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WO2018130848A1 - Oligonucleotide ligation - Google Patents

Oligonucleotide ligation 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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WIPO (PCT)
Prior art keywords
oligonucleotide
formula
phosphodiester backbone
nucleotide
alkyl
Prior art date
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PCT/GB2018/050091
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French (fr)
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

The present invention relates to a process for preparing certain oligonucleotides, in particular a process of ligating a first and a second oligonucleotide together. The present invention also relates to oligonucleotides obtained from this process and to the use of said olifonucleotides in PCR, replication, transcription, reverse transcription, translation and CRISPR-Cas processes.

Description

OLIGONUCLEOTIDE LIGATION
INTRODUCTION
[0001] 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.
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] The use of enzymatic ligation to synthesise long DNA and RNA constructs also has many associated problems, one of which being the incompatibility of these enzymatic processes with multiple modifications at the sugar, base and/or phosphate of the oligonucleotide units. Furthermore, these enzymatic ligation methodologies are often extremely sensitive to the medium in which they are carried out and often require specific buffering conditions in order for the ligation reactions to proceed.
[0004] In an attempt to overcome this long standing problem in the art, multiple attempts have been made to use chemical ligation as a means of providing ligated oligonucleotide constructs [see, for example, International Patent Publication No. W02008/120016; Kumar et al. 2007, J Am Chem Soc 129, 6859-6864; Kocalka et al. 2008, Chem Bio Chem, 9, 1280-1285, and El-Sagheer et al. 2009, J.Am. Chem. Soc. 131 (11), 3958-3964]. However, success in using chemical ligation has been limited due, in part, to the fact that the unnatural linkages employed were found to be incompatible with DNA and RNA polymerases, meaning that the nucleotide sequences of these oligonucleotides could not be read accurately, resulting in the mis-reading and/or skipping of certain nucleotides when the sequences were attempted to be replicated.
[0005] Despite the difficulties associated with chemical ligation techniques, some success has been achieved by chemically ligating oligonucleotides by using certain triazole-based backbone mimics [see US Patent No. 8,846,883]. However, to further advance the use of oligonucleotides, in particular modified oligonucleotides, in synthetic biology, and to provide greater access into long DNA and RNA molecules for biological and nanotechnological application, there remains a need for new, improved and orthogonal methodologies that can be used on an industrial scale to synthesise lengthened DNA and RNA constructs that is capable of being read correctly by DNA and RNA polymerase.
[0006] The present invention was devised with the foregoing in mind.
SUMMARY OF THE INVENTION
[0007] According to one aspect of the present invention, there is provided a process for ligating a first and a second oligonucleotide together as defined herein.
[0008] According to a second aspect of the present invention, there is provided an oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C as defined herein.
[0009] According to a third aspect of the present invention, there is provided a 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).
[0010] According to a fourth aspect of the present invention, there is provided a 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.
[0011] According to a fifth aspect of the present invention, there is provided 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 a corresponding RNA transcript, or as a template in a reverse transcription process to provide a corresponding DNA transcript.
[0012] According to a sixth aspect of the present invention, there is provided 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.
[0013] According to a seventh aspect of the present invention, there is provided a method for amplifying an oligonucleotide sequence as defined herein.
[0014] According to an eighth aspect of the present invention, there is provided a method for replicating an oligonucleotide sequence as defined herein. [0015] According to a ninth aspect of the present invention, there is provided a method for producing a ribonucleic acid (RNA) sequence or deoxyribonucleic acid (DNA) sequence as defined herein.
[0016] According to a tenth aspect of the present invention, there is provided a method for preparing a protein or peptide as defined herein.
[0017] According to an eleventh aspect of the present invention, there is provided a use of 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).
[0018] According to a twelfth aspect of the present invention, there is provided 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 (e.g. in a CRISPR-Cas9 mediated homology directed repair (HDR) process).
[0019] According to a thirteenth aspect of the present invention, there is provided 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).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0020] Unless otherwise stated, the following terms used in the specification and claims have the following meanings set out below.
[0021] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of them mean "including but not limited to", and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
[0022] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or examples of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.
[0023] The term "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. For example, "(1-6C)alkyl" includes (1-4C)alkyl, (1-3C)alkyl, propyl, isopropyl and f-butyl. A similar convention applies to other radicals, for example "phenyl(1- 6C)alkyl" includes phenyl(1-4C)alkyl, benzyl, 1-phenylethyl and 2-phenylethyl.
[0024] The term "(m-nC)" or "(m-nC) group" used alone or as a prefix, refers to any group having m to n carbon atoms.
[0025] "(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.
[0026] The term "halo" refers to fluoro, chloro, bromo and iodo.
[0027] The term "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). Suitably, any given "haloalkyl" is a "fluoroalkyl" in which one or more hydrogen atoms have been replaced by fluorine atoms. Examples of fluoroalkyl groups include -CHF2, -CH2CF3, or perfluoroalkyl groups such as -CF3 or -CF2CF3. An analogous definition applies to the term "haloalkoxy".
[0028] The term "heterocyclyl", "heterocyclic" or "heterocycle" 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. Examples of 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. Other heterocycles include dihydro-oxathiolyl, tetrahydro-oxazolyl, tetrahydro-oxadiazolyl, tetrahydrodioxazolyl, tetrahydro-oxathiazolyl, hexahydrotriazinyl, tetrahydro-oxazinyl, morpholinyl, thiomorpholinyl, tetrahydropyrimidinyl, dioxolinyl, octahydrobenzofuranyl, octahydrobenzimidazolyl, and octahydrobenzothiazolyl. For heterocycles containing sulfur, 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. A suitable value for a heterocyclyl group which bears 1 or 2 oxo (=0) or thioxo (=S) substituents is, for example, 2-oxopyrrolidinyl, 2-thioxopyrrolidinyl, 2-oxoimidazolidinyl, 2-thioxoimidazolidinyl, 2-oxopiperidinyl, 2,5-dioxopyrrolidinyl, 2,5-dioxoimidazolidinyl or 2,6-dioxopiperidinyl. Particular 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. As the skilled person would appreciate, any heterocycle may be linked to another group via any suitable atom, such as via a carbon or nitrogen atom. However, reference herein to piperidino or morpholino refers to a piperidin-1-yl or morpholin-4-yl ring that is linked via the ring nitrogen.
[0029] The term "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. Examples of 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. Typically the heteroaryl ring will contain up to 3 heteroatoms, more usually up to 2, for example a single heteroatom. In one embodiment, 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.
[0030] Examples of heteroaryl 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, naphthyridinyl, carbazolyl, phenazinyl, benzisoquinolinyl, pyridopyrazinyl, thieno[2,3-b]furanyl, 2H-furo[3,2-b]-pyranyl, 5H-pyrido[2,3-d]-o-oxazinyl, 1 H-pyrazolo[4,3-d]-oxazolyl, 4H-imidazo[4,5-d]thiazolyl, pyrazino[2,3-d]pyridazinyl, imidazo[2, 1-b]thiazolyl, imidazo[1 ,2-b][1 ,2,4]triazinyl. "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. Examples of 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,
1 ,2,3,4-tetrahydro-1 ,8-naphthyridinyl, 1 ,2,3,4-tetrahydropyrido[2,3- 5]pyrazinyl and
3,4-dihydro-2/-/-pyrido[3,2-£>][1 ,4]oxazinyl.
[0031] Examples of five membered heteroaryl groups include but are not limited to pyrrolyl, furanyl, thienyl, imidazolyl, furazanyl, oxazolyl, oxadiazolyl, oxatriazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, triazolyl and tetrazolyl groups.
[0032] Examples of six membered heteroaryl groups include but are not limited to pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl and triazinyl.
[0033] Examples of 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.
[0034] Examples of 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.
[0035] The term "aryl" means a cyclic or polycyclic aromatic ring having from 5 to 12 carbon atoms. The term 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.
[0036] Where optional substituents are chosen from "one or more" groups it is to be understood that this definition includes all substituents being chosen from one of the specified groups or the substituents being chosen from two or more of the specified groups. [0037] The phrase "compound of the invention" means those compounds which are disclosed herein, both generically and specifically.
[0038] The terms "oligonucleotide analogue" and "nucleotide analogue" refer to any modified synthetic analogues of oligonucleotides or nucleotides respectively that are known in the art. Examples of oligonucleotide analogues include peptide nucleic acids (PNAs), morpholino oligonucleotides, phosphorothioate oligonucleotides, phosphorodithioate oligonucleotides, alkylphosphonate oligonucleotides, acylphosphonate oligonucleotides and phosphoramidate oligonucleotides.
[0039] The term "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).
Process of the invention
[0040] According to one aspect of the present invention, there is provided a process for ligating a first and a second oligonucleotide together, wherein the process comprises reacting:
a) a first oligonucleotide comprising a terminal functional group of Formula A shown below:
Figure imgf000009_0001
Formula A
wherein
» w r denotes the point of attachment to the oligonucleotide backbone; and X is a leaving group optionally selected from halo, OSO2R, (1-2C)haloalkyl, (1- 2C)haloalkoxy, OR1 , heteroaryl, wherein R and R1 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 R1 are a solid support to which the one or more oligonucleotides are attached; V is selected from O, S or NRX, wherein Rx is selected from hydrogen or (1-4C)alkyl; Q is O or S;
Ra and Rb 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; and
q is an integer selected from 0 to 1 ;
with
a second oligonucleotide comprising a terminal functional group of Formula B, shown below:
Figure imgf000010_0001
Formula B
wherein:
Rc and Rd are independently selected from hydrogen or (1-4C)alkyl;
Y is selected from O or NH;
W is selected from NRe or SH, wherein Re 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 ;
or
b) reacting a second oligonucleotide comprising a terminal functional group of Formula B, as defined above, with a further second oligonucleotide comprising a terminal functional group of Formula B, as defined above, together with a coupling agent of Formula D shown below:
Figure imgf000011_0001
Forrmula D
wherein:
Q1 is selected from O or S; and
LG1 and LG2 are each independently a leaving group (e.g. halo, imidazolyl or haloalkoxy);
and wherein the reaction is optionally conducted in the presence of one or more of the following:
i) a template oligonucleotide;
ii) one or more peptide coupling reagents;
iii) one or more activating agents; and
iv) a catalyst;
and with the proviso that:
1) the sum of integers m, n, p and q is equal to or greater than 2; and
2) when p is 1 , q is 0.
[0041] 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. Furthermore, 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. Long DNA and RNA constructs are otherwise difficult to synthesise using standard solid-phase synthesis. Additionally, the process of the present invention is amenable to scale-up, and therefore allows oligonucleotides to be produced on a large scale, something which is challenging to achieve using current enzymatic ligation methodology.
[0042] It will be appreciated that any suitable reaction conditions may be used in the process defined hereinabove. Furthermore, it will be understood that the 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. A person skilled in the art will be able to select suitable reaction conditions (e.g. temperature, pressures, reaction times, concentration etc.) to use in the present process.
[0043] In an embodiment, the process of the present invention is conducted at a temperature of between 0 °C and 150 °C. Suitably, 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.
[0044] In an embodiment, 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.
[0045] In another embodiment, the process of the present invention is carried out in an aqueous medium at a pH within the range of 5 to 9. Suitably, the process of the present invention is carried out at a pH within the range of 6 to 8. Most suitably, the process of the present invention is carried out at a pH within the range of 6.5 to 7.5.
[0046] In an embodiment, 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.
