• fluorescent labels are preferred, other forms of detectable labels will be apparent as useful to those of ordinaiy skill in the art.
• microparticles including quantum dots (Empodocles et al., Nature 399: 126-130, 1999), gold nanoparticles (Reichcrt et al., Anal. Chem. 72:6025-6029, 2000) and microbeads (Lacoste et al, Proc. Natl. Acad. Sci USA 97( 17 ):9461 -9466, 2000) can all be used.
• Multi-component labels can also be used in the present application.
• a multi- component label is one which is dependent on the interaction with a further compound for detection.
• the most common multi-component label used in biology is the biotin-streptavidin system. Biotin is used as the label attached to the nucleotide base. Streptavidin is then added separately to enable detection to occur.
• Other multi-component systems are available. For example, dinitrophenol has a commercially available fluorescent antibody that can be used for detection.
• nucleotides are also intended to be applicable to nucleosides.
• the present application will also be further described with reference to DNA, although the description will also be applicable to RNA, PNA, and other nucleic acids, unless otherwise indicated.
• the purine or pyrimidinc base of the modified nucleotide or nucleoside molecules can be linked to a detectable label as described above.
• the linkers used are cleavable. The use of a cleavable linker ensures that the label can, if required, be removed after detection, avoiding any interfering signal with any labeled nucleotide or nucleoside incorporated subsequently.
• the linkers used are non-cleavable. Since in each instance where a labeled nucleotide of the invention is incorporated, no nucleotides need to be subsequently incorporated and thus the label need not be removed from the nucleotide.
• Sanger and Sanger-type methods generally operate by the conducting of an experiment in which eight types of nucleotides are provided, four of which contain a 3 '-OH group; and four of which omit the OH group and which are labeled differently from each other.
• the nucleotides used which omit the 3'-OH group - dideoxy nucleotides (ddNTPs).
• ddNTPs dideoxy nucleotides
• nucleotides of the present application may be of utility in Sanger methods and related protocols since the same effect achieved by using ddNTPs may be achieved by using the 3'-OH protecting groups described herein: both prevent incorporation of subsequent nucleotides.
• monitoring of the incorporation of 3'-OH protected nucleotides may be determined by use of radioactive ⁇ P in the phosphate groups attached. These may be present in either the ddNTPs themselves or in the primers used for extension.
• Cleavable linkers are known in the art, and conventional chemistry can be applied to attach a linker to a nucleotide base and a label.
• the linker can be cleaved by any suitable method, including exposure to acids, bases, nucleophi!es, electrophilcs, radicals, metals, reducing or oxidizing agents, light, temperature, enzymes etc.
• the linker as discussed herein may also be cleaved with the same catalyst used to cleave the 3'-0-protccting group bond.
• Suitable linkers can be adapted from standard chemical protecting groups, as disclosed in Greene & Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, Further suitable cleavable linkers used in solid- phase synthesis are disclosed in Guillier et al. (Chem. Rev. 100:2092-2157, 2000).
• nucleoside cleavage site can be located at a position on the linker that ensures that part of the linker remains attached to the nucleotide base after cleavage.
• the linker can be attached at any position on the nucleotide base provided that Watson-Crick base pairing can still be carried out.
• the linker is attached via the 7-position of the purine or the preferred deazapurine analogue, via an 8-modified purine, via an N-6 modified adenosine or an N-2 modified guanine.
• attachment is preferably via the 5-positioii on cytosine, thymidine or uracil and the N-4 position on cytosine.
• Electrophilically cleaved linkers are typically cleaved by protons and include cleavages sensitive to acids.
• Suitable linkers include the modified benzylic systems such as trityl, p- alkoxybenzyl esters and p-alkoxybenzyl amides.
• Other suitable linkers include tert- butyloxycarbonyl (Boc) groups and the acetal system.
• thiophilic metals such as nickel, silver or mercury
• tliioacetal or other sulfur-containing protecting groups can also be considered for the preparation of suitable linker molecules.
• Nuclcophilic cleavage is also a well recognised method in the preparation of linker molecules.
• Groups such as esters that are labile in water (i.e., can be cleaved simply at basic pH) and groups that are labile to non-aqueous nucleophiles, can be used.
• Fluoride ions can be used to cleave silicon-oxygen bonds in groups such as triisopropyl silane (TIPS) or t-butyldimethyl silane (TBDMS).
• Photocleavable linkers have been used widely in carbohydrate chemistry. It is preferable that the light required to activate cleavage does not affect the other components of the modified nucleotides. For example, if a f!uorophore is used as the label, it is preferable if this absorbs light of a different wavelength to that required to cleave the linker molecule.
• Suitable linkers include those based on O-nitrobenzyl compounds and nitroveratryl compounds. Linkers based on benzoin chemistry can also be used (Lee et ah, J. Org. Chem. 64:3454-3460, 1999).
• Oxidation-based approaches are well known in the art. These include oxidation of p-alkoxybenzyl groups and the oxidation of sulfur and selenium linkers. The use of aqueous iodine to cleave disulfides and other sulfur or selenium-based linkers is also within the scope of the invention. F. Safety-catch linkers
• Safety-catch linkers are those that cleave in two steps.
• the first step is the generation of a reactive nucleophilic center followed by a second step involving an intra-molecular cyclization that results in cleavage.
• levulinic ester linkages can be treated with hydrazine or photochemistry to release an. active amine, which can then be cyclised to cleave an ester elsewhere in the molecule (Burgess et at, J. Org. Chem. 62:5165-5168, 1997).
• Elimination reactions can also be used.
• groups such as Fmoc and cyanoethyi, and palladium-catalysed reductive elimination, of allylic systems, can be used.
• the linker can comprise a spacer unit.
• the length of the linker is unimportant provided that the label is held a sufficient distance from the nucleotide so as not to interfere with any interaction between the nucleotide and an enzyme.
• the linker may consist of the similar functionality as the 3' -OH protecting group. This will make the deprotection and deprotecting process more efficient, as only a single treatment will be required to remove both the label and the protecting group.
• Particularly preferred linkers are phosphine-c!eavable azide containing linkers.
• modified nucleosides or nucleotides described herein can be used in conjunction with a variety of sequencing techniques.
• the process to determine the nucleotide sequence of a target nucleic acid can be an automated process.
• the nucleotide analogues presented herein can be used in a sequencing procedure, such as a sequencing-by-synthesis (SBS) technique.
• SBS can be initiated by contacting the target nucleic acids with one or more labeled nucleotides, DNA polymerase, etc.
• Those features where a primer is extended using the target nucleic acid as template will incorporate a labeled nucleotide that can be detected.
• the labeled nucleotides can further include a reversible termination property that terminates further primer extension once a nucleotide has been added to a primer.
• a nucleotide analog having a reversible terminator moiety can be added to a primer such that subsequent extension cannot occur until a deblocking agent is delivered to remove the moiety.
• a deblocking reagent can be delivered to the flow cell (before or after detection occurs). Washes can be carried out between the various delivery steps. The cycle can then be repeated n times to extend the primer by n nucleotides, thereby detecting a sequence of length n.
• Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into a nascent nucleic acid strand (Ronaghi, et al., Analytical Biochemistry 242(1), 84-9 (1996); Ronaghi, Genome Res. 1 1 ( 1 ), 3- 1 1 (2001); Ronaghi et al. Science 281 (5375), 363 (1998); US Pat. Nos. 6,210,891 ; 6,258,568 and 6,274,320, each of which is incorporated herein by reference).
• PPi inorganic pyrophosphate
• pyrosequencing In pyrosequencing, released PPi can be detected by being converted to adenosine triphosphate (ATP) by ATP sulfurylase, and the resulting ATP can be detected via luciferasc-produccd photons.
• the sequencing reaction can be monitored via a luminescence detection system.
• Excitation radiation sources used for fluorescence based detection systems are not necessary for pyrosequencing procedures.
• Useful fluidic systems, detectors and procedures that can be used for application of pyrosequencing to arrays of the present disclosure are described, for example, in WIPO Pat. App. Ser. No. PCT/US l 1 /571 1 1 , US Pat. App. Pub. No. 2005/0191698 A l . US Pat. No. 7,595,883, and US Pat. No. 7.244,559, each of which is incorporated herein by reference.
• Sequencing-by-ligation reactions are also useful including, for example, those described in Shendure et al. Science 309: 1728- 1 732 (2005); US Pat. No. 5,599,675; and US Pat. No. 5,750,341 , each of which is incorporated herein by reference.
• Some embodiments can include sequencing-by-hybridization procedures as described, for example, in Bains et al.. Journal of Theoretical Biology 135(3), 303-7 (1988); Drmanac et al., Nature Biotechnology 16, 54-58 (1998); Fodor et al.. Science 2 1 (4995), 767-773 ( 1995); and WO 1989/10977, each of which is incorporated herein by reference.
• nucleic acids that are present in gel-containing wells are subjected to repeated cycles of oligonucleotide delivery and detection.
• Fluidic systems for SBS methods as set forth herein, or in references cited herei can be readily adapted for delivery of reagents for sequencing-by-ligation or sequencing-by-hybridization procedures.
• the oligonucleotides are fluorescently labeled and can be detected using fluorescence detectors similar to those described with regard to SBS procedures herein or in references cited herein.
• Some embodiments can utilize methods involving the real-time monitoring of DNA polymerase activity.
• nucleotide incorporations can be detected through fluorescence resonance energy transfer (FRET) interactions between a fluorophore-bearing polymerase and ⁇ -phosphate-labeled nucleotides, or with zeromode waveguides.
• FRET fluorescence resonance energy transfer
• Techniques and reagents for FRET-based sequencing are described, for example, in Levene et al. Science 299, 682- 686 (2003); Limdquist et al. Opt Lett. 33, 1026-1028 (2008); Korlacli et al. Proc. Natl Acad. Sci. USA 105, 1 176-1 181 (2008), the disclosures of which are incorporated herein by reference.
• Some SBS embodiments include detection of a proton released upon incorporation of a nucleotide into an extension product.
• sequencing based on detection of released protons can use an electrical detector and associated techniques that are commercially available from Ion Torrent (Guilford, CT, a Life Technologies subsidiary) or sequencing methods and systems described in US Pat. App. Pub. Nos. 2009/0026082 Al ; 2009/0127589 Al ; 2010/0137143 Al ; or 2010/0282617 A l , each of which is incorporated herein by reference.
• Scheme 1 illustrates a synthetic route for the preparation of the modified nucleotides with monofluoromethyl substituted azidomethyl as 3'-OH protecting groups.
• Compounds la-lf employ a modified thymine (T-PA) as the base.
• T-PA modified thymine
• Other non-limiting examples of the bases that can be used include Cbz-PA, ADMF-PA, and GPac-PA, the stmctures of which are shown above in Scheme 1.
• Crude product lc was purified by Biotag silica gel column (50g) using a gradient of petroleum ether to petroleum cthenEtOAc 1 : 1 (v/v) to afford 1 .6 g nucleoside lb as a pale yellow foam (diastereomers, 82% yield).
• Scheme 2 illustrates a synthetic route for the preparation of the modified nucleotides with C-amido substituted azidomethyl as 3'-OH protecting groups.
• Compounds 2a-2i employ a modiiied thymine (T-PA) as the base.
• T-PA modiiied thymine
• Other non-limiting examples of the bases that can be used include Cbz-PA, ADMF-PA, and GPac-PA, the structures of which are shown above in Scheme 1.
• Crude product 2b was purified by a Biotag silica gel column (100 g) using a gradient of petroleum ether to petroleum ether: EtOAc 2:8 (v/v) as a pale yellow foam (4.1 7g, yield: 74%, diastereoisomers).
• the organic layer was evaporated under reduced pressure without drying over MgS0 4 in order to prevent the product from precipitating out.
• the crude product 2e was purified by a Biotag silica gel column (10 g) using a gradient of petroleum ether: EtOAc (1 : 1) (v/v) to EtOAc to MeOH: EtOAc (1 :9) as a pale yellow foam (isomer A, 482 mg, 68% yield).
• Scheme 3 illustrates a synthetic route for the preparation of modified nucleotides with difluoromethyl substituted azidomethyl 3'-OH protecting groups.
• Compounds 3a-3i employ a modified thymine (T-PA) as the base.
• T-PA modified thymine
• Other non-limiting examples of the bases that can be used include Cbz-PA, ADMF-PA, and GPac-PA, the structures of which are shown above in Scheme 1. The procedure for the synthesis of 3b, 3c and 3d were described i Example 2.
• the reaction mixture was partitioned between DCMiNaHCO ? (1 : 1) (50 mL). The aqueous layer was further extracted into DCM (2x30 mL). The organic extractions were combined, dried (MgS0 4 ), filtered, and evaporated to give a yellow oil.
• the crude product 3c was purified by a Biotag silica gel column (10 g) using a gradient of petroleum ether: EtOAc (8:2) (v/v) to petroleum ether: EtOAc (2:8) (v/v) as a pale yellow foam (isomer A, 250 mg, 51% yield).
• the organic extractions were combined, dried (MgS0 4 ), filtered, and evaporated to give a yellow oil.
• the erode product 3f was purified by a Biotag silica gel column (10 g) using a gradient of petroleum ether: EtOAc (9: 1 ) (v/v) to petroleum ether : EtOAc (2:8) (v/v) as a pale yellow foam (isomer A, 100 mg, 28%).
• Table 1 shows various nucleotide combinations with A and B isomers of mono-F substituted azidomethyl that were evaluated in regard to phasing and pre-phasing impacts. In all cases, the pre-phasing values were substantially lower than the control that standard V2 Miseq kits nucleotides used (FIG. 3).

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