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WO2018130848A1 - Ligature d'oligonucléotides - Google Patents

Ligature d'oligonucléotides Download PDF

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Publication number
WO2018130848A1
WO2018130848A1 PCT/GB2018/050091 GB2018050091W WO2018130848A1 WO 2018130848 A1 WO2018130848 A1 WO 2018130848A1 GB 2018050091 W GB2018050091 W GB 2018050091W WO 2018130848 A1 WO2018130848 A1 WO 2018130848A1
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Prior art keywords
oligonucleotide
formula
phosphodiester backbone
nucleotide
alkyl
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PCT/GB2018/050091
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Inventor
Tom Brown
Arun SHIVALINGAM
Afaf Helmy El-Sagheer
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Oxford University Innovation Ltd
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Oxford University Innovation Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C12N2310/3181—Peptide nucleic acid, PNA

Definitions

  • the present invention relates to a process for ligating oligonucleotides.
  • the present invention also relates to oligonucleotides obtained by this process and to the use of these oligonucleotides in PCR, replication, transcription, reverse transcription, translation and CRISPR- Cas processes.
  • Oligonucleotides are fundamental to many areas of molecular biology and are essential tools in technologies such as DNA sequencing, forensic and genetic analysis. They are often produced by automated solid-phase phosphoramidite synthesis. However, this process can only assemble DNA strands up to about 150 bases in length. Furthermore, the synthesis of long RNA strands is also a challenging task owing mainly to problems caused by the presence of the 2'- hydroxyl group of ribose, which typically requires selective protection during oligonucleotide assembly. Such protection of the 2'-hydroxyl groups of ribose consequently reduces the coupling efficiency of RNA phosphoramidite monomers due to increased steric hindrance.
  • an oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C as defined herein.
  • oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C as defined herein, as a template for amplification in a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C, as defined herein, as a template in a DNA replication process.
  • oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C as defined herein, as a template in a transcription process to provide a corresponding RNA transcript, or as a template in a reverse transcription process to provide a corresponding DNA transcript.
  • oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C as defined herein, as template in a translation process to produce a corresponding protein or peptide.
  • a seventh aspect of the present invention there is provided a method for amplifying an oligonucleotide sequence as defined herein.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • an oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C, as defined herein, as a guide RNA (gRNA) in a CRISPR-Cas process (e.g. in a CRISPR-Cas9 gene editing process).
  • gRNA guide RNA
  • oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C as defined herein, as a donor DNA template in a CRISPR-Cas mediated homology directed repair (HDR) process (e.g. in a CRISPR-Cas9 mediated homology directed repair (HDR) process).
  • HDR homology directed repair
  • a method of using an oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C as defined herein, in a CRISPR-Cas process e.g. as a donor DNA template and/or as a guide RNA.
  • alkyl includes both straight and branched chain alkyl groups. References to individual alkyl groups such as “propyl” are specific for the straight chain version only and references to individual branched chain alkyl groups such as “isopropyl” are specific for the branched chain version only.
  • (1-6C)alkyl includes (1-4C)alkyl, (1-3C)alkyl, propyl, isopropyl and f-butyl.
  • phenyl(1- 6C)alkyl includes phenyl(1-4C)alkyl, benzyl, 1-phenylethyl and 2-phenylethyl.
  • (m-nC) or "(m-nC) group” used alone or as a prefix, refers to any group having m to n carbon atoms.
  • (3-10C)cycloalkyl means a hydrocarbon ring containing from 3 to 10 carbon atoms, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or bicycle[2.2.2]octane, bicycle[2.1.1]hexane, bicycle[1.1.1]pentane, adamantane and bicyclo[2.2.1]heptyl.
  • halo refers to fluoro, chloro, bromo and iodo.
  • haloalkyl is used herein to refer to an alkyl group in which one or more hydrogen atoms have been replaced by halogen atoms (e.g. fluorine atoms).
  • halogen atoms e.g. fluorine atoms
  • any given "haloalkyl” is a "fluoroalkyl” in which one or more hydrogen atoms have been replaced by fluorine atoms.
  • fluoroalkyl groups include -CHF2, -CH2CF3, or perfluoroalkyl groups such as -CF3 or -CF2CF3.
  • An analogous definition applies to the term "haloalkoxy”.
  • heterocyclyl means a non-aromatic saturated or partially saturated monocyclic, fused, bridged, or spiro bicyclic heterocyclic ring system(s).
  • Monocyclic heterocyclic rings contain from about 3 to 12 (suitably from 3 to 7) ring atoms, with from 1 to 5 (suitably 1 , 2 or 3) heteroatoms selected from nitrogen, oxygen or sulfur in the ring.
  • Bicyclic heterocycles contain from 7 to 17 member atoms, suitably 7 to 12 member atoms, in the ring.
  • Bicyclic heterocyclic(s) rings may be fused, spiro, or bridged ring systems.
  • heterocyclic groups include cyclic ethers such as oxiranyl, oxetanyl, tetrahydrofuranyl, dioxanyl, and substituted cyclic ethers.
  • Heterocycles containing nitrogen include, for example, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, tetrahydrotriazinyl, tetrahydropyrazolyl, and the like.
  • Typical sulfur containing heterocycles include tetrahydrothienyl, dihydro-1 ,3-dithiol, tetrahydro-2/-/- thiopyran, and hexahydrothiepine.
  • heterocycles include dihydro-oxathiolyl, tetrahydro-oxazolyl, tetrahydro-oxadiazolyl, tetrahydrodioxazolyl, tetrahydro-oxathiazolyl, hexahydrotriazinyl, tetrahydro-oxazinyl, morpholinyl, thiomorpholinyl, tetrahydropyrimidinyl, dioxolinyl, octahydrobenzofuranyl, octahydrobenzimidazolyl, and octahydrobenzothiazolyl.
  • the oxidized sulfur heterocycles containing SO or SO2 groups are also included.
  • examples include the sulfoxide and sulfone forms of tetrahydrothienyl and thiomorpholinyl such as tetrahydrothiene 1 , 1 -dioxide and thiomorpholinyl 1 , 1 -dioxide.
  • heterocyclyl groups are saturated monocyclic 3 to 7 membered heterocyclyls containing 1 , 2 or 3 heteroatoms selected from nitrogen, oxygen or sulfur, for example azetidinyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, morpholinyl, tetrahydrothienyl, tetrahydrothienyl 1 , 1 -dioxide, thiomorpholinyl, thiomorpholinyl 1 , 1-dioxide, piperidinyl, homopiperidinyl, piperazinyl or homopiperazinyl.
  • any heterocycle may be linked to another group via any suitable atom, such as via a carbon or nitrogen atom.
  • reference herein to piperidino or morpholino refers to a piperidin-1-yl or morpholin-4-yl ring that is linked via the ring nitrogen.
  • heteroaryl or “heteroaromatic” means an aromatic mono-, bi-, or polycyclic ring incorporating one or more (for example 1-4, particularly 1 , 2 or 3) heteroatoms selected from nitrogen, oxygen or sulfur.
  • heteroaryl groups are monocyclic and bicyclic groups containing from five to twelve ring members, and more usually from five to ten ring members.
  • the heteroaryl group can be, for example, a 5- or 6-membered monocyclic ring or a 9- or 10- membered bicyclic ring, for example a bicyclic structure formed from fused five and six membered rings or two fused six membered rings.
  • Each ring may contain up to about four heteroatoms typically selected from nitrogen, sulfur and oxygen.
  • the heteroaryl ring will contain up to 3 heteroatoms, more usually up to 2, for example a single heteroatom.
  • the heteroaryl ring contains at least one ring nitrogen atom.
  • the nitrogen atoms in the heteroaryl rings can be basic, as in the case of an imidazole or pyridine, or essentially non-basic as in the case of an indole or pyrrole nitrogen. In general the number of basic nitrogen atoms present in the heteroaryl group, including any amino group substituents of the ring, will be less than five.
  • heteroaryl examples include furyl, pyrrolyl, thienyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1 ,3,5-triazenyl, benzofuranyl, indolyl, isoindolyl, benzothienyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzothiazolyl, indazolyl, purinyl, benzofurazanyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, cinnolinyl, pteridinyl, naphthy
  • Heteroaryl also covers partially aromatic bi- or polycyclic ring systems wherein at least one ring is an aromatic ring and one or more of the other ring(s) is a non-aromatic, saturated or partially saturated ring, provided at least one ring contains one or more heteroatoms selected from nitrogen, oxygen or sulfur.
  • partially aromatic heteroaryl groups include for example, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 2-oxo-1 ,2,3,4-tetrahydroquinolinyl, dihydrobenzthienyl, dihydrobenzfuranyl, 2,3-dihydro-benzo[1 ,4]dioxinyl, benzo[1 ,3]dioxolyl, 2,2- dioxo-1 ,3-dihydro-2-benzothienyl, 4,5,6,7-tetrahydrobenzofuranyl, indolinyl,
  • heteroaryl groups examples include but are not limited to pyrrolyl, furanyl, thienyl, imidazolyl, furazanyl, oxazolyl, oxadiazolyl, oxatriazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, triazolyl and tetrazolyl groups.
  • heteroaryl groups include but are not limited to pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl and triazinyl.
  • bicyclic heteroaryl groups containing a six membered ring fused to a five membered ring include but are not limited to benzofuranyl, benzothiophenyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, isobenzofuranyl, indolyl, isoindolyl, indolizinyl, indolinyl, isoindolinyl, purinyl (e.g., adeninyl, guaninyl), indazolyl, benzodioxolyl, pyrrolopyridine, and pyrazolopyridinyl groups.
  • bicyclic heteroaryl groups containing two fused six membered rings include but are not limited to quinolinyl, isoquinolinyl, chromanyl, thiochromanyl, chromenyl, isochromenyl, chromanyl, isochromanyl, benzodioxanyl, quinolizinyl, benzoxazinyl, benzodiazinyl, pyridopyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl and pteridinyl groups.
  • aryl means a cyclic or polycyclic aromatic ring having from 5 to 12 carbon atoms.
  • aryl includes both monovalent species and divalent species. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl and the like. In particular embodiment, an aryl is phenyl.
  • oligonucleotide analogue and “nucleotide analogue” refer to any modified synthetic analogues of oligonucleotides or nucleotides respectively that are known in the art.
  • oligonucleotide analogues include peptide nucleic acids (PNAs), morpholino oligonucleotides, phosphorothioate oligonucleotides, phosphorodithioate oligonucleotides, alkylphosphonate oligonucleotides, acylphosphonate oligonucleotides and phosphoramidate oligonucleotides.
  • PNAs peptide nucleic acids
  • morpholino oligonucleotides include morpholino oligonucleotides, phosphorothioate oligonucleotides, phosphorodithioate oligonucleotides, alkylphosphonate oligonucle
  • nucleobase analogue refers to any analogues of nucleobases known in the art.
  • the skilled person will appreciate there to be numerous natural and synthetic nucleobase analogues available in the art which could be employed in the present invention. As such, the skilled person will readily be able to identify suitable nucleobase analogues for use in the present invention.
  • Commonly available nucleobase analogues are commercially available from a number of sources (for example, see the Glen Research catalogue (http://www.qlenresearch.com/Catalog/contents.php). It will also be appreciated that the term “nucleobase analogue” covers: universal/degenerate bases (e.g.
  • 3-nitropyrrole, 5-nitroindole and hypoxanthine fluorescent bases (e.g. tricyclic cytosine analogues (tCO, tCS) and 2- aminopurine); base analogues bearing reactive groups selected from alkynes, thiols or amines; and base analogues that can crosslink oligonucleotides to DNA, RNA or proteins (e.g. 5- bromouracil or 3-cyanovinyl carbazole).
  • fluorescent bases e.g. tricyclic cytosine analogues (tCO, tCS) and 2- aminopurine
  • base analogues bearing reactive groups selected from alkynes, thiols or amines e.g. 5- bromouracil or 3-cyanovinyl carbazole.
  • a process for ligating a first and a second oligonucleotide together comprising reacting:
  • w r denotes the point of attachment to the oligonucleotide backbone
  • X is a leaving group optionally selected from halo, OSO2R, (1-2C)haloalkyl, (1- 2C)haloalkoxy, OR 1 , heteroaryl, wherein R and R 1 are independently selected from H, (1-6C)alkyl, (1-6C)alkanoyl, cycloalkyi, heteroalkyi, aryl, heteroaryl, (1-2C)haloalkyl, and wherein each of (1-6C)alkyl, cycloalkyi, heteroalkyi, aryl, heteroaryl are optionally further substituted with one or more groups selected from (1-4C)alkyl, halo, cyano, nitro or (1-2C)haloalkyl; or
  • R and R 1 are a solid support to which the one or more oligonucleotides are attached;
  • V is selected from O, S or NR X , wherein R x is selected from hydrogen or (1-4C)alkyl;
  • Q is O or S;
  • R a and R b are independently selected from hydrogen or (1-4C)alkyl, wherein each (1- 4C)alkyl is optionally substituted with one or more NH2, OH or SH;
  • n is an integer selected from 0 to 2;
  • q is an integer selected from 0 to 1 ;
  • R c and R d are independently selected from hydrogen or (1-4C)alkyl
  • Y is selected from O or NH
  • W is selected from NR e or SH, wherein R e is selected from hydrogen or (1-4C)alkyl; m is an integer selected from 0 to 2; and
  • p is an integer selected from 0 or 1 ;
  • Q 1 is selected from O or S
  • LG 1 and LG 2 are each independently a leaving group (e.g. halo, imidazolyl or haloalkoxy);
  • reaction is optionally conducted in the presence of one or more of the following:
  • the inventors have surprisingly discovered that the process of the present invention allows for the preparation of oligonucleotides comprising polymerase-compatible artificial backbones.
  • the oligonucleotides prepared by the present invention show fast read-through and good fidelity with both DNA and RNA polymerases.
  • the process of the present invention provides a cheap and highly efficient process for oligonucleotide synthesis.
  • Chemical ligation also enables long DNA and RNA constructs (i.e. DNA and RNA constucts comprising 20 or more, 50 or more, 100 or more or 200 or more nucleotide and/or nucleotide analogue monomers) to be formed.
  • reaction conditions may be used in the process defined hereinabove.
  • reaction conditions used in the present process will vary according to the specific oligonucleotide and/or functional groups of Formula A and B that are used.
  • suitable reaction conditions e.g. temperature, pressures, reaction times, concentration etc.
  • the process of the present invention is conducted at a temperature of between 0 °C and 150 °C.
  • the process of the present invention is conducted at a temperature of between 0 °C and 100 °C. More suitably, the process of the present invention is conducted at a temperature of between 0 °C and 75 °C. Most suitably, the process of the present invention is conducted at a temperature of between 4 °C and 70 °C.
  • the process of the present invention is carried out in a polar solvent.
  • the polar solvent may be used to solubilise the oligonucleotides comprising functional groups of Formulae A and B and thereby facilitate reaction therebetween. Accordingly, it will be understood that the polar solvent selected will depend on the specific oligonucleotides selected.
  • Suitable polar solvents may include, but are not limited to, water, an aquous buffered solution (e.g. a solution of sodium phosphate or sodium carbonate), DMF, DMSO, acetonitrile, tetrahydrofuran (THF) and mixtures thereof with aqueous salt solutions.
  • the process of the present invention is carried out in an aqueous medium at a pH within the range of 5 to 9.
  • the process of the present invention is carried out at a pH within the range of 6 to 8.
  • the process of the present invention is carried out at a pH within the range of 6.5 to 7.5.
  • a suitable buffer is present to maintain the reaction medium within the pH range 5 to 9. In a further embodiment, the buffer maintains the reaction medium within the pH range 6 to 8. In another embodiment, the buffer maintains the reaction medium within the pH range 6.5 to 7.5.
  • the buffer is selected from the group comprising: phosphate, acetate, borate, citrate, sulfonic acid, ascorbate, linolenate, carbonate and bicarbonate based buffers.
  • the buffer is selected from the group comprising: phosphate, acetate, carbonate and bicarbonate based buffers.
  • the buffer is sodium phosphate or sodium carbonate.
  • the resultant oligonucleotides formed by the process of the present invention may be isolated and purified using any suitable techniques known in the art.
  • the resultant oligonucleotides formed by the process of the present invention may be isolated and purified using column chromatography, for example, using sephadex columns.
  • the process of the present invention is conducted in the presence of a salt (e.g. NaCI).
  • a salt e.g. NaCI
  • Any suitable concentration of salt may be used.
  • the salt is present in a concentration of between 20 mM and 500 mM. More suitably, the salt is present in a concentration between 50 mM and 300 mM. Yet more suitably, the salt is present in a concentration between 100 mM and 250 mM.
  • one of the first or second oligonucleotides is present in an excess.
  • the process of the present invention is conducted in the presence of a template oligonucleotide.
  • a template oligonucleotide will vary in accordance with the first and second oligonucleotide that is used.
  • a person skilled in the art will be able to select a suitable template oligonucleotide having a suitable size and sequence to hybridise with the first and second oligonucleotides of the present process.
  • the template oligonucleotide may also comprise synthetic oligonucleotide analogues, such as, for example, peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the template oligonucleotide is a single stranded oligonucleotide or oligonucleotide analogue.
  • the template oligonucleotide form a duplex with the terminii bearing the group of formula A of the first oligonucleotide and the group of formula B of the second oligonucleotide adjacent to one another.
  • the chemical ligation process of the present invention may then be conducted to ligate the first and second oligonucleotides together. It will be appreciated that in embodiments where a template oligonucleotide is utilised, and once the ligation is complete, the process of the invention may additionally comprise a step of separating the template oligonucleotide from the ligated oligonucleotide formed by the process of the present invention.
  • the template oligonucleotide comprises between 2 and 100 nucleotide monomer units.
  • the template oligonucleotide comprises between 10 and 100 nucleotide monomer units. More suitably, the template oligonucleotide comprises between 15 and 75 nucleotide monomer units. Most suitably, the template oligonucleotide comprises between 25 and 50 nucleotide monomer units.
  • a catalyst may be any suitable reagent that helps to promote the rate of the reaction between the first and second oligonucleotide.
  • the catalyst is an acid and/or a base.
  • the catalyst is a base.
  • suitable bases include NaOH, trimethylamine, diisopropylethylamine and N-methylmorpholine.
  • the process of the present invention is carried out in the presence of one or more peptide coupling agents.
  • Any suitable peptide coupling reagent capable of enhancing the reaction between the functional group of Formula A of the first oligonucleotide and the functional group of Formula B of the second oligonucleotide may be used. It will be understood that the peptide coupling agent is preferably present when X is OH (i.e. the functional group of Formula A comprises a caboxy group).
  • the peptide coupling reagent is a carbodiimide-based coupling reagent.
  • the peptide coupling reagent is selected from 1- [Bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), 2-(1 H-benzotriazol-1-yl)-1 ,1 ,3,3-tetramethyluronium hexafluorophosphate (HBTU), (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 4-(4,6- Dimethoxy-1 ,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), N-Ethoxycarbonyl-2- ethoxy-1 ,2-dihydroquinoline (EEDQ), ⁇
  • the coupling reagent is selected from ⁇ , ⁇ '-dicyclohexylcarbodiimide (DCC), ⁇ , ⁇ '- diisopropylcarbodiimide (DIC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI).
  • the coupling reagent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI).
  • Additional activating agents such as, for example, hydroxybenzotriazole (HOBt), N- hydroxy 2-phenyl benzimidazole (HOBI), 1-hydroxy-7-azabenzotriazole (HOAt), N- hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (Sulfo-NHS), 4-dimethylaminopyridine (DMAP) and ethyl cyano(hydroxyimino)acetate (Oxyma Pure ® ) may also be used together with the peptide coupling reagent defined hereinabove, to further enhance reactivity between the functional group of Formula A of the first oligonucleotide and the functional group of Formula B of the second oligonucleotide.
  • HOBt hydroxybenzotriazole
  • HOBI N- hydroxy 2-phenyl benzimidazole
  • HOAt 1-hydroxy-7-azabenzotriazole
  • NHS N- hydroxysuccinimide
  • the activating agent is N-hydroxysuccinimde (NHS), N- hydroxysulfosuccinimide (Sulfo-NHS) or ethyl cyano(hydroxyimino)acetate (Oxyma Pure ® ).
  • the activating agent is N-hydroxysuccinimde (NHS).
  • the process of the present invention is carried out in the presence of both a peptide coupling agent (e.g. EDCI) and an activating agent (e.g. NHS).
  • a peptide coupling agent e.g. EDCI
  • an activating agent e.g. NHS
  • the ratio of peptide coupling agent (e.g. EDCI) to activating agent (e.g. NHS) is from between 10: 1 to 1 :1. More suitably, the ratio of peptide coupling agent (e.g. EDCI) to activating agent (e.g. NHS) is from between 6: 1 to 1 : 1. Most suitably, the ratio of peptide coupling agent (e.g. EDCI) to activating agent (e.g. NHS) is 4:1.
  • the sum of integers m, n, p and q is 2, 3 or 4.
  • the sum of integers m, n, p and q is 2 or 3.
  • the sum of integers m, n, p and q is 2.
  • one or more of the following proivos may apply:
  • each of X, V, Q, R a , R b , n and q of the terminal functional group of Formula A has any one of the meanings defined hereinabove or any one of the meanings defined in any of paragraphs (1) to (21) hereinafter: -
  • X is selected from halo, OS0 2 R, (1-2C)haloalkyl, (1-2C)haloalkoxy, OR 1 , 5-membered heteroaryl, wherein R and R 1 are independently selected from H, (1-6C)alkyl, (1- 6C)alkonyl, aryl or (1-2C)haloalkyl, and wherein each of (1-6C)alkyl or aryl is optionally further substituted with one or more groups selected from (1-4C)alkyl, halo, cyano, nitro or (1-2C)haloalkyl; or
  • R and R 1 are a solid support to which the one or more oligonucleotides are attached;
  • X is selected from halo, OS0 2 R, (1-2C)haloalkyl, (1-2C)haloalkoxy, OR 1 , triazolyl,
  • R and R 1 are independently selected from H, (1-6C)alkyl, (1-6C)alkonyl, aryl or (1-2C)haloalkyl, and wherein each of (1-6C)alkyl or aryl is optionally further substituted with one or more groups selected from (1-4C)alkyl, halo, cyano, nitro or (1-2C)haloalkyl;
  • X is selected from halo, OS0 2 R, (1-2C)haloalkyl, (1-2C)haloalkoxy, OR 1 , triazolyl,
  • R and R 1 are independently selected from H, (1-6C)alkyl, (1-6C)alkonyl, phenyl or (1-2C)haloalkyl;
  • X is selected from halo, (1-2C)haloalkyl or OR 1 , wherein R and R 1 are independently selected from H, (1-6C)alkyl or a (1-6C)alkonyl;
  • R and R 1 are a solid support to which the one or more oligonucleotides are attached
  • X is selected from halo, (1-2C)haloalkyl or OR 1 , wherein R and R 1 are independently selected from H, (1-6C)alkyl or a (1-6C)alkonyl;
  • X is selected from OR 1 , wherein R and R 1 are independently selected from H or (1- 6C)alkyl;
  • X is OH
  • V is selected from O or NR X , wherein R x is selected from hydrogen or (1-4C)alkyl;
  • V is O; (10) V is NR X , wherein R x is selected from hydrogen or methyl;
  • V is NH
  • R a and R b are independently selected from hydrogen or (1-4C)alkyl, wherein each (1- 4C)alkyl is optionally substituted with one or more OH;
  • R a and R b are independently selected from hydrogen or (1-4C)alkyl
  • R a and R b are independently selected from hydrogen or (2-4C)alkyl
  • R a and R b are independently selected from hydrogen or methyl
  • R a and R b are hydrogen
  • n 1 ;
  • a heteroaryl or heterocyclyl group as defined herein is a monocyclic heteroaryl or heterocyclyl group comprising one, two or three heteroatoms selected from N, O or S.
  • a heteroaryl is a 5-membered heteroaryl ring comprising one, two or three heteroatoms selected from N, O or S.
  • a heteroaryl is a 5-membered heteroaryl ring comprising one, two or three nitrogen heteroatoms.
  • a heterocyclyl group is a 4-, 5- or 6-membered heterocyclyl ring comprising one, two or three heteroatoms selected from N, O or S.
  • a heterocyclyl group is a 5-, 6- or 7-membered ring comprising one, two or three heteroatoms selected from N, O or S [e.g. morpholinyl (e.g. 4-morpholinyl), pyridinyl, piperazinyl, homopiperazinyl or pyrrolidinonyl].
  • an aryl group is phenyl
  • X is as defined in any one of paragraphs (1) to (7) above. Most suitably, X is as defined in paragraph (7) above.
  • V when present, is as defined in any one of paragraphs (8) to (11) above. Most suitably, V, when present, is NH.
  • the first oligonucleotide comprises a terminal functional group of Formula A1 , shown below
  • the first oligonucleotide comprises a terminal functional group of Formula A2, shown below:
  • R a , R b , X and n are as defined hereinabove.
  • the first oligonucleotide comprises a terminal functional group of Formula A3, shown below:
  • the first oligonucleotide comprises a terminal functional group of Formula A4, shown below:
  • the first oligonucleotide comprises a terminal functional group of Formula A5, shown below:
  • R a , R b , V, X, and n are as defined hereinabove.
  • the terminal functional group of Formula A (including A1 , A2, A3, A4 and A5) is attached to the 3' position of the first oligonucleotide.
  • the functional group of Formula B (including A1 , A2, A3, A4 and A5) is attached to the 3' position of the first oligonucleotide.
  • each of Y, W, R c , R d , R e , m and p of the terminal functional group of Formula B has any one of the meanings defined hereinabove or any one of the meanings defined in any of paragraphs (A) to (I) hereinafter: -
  • (C) W is NR e , wherein R e is selected from hydrogen or (1-4C)alkyl;
  • R c and R d are independently selected from hydrogen or methyl
  • R c and R d are hydrogen
  • m is 1 and p is 0. In another embodiment, m is 1 and p is 1 and Y is as defined in paragraph (A) or (B) above.
  • W is as defined in any one of paragraphs (C) or (D) above. Most suitably, W is
  • the second oligonucleotide comprises a terminal functional group of Formula B1 , shown below:
  • the second oligonucleotide comprises a terminal functional group of Formula B2, shown below:
  • the second oligonucleotide comprises a terminal functional group of Formula B3, shown below:
  • the second oligonucleotide comprises a terminal functional group of Formula B4, shown below:
  • R c , R d , Y and m are as defined hereinabove.
  • amino group of the functional group of Formula B, B1 , B2, B3 or B4 may be protonated and thus present as ammoninum salt group of the formula -NhVX " , wherein X " is a suitable counterion (e.g. CI " )-
  • the process of the present invention may be carried out in the presence of a base (e.g. NaOH).
  • a base e.g. NaOH
  • the terminal functional group of Formula B, B1 , B2, B3 or B4 is attached to the 5' position of the second oligonucleotide.
  • the process of the present invention comprises reacting together two "second" oligonucleotides comprising terminal functional groups of Formula B, as defined herein, together with a coupling agent of Formula D.
  • the coupling agent will be understood to react with the two functional groups of Formula B present on each oligonucleotide so as to form a covalent attachment therebetween.
  • one group of formula B is attached to the 5' end of one oligonucleotide and a second group of formula B is attached to the 3' end of the oligonucleotide to be ligated.
  • Q 1 of the coupling agent is oxygen.
  • LG 1 and LG 2 may independently be selected from any suitable leaving group.
  • suitable leaving groups include halo, heteroaryl, alkoxy, haloalkyl or haloalkoxy.
  • the LG 1 and LG 2 are both selected from halo (e.g. CI), heteroaryl (e.g. imidazolyl) or haloalkoxy (e.g. OCCU).
  • the coupling agent is selected from phosgene, triphosgene or carbonyldiimidazole.
  • the coupling agent is carbonyldiimidazole.
  • first and second oligonucleotides of the present process may independently comprise any suitable number and/or type of nucleotide and/or nucleotide analogue monomers.
  • the first and second oligonucleotides of the present process independently comprise between 10 and 200 nucleotide and/or nucleotide analogue monomers. In an embodiment, the first and second oligonucleotides of the present process independently comprise between 10 and 100 nucleotide and/or nucleotide analogue monomers. In another embodiment, the first and second oligonucleotides of the present process independently comprise between 10 and 75 nucleotide and/or nucleotide analogue monomers. In a further embodiment, the first and second oligonucleotides of the present process independently comprise between 10 and 50 nucleotide and/or nucleotide analogue monomers. In yet another embodiment, the first and second oligonucleotides of the present process independently comprise between 20 and 50 nucleotide and/or nucleotide analogue monomers.
  • oligonucleotides described herein encompass all suitable salt, hydrate and/or solvate forms thereof.
  • the first oligonucleotide comprises a terminal nucleotide analogue select
  • R a and R b are as defined herein;
  • R a ' and R b' are independently selected from hydrogen or (1-4C)alkyl;
  • Z is selected from hydrogen, halo, (1-4C)alkyl, (1-2C)haloalkyl, OR 2 wherein R 2 is selected from hydrogen, (1-4C)alkyl, (2-4C)alkenyl or 4C)alkynyl; and
  • B is a nucleobase or nucleobase analogue.
  • the first oligonucleotide comprises a terminal nucleotide analogue of the formula:
  • R a , R b , Z and B are as defined hereinabove.
  • the second oligonucleotide comprises a terminal nucleotide analogue selected from one of the following:
  • R c , R d and R e are each as defined herein;
  • R c' and R d' are independently selected from hydrogen or (1-4C)alkyl
  • Z is selected from hydrogen, halo, (1-4C)alkyl, (1-2C)haloalkyl, OR 2 or NH 2 , wherein R 2 is selected from hydrogen, (1-4C)alkyl, (2-4C)alkenyl or (2- 4C)alkynyl; and
  • B is a nucleobase or nucleobase analogue.
  • the second oligonucleotide comprises a terminal nucleotide analogue of the
  • R c , R d , R e , Z and B are as defined herein.
  • terminal nucleotide analogues of the first and second oligonucelotides of the present invention comprise a terminal functional group of Formula A and B respectively (i.e. as per step a) of the process of the present invention).
  • terminal nucleotide analogues of the first and second oligonucelotides of the present invention both comprise a terminal functional group of Formula B (i.e. as per step b) of the process of the present invention).
  • B can be any suitable nucleobase (e.g. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)) or any suitable modified analogue thereof.
  • B is a nucleobase selected from A, G, C, T or U.
  • Z is selected from hydrogen, halo, OR 2 or NH2, wherein R 2 is selected from hydrogen, (1-4C)alkyl, (2-4C)alkenyl or (2-4C)alkynyl.
  • R 2 is selected from hydrogen, halo, OR 2 or NH2, wherein R 2 is selected from hydrogen or (1-4C)alkyl. More suitably, Z is selected from hydrogen, fluoro, OH, OMe or NH2.
  • each of R a' , R b' , R c' and R d' are independently selected from hydrogen or methyl.
  • each of R a' , R b' , R c' and R d' are hydrogen.
  • an oligonucleotide obtainable by, obtained by or directly obtained by the process of the present invention.
  • an oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C shown below:
  • R a and R b are independently selected from hydrogen or (1-4C)alkyl, wherein each (1-4C)alkyl is optionally substituted with one or more NH2, OH or SH;
  • R c , R d and R e are independently selected from hydrogen or (1-4C)alkyl
  • Y is selected from O or NH
  • V is selected from O, S or NR X , wherein R x is selected from hydrogen or (1- 4C)alkyl;
  • Q is O or S
  • n and n are integers independently selected from 0 to 2;
  • p and q are integers independently selected from 0 to 1 ;
  • the oligonucleotide comprises greater than or equal to 20 nucleotide and/or nucleotide analogue monomers
  • the phosphodiester backbone mimics are separated by at least 10 nucleotide and/or nucleotide analogue monomers.
  • each of R a , R b , R c , R d , R e , Y, V, Q, m, n, p and q are as defined hereinbefore in relation to the process of the present invention.
  • the oligonucleotide comprises one or more phosphodiester backbone mimics of Formula C1 shown below:
  • R a , R b , R c , R d , R e and R x are independently selected from hydrogen or (1-4C)alkyl; n and m are integers independently selected from 0 to 2; and
  • q is an interger from 0 to 1 ;
  • the oligonucleotide comprises greater than or equal to 20 nucleotide and/or nucleotide analogue monomers;
  • the oligonucleotide comprises two or more phosphodiester backbone mimics of Formula C1 , the phosphodiester backbone mimics are separated by at least 10 nucleotide and/or nucleotide analogue monomers.
  • the oligonucleotide comprises one or more phosphodiester backbone mimics of Formula C2 shown below :
  • R a , R b , R c , R d and R e are independently selected from hydrogen or (1-4C)alkyl;
  • the oligonucleotide comprises only one phosphodiester backbone mimic of Formula C2, the oligonucleotide comprises greater than or equal to 20 nucleotide and/or nucleotide analogue monomers; or
  • the oligonucleotide comprises two or more phosphodiester backbone mimics of Formula C2
  • the phosphodiester backbone mimics are separated by at least 10 nucleotide and/or nucleotide analogue monomers.
  • the oligonucleotide comprises one or more phosphodiester backbone mimics of Formula C3 shown below:
  • R a and R b are independently selected from hydrogen or (1-4C)alkyl, wherein each (1-4C)alkyl is optionally substituted with one or more NH2, OH or SH;
  • R c , R d and R e are independently selected from hydrogen or (1-4C)alkyl
  • Y is selected from O or NH
  • V is selected from O, S or NR X , wherein R x is selected from hydrogen or (1- 4C)alkyl;
  • n and n are integers independently selected from 0 to 2;
  • p and q are integers independently selected from 0 to 1 ;
  • the oligonucleotide comprises greater than or equal to 20 nucleotide and/or nucleotide analogue monomers
  • the oligonucleotide comprises two or more phosphodiester backbone mimics of Formula C3
  • the phosphodiester backbone mimics are separated by at least 10 nucleotide and/or nucleotide analogue monomers.
  • an oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C4 shown below:
  • R a and R b are independently selected from hydrogen or (1-4C)alkyl, wherein each (1-4C)alkyl is optionally substituted with one or more NH2, OH or SH;
  • R c , R d and R e are independently selected from hydrogen or (1-4C)alkyl
  • V is selected from O, S or NR X , wherein R x is selected from hydrogen or (1- 4C)alkyl;
  • n and n are integers independently selected from 0 to 2;
  • the oligonucleotide comprises only one phosphodiester backbone mimic of Formula C4, the oligonucleotide comprises greater than or equal to 20 nucleotide and/or nucleotide analogue monomers; or
  • the oligonucleotide comprises two or more phosphodiester backbone mimics of Formula C4, the phosphodiester backbone mimics are separated by at least 10 nucleotide and/or nucleotide analogue monomers.
  • oligonucleotides comprising one or more phosphodiester backbone mimic of Formula C
  • the sum of integers m, n, p and q equals 2, 3 or 4, suitably 2 or 3, and most suitably 2.
  • oligonucleotides comprising one or more phosphodiester backbone mimic of Formula C1
  • the sum of integers m, n and q equals 2 or 3, suitably 2.
  • oligonucleotides comprising one or more phosphodiester backbone mimic of Formula C2
  • the sum of integers m and n equals 2.
  • oligonucleotides comprising one or more phosphodiester backbone mimic of Formula C3, the sum of integers m, n, p and q equals 2, 3 or 4, suitably 2 or 3, and most suitably 2.
  • oligonucleotides comprising one or more phosphodiester backbone mimic of Formula C4, the sum of integers m and n equals 1 or 2.
  • the oligonucleotides of the present invention may be formed from either: i) ligating one or more first oligonucleotides as defined hereinabove, with one or more second oligonucleotide as defined hereinabove; or ii) ligating together two or more second oligonucleotides as defined hereinabove, in the presence of one or more coupling agents of Formula D as defined hereinabove.
  • oligonucleotides of the present invention may be long oligonucleotides comprising, for example, greater than or equal to 20, 30, 50, 100, 150 200, 400, 500 or 1000 nucleotide and/or nucleotide analogue monomers in length.
  • oligonucleotides of the present invention may synomously be referred to as "polynucleotides”.
  • the oligonucleotides of the present invention comprise between 20 and 2000 nucleotide and/or nucleotide analogue monomers. In another embodiment, the oligonucleotides of the present invention comprise betweren 20 and 500 nucleotide and/or nucleotide analogue monomers. In another embodiment, the oligonucleotides of the present invention comprise betweren 20 and 200 nucleotide and/or nucleotide analogue monomers. In yet another embodiment, the oligonucleotides of the present invention comprise betweren 20 and 100 nucleotide and/or nucleotide analogue monomers.
  • the oligonucleotide comprises only one phosphodiester backbone mimic of Formula C, C1 , C2, C3 or C4
  • the oligonucleotide comprises greater than or equal to 30 nucleotide and/or nucleotide analogue monomers, suitably greater than or equal to 40 nucleotide and/or nucleotide analogue monomers, and most suitably, greater than or equal to 50 nucleotide and/or nucleotide analogue monomers.
  • the oligonucleotide comprises two or more phosphodiester backbone mimics of Formula C, C1 , C2, C3 or C4
  • the phosphodiester backbone mimics are separated by at least 15 nucleotide and/or nucleotide analogue monomers, suitably by at least 25 nucleotide and/or nucleotide analogue monomers, more suitably, by at least 40 nucleotide and/or nucleotide analogue monomers and most suitably, by at least 50 nucleotide and/or nucleotide analogue monomers.
  • the one or more phosphodiester backbone mimics is selected from one of the following:
  • the one or more phosphodiester backbone selected from one of the following:
  • the one or more phosphodiester backbone mimics is selected from one of the following:
  • the one or more phosphodiester backbone mimics is selected from one of the following:
  • the one or more phosphodiester backbone mimics is selected from one of the following:
  • the one or more phosphodiester backbone mimics is selected from one of the following:
  • the one or more phosphodiester backbone mimics is selected from one of the following:
  • the one or more phosphodiester backbone mimics is selected from one of the following:
  • terminal nucleotides of the first and second oligonucleotides and the phosphodiester backbone mimic of formula C is selected from one of the following formulae:
  • B and B' are each independently a nucleobase or nucleobase analogue.
  • terminal nucleotides of the first and second oligonucleotides and the phosphodiester backbone mimic of formula C is selected from one of the following formulae:
  • each of Z, Z', B and B' correspond with any one of the defintions of Z, Z', B and B' set out above in relation to the process of the invention.
  • terminal nucleotides of the first and second oligonucleotides and the phosphodiester backbone mimic is selected from one of the following formulae:
  • terminal nucleotides of the first and second oligonucleotides and the phosphodiester backbone mimic is selected from one of the following formulae:
  • terminal nucleotides of the first and second oligonucleotides and the phosphodiester backbone mimic is selected from one of the following formulae:
  • terminal nucleotides of the first and second oligonucleotides and the phosphodiester backbone mimic is selected from one of the following formulae:
  • terminal nucleotides of the first and second oligonucleotides and the phosphodiester backbone mimic is a group of the formula:
  • the present invention provides access to ligated DNA and RNA oligonucleotides or oligonucleotide analogues which are capable of being read by DNA and RNA polymerase.
  • the process of the present invention enables the synthesis of DNA and RNA constructs containing modified nucleobases for application in, for example, altered gene expression and mutagenic modifications which may allow for the synthesis of altered proteins and/or suitable fluorescent tags to visualise DNA in cells.
  • the present invention also provides the use of an oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C, as defined herein, as a template for amplification in a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the present invention also provides the use of an oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C, as defined herein, as a template in a DNA replication process.
  • the present invention also provides a use of an oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C, as defined herein, as a template in a transcription process to provide the corresponding RNA transcript, or as a template in a reverse transcription process to provide the corresponding DNA transcript.
  • the present invention provides a use of an oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C, as defined herein, as template in a translation process to produce a corresponding protein or peptide.
  • the present invention provides a method for amplifying an oligonucleotide sequence as defined herein.
  • the present invention also provides a method of replicating an oligonucleotide sequence as defined herein.
  • the present invention provides a method for producing a ribonucleic acid (RNA) sequence or deoxyribonucleic acid (DNA) sequence as defined here.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • oligonucleotides comprising one or more phosphodiester backbone mimics of Formula C of the present invention is in the recently developed CRISPR (clustered regularly interspaced short palindromic repeats)/Cas technology (see, for example, J. A. Doudna and E. Charpentier, Science, 2014, 346, 12580961 - 12580969).
  • the CRISPR/Cas system is a prokaryotic adaptive immune response system that uses non-coding RNAs to guide Cas nucleases to induce site-specific nucleic acid cleavage.
  • the subsequently damaged (cleaved) nucleic acid is either functionally altered (i.e. for RNA targets) or may then be repaired using various cellular repair mechanisms, such as the non-homologous end joining DNA repair pathway (NHEJ) or the homology directed repair (HDR) pathway (i.e. for DNA targets).
  • NHEJ non-homologous end joining DNA repair pathway
  • HDR homology directed repair
  • a single "guide" RNA gRNA
  • the mammalian cell Upon breaking (cleavage) of the double- stranded DNA, the mammalian cell then utilizes one of the above noted endogenous mechanisms to repair the break.
  • the double strand break can selectively be repaired by utilisation of the HDR pathway.
  • the template can impart a desired genomic alteration into the target DNA (e.g. an insertion, a removal and/or a replacement or part of the genome).
  • the present invention may also provide the use of an oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C, as defined herein, as a guide RNA in a CRISPR-Cas process.
  • the present invention provides a use of an oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C, as defined herein, as a guide RNA in CRISPR-Cas9, CRISPR-Cas12a and/or CRISPR-Cas13a process.
  • the present invention provides a use of an oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C, as defined herein, as a guide RNA in a CRISPR-9 genome editing process.
  • the present invention provides a use of an oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C, as defined herein, as a donor DNA template in a CRISPR-Cas mediated homology directed repair (HDR) process.
  • the present invention provides a use of an oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C, as defined herein, as a donor DNA template in a CRISPR-Cas9 mediated homology directed repair (HDR) process.
  • the present invention also provides a method of using an oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C as defined herein, in a CRISPR-Cas process (e.g. in a CRISPR-Cas9, CRISPR-Cas 12a and/or CRISPR-Cas 13 process).
  • a CRISPR-Cas process e.g. in a CRISPR-Cas9, CRISPR-Cas 12a and/or CRISPR-Cas 13 process.
  • the present invention provides a method of using an oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C as defined herein, in a CRISPR-Cas9 gene editing process.
  • Figure 1 shows a schematic representation of how the backbone modifications are introduced via post-synthetic chemical oligonucleotide ligation (templated or untemplated).
  • Figure 2 shows the optimisation of oligonucleotide ligation using amide coupling to give the artificial backbone Ami .
  • Samples were analysed by denaturing PAGE post-stained with 1x SYBR gold.
  • the final EDC:NHS concentration was kept constant at 1000:250 ⁇ with amine, carboxylic acid and splint oligonucleotide concentrations fixed at 1 , 1.5 and 1.5 ⁇ respectively.
  • Certain reactions were performed with pre-activation of the carboxylic acid separately before addition of the amine and splint in buffer. Un-buffered conditions gave the strongest product bands within 10 min. Full buffer composition can be found in the 'Amide oligonucleotide ligation' section.
  • C negative control (i.e. no coupling reagents).
  • Figure 3 shows the optimisation of one-pot oligonucleotide ligation using amide coupling to give the artificial backbone Ami .
  • Figure 4 shows the kinetics of backbone read-through. Templates were amplified by PCR and extension times varied (0.5 - 8 min) using hot-start Taq (exo-) and Phusion (exo+) polymerases. Templates are color-coded. Lines of best fit are for trend visualization. The pentagon represents the site of the modified linkage. Full amplification curves and duplex melting curves confirming product formation can be found in Figures 4a and 4b.
  • Figures 5a and 5b show the qPCR amplification curves for different backbone modifications as a function of PCR extension time (0.5 - 8 min, light blue to dark blue) and polymerase. Melting curves demonstrate single product formation. Ct values were determined at a fixed threshold of 300 fluorescence units, which is in the exponential region of amplification for all qPCR curves.
  • Figures 6a and 6b show the profile of mutations generated by DNA polymerases upon replication of modified backbones using lllumina deep sequencing.
  • B Correlation between the unique mutated sequences observed (frequency>0.005) and polymerases, with the frequency of observation color-coded. Conformation of PCR and linear extension products used for library preparation can be found in Figures 6a to 6e.
  • m CX m CG- clamp. 42
  • Figures 7a to 7e show the bioanalyzer DNA 1000 electrophoretograms of PCR and linear extension products from different polymerases for sequencing analysis. Single major products of the expected size were observed for all templates and polymerases. Differences in size between templates are due to slight differences in primer size.
  • Figure 8 shows the 8% denaturing polyacrylamide gel for transcription of amide template ODN5 and control template ODN6. Note that control template is longer than amide template. Full length product was confirmed by mass spectrometry.
  • Figure 9 shows primer extension time course experiments (5 - 240 min) using Klenow large fragment DNA polymerase I (0.1 U/ ⁇ ) at 37 °C. Samples were analysed by denaturing PAGE and fluorescent visualisation of the FAM labelled primers
  • Reactions were monitored by thin layer chromatography (TLC) using Merck Kieselgel 60 F24 silica gel plates (0.22 mm thickness, aluminium backed). The compounds were visualised by UV irradiation at 254/365 nm and by staining in p-anisaldehyde solution or KMnO4 (10% aq.). Column chromatography was carried out using Merck Geduran 60 A (40-63 micron) silica.
  • the reaction was cooled to 4 °C and concentrated NH 3 added (16 ml_) before bringing the reaction to room temperature and stirring for 17 h.
  • the reaction mixture was concentrated in vacuo before additional pyridine (20 ml_) and concentrated NH 3 (16 ml_) were added, and the reaction stirred for 5 h. After evaporating to dryness, the residue was extracted using DCM and water. The aqueous layer was extracted three times with DCM. The organic fractions were then combined and washed with a saturated KCI solution before evaporating to dryness.
  • Pentachlorophenol (0.12 g, 0.45 mmol) was added and the slurry rotated for a further 1 h before washing with pyridine, DCM, Et20 and DMF (three time each). The resin was then washed with 10 % piperidine in DMF and incubated with the same solution for 1 min. After washing with DMF, DCM and Et20 (three times each) and drying in vacuo, the resin was incubated with a 1 :1 mix of oligonucleotide synthesis grade Cap Mix A (8: 1 : 1 acetic anhydride/pyridine/THF, 2.5 ml_) and Cap Mix B (84: 16 THF/ N-methylimidazole, 2.5 ml_) and rotated for 60 min.
  • Cap Mix A 8: 1 : 1 acetic anhydride/pyridine/THF, 2.5 ml_
  • Cap Mix B 84: 16 THF/ N-methylimidazole, 2.5 ml_
  • Custom Primer Support Amino 200 (9, 750 mg, 0.15 mmol of amine, GE Healthcare) was activated with 3% TCA in DCM for 1 h.
  • the solid support was washed with NEt3 / A/,A/-diisopropylethylamine (9: 1), DCM and Et20 before drying in vacuo for 1 h.
  • the resin was soaked in dry pyridine for 10 min.
  • Solid support 10 (0.3 g) was soaked in pyridine for 10 min.
  • DNA reagents including standard DNA phosphoramidites and solid supports (CPG resin) were purchased from Link Technologies Ltd. Oligonucleotides were synthesised on an Applied Biosystems 394 automated DNA/RNA synthesiser. Phosphoramidite cycles, including acid-catalysed detritylation, coupling, capping and iodine oxidation steps, were undertaken in 0.2 or 1.0 ⁇ scale. Standard ⁇ -cyanoethyl phosphoramidite monomers were used for all the oligonucleotide sequences. Coupling efficiencies and overall oligonucleotide yields were determined by the automated trityl cation conductivity monitoring facility of the synthesiser and were ⁇ 98.0% for all cases.
  • Standard phosphoramidite monomers were dissolved in anhydrous acetonitrile to a concentration of 0.1 M immediately prior to use.
  • the coupling time for A, G, C and T monomers was set to 60 s, and for modified monomers 600 s.
  • the oligonucleotides were then cleaved and deprotected by exposure to concentrated aqueous ammonium hydroxide for 60 min at room temperature followed by heating in a sealed tube for 5 h at 55 °C. The ammonia was then allowed to evaporate under normal pressure for 4 - 5 h.
  • oligonucleotides For phosphorothioate and phosphorodithioate oligonucleotides, tetraethyliuram disulfide in acetonitrile was used in the oxidation step instead of the standard iodine oxidizer for 900 s instead of 20 s. These oligonucleotides were deprotected using concentrated ammonium hydroxide / ethanol (3:1 v/v) containing dithiothreitol (20 mM) for 17 h at 55 °C.
  • NAP-25 columns G.E. Healthcare Life Sciences, cat. no. 17-0854-01 were used.
  • EDC HCI A/-(3-dimethylaminopropyl)-A/'-ethylcarbodiimide hydrochloride
  • NHS /V-hydroxysuccinimide
  • the azide and alkyne oligonucleotides (10 nmol each) were freeze dried together, re-suspended in water (80 ⁇ ), and heated to 95 °C (5 min) before cooling to room temperature rapidly on ice to avoid unexpected base pairing. The sample was then degassed using argon for 5 min. Separately, CuS0 4 .H 2 0 (1 ⁇ , 0.1 M) and tris- (hydroxypropyltriazolylmethyl)amine (0.3 mg) were mixed in water (17 ⁇ ) before the addition of sodium ascorbate (2 ⁇ , 0.5 M) under argon. The two solutions were mixed and incubated at room temperature for 2 - 3 h before purification by denaturing PAGE.
  • the azide, alkyne and complementary splint oligonucleotides (5 nmol each) were dissolved in an aqueous solution of NaCI (0.2 M, 100.0 ⁇ _) and annealed by heating at 90 °C for 5 min before cooling slowly (2 - 3 h) to room temperature. The sample was then degassed using argon for 5 min.
  • qPCR reactions were performed using hot-start Taq (NEB, cat. no. M0495S) or hot-start flex Phusion (NEB, cat. no. M0535S) DNA polymerases on a Bio-Rad CFX96. Master- mixes composed of either Phusion HF buffer (5x, 4 ⁇ ), EvaGreen (Biotium, cat. no.
  • PCR thermal cycling conditions consisted of thermal activation (120 s, 95 °C), and 31 cycles of denaturation (15 s, 95 °C) and annealing/extension (30, 60, 120, 180, 240, 360 or 480 s, 60 °C), with emission recorded at the end of each extension step.
  • Samples were excited at 450-490 nm and emission monitored at 510-530 nm.
  • melt curves analysis samples were heated from 60 to 90 °C post-PCR with emission recorded every 0.5 °C using a ramp rate of 6 °C/min. Single products were confirmed by single peaks in 3F/3T vs. T plots.
  • Amplifications curves were baseline corrected using the CFX96 internal analysis software, before linear interpolation of the data at 0.1 cycle intervals. Threshold cycles were determined at 300 RFU, where all reactions are in the exponential phase of growth.
  • PCR reactions were performed using GoTaq (Promega, cat. no. M3001), KOD XL (Merck Millipore, cat. no. 71087) or Phusion (NEB, cat. no. M0530S) DNA polymerase on a Bio- Rad CFX96.
  • PCR thermal cycling conditions consisted of thermal activation (120 s, 95 °C), and 26 cycles of denaturation (15 s, 95 °C), annealing (20 s, 54 °C) and extension (30 s, 72 °C). Each reaction was then mixed with phenol-chloroform (20 ⁇ , ThermoFisher Scientific, cat. no. 15593031), vortexed for 30 s and centrifuged (1000 rpm, 5 min).
  • the aqueous layer was then transferred to a new tube, mixed with sodium acetate (2 ⁇ , 3 M, pH 5.2) and ethanol (66 ⁇ ), before incubating at -80 °C overnight.
  • the samples were then centrifuged (13,000 rpm, 20 min), the supernatant removed and the pellet re-dissolved in water (20 ⁇ ).
  • Primer extension reactions for the artificial templates were performed using Klenow large fragment DNA polymerase I (NEB, cat. no. M0210S) and primers in Table 3 below on a Bio-Rad T100.
  • lllumina sequencing libraries were prepared using the TruSeq DNA PCR-free Library Preparation Kit (lllumina, cat. no. FC-121-3001) starting from the 'Adenylate 3' ends' step.
  • TruSeq DNA PCR-free Library Preparation Kit lllumina, cat. no. FC-121-3001
  • the lllumina recommended protocol was followed. The only exception is the volume of SPB beads (provided by lllumina) to DNA used, where a 1.5: 1 ratio was used to facilitate recovery of shorter DNA fragments.
  • the sorted reads were counted for unique reads that occur more than 0.5% of the total reads. These reads were then pair-wise aligned to the expected template using the EMBOSS needle 62 settings above in order to identify unique sequences in the region local to the modification (four and eight bases to 5V3'-sides) before correlation of unique templates between the different polymerases used.
  • Oligonucleotides used for chemical ligation Oligonucleotides were purified by denaturing PAGE or HPLC. If purified by HPLC, purity was confirmed by running analytical denaturing PAGE. Terminal base modification nomenclature is depicted at the end of the table and the synthesis described in the Oligonucleotide synthetic and purification procedures' section.
  • 24483 24484 P (com.) (UU) CACTGACAATACACCACAACTCAGAC

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Abstract

La présente invention concerne un procédé de préparation de certains oligonucléotides, en particulier un procédé de ligature d'un premier et d'un second oligonucléotide ensemble. La présente invention concerne également des oligonucléotides obtenus à partir de ce procédé et l'utilisation desdits oligonucléotides dans des procédés de PCR, de réplication, de transcription, de transcription inverse, de traduction et CRISPR-Cas.
PCT/GB2018/050091 2017-01-13 2018-01-12 Ligature d'oligonucléotides Ceased WO2018130848A1 (fr)

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GBGB1700595.0A GB201700595D0 (en) 2017-01-13 2017-01-13 Oligonucleotide ligation
GB1700595.0 2017-01-13

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US5663312A (en) * 1993-03-31 1997-09-02 Sanofi Oligonucleotide dimers with amide linkages replacing phosphodiester linkages
EP0714907A1 (fr) * 1994-11-30 1996-06-05 F. Hoffmann-La Roche Ag Oligonucléotides avec squelette d'amides
WO1998000434A1 (fr) * 1996-06-28 1998-01-08 Novartis Ag Oligonucleotides modifies

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