[0047] It will be understood that any suitable buffer may be used. In an embodiment, the buffer is selected from the group comprising: phosphate, acetate, borate, citrate, sulfonic acid, ascorbate, linolenate, carbonate and bicarbonate based buffers. In a further embodiment, the buffer is selected from the group comprising: phosphate, acetate, carbonate and bicarbonate based buffers. In a particular embodiment, the buffer is sodium phosphate or sodium carbonate.
[0048] The resultant oligonucleotides formed by the process of the present invention may be isolated and purified using any suitable techniques known in the art. Suitably, the resultant oligonucleotides formed by the process of the present invention may be isolated and purified using column chromatography, for example, using sephadex columns.
[0049] In an embodiment, the process of the present invention is conducted in the presence of a salt (e.g. NaCI). Any suitable concentration of salt may be used. Suitably, 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.
[0050] In another embodiment, one of the first or second oligonucleotides is present in an excess.
[0051] In yet another embodiment, the process of the present invention is conducted in the presence of a template oligonucleotide. It will be appreciated that the 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. It will be understood that the template oligonucleotide may also comprise synthetic oligonucleotide analogues, such as, for example, peptide nucleic acid (PNA).
[0052] In an embodiment, the template oligonucleotide is a single stranded oligonucleotide or oligonucleotide analogue.
[0053] It will be understood that 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.
[0054] In yet another embodiment, the template oligonucleotide comprises between 2 and 100 nucleotide monomer units. Suitably, 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.
[0055] In a further embodiment, the process of the present invention is carried out in the presence of a catalyst. It will be understood that a catalyst may be any suitable reagent that helps to promote the rate of the reaction between the first and second oligonucleotide. Suitably, the catalyst is an acid and/or a base. Most suitably, the catalyst is a base. Non-limiting examples of suitable bases include NaOH, trimethylamine, diisopropylethylamine and N-methylmorpholine.
[0056] In yet a further embodiment, 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).
[0057] In an embodiment, the peptide coupling reagent is a carbodiimide-based coupling reagent.
[0058] Suitably, 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), Ν,Ν'-dicyclohexylcarbodiimide (DCC), Ν,Ν'- diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), N- cyclohexyl-N'-isopropylcarbodiimide (CIC) or Ν,Ν'-dicyclopentylcarbodiimide (CPC). More suitably, the coupling reagent is selected from Ν,Ν'-dicyclohexylcarbodiimide (DCC), Ν,Ν'- diisopropylcarbodiimide (DIC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI). Most suitably, the coupling reagent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI).
[0059] 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.
[0060] In an embodiment, the activating agent is N-hydroxysuccinimde (NHS), N- hydroxysulfosuccinimide (Sulfo-NHS) or ethyl cyano(hydroxyimino)acetate (Oxyma Pure®). Suitably, the activating agent is N-hydroxysuccinimde (NHS).
[0061] In a further embodiment, 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). Suitably, 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. [0062] In yet a further embodiment, the the sum of integers m, n, p and q is 2, 3 or 4. Suitably, the the sum of integers m, n, p and q is 2 or 3. Most suitably, the the sum of integers m, n, p and q is 2.
[0063] In certain embodiments, one or more of the following proivos may apply:
i) when Q is S, V and W are not S; and
ii) when Q1 is S, W is not S.
The terminal functional group of Formula A
[0064] In certain embodiments, each of X, V, Q, Ra, Rb, 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: -
(1) X is selected from halo, OS02R, (1-2C)haloalkyl, (1-2C)haloalkoxy, OR1 , 5-membered heteroaryl, wherein R and R1 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 R1 are a solid support to which the one or more oligonucleotides are attached;
(2) X is selected from halo, OS02R, (1-2C)haloalkyl, (1-2C)haloalkoxy, OR1 , triazolyl,
wherein R and R1 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;
(3) X is selected from halo, OS02R, (1-2C)haloalkyl, (1-2C)haloalkoxy, OR1 , triazolyl,
wherein R and R1 are independently selected from H, (1-6C)alkyl, (1-6C)alkonyl, phenyl or (1-2C)haloalkyl;
(4) X is selected from halo, (1-2C)haloalkyl or OR1 , wherein R and R1 are independently selected from H, (1-6C)alkyl or a (1-6C)alkonyl;
or
R and R1 are a solid support to which the one or more oligonucleotides are attached
(5) X is selected from halo, (1-2C)haloalkyl or OR1 , wherein R and R1 are independently selected from H, (1-6C)alkyl or a (1-6C)alkonyl;
(6) X is selected from OR1 , wherein R and R1 are independently selected from H or (1- 6C)alkyl;
(7) X is OH;
(8) V is selected from O or NRX, wherein Rx is selected from hydrogen or (1-4C)alkyl;
(9) V is O; (10) V is NRX, wherein Rx is selected from hydrogen or methyl;
(1 1) V is NH;
(12) Q is O;
(13) Q is S;
(14) Ra and Rb are independently selected from hydrogen or (1-4C)alkyl, wherein each (1- 4C)alkyl is optionally substituted with one or more OH;
(15) Ra and Rb are independently selected from hydrogen or (1-4C)alkyl;
(16) Ra and Rb are independently selected from hydrogen or (2-4C)alkyl;
(17) Ra and Rb are independently selected from hydrogen or methyl;
(18) Ra and Rb are hydrogen;
(19) n is 1 ;
(20) q is 1 ;
(21) q is O.
[0065] Suitably, 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.
[0066] Suitably, a heteroaryl is a 5-membered heteroaryl ring comprising one, two or three heteroatoms selected from N, O or S. Most suitably, a heteroaryl is a 5-membered heteroaryl ring comprising one, two or three nitrogen heteroatoms.
[0067] Suitably, a heterocyclyl group is a 4-, 5- or 6-membered heterocyclyl ring comprising one, two or three heteroatoms selected from N, O or S. Most suitably, 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].
[0068] Suitably an aryl group is phenyl.
[0069] Suitably, X is as defined in any one of paragraphs (1) to (7) above. Most suitably, X is as defined in paragraph (7) above.
[0070] Suitably, V, when present, is as defined in any one of paragraphs (8) to (11) above. Most suitably, V, when present, is NH.
[0071] In a particular embodiment, the first oligonucleotide comprises a terminal functional group of Formula A1 , shown below
Figure imgf000016_0001
Formula A1 wherein each of Ra, Rb, X, n and q are as defined hereinabove.
[0072] In a particular embodiment, the first oligonucleotide comprises a terminal functional group of Formula A2, shown below:
Figure imgf000017_0001
Formula A2
wherein each of Ra, Rb, X and n are as defined hereinabove.
[0073] In a particular embodiment, the first oligonucleotide comprises a terminal functional group of Formula A3, shown below:
Figure imgf000017_0002
Formula A3
wherein X is as defined hereinabove.
[0074] In a specific embodiment, the first oligonucleotide comprises a terminal functional group of Formula A4, shown below:
Figure imgf000017_0003
Formula A4
[0075] In yet another embodiment, the first oligonucleotide comprises a terminal functional group of Formula A5, shown below:
Figure imgf000017_0004
Formula A5
wherein each of Ra, Rb, V, X, and n are as defined hereinabove.
[0076] In another embodiment, 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
[0077] In certain embodiments, each of Y, W, Rc, Rd, Re, 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: -
(A) Y is O;
(B) Y is NH;
(C) W is NRe, wherein Re is selected from hydrogen or (1-4C)alkyl;
(D) W is NH;
(E) Rc and Rd are independently selected from hydrogen or methyl;
(F) Rc and Rd are hydrogen;
(G) m is 1 ;
(H) p is O;
(I) p is 1.
[0078] In an embodiment, 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.
[0079] Suitably, W is as defined in any one of paragraphs (C) or (D) above. Most suitably, W is
NH.
[0080] In a specific embodiment, the second oligonucleotide comprises a terminal functional group of Formula B1 , shown below:
Figure imgf000018_0001
Formula B1
wherein each of Y, Rc, Rd, m and p are as defined hereinabove.
[0081] In a specific embodiment, the second oligonucleotide comprises a terminal functional group of Formula B2, shown below:
Figure imgf000018_0002
Formula B2 wherein each of Rc, Rd and m are as defined hereinabove.
[0082] In a specific embodiment, the second oligonucleotide comprises a terminal functional group of Formula B3, shown below:
Figure imgf000019_0001
Formula B3
[0083] In another specific embodiment, the second oligonucleotide comprises a terminal functional group of Formula B4, shown below:
Formula B4
wherein each of Rc, Rd, Y and m are as defined hereinabove.
[0084] Furthermore, it will be appreciated that the 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")-
[0085] The process of the present invention may be carried out in the presence of a base (e.g. NaOH).
[0086] In another embodiment, the terminal functional group of Formula B, B1 , B2, B3 or B4 is attached to the 5' position of the second oligonucleotide.
Coupling agent of formula D
[0087] In certain embodiments, 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.
[0088] 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.
[0089] Suitably, 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.
[0090] In an embodiment, Q1 of the coupling agent is oxygen. [0091] It will be appreciated that LG1 and LG2 may independently be selected from any suitable leaving group. Non-limiting examples of suitable leaving groups include halo, heteroaryl, alkoxy, haloalkyl or haloalkoxy. Suitably, the LG1 and LG2 are both selected from halo (e.g. CI), heteroaryl (e.g. imidazolyl) or haloalkoxy (e.g. OCCU).
[0092] In a particular embodiment, the coupling agent is selected from phosgene, triphosgene or carbonyldiimidazole. Suitably, the coupling agent is carbonyldiimidazole.
First and second oligonucelotides
[0093] It will be appreciated that the first and second oligonucleotides of the present process may independently comprise any suitable number and/or type of nucleotide and/or nucleotide analogue monomers.
[0094] In an embodiment, 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.
[0095] It will be understood that the oligonucleotides described herein encompass all suitable salt, hydrate and/or solvate forms thereof.
[0096] In an embodiment, the first oligonucleotide comprises a terminal nucleotide analogue select
Figure imgf000020_0001
wherein:
Ra and Rb are as defined herein;
Ra' and Rb' are independently selected from hydrogen or (1-4C)alkyl; Z is selected from hydrogen, halo, (1-4C)alkyl, (1-2C)haloalkyl, OR2 wherein R2 is selected from hydrogen, (1-4C)alkyl, (2-4C)alkenyl or 4C)alkynyl; and
B is a nucleobase or nucleobase analogue.
[0097] Suitably, the first oligonucleotide comprises a terminal nucleotide analogue of the formula:
Figure imgf000021_0001
wherein each of Ra, Rb, Z and B are as defined hereinabove.
[0098] In another embodiment, the second oligonucleotide comprises a terminal nucleotide analogue selected from one of the following:
Figure imgf000021_0002
wherein:
Rc, Rd and Re are each as defined herein;
Rc' and Rd' are independently selected from hydrogen or (1-4C)alkyl;
Z is selected from hydrogen, halo, (1-4C)alkyl, (1-2C)haloalkyl, OR2 or NH2, wherein R2 is selected from hydrogen, (1-4C)alkyl, (2-4C)alkenyl or (2- 4C)alkynyl; and
B is a nucleobase or nucleobase analogue.
Suitably, the second oligonucleotide comprises a terminal nucleotide analogue of the
Figure imgf000022_0001
wherein each of Rc, Rd, Re, Z and B are as defined herein.
[00100] In certain embodiments, it will be appreciated that the 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).
[00101] In other embodiments, it will be appreciated that the 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).
[00102] It will be appreciated that 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. In an embodiment, B is a nucleobase selected from A, G, C, T or U.
[00103] In an embodiment, Z is selected from hydrogen, halo, OR2 or NH2, wherein R2 is selected from hydrogen, (1-4C)alkyl, (2-4C)alkenyl or (2-4C)alkynyl. Suitably, Z is selected from hydrogen, halo, OR2 or NH2, wherein R2 is selected from hydrogen or (1-4C)alkyl. More suitably, Z is selected from hydrogen, fluoro, OH, OMe or NH2.
[00104] In another embodiment, each of Ra', Rb', Rc' and Rd' are independently selected from hydrogen or methyl. Suitably, each of Ra', Rb', Rc' and Rd' are hydrogen.
[00105] In a further aspect of the present invention there is provided an oligonucleotide obtainable by, obtained by or directly obtained by the process of the present invention.
Oligonucleotides
[00106] According to another aspect of the present invention, there is provided an oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C shown below:
Figure imgf000022_0002
Formula C wherein:
Ra and Rb 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;
Rc, Rd and Re are independently selected from hydrogen or (1-4C)alkyl;
Y is selected from O or NH;
V is selected from O, S or NRX, wherein Rx is selected from hydrogen or (1- 4C)alkyl;
Q is O or S;
m and n are integers independently selected from 0 to 2; and
p and q are integers independently selected from 0 to 1 ;
with the proviso that:
1) the sum of integers m, n, p and q is equal to or greater than 2;
2) when p is 1 , q is 0;
3) when q and p are 0 and the oligonucleotide comprises only one phosphodiester backbone mimic of Formula C, the oligonucleotide comprises greater than or equal to 20 nucleotide and/or nucleotide analogue monomers; and
4) when q and p are 0 and the oligonucleotide comprises two or more phosphodiester backbone mimics of Formula C, the phosphodiester backbone mimics are separated by at least 10 nucleotide and/or nucleotide analogue monomers.
[00107] In Formula C above, each of Ra, Rb, Rc, Rd, Re, Y, V, Q, m, n, p and q are as defined hereinbefore in relation to the process of the present invention.
[00108] In an embodiment, the oligonucleotide comprises one or more phosphodiester backbone mimics of Formula C1 shown below:
Figure imgf000023_0001
Formula C1
wherein:
Ra, Rb, Rc, Rd, Re and Rx 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 ;
with the proviso that n + m + q = 2 or 3;
and with the proviso that:
a) when q is 0 and the oligonucleotide comprises only one phosphodiester backbone mimic of Formula C1 , the oligonucleotide comprises greater than or equal to 20 nucleotide and/or nucleotide analogue monomers; or
b) when q is 0 and 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.
[00109] In an embodiment, the oligonucleotide comprises one or more phosphodiester backbone mimics of Formula C2 shown below :
Figure imgf000024_0001
Formula C2
wherein:
Ra, Rb, Rc, Rd and Re are independently selected from hydrogen or (1-4C)alkyl; and
n and m are integers independently selected from 0 to 2, with the proviso that n + m = 2 or 3;
and with the proviso that:
a) when 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
b) when 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.
[00110] In an embodiment, the oligonucleotide comprises one or more phosphodiester backbone mimics of Formula C3 shown below:
Figure imgf000025_0001
Formula C3
wherein:
Ra and Rb 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;
Rc, Rd and Re are independently selected from hydrogen or (1-4C)alkyl;
Y is selected from O or NH;
V is selected from O, S or NRX, wherein Rx is selected from hydrogen or (1- 4C)alkyl;
m and n are integers independently selected from 0 to 2; and
p and q are integers independently selected from 0 to 1 ;
with the proviso that:
1) the sum of integers m, n, p and q is equal to or greater than 1 ;
2) when p is 1 , q is 0;
3) at least one of p and/or q is 1 ;
4) when q and p are 0 and the oligonucleotide comprises only one phosphodiester backbone mimic of Formula C3, the oligonucleotide comprises greater than or equal to 20 nucleotide and/or nucleotide analogue monomers; and
5) when q and p are 0 and 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.
[00111] In an embodiment, there is provided an oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C4 shown below:
Figure imgf000025_0002
Formula C4 wherein:
Ra and Rb 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;
Rc, Rd and Re are independently selected from hydrogen or (1-4C)alkyl;
V is selected from O, S or NRX, wherein Rx is selected from hydrogen or (1- 4C)alkyl;
m and n are integers independently selected from 0 to 2; and
with the proviso that:
1) the sum of integers m and n is equal to or greater than 1 ;
2) when 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
3) when 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.
[00112] In an embodiment of the 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.
[00113] In another embodiment of the 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.
[00114] In another embodiment of the oligonucleotides comprising one or more phosphodiester backbone mimic of Formula C2, the sum of integers m and n equals 2.
[00115] In an embodiment of the 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.
[00116] In another embodiment of the oligonucleotides comprising one or more phosphodiester backbone mimic of Formula C4, the sum of integers m and n equals 1 or 2.
[00117] It will be appreciated that 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. [00118] It will also be further appreciated that the 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.
[00119] It will also be understood that in certain embodiments, the oligonucleotides of the present invention may synomously be referred to as "polynucleotides".
[00120] In an embodiment, 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.
[00121] In another embodiment, when 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.
[00122] In another embodiment, when 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.
[00123] In yet another embodiment, the one or more phosphodiester backbone mimics is selected from one of the following:
-CH2-C(0)-NH-CH2-;
-C(0)-NH-CH2-CH2-;
-CH2-CH2-C(0)-NH-;
-CH2-NH-C(0)-CH2;
-NH-C(0)-CH2-CH2-;
-CH2-C(0)-NH-0-CH2;
-0-NH-C(0)-CH2-; -NH-C(0)-0-CH2-;
-0-C(0)-NH-CH2-;
-C(0)-NH-NH-CH2-;
-CH2-C(0)-NH-NH-CH2-;
-CH2-NH-NH-C(0)-;
-NH-NH-C(0)-CH2-;
-NH-C(0)-NH-CH2-;
-S-C(0)-NH-CH2-;
-NH-C(0)-S-CH2-; or
-NH-C(S)-NH-CH2-.
[00124] In still another embodiment, the one or more phosphodiester backbone selected from one of the following:
-CH2-C(0)-NH-CH2-;
-C(0)-NH-CH2-CH2-;
-CH2-CH2-C(0)-NH-;
-CH2-NH-C(0)-CH2;
-NH-C(0)-CH2-CH2-;
-CH2-C(0)-NH-0-CH2;
-0-NH-C(0)-CH2-;
-NH-C(0)-0-CH2-;
-0-C(0)-NH-CH2-;
-C(0)-NH-NH-CH2-;
-CH2-C(0)-NH-NH-CH2-;
-CH2-NH-NH-C(0)-;
-NH-NH-C(0)-CH2-; or
-NH-C(0)-NH-CH2-. [00125] In a further embodiment, the one or more phosphodiester backbone mimics is selected from one of the following:
-CH2-C(0)-NH-CH2-;
-C(0)-NH-CH2-CH2-;
-CH2-CH2-C(0)-NH-; or
-NH-C(0)-NH-CH2-.
[00126] In still a further embodiment, the one or more phosphodiester backbone mimics is selected from one of the following:
-CH2-C(0)-NH-CH2-;
-C(0)-NH-CH2-CH2-; or
-CH2-CH2-C(0)-NH-.
[00127] In yet a further embodiment, the one or more phosphodiester backbone mimics is selected from one of the following:
-CH2-C(0)-NH-CH2-; or
-NH-C(0)-NH-CH2-.
[00128] In yet another embodiment, the one or more phosphodiester backbone mimics is selected from one of the following:
-CH2-C(0)-NH-0-CH2;
-0-NH-C(0)-CH2-;
-NH-C(0)-0-CH2-;
-0-C(0)-NH-CH2-;
-C(0)-NH-NH-CH2-;
-CH2-C(0)-NH-NH-CH2-;
-CH2-NH-NH-C(0)-;
-NH-NH-C(0)-CH2-;
-NH-C(0)-NH-CH2-;
-S-C(0)-NH-CH2-;
-NH-C(0)-S-CH2-; or
-NH-C(S)-NH-CH2-. [00129] In yet another embodiment, the one or more phosphodiester backbone mimics is selected from one of the following:
-CH2-C(0)-NH-0-CH2;
-0-NH-C(0)-CH2-;
-NH-C(0)-0-CH2-;
-0-C(0)-NH-CH2-;
-C(0)-NH-NH-CH2-;
-CH2-C(0)-NH-NH-CH2-;
-CH2-NH-NH-C(0)-;
-NH-NH-C(0)-CH2-; or
-NH-C(0)-NH-CH2-.
[00130] In yet another embodiment, the one or more phosphodiester backbone mimics is selected from one of the following:
-NH-C(0)-0-CH2-;
-0-C(0)-NH-CH2-; or
-NH-C(0)-NH-CH2-.
[00131] In yet another embodiment, the terminal nucleotides of the first and second oligonucleotides and the phosphodiester backbone mimic of formula C is selected from one of the following formulae:
Figure imgf000030_0001
Figure imgf000031_0001
4C)alkenyl or (2-4C)alkynyl; and
B and B' are each independently a nucleobase or nucleobase analogue.
[00132] In yet another embodiment, the terminal nucleotides of the first and second oligonucleotides and the phosphodiester backbone mimic of formula C is selected from one of the following formulae:
Figure imgf000031_0002
Figure imgf000032_0001
wherein each of Z, Z', B and B' are as defined hereinabove.
[00133] Suitably, 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.
[00134] In another embodiment, the terminal nucleotides of the first and second oligonucleotides and the phosphodiester backbone mimic is selected from one of the following formulae:
Figure imgf000032_0002
wherein each of Z, Z', B and B' are as defined hereinabove.
[00135] In yet another embodiment, the terminal nucleotides of the first and second oligonucleotides and the phosphodiester backbone mimic is selected from one of the following formulae:
Figure imgf000033_0001
wherein each of Z, Z', B and B' are as defined hereinabove. [00137] In yet another embodiment, the terminal nucleotides of the first and second oligonucleotides and the phosphodiester backbone mimic is selected from one of the following formulae:
Figure imgf000034_0001
wherein each of Z, Z', B and B' are as defined hereinabove.
[00138] Suitably, the terminal nucleotides of the first and second oligonucleotides and the phosphodiester backbone mimic is selected from one of the following formulae:
Figure imgf000034_0002
wherein each of Z, Z', B and B' are as defined hereinabove.
[00139] Most suitably, the terminal nucleotides of the first and second oligonucleotides and the phosphodiester backbone mimic is a group of the formula:
Figure imgf000034_0003
wherein each of Z, Z\ B and B' are as defined hereinabove APPLICATIONS
[00140] 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.
[00141] Accordingly, 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.
[00142] 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).
[00143] 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.
[00144] 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.
[00145] Furthermore, 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.
[00146] Additionally, the present invention provides a method for amplifying an oligonucleotide sequence as defined herein.
[00147] The present invention also provides a method of replicating an oligonucleotide sequence as defined herein.
[00148] Furthermore, the present invention provides a method for producing a ribonucleic acid (RNA) sequence or deoxyribonucleic acid (DNA) sequence as defined here.
[00149] Another possible application for the 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).
[00150] As the skilled person will duly appreciate, 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).
[00151] For example, in the CRISPR-Cas9 / HDR process, it will be understood that a single "guide" RNA (gRNA) is used to direct the Cas9 nuclease to a specific location of the target genome, which is subsequently broken (cleaved). Upon breaking (cleavage) of the double- stranded DNA, the mammalian cell then utilizes one of the above noted endogenous mechanisms to repair the break. In the presence of a donor (or template) DNA with sufficient homology to the regions of the target DNA flanking the break, the double strand break can selectively be repaired by utilisation of the HDR pathway. A consequence of this is that 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).
[00152] Thus, it will be appreciated that 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. Suitably, 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. Most suitably, 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.
[00153] Furthermore, it will also be appreciated that 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. In an embodiment, 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.
[00154] Furthermore, 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). Suitably, 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. EXAMPLES
[00155] Embodiments of the invention will be described, by way of example only, with reference to the accompanying drawings, in which:
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 μΜ with amine, carboxylic acid and splint oligonucleotide concentrations fixed at 1 , 1.5 and 1.5 μΜ 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 . Samples were analysed by denaturing PAGE UV visualisation and mass spectroscopy. Reactions were left for 2 h at room temperature. The final concentration of amine, carboxylic acid and splint oligonucleotides was fixed at 1.0, 1.5 and 1.5 μΜ respectively. All conditions gave the desired product with minor variation in yield between them. Full buffer composition can be found in the 'Amide oligonucleotide ligation' section. * = pre-activation of the carboxylic acid. 100 eq EDC:NHS = 100:25 μΜ.
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. Median reads = 158,147; lower/upper quartile = 35,746/290,696. A. Mutational profile obtained by global alignment of sequences to a master template, with all templates aligned relative to the backbone modification (black line) in the image. The main error at each position is assigned as a substitution (green), insertion (blue) or deletion (red), where the color intensity corresponds to the frequency of observation. 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. mCX = mCG- 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/μΙ) at 37 °C. Samples were analysed by denaturing PAGE and fluorescent visualisation of the FAM labelled primers
Synthetic Procedures
[00156] All chemical reagents were purchased from Sigma-Aldrich, Alfa Aesar, Acros Organics or Fisher Scientific and used without further purification. THF (from Na and benzophenone), DCM, triethylamine, A/./V-diisopropylethylamine and pyridine (from Cahb) were distilled prior to use, or dried over 3 A activated molecular sieves for 24 h. When mentioned, deoxygenated solvents were prepared by bubbling argon through the solvent for 15 min. All air/moisture sensitive reactions were carried out under inert atmosphere (Ar) in flame-dried glassware. 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.
[00157] 1 H, 13C, and 31 P NMR spectra were recorded using Bruker AV300 (300, 75 and 121 MHz respectively), AVIII 400 (400 and 101 MHz respectively) and AVII 500 (500, 126 and 202 MHz respectively) spectrometers. Chemical shifts (δ) are given in ppm and were internally referenced to the appropriate residual solvent signal, all coupling constant (J) are quoted in Hertz (Hz). Assignment of compounds was aided by COSY, HSQC, DEPT-135 and HMBC experiments.
[00158] Low-resolution mass spectra were measured on a Waters LCT premier mass spectrometer for +/- electrospray ionisation (ESI). High-resolution mass spectra were recorded on a Bruker 9.4T FT-ICR-MS mass spectrometer. Samples were run in MeOH or MeCN.
[00159] Compounds 1 ,1 2,2 8, 3 12, 4 14,5 tris-(benzyltriazolylmethyl)amine,6 the Ami- containing3 and the Tz2-containing7 T-T phosphoramidite dimers were prepared as previously reported.
Synthesis of comparative phosphodiester backbone mimics
5'-0-(4,4'-dimethoxytrityl)-triaz
Figure imgf000039_0001
C43H45N7O10
(819.32)
Compound 3
[00160] To a solution of 3'-azido-5'-0-(4,4'-dimethoxytrityl)-3'-deoxythymidine 1 (0.750 g, 1.32 mmol), 5'-ethynyl-5'-deoxythymidine 2 (0.300 g, 1.20 mmol), and tris- (benzyltriazolylmethyl)amine (0.320 g, 0.603 mmol) in DMF (5 mL), was added a solution of sodium ascorbate (0.480 g, 2.42 mmol) in H2O (4 mL). Following this, a solution of CuS04-5H20 (0.150 g, 0.601 mmol) in H2O (4 mL) was added and the reaction was stirred at rt for 2 h. The solvent was subsequently removed in vacuo and the residue was dissolved in a 10:90 MeOH/DCM (55 mL) mixture. The solution was washed with a 5% aqueous solution of disodium ethylenediaminetetraacetate (3 X 55 mL), H2O and brine. The organic layer was then dried over Na2S04 and concentrated in vacuo. The residue was purified by column chromatography on silica gel (4.75:0.5:94.75 to 7:0.5:92.5 MeOH/pyridine/DCM) to give a beige solid of 3-pyridine. Yield: 0.780 g, 0.952 mmol, 79%; 1 H NMR (CDC , 400 MHz): δΗ 9.52 (br s, 1 H, NH-3), 9.40 (br s, 1 H, NH-3), 7.61 (s, 1 H, H-6), 7.50 (s, 1 H, H-16), 7.44 - 7.34 (m, 2 H, H-13), 7.33 - 7.21 (m, 7 H, H- 15, H-14, H-9), 7.17 (s, 1 H, H-6), 6.93 - 6.78 (m, 4 H, H-10), 6.48 (app. t, J = 5.8 Hz, 1 H, H- 1 Ά), 6.17 (app. t, J = 6.9 Hz, 1 H, H-1'B), 5.37 (app. dt, J = 8.6, 5.8 Hz, 1 H, Η-3Ά), 4.49 (br s, 1 H, OH-3'), 4.46 - 4.37 (m, 2 H, Η-4Ά, H-3'B), 4.07 (app. dt, J = 7.2, 5.5 Hz, 1 H, H-4'B), 3.80 (s, 6 H, OCH3), 3.67 (dd, J = 10.8, 3.0 Hz, 1 H, Η-5Ά), 3.38 (dd, J = 10.8, 2.8 Hz, 1 H, Η-5Ά), 3.16 (dd, J = 15.3, 5.5 Hz, 1 H, H-5'B), 3.08 (dd, J = 15.3, 7.2 Hz, 1 H, H-5'B), 3.00 (dt, J = 14.1 , 5.8 Hz, 1 H, Η-2Ά), 2.75 (ddd, J = 14.1 , 8.6, 5.8 Hz, 1 H, Η-2Ά), 2.45 (app. dt, J = 13.4, 6.9 Hz, 1 H, H-2'B), 2.33 (app. dt, J = 13.4, 6.9 Hz, 1 H, H-2'B), 1.91 (s, 3 H, CH3-f 7ym/>7e), 1.58 (s, 3 H, CH3- thymine); 13C NMR (CDC , 101 MHz): 5C 163.8 (C-4), 163.7 (C-4), 158.8 (C-1 1), 150.5 (C-2), 150.4 (C-2), 144.1 (C-12), 143.8 (C-17), 135.3 (C-6A, C-6B), 135.1 (C-8), 135.0 (C-8), 130.0 (C- 9), 128.1 (C-14 or C-13), 128.0 (C-14 or C-13), 127.3 (C-15), 121.8 (C-16), 113.4 (C-10), 1 11.7 (C-5), 111.2 (C-5), 87.2 (C-7), 85.3 (C-1 'A), 85.0 (C-1 'B), 84.3 (C-4'A), 83.7 (C-4'B), 73.7 (C-3'B), 62.7 (C-5'A), 60.2 (C-3'A), 55.3 (OCH3), 39.5 (C-5'B), 38.3 (C-2'A), 29.4 (C-2'B), 12.6 (CH3- thymine), 12.0 (CHs- ym/ne); ESI-TOF m/z C43H45N7Oio+H+ Calc. 820.3301 , Found 820.3299 a.m.u.
5'-0-(4,4'-dimethoxytrityl)-triazole thymidine dimer hosphoramidite (4)
Figure imgf000040_0001
C52H62NgO-| 1 P
(1020.08)
Compound 4
[00161] 5'-0-(4,4'-dimethoxytrityl)-triazole thymidine dimer 3 (0.400 g, 0.488 mmol) was co-evaporated with dry pyridine (3 X 10 mL) and dissolved in anhydrous DCM (5 mL) under an argon atmosphere. A/,A/-diisopropylethylamine (0.22 mL, 1.26 mmol) was added, followed by the dropwise addition of 2-cyanoethyl-A/,A/-diisopropylchlorophosphoramidite (0.14 mL, 0.633 mmol). The reaction was stirred at rt for 2 h and diluted with DCM (10 mL). The solution was washed with degassed saturated aqueous KCI (10 mL), dried over Na2S04 and concentrated in vacuo. The residue was purified by column chromatography under argon pressure eluting with degassed EtOAc. 5'-0-(4,4'-dimethoxytrityl)-triazole thymidine dimer phosphoramidite was obtained as a white solid of 1 : 1 mixture of diastereomers (4i/4ii). Yield: 0.298 g, 0.292 mmol, 60%; 1 H NMR (CDsCN, 300MHz): δΗ 9.22 (s, 2 H, NH-3), 7.68 (d, J = 8.5 Hz, 1 H, H-15), 7.55 (t, J = 1.5 Hz, 1 H, H-16), 7.46 (s, 1 H, H-6), 7.43 (s, 1 H, H-6), 7.41 - 7.19 (m, 8 H, H-14, H-13, H-9), 6.92 - 6.83 (m, 4 H, H-10), 6.43 (app. t, J = 6.8 Hz, 1 H, Η-1 Ά), 6.19 (app. t, J = 7.0 Hz, 0.5 H, H-1'B i or ii), 6.15 (app. t, J = 6.8 Hz, 0.5 H, H-1'B i or ii) 5.46 - 5.35 (m, 1 H, Η-3Ά), 4.57 - 4.44 (m, 1 H, H- 3'B), 4.36 (app. td, J = 6.8, 3.3 Hz, 0.5 H, H-4' i or ii), 4.34 (app. td, J = 6.6, 3.3 Hz, 0.5 H, H-4' i or ii), 4.29 - 4.22 (m, 0.5 H, H-4' i or ii), 4.22 - 4.14 (m, 0.5 H, H-4' i or ii) 3.88 - 3.72 (m, 2 H, H- 18), 3.78 (s, 6 H, OCH3), 3.65 (spt, J = 6.6 Hz, 1 H, H-20), 3.56 (spt, J = 7.0 Hz, 1 H, H-20), 3.45 (dd, J = 11.0, 3.2 Hz, 1 H, Η-5Ά), 3.32 (dd, J = 11.0, 2.9 Hz, 1 H, Η-5Ά), 3.14 (ddd, J = 15.1 , 4.5, 1.5 Hz, 1 H, H-5'B), 3.04 (ddd, J = 15.1 , 7.5, 1.5 Hz, 1 H, H-5'B), 2.86 (ddd, J = 13.4, 6.8, 3.1 Hz, 0.5 H, Η-2Ά i or ii), 2.83 (ddd, J = 9.5, 6.8, 3.1 Hz, 0.5 H, Η-2Ά i or ii), 2.75 (ddd, J = 9.5, 6.8, 2.7 Hz, 0.5 H, Η-2Ά i or ii), 2.72 (ddd, J = 13.4, 6.8, 3.20 Hz, 0.5 H, Η-2Ά i or ii), 2.67 (t, J = 5.9 Hz, 2 H, H-19), 2.47 - 2.21 (m, 2 H, H-2'B), 1.83 (d, J = 1.2 Hz, 3 H, CH3-f 7ym/>7e), 1.61 (s, 3 H, CH3-f 7ym/>7e), 1.27 - 1.10 (m, 12 H, H-21); 31 P NMR (121 MHz, CD3CN): δΡ 149.32, 142.24.
5'-0-(4,4'-dimethoxytrityl)-3'-azid -methylcytidine (5)
Figure imgf000041_0001
C3.H3SN60;i
(568.62)
Compound 5
[00162] 3'-azido-5'-0-(4,4'-dimethoxytrityl)-3'-deoxythymidine 1 (1.30 g, 2.28 mmol) was co-evaporated twice with dry pyridine (10 ml_) before dissolution in dry pyridine (20 ml_). N- methylimidazole (2.3 ml_) was added under argon and left to stir at 4 °C for 20 min. Phosphorous oxychloride (0.83 ml_) was added to the resulting yellow suspension at 4 °C over 20 min. After stirring for a further 30 min at 4 °C, the reaction was brought to room temperature and stirred for 30 min. The reaction was cooled to 4 °C and concentrated NH3 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 NH3 (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. The residue was then purified by column chromatography on silica gel eluting with MeOH:pyridine:DCM (0.5:0.5:99 to 4.5:0.5:95) to give 5 as a white solid. Yield: 0.850 g, 1.49 mmol, 65%; 1 H NMR (CDCIa, 400 MHz): δΗ 7.73 (s, 1 H, H-6), 7.44 - 7.38 (m, 2 H, Ar-H), 7.34 - 7.21 (m, 7 H, Ar-H), 6.88 - 6.80 (m, 4 H, Ar-H), 6.23 (dd, J = 6.3, 5.3 Hz, 1 H, H-1 '), 4.28 (dt, J = 7.4, 5.8 Hz, 1 H, H- 3'), 3.96 (dt, J = 5.8, 2.8 Hz, 1 H, H-4'), 3.80 (s, 6 H, OCH3), 3.58 (dd, J = 1 1.1 , 2.8 Hz, 1 H, H- 5'), 3.31 (dd, J = 1 1.1 , 2.8 Hz, 1 H, H-5'), 2.57 (m, 1 H, H-2'), 2.40 (m, 1 H, H-2'), 1.52 (s, 3 H, CH3-cyfos/>7e); 13C NMR (CDCI3, 126 MHz): 5C 165.6 (Cq), 158.8 (Cq), 155.8 (Cq), 144.3 (Cq), 138.1 (C-6), 135.3 (Cq), 130.1 (Ar-C), 128.1 (Ar-C), 127.2 (Ar-C), 113.3 (Ar-C), 101.5 (Cq), 86.9 (Cq), 85.6 (C-r), 83.5 (C-4'), 62.4 (C-5'), 59.7 (C-3'), 55.3 (OCH3), 38.8 (C-2'), 12.7 (CH3- cytosine); ESI-TOF m/z Ο^ ΗκΝβΟδ-Η* Calc. 567.2361 , Found 567.2360 a.m.u. Succinylated resin
Figure imgf000042_0001
[00163] AM polystyrene (1 g, 40
Figure imgf000042_0002
loading, Applied Biosystems) was washed with 3% TCA in DCM before standing in fresh 3% TCA in DCM for 1 h. The solid support was then washed with ΝΕίβ / A/,A/-diisopropylethylamine (9: 1), DCM and Et20 before drying in vacuo. Next succinic anhydride (0.25 g, 2.5 mmol) and 4-(dimethylamino)pyridine (0.05 g, 0.41 mmol) dissolved in pyridine (7 ml_) was added to the resin and rotated for 20 h. The resin was washed with pyridine, DCM and Et20 (three times each) before drying in vacuo to give the succinylated resin.
5'-0-(4,4'-dimethoxytrityl)-3'-azide 5-methylcytidine functionalised resin (6)
Figure imgf000042_0003
[00164] A solution of pyridine (4 ml_), Λ/,Λ/'-diisopropylcarbodiimide (0.245 ml_, 1.582 mmol), 1-hydroxybenzotriazole (0.245 g, 1.813 mmol) and 5'-0-(4,4'-dimethoxytrityl)-3'-azido 5- methylcytidine 5 (0.075 g, 0.132 mmol) was added to the succinylated resin (1.0 g) and rotated for 20 h. 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. The resin was washed with THF, pyridine, DCM and Et20 (four times each) before drying in vacuo overnight. Loading of solid support was 30
Figure imgf000042_0004
(determined from the cleaved DMT group8). *Note: During treatment of the support with piperidine to cap the unreacted succinic acid groups the loading of nucleoside decreased with time due to cleavage of the succinyl linkage. Therefore, only a brief piperidine treatment (1 min) was carried out. 5'-ethynyl-5'-deoxythymidine phosphoramidite (7)
Figure imgf000043_0001
(450.47)
Compound 7
[00165] 5'-ethynyl-5'-deoxythymidine 2 (0.481 g, 1.92 mmol) was dissolved in anhydrous THF (20 mL) under an argon atmosphere. A/,A/-diisopropylethylamine (1.22 mL, 7.00 mmol) was added, followed by the dropwise addition of 2-cyanoethyl-A/,A/-diisopropylchlorophosphoramidite (0.63 mL, 2.82 mmol). The reaction was stirred at rt for 3.5 h and diluted with DCM (40 mL). The solution was washed with degassed saturated aqueous KCI (20 mL), dried over Na2S04 and concentrated under vacuum. The residue was purified by column chromatography under argon pressure eluting with degassed EtOAc:hexane: pyridine (49.75:49.75:0.5 to 79.75: 19.75:0.5). 5'- ethynyl-5'-deoxythymidine phosphoramidite was obtained as a white solid of 1 :1 mixture of diastereomers (7i/7ii). Yield: 0.631 g, 1.40 mmol, 73%; 1 H NMR (CD3CN, 400 MHz): δΗ 9.02 (s, 1 H, NH-3), 7.44 (q, J = 1.2 Hz, 0.5 H, H-6 i or ii), 7.43 (q, J = 1.2 Hz, 0.5 H, H-6 i or ii), 6.20 (app. t, J = 6.6 Hz, 1 H, H-1 '), 4.55 - 4.44 (m, 1 H, H-3'), 4.11 - 3.98 (m, 1 H, H-4'), 3.90 - 3.70 (m, 2 H, H-9), 3.70 - 3.52 (m, 2 H, H-1 1), 2.74 - 2.52 (m, 4 H, H-5' and H-10), 2.45 - 2.20 (m, 3 H, H-2' and H-8), 1.83 (d, J = 1.2 Hz, 3 H, CHs- ym/ne), 1.20 - 1.10 (d and d, J = 6.8 Hz, 12 H, H-12); 13C NMR (CD3CN, 126 MHz): 5C 163.2 (C-4), 150.1 (C-2), 135.4 (C-6), 110.1 (C-5), 83.9 (C-r), 82.6 (C-4', i or ii), 82.4 (C-4', i or ii), 80.1 (C-8, i or ii), 79.9 (C-8, i or ii), 74.7 (C-5', i or ii), 74.5 (C-5', i or ii), 71.1 (C-7), 58.2 (C-9, i or ii), 58.0 (C-9, i or ii), 42.8 (C-11 , i or ii), 42.7 (C- 1 1 , i or ii), 37.9 (C-2'), 23.5 (C-12, i or ii), 23.6 (C-12, i or ii), 22.0 (C-5' or C-10), 19.7 (C-5' or C-10), 1 1.2 (CH3-f 7ym/>7e); 31P NMR (CD3CN, 121 MHz): δΡ 149.61 , 149.52; ESI-TOF m/z C2iH3iN405Pi+Na+ Calc. 473.1924, Found 473.1919 a.m.u.
Synthesis of phosphodiester backbone mimics of the present invention
Alcohol functionalized resin (10).
Figure imgf000043_0002
[00166] 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. Next, the resin was soaked in dry pyridine for 10 min. A solution of 6-O-DMT-hexanoic acid (100 mg, 0.23 mmol), A/,A/-diisopropylcarbodiimide (70 μΙ_, 0.45 mmol) and 1-hydroxybenzotriazole (70 mg, 0.52 mmol) dissolved in pyridine (1 mL) was then added and the mixture rotated for 20 h. The solvent was removed and the solid support washed with pyridine, DCM and Et20. After capping using a 1 : 1 mix of oligonucleotide synthesis grade Cap Mix A (8: 1 : 1 acetic anhydride/pyridine/THF, 5 mL) and Cap Mix B (84: 16 THF/ N-methylimidazole, 5 mL) for 1 h with rotational mixing, the support was washed with pyridine, DCM and Et20 and left to dry in vacuo overnight. Loading of 6-0- DMT-hexanoic acid solid support was 120 μΓΤΐοΙ/g (determined from the cleaved DMT group8). Next, all of the DMT functionalized solid support was deprotected with 3% TCA in DCM (5 mL). After washing with DCM and Et20 followed by drying in vacuo, solid support 10 was obtained.
3'-Deoxythymidine-3'-acetic acid functionalized resin (11).
Figure imgf000044_0001
[00167] Solid support 10 (0.3 g) was soaked in pyridine for 10 min. A solution of Λ/-(3- dimethylaminopropyl)-/V'-ethylcarbodiimide hydrochloride (288 mg, 1.50 mmol), 4- (dimethylamino)pyridine (12 mg, 0.098 mmol), NEt3 (60 μΐ, 0.43 mmol) and 5'-0-tert- butyldimethylsilyl-3'-deoxythymidine-3'-acetic acid (8, 50 mg, 0.13 mmol) in dry pyridine (5 mL) was added and the reaction slurry rotationally mixed for 20 h. The solvent was removed by filtration and the support was washed with pyridine, DCM and Et20. After capping using a 1 : 1 mix of oligonucleotide synthesis grade Cap Mix A (8: 1 : 1 acetic anhydride/pyridine/THF, 5 mL) and Cap Mix B (84: 16 THF/ N-methylimidazole, 5 mL) for 1 h with rotational mixing, the support was washed with pyridine, DCM and Et20 and left to dry in vacuo overnight. Tetrabutylammonium flouride (1.0 M, THF) was added for 4 h at rt to remove the 5'-TBDMS group. The solvent was then removed and the solid support 1 1 washed with THF, DMF, pyridine, DCM and Et20. The solid support was dried in vacuo and used in oligonucleotide synthesis. Note that after oligonucleotide synthesis, the resin requires non-standard conditions for cleavage as detailed in "General oligonucleotide synthetic and purification procedures". Oligonucleotide synthesis and purification procedures.
[00168] 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 μηιοΙ scale. Standard β-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.
[00169] 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.
[00170] For terminal 5'-amino-dT modifications, the commercially available phosphoramidite was purchased from Glen Research (cat. no. 10-1932-90).
[00171] For terminal 3'-carboxylic acid oligonucleotide deprotection, the resin was treated with 1 mL methanol / water (4: 1 v/v) containing sodium hydroxide (0.4 M) for 6 h. Then 1 mL of methanol / water (1 :4 v/v) containing sodium hydroxide (0.4 M) was added and the resin left for a further 17 h. The solution was neutralised using TEAA buffer (1 M, pH 7.4, 2 mL), concentrated in vacuo and desalted as detailed below. For terminal 3'-0-propargyl/2'-hydroxy modifications, commercially available resins (rC = cat. no. N-9882-10; rG = cat. no. N-9883-10; rU = cat. no. N- 9884-05) from Chem Genes were purchased as well as 3'-0-propargyl/2'-hydroxy rU modifications (cat. no. N-8940-05).
[00172] For desalting of oligonucleotides (primers only), NAP-25 columns (G.E. Healthcare Life Sciences, cat. no. 17-0854-01) were used.
[00173] For RP-HPLC purification of oligonucleotides, a Gilson HPLC system with ABI Aquapore C8 column (8 mm x 250 mm, pore size 300 A) with a gradient of acetonitrile in triethylammonium bicarbonate (0 to 50% buffer B over 20 min, flow rate 4 mL/min) was used. The following elution buffers were used: buffer A (0.1 M triethylammonium bicarbonate, pH 7.5), buffer B (0.1 M triethylammonium bicarbonate, pH 7.5, mixed with 50% acetonitrile). The fractions from HPLC were evaporated without additional desalting and purity re-checked using PAGE.
[00174] For denaturing PAGE purification of oligonucleotides, samples were mixed with formamide (50 v/v) and loaded on 8 - 12 % denaturing polyacrylamide gels (1x TBE, 7 M urea, W x D x H = 18 x 0.2 x 24.4 cm) at 20 W for 2 - 3 h. Oligonucleotide bands were then visualized using a UV lamp and the desired bands excised, crushed and soaked in water (ca. 15 ml) overnight at 37 °C. After evaporation of the solvent, samples were desalted using two NAP-25 columns (G.E. Healthcare Life Sciences, cat. no. 17-0852-01).
[00175] All oligonucleotides were characterized by negative-mode electrospray HPLC- mass spectrometry, using a Bruker micrOTOF mass spectrometer and an Acquity UPLC system with a BEH C18 1.7 μηι column (Waters). Raw data were processed and deconvoluted using the Data Analysis function of the Bruker Daltronics Compass™ 1.3 software package.
Amide oligonucleotide ligation.
[00176] The reaction mixture consisted of:
Figure imgf000046_0001
* ODNs are listed in Supplementary Table 1.
[00177] The A/-(3-dimethylaminopropyl)-A/'-ethylcarbodiimide hydrochloride (EDC HCI) and /V-hydroxysuccinimide (NHS) solutions were prepared separately in water and pre-mixed to the initial concentration solutions used in the table. Stock solutions of EDC and NHS were kept at -80 °C. The 2x buffers tested include:
Figure imgf000046_0002
[00178] For pre-activation of the carboxylic acid oligonucleotide prior to ligation, the CA- ODN, water and EDC/NHS were mixed together for 15 min at room temperature before adding this solution to a mixture of the other components. Reaction times ranged from 10 min to 18 h at room temperature. [00179] For the one-pot procedure, all components were mixed together expect the EDC/NHS solution which was added last. The reaction was then left at room temperature for 2 h.
[00180] After the reaction, excess reagents were removed using NAP-10 columns (G.E. Healthcare Life Sciences 17-0854-01). For mass spectroscopy and denaturing PAGE UV visualization, the reactions were scaled up 30-fold. For fluorescence visualization, the gels were stained with SYBR gold for 10 min and imaged using a Syngene G:Box imager.
[00181] Of the conditions tested, optimal amide ligation was observed in un-buffered solutions containing NaCI (0.1 M), Amine-ODN (0.9 nmol, 1.5 μΜ), Am-Splint (0.9 nmol, 1.5 μΜ) and CA-ODN (0.6 nmol, 1 μΜ), to which EDC:NHS (60: 15 nmol, 100:25 μΜ) was added for 2 h at room temperature (i.e. a one-pot procedure). The total volume of the reaction was 600 μΙ.
Urea oligonucleotide ligation
[00182] For urea ligation, 10 nmol of each of the required oligonucleotides (res5873-1 [145.4 μΜ, 68.8 μΙ], res5871 [66.4 μΜ, 150.7 μΙ], res6044-p2 [90 μΜ, 11 1.1 μΙ] - see Table A below) were mixed with water (169.5 μΙ) and a sodium chloride solution (4 M, 12.5 μΙ). The coupling reagent, Λ/,Λ/'-carbonyldiimidazole (2 mg) was added to the oligonucleotide solution and the reaction left at room temperature for 1 h before desalting using a NAP-10 column (G.E. Healthcare Life Sciences, cat. no. 17-0852-01). Samples were then mixed with equal volume formamide and loaded on an 8% denaturing polyacrylamide gels (1x TBE, 7 M urea, W x D x H = 18 x 0.2 x 24.4 cm) at 20 W for 2 h. Oligonucleotide bands were then visualized using a UV lamp and the desired bands excised, crushed and soaked in buffer (50 mM Tris-HCI, pH 7.5, 25 mM NaCI, ca. 15 ml) overnight at 37 °C. After evaporation of the solvent, samples were desalted using two NAP-25 columns (G.E. Healthcare Life Sciences, cat. no. 17-0852-01).
Table A. Oligonucleotides used for urea chemical ligation. x = urea linkage site.
Mol. Weight
Res
Sequence (5' - 3') (g/mol)
Expected Found
H2N-TAGCACACAATCTCACACTCTGGAATTCA
5873-1 17619 17620
CACTGACAATACTGCAAGACACACCACTC
5871 TAGAGGAAGACAGCCTCGATCT-NH2 6767 6768
6044 GAGATTGTGTGCTAAGATCGAGGCTGTC 8699 8701
TAGAGGAAGACAGCCTCGATCTxTAGCACAC
5872 24414 24412
AATCTCACACTCTGGAATTCACACTGACAATAC TGCAAGACACACCACTC Comparative CuAAC 'click' oligonucleotide ligation
[00183] For un-templated ligation, the azide and alkyne oligonucleotides (10 nmol each) were freeze dried together, re-suspended in water (80 μΙ), 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, CuS04.H20 (1 μΙ, 0.1 M) and tris- (hydroxypropyltriazolylmethyl)amine (0.3 mg) were mixed in water (17 μΙ) before the addition of sodium ascorbate (2 μΙ, 0.5 M) under argon. The two solutions were mixed and incubated at room temperature for 2 - 3 h before purification by denaturing PAGE.
[00184] For templated ligation, the azide, alkyne and complementary splint oligonucleotides (5 nmol each) were dissolved in an aqueous solution of NaCI (0.2 M, 100.0 μΙ_) 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. Separately, CuS04.H20 (2.0 μΙ, 0.2 μηιοΙ), tris-(hydroxypropyltriazolylmethyl)amine (94 μΙ_, 1.4 μηιοΙ) and sodium ascorbate (4 μΙ_, 2 μηιοΙ) were mixed and placed under argon; each were dissolved in an aqueous solution of NaCI (0.2 M). The two solutions were mixed and incubated at room temperature for 2 - 3 h. Reagents were removed using NAP-25 columns (G.E. Healthcare Life Sciences, cat. no. 17-0852-01), analyzed and purified by denaturing PAGE.
UV melting analysis.
[00185] Thermal denaturation of DNA duplexes was measured at 260 nm using 1 cm path length cuvette on a Varian Cary 4000 Scan UV-Visible spectrophotometer. Total duplex concentration was 1 μΜ in phosphate buffer (10 mM, pH 7.0) containing sodium chloride (200 mM). Samples were initially rapidly denatured (20→ 85 °C, 10 °C/min), before three successive cycles of heating (20→ 85 °C, 1 °C/min) and cooling (85→ 20 °C, 1 °C/min). Tm values were calculated from the derivative of the 1 °C/min cycles using the Cary Win UV thermal application software, before averaging and determination of the standard deviation (± 0.3 °C). gPCR kinetics.
[00186] 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 μΙ), EvaGreen (Biotium, cat. no. 31000, 20x, 1 μΙ), dNTPs (10 mM, 0.4 μΙ), polymerase (2 ΙΙ/μΙ, 0.25 μΙ) and water (11.35 μΙ), or standard Taq buffer (10x, 2 μΙ), EvaGreen (20x, 1 μΙ), dNTPs (10 mM, 0.4 μΙ), polymerase (5 ΙΙ/μΙ, 0.1 μΙ) and water (13.5 μΙ) were prepared. This mix (17 μΙ) was then added to a solution of forward primer (10 μΜ, 1 μΙ), reverse primer (10 μΜ, 1 μΙ) and template (18.7 pM, 1 μΙ). Samples for all qPCR extension times were prepared together and stored at 4 °C before immediate sequential use. 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. For 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 and linear extension for sequencing.
[00187] 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. Master-mixes composed of either GoTaq buffer (5x, 4 μΙ), dNTPs (2 mM, 2 μΙ), GoTaq DNA polymerase (5 ΙΙ/μΙ, 0.1 μΙ) and water (10.9 μΙ), or KOD XL buffer (1 Ox, 2 μΙ), dNTPs (2 mM, 2 μΙ), KOD XL DNA polymerase (2.5 ΙΙ/μΙ, 0.2 μΙ) and water (12.8 μΙ), or Phusion HF buffer (5x, 4 μΙ), dNTPs (2 mM, 2 μΙ), Phusion DNA polymerase (2 ΙΙ/μΙ, 0.25 μΙ) and water (10.75 μΙ) were prepared. This mix (17 μΙ) was then added to a solution of the forward primer (10 μΜ, 1 μΙ), reverse primer (10 μΜ, 1 μΙ) and template (0.187 μΜ, 1 μΙ). Note PCR was performed for each individual template separately. 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 μΙ, 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 μΙ, 3 M, pH 5.2) and ethanol (66 μΙ), 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 μΙ).
[00188] For linear extension, master-mixes consisted of NEB buffer 2 (10x, 2 μΙ), dNTPs (2 mM, 2 μΙ), Klenow large fragment DNA polymerase I (5 ΙΙ/μΙ, 0.4 μΙ, NEB, cat. no. M0210S) and water (7.6 μΙ). This mix (12 μΙ) was then added to a solution of reverse primer (10 μΜ, 3 μΙ) and template (12 μΜ, 5 μΙ). The extension was performed at 25 or 37 °C for 1 h before heat denaturing the polymerase at 75 °C for 20 min and cooling to room temperature.
[00189] Each PCR and linear extension was analyzed by running 1 μΙ of sample on an Agilent 2100 Bioanalyzer using a DNA 1000 kit (Agilent, cat. no. 5067-1504) to confirm product formation. The entire process was repeated twice independently for two independent sequencing runs.
[00190] 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. A master-mix composed of NEB buffer 2 (10x, 1 μΙ), dNTPs (10 mM, 0.2 μΙ), polymerase (5 U/μΙ, 0.2 μΙ) and water (3.6 μΙ) was prepared. This mix (5 μΙ) was then added to a solution of reverse primer (10 μΜ, 1.5 μΙ), template (10 μΜ, 2.3 μΙ) and water (1.2 μΙ). Practically, oligonucleotide solutions and the master mix were kept on ice before mixing on ice and immediately incubating on a pre-warmed thermocycler at 37 °C for the specified times. Reactions were quenched at 37 °C using 20 μΙ of stop solution (1 :3 mix of 1 mM EDTA, pH 8.0 and formamide) before storage at -20 °C. Once all samples were collected, the reaction mixtures products were resolved on using 8% denaturing polyacrylamide gels (1x TBE, 7 M urea, W x D x H = 18 x 0.2 x 24.4 cm) at 20 W for 1.5 h.
Sequencing library preparation.
[00191] Equal volumes of PCR / linear extension reactions for each template were mixed based on the polymerase used (i.e. GoTaq, Phusion, KOD XL, Klenow 25 °C or Klenow 37 °C) to give five mixtures. For each mixture, 27 μΙ was mixed with shrimp alkaline phosphatase (1 μΙ, 1 U/μΙ, NEB, cat. no. M0371 S) and incubated at 37 °C for 45 min to dephosphorylate any excess dNTPs. The phosphatase was then heat inactivated (65 °C, 6 min) and the samples cooled to 4 °C. Next dithiothreitol (0.5 μΙ, 150 mM), ATP (2.1 μΙ, 10 mM) and T4 polynucleotide kinase (1 μΙ, 10 U/μΙ, NEB, cat. no. M0201S) were added to phosphorylate the PCR / linear extension products. The mixtures were incubated at 37 °C for 30 min before the kinase was heat inactivated (65 °C, 20 min) and the samples cooled to 4 °C.
[00192] 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. For PCR mixtures 17.5 μΙ was used directly as the starting input. For linear extension mixtures 14 μΙ was mixed with NEB buffer 2 (1x, 3.5 μΙ). These volumes correspond to approximately 5 - 6 pmol DNA if polymerase reactions are perfectly efficient. From the 'Adenylate 3' ends' step onwards 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. Libraries were quantified using the KAPA Library quantification kit (cat. no. KK4844) on a Bio-Rad CFX96. 18 pM libraries were loaded on a MiSeq cartridge (600- cycle, v3, cat. no. MS-102-3003, including a 10% by volume PhiX control) and run on an lllumina MiSeq. Library preparation was repeated twice independently using two independently prepared PCR/linear extension products. Next-generation sequencing data analysis.
[00193] The below analysis was performed using custom MatLab (2014a) scripts unless otherwise stated and is available on request. Samples were separated by polymerase using lllumina adapter indexes. Paired-end reads were merged to get a consensus sequence with adjusted quality scores using the vsearch program's merge pairs command and allowing for sequence staggering. Bases with a quality score of less than 30 were masked with an 'N' and reads with a length greater than 140 (concatamers) or >50% 'NTs removed. To sort reads by template, the first and last 18 bases were compared to the expected template sequence using the Levenshtein distance. These regions are unique per template and act as barcodes. Reads with a Levenshtein distance below six for PCR using GoTaq, KOD XL and Phusion, and four for linear extensions using Klenow were retained. For PCR, sequences complementary to the expected template were reverse complemented. For linear extension, sequences that are complementary to the expected template are generated by Klenow, whilst the expected template sequences are generated using the lllumina bridge amplification polymerase of the original modified template. For the Klenow reads, sequences were reverse complemented. For the lllumina polymerase reads, samples from Klenow extension at 25 and 37 °C were combined.
[00194] For mutational profile analysis, sorted reads were aligned using the Needleman- Wunsch global pair-wise alignment (EMBOSS needle)62 to the expected templates, with gap open (10.0), gap extension (0.5), end open (10.0) and end extend (0.5) penalties. These pair- wise alignments were then converted to an alignment of all reads to a single master sequence. This was done by indexing the position and size of insertions for all templates in the pair-wise alignments and iteratively placing the appropriate number of insertions at the specified position using '-' for each individual pair-wise alignments that does not contain the insertions. This ultimately yields a single consensus that better takes into account multi-base insertions. Next, the statistical significance of observing a base given the total number of all base observations at a position of the template was tested using a negative binomial test (p>0.005). Any base occurring less than ~2-3x the reported oligonucleotide synthesis error rates (0.0009) were also masked as 'N'. When no base is statically significant at a position, this insertion was removed. A sequence profile was generated by calculating the frequency of each base at each position of the template, with masked 'NTs ignored. Each position is classified as mainly substitutions (frequency other bases > expected base, and other bases > '-'), insertions (frequency other bases > expected base, and '-' > other bases) or deletions (frequency '-' > expected base).
[00195] For unique sequence analysis, 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 needle62 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.
Table 1. 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.
Modified Mol. Weight Oligo. u^cd Sequence (5' - 3') (g/mol) Code Expected Found
CA-ODN CCATTCATCCTCA 3269 3268
Amine- amTCGGTCCGTGT 3338 3337
Am1-Lig ODN
Am-
TTTTACACGGACCGAAGGATGAATGGTTTT 9276 9276 Splint
Azide-
GCATTCGAGCAACGTAAGATCCmCAZ-A 7058 7057 ODN1
Alkyne- aik-ATACCACACAATCTCACACTCTGGAATTC
Tz3 (mCT) 17632 17631
ODN1 ACACTGACAATACTGCCGACACACATAACC
Tria-
TGTGTGGTAGGGATCTTA 5601 5601 Splint
Alkyne-
TGACAGGGAGCTAACCAGATCrUaik-B 6809 6809
Tz2M ODN1
(rUT) Azide- az-BTAGCACACAATCTCACACTCTGGAATTC
17646 17646 ODN2 ACACTGACAATACTTCAGACACCAGCACAC
Alkyne-
CCGGGAGTCCATAAGAAGATCrUaik-c 6809 6808 ODN2
Tz2 (rUT)
Azide- az-BTAGCACACAATCTCACACTCTGGAATTC
17646 17646 ODN2 ACACTGACAATACTTCAGACACCAGCACAC
Alkyne-
GATATCGAACCAGGCGAGATCrGaik-c 6849 6847 ODN3
Tz2 (rGT)
Azide- az-BTAGCACACAATCTCACACTCTGGAATTC
17646 17646 ODN2 ACACTGACAATACTTCAGACACCAGCACAC
Alkyne-
GTAAGACGCTGCAACAGGATCrCaik-c 6808 6807 ODN4
Tz2 (rCC)
Azide- az-BCAGCACACAATCTCACACTCTGGAATTC
17631 17630 ODN3 ACACTGACAATACTGCCGACACACATAACC
Alkyne- 7110 7109
GCATTCGAGCAACGTAAGATCGmCaik-D
Tz2 ODN5
(mCG- az-B(G-clamp)AGCACACAATCTCACAC 17779 17778
Azide- clamp) TCTGGAATTCACACTGACAATACTGCCGAC
ODN4
ACACATAACC
Alkyne-
GCATTCGAGCAACGTAAGATCmCaik-D 6781 6780
Tz2 ODN6
(mCC) Azide- az-BCAGCACACAATCTCACACTCTGGAATTC
17631 17630 ODN3 ACACTGACAATACTGCCGACACACATAACC Alkyne-
GCATTCGAGCAACGTAAGATCmCaik-D 6781 6780
Tz2 ODN6
(mCU) Azide- az-BUAGCACACAATCTCACACTCTGGAATTC
17632 17632 ODN4 ACACTGACAATACAACCCGATCCGTCAAAC
Azide-
GCATTCGAGCAACGTAAGATCCmCaz-A 7058 7057 ODN1
Tz1 (mCT)
Alkyne- aik-ETACCACACAATCTCACACTCTGGAATT
17632 17631 0DN7 CACACTGACAATACTGCCGACACACATAACC
Terminal oligonucleotide modification nomenclature (B = any base)
Figure imgf000053_0001
az-A aik-8 afk-C alk-D
Figure imgf000053_0002
am aik-A alk-E 82-B
Table 2. Templates used for kinetics and sequencing assays. Oligonucleotides were purified by denaturing PAGE or HPLC. If purified by HPLC, purity was confirmed by running analytical denaturing PAGE. Oligonucleotides used to assemble templates by chemical ligation are listed in Supplementary Table 1. x = site of modified backbone where the code (e.g. PDS) indicates the modification; the structure of the backbone modifications are listed at the bottom of the table. P = phosphoramidite. O = Sulfurizing oxidizer. P () = phosphoramidite monomer (compound no. or commercial). PD () = phosphoramidite dimer (compound no.), com. = commercial. CL = Un- templated CuAAC. CL-T= Templated CuAAC.
Modified Mol. Weight (g/mol) Synthesis
Sequence (5' - 3')
Oligo. Code Expected Found Method
ATTAGCGACGAGAGCCAGATCTxTxAG
CA P (com.)
2x PDS (TT) CACACAATCTCACACTCTGGAATT 24515 24515
CACTGACAATACAACCTGCACCCAAC and O
GAT
GAAGAGGCAATCTCAGCGATCTxTAG
CACACAATCTCACACTCTGGAATTCA P (com.)
1x PDS (TT) 24483 24484
CACTGACAATACGACTCAACCGCCAA and O
TCA CGCGAGCGTAACAAGATGATCTxTxAx
GCACACAATCTCACACTCTGGAATTC
3x PS (TTA) 24499 24500 0
ACACTGACAATACAATCCTACCGGAC CCAA
AAGAACATCCGGGTCGAGATCTxTAG
CACACAATCTCACACTCTGGAATTCA
1x PS (TT) 24467 24469 0
CACTGACAATACCACGATACCACACG CAT
AAGGCTCAGCGGAAATCGATCUxUxAG
2x 2'-0Me CACACAATCTCACACTCTGGAATTCA
24483 24484 P (com.) (UU) CACTGACAATACACCACAACTCAGAC
TGC
GCGCGGTAAACATGACAGATCUxTAG
1x 2'-0Me CACACAATCTCACACTCTGGAATTCA
24467 24467 P (com.) (UT) CACTGACAATACACGACCAAATCCGA
TCC
AGCAACAGCTGGTAGACGATCTxTAG
CACACAATCTCACACTCTGGAATTCA
Ami (TT) 24413 24413 PD (ref.3)
CACTGACAATACACCACAAATCCGTG CCA
GCATTCGAGCAACGTAAGATCTxTAGC
ACACAATCTCACACTCTGGAATTCAC
Tz2+ (TT) 24443 24442 PD (19)
ACTGACAATACTGCCGACACACATAA CC
GCATTCGAGCAACGTAAGATCCmCxTA
CCACACAATCTCACACTCTGGAATTC
Tz3 (mCT) 24646 24644 CL-T
ACACTGACAATACTGCCGACACACAT AACC
GCGTACGAGAACCGAATGATCTxTAG
CACACAATCTCACACTCTGGAATTCA
Tz3 (TT) 24423 24423 PD (4)
CACTGACAATACGTCGCACACAACAA TCC
TGACAGGGAGCTAACCAGATCrUxTAG
CACACAATCTCACACTCTGGAATTCA
Tz2M (rUT) 24455 24455 CL
CACTGACAATACTTCAGACACCAGCA CAC
CCGGGAGTCCATAAGAAGATCrUxTAG
CACACAATCTCACACTCTGGAATTCA
Tz2 (rUT) 24455 24454 CL
CACTGACAATACTTCAGACACCAGCA CAC
GATATCGAACCAGGCGAGATCrGxTAG
CACACAATCTCACACTCTGGAATTCA
Tz2 (rGT) 24494 24494 CL
CACTGACAATACTTCAGACACCAGCA CAC
GTAAGACGCTGCAACAGGATCrCxCAG
CACACAATCTCACACTCTGGAATTCA
Tz2 (rCC) 24439 24439 CL
CACTGACAATACTGCCGACACACATA ACC
GCATTCGAGCAACGTAAGATCGmCxYA
Tz2
GCACACAATCTCACACTCTGGAATTC
(mCY where 24889 24889 CL
ACACTGACAATACTGCCGACACACAT
Y=G-clamp)
AACC
GCATTCGAGCAACGTAAGATCmCxCA
Tz2 (mCC) 24412 2441 1 CL
GCACACAATCTCACACTCTGGAATTC
Figure imgf000055_0001
Table 3. Primers used for kinetic assays. Primers were purified using NAP-10 columns (G.E. Healthcare Life Sciences, cat. no. 17-0854-01).
Modified Oligo. Drimar CQ ,1 1Q i„Q lc-, ,„
Code Primer bequence (5 — 3 )
F GAAGAGGCAATCTCAGCG
1x PDS (TT)
R TGATTGGCGGTTGAGTCG
F AAGAACATCCGGGTCGAG
1x PS (TT)
R ATGCGTGTGGTATCGTGG
F AGCAACAGCTGGTAGACG
Ami (TT)
R TGGCACGGATTTGTGGTG
F GCATTCGAGCAACGTAAG
Tz2+ (TT)
R GGTTATGTGTGTCGGCAG
F GCATTCGAGCAACGTAAG
Tz3 (mCT)
R GGTTATGTGTGTCGGCAG
Tz3 (TT) F GCGTACGAGAACCGAATG R GGATTGTTGTGTGCGACG
F TGACAGGGAGCTAACCAG
Tz2M (rUT)
R GTGTGCTGGTGTCTGAAG
F CCGGGAGTCCATAAGAAG
Tz2 (rUT)
R GTGTGCTGGTGTCTGAAG
F GATATCGAACCAGGCGAG
Tz2 (rGT)
R GTGTGCTGGTGTCTGAAG
F GTAAGACGCTGCAACAGG
Tz2 (rCC)
R GGTTATGTGTGTCGGCAG
F GCATTCGAGCAACGTAAG
Tz2 (mCC)
R GGTTATGTGTGTCGGCAG
F GCATTCGAGCAACGTAAG
Tz2 (mCU)
R GTTTGACGGATCGGGTTG
F GCTTACGACGAAGAACGG
Tz2 (TT)
R TGTGTGTATTGGCCGAGG
F GCATTCGAGCAACGTAAG
Tz1 (mCT)
R GGTTATGTGTGTCGGCAG
F ACGTTAGCACGAAGAGGC
Control (TT)
R ATTGGGTGTGTTCGCGAG
Table 4. Primers used for sequencing assays. Primers were purified using NAP-10 columns (G.E. Healthcare Life Sciences, cat. no. 17-0854-01).
Modified Oligo. . _ ._,
£ocie a Primer Sequence (5' - 3')
F ATTAGCGACGAGAGCCAG
2x PDS (TT)
R ATCGTTGGGTGCAGGTTG
F GAAGAGGCAATCTCAGCG
1x PDS (TT)
R TGATTGGCGGTTGAGTCG
F CGCGAGCGTAACAAGATG
3x PS (TTA)
R TTGGGTCCGGTAGGATTG
F AAGAACATCCGGGTCGAG
1x PS (TT)
R ATGCGTGTGGTATCGTGG
F AAGGCTCAGCGGAAATCG
2x 2'-OMe (UU)
R GCAGTCTGAGTTGTGGTG
F GCGCGGTAAACATGACAG
1x 2'-OMe (UT)
R GGATCGGATTTGGTCGTG
F AGCAACAGCTGGTAGACG
Ami (TT)
R TGGCACGGATTTGTGGTG
F GCATTCGAGCAACGTAAG
Tz2+ (TT)
R GGTTATGTGTGTCGGCAG
F ATACAGGAAGCATTCGAGCAACGTAAG
Tz3 (mCT)
R CTAGGTGTCGGTTATGTGTGTCGGCAG
F GCGTACGAGAACCGAATG
Tz3 (TT)
R GGATTGTTGTGTGCGACG
F TGACAGGGAGCTAACCAG
Tz2M (rUT)
R ATTGCGTAACGTGTGCTGGTGTCTGAAG
F CCGGGAGTCCATAAGAAG
Tz2 (rUT)
R GTCGTAGTTGGTGTGCTGGTGTCTGAAG
F GATATCGAACCAGGCGAG
Tz2 (rGT)
R CTAAGTCATGGTGTGCTGGTGTCTGAAG F GTAAGACGCTGCAACAGG
Tz2 (rCC)
R ATAACGCGAGGTTATGTGTGTCGGCAG
F GAAAGCAGTGCATTCGAGCAACGTAAG
Tz2 (mC-G-clamp)
R GTATGCGGTGGTTATGTGTGTCGGCAG
F GCAGTGACCAGCATTCGAGCAACGTAAG
Tz2 (mCC)
R GTCTATGGATGGTTATGTGTGTCGGCAG
F ATACACTACGCATTCGAGCAACGTAAG
Tz2 (mCU)
R GTTTGACGGATCGGGTTG
F GCTTACGACGAAGAACGG
Tz2 (TT)
R TGTGTGTATTGGCCGAGG
F AGCGCTACGGCATTCGAGCAACGTAAG
R TGTAAGCGTGGTTATGTGTGTCGGCAG
F ACGTTAGCACGAAGAGGC
Control (TT)
R ATTGGGTGTGTTCGCGAG
Table 5. Thermal stability of duplexes formed using backbone modified oligonucleotides (Supplementary Table 2) and control unmodified oligonucleotides (Supplementary Table 6) determined by UV melting assays. The complementary oligonucleotides (Supplementary Table 6) used for the control unmodified templates are the same as those for the modified templates. The difference in stability between the two is referred to as the AJm. Oligonucleotide concentrations were 1 μΜ (except * = 0.2 μΜ).
Unmodified
Modified Template Tm (°C) ATm (°C) Tm (°C)
Template
2x PDS TT 53.6 -2.4 56.0 Control (TT)
1x PDS TT 54.3 -1.7 56.0 Control (TT)
3x PS TTA 54.3 -1.7 56.0 Control (TT)
1x PS TT 54.9 -1.1 56.0 Control (TT)
2x 2'-OMe UU 53.2 -2.8 56.0 Control (TT)
1x 2'-OMe UT 53.0 -3.0 56.0 Control (TT)
Ami TT 54.8 -1.2 56.0 Control (TT)
Tz2+ TT 49.5 -6.5 56.0 Control (TT) mCT 48.9* -6.6* 55.5* Control (CCT)
Tz3
TT 48.5 -7.5 56.0 Control (TT)
Tz2M rUT 47.5 -8.5 56.0 Control (TT) rUT 48.4 -7.6 56.0 Control (TT) rGT 53.6 -6.3 59.9 Control (GT) rCC 56.5 -5.5 62.0 Control (CC)
Tz2
mCC 59.2 -2.8 62.0 Control (CC) mCU 55.8 -2.9 58.7 Control (CT)
TT 50.1 -5.9 56.0 Control (TT)
Tz1 mCT 51.8 -6.7 58.5 Control (CCT) Table 6. Additional control unmodified oligonucleotides used for UV melting assays and their complements. Longer control oligonucleotides (~80-mers) were purified by HPLC and the purity confirmed by denaturing PAGE. Shorter complementary oligonucleotides were purified by HPLC. The site of complementary oligonucleotide binding on the control oligonucleotide is highlighted in bold and underlined.
Figure imgf000058_0001
Transcription of the amide linkage using short coding strands
Dual amide ligation to form the transcription template (ODN5)
[00196] The three oligonucleotides (3'-carboxy dT, ODN1), (di-modified 5'-amino dT and 3'-carboxy dT, ODN2), (5'-amino dC, ODN3) , and the complementary splint (6.8 nmole of each) in 0.2 M NaCI (640 μί) were annealed by heating at 90 °C for 5 min then cooled down slowly to room temperature. EDC:NHS solution (200:40 mM, 3.4 μΙ) was added to the annealed oligonucleotides and the mixture was kept at room temperature for 2 hr. Reagents were removed by NAP-10 gel-filtration and samples were then mixed with equal volume of formamide and loaded on an 8% denaturing polyacrylamide gel (1x TBE, 7 M urea) at 20 W for 2 h. Oligonucleotide bands were then visualized using a UV lamp and the desired bands excised, crushed and soaked in buffer (50 mM Tris-HCI, pH 7.5, 25 mM NaCI, ca. 15 ml) overnight at 37 °C. After evaporation of the solvent, samples were desalted using 2X NAP-25 columns (G.E. Healthcare Life Sciences).
Transcription of double modified amide template (ODN5)
[00197] MegaScript T7 Transcription Kit (ThermoFisher Scientific, cat. no. AM 1333) was used according to the manufacturer recommended protocol. Reaction mixtures were prepared in the following order at room temperature: amide template ODN5 (1 μΜ, 2.5 μΙ), coding strand ODN7 (1 μΜ, 2.75 μΙ), water (14.75 μΙ), reaction buffer (10x, 5 μΙ), ATP (5 μΙ), CTP (5 μΙ), GTP (5 μΙ), UTP (5 μΙ) and enzyme mix (5 μΙ). The reaction mixture was then incubated at 37 °C and 10 μΙ aliquots removed at the specified times and mixed with an equal volume of formamide before storing at -80 °C. Samples were then loaded on 8% denaturing polyacrylamide gel (1x TBE, 7 M urea, W x D x H = 18 x 0.2 x 24.4 cm) at 20 W for 2 h. Oligonucleotide bands were then visualized using a UV lamp and the desired bands excised, crushed and soaked in buffer (50 mM Tris-HCI, pH 7.5, 25 mM NaCI, ca. 15 ml) overnight at 37 °C. After evaporation of the solvent, samples were desalted using two NAP-25 columns (G.E. Healthcare Life Sciences, cat. no. 17-0852-01).
Table 7. Oligonucleotides used in transcription study.
cT=3'-Carboxy methyl dT (1), NT=5'-amino dT, NC=5'-amino dC, X= amide linkage
Figure imgf000059_0001
[00198] While specific embodiments of the invention have been described for the purpose of reference and illustration, various modifications will be apparent to a person skilled in the art without departing from the scope of the invention as defined by the appended claims.

Claims

1. A process for ligating a first and a second oligonucleotide together, wherein the process comprises reacting:
a) a first oligonucleotide comprising a terminal functional group of Formula A shown below:
Figure imgf000060_0001
Formula A
wherein:
^ wv denotes the point of attachment to the oligonucleotide backbone; and
X is a leaving group optionally selected from halo, OSO2R, (1-2C)haloalkyl, (1- 2C)haloalkoxy, OR1 , heteroaryl, wherein R and R1 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 each of R and R1 are a solid support to which the one or more oligonucleotides are attached;
V is selected from O, S or NRX, wherein Rx is selected from hydrogen or (1-4C)alkyl; Q is selected from O or S;
Ra and Rb 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; and
q is an integer selected from 0 to 1 ;
with
a second oligonucleotide comprising a terminal functional group of Formula B, shown below:
Figure imgf000060_0002
Formula B wherein:
Rc, Rd and Re are independently selected from hydrogen or (1-4C)alkyl;
Y is selected from O or NH;
W is selected from NRe or SH, wherein Re 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 ;
or
b) reacting a second oligonucleotide comprising a terminal functional group of Formula B, as defined above, with a further second oligonucleotide comprising a terminal functional group of Formula B, as defined above, together with a coupling agent of formula D shown below:
Figure imgf000061_0001
Formula D
wherein:
Q1 is selected from O or S; and
LG1 and LG2 are each independently a leaving group (e.g. halo, imidazolyl or haloalkoxy);
and wherein the reaction is optionally conducted in the presence of one or more of the following:
i) a template oligonucleotide;
ii) one or more peptide coupling reagents;
iii) one or more activating agents; and
iv) a catalyst;
and with the proviso that:
1) the sum of integers m, n, p and q is equal to or greater than 2; and
2) when p is 1 , q is 0. A process according to claim 1 , wherein q is 0.
A process according to claims 1 or 2, wherein X is selected from halo, OSO2R, (1- 2C)haloalkyl, (1-2C)haloalkoxy or OR1 , wherein R and R1 are independently selected from H or (1-6C)alkyl.
A process according to any one of claims 1 to 3, wherein X is OH.
A process according to claim 4, wherein the first oligonucleotide comprises a terminal nucleotide analogue selected from one of the following:
Figure imgf000062_0001
wherein:
Ra and Rb are as defined in claim 1 ;
Ra' and Rb' are independently selected from hydrogen or (1-4C)alkyl;
Z is selected from hydrogen, halo, (1-4C)alkyl, (1-2C)haloalkyl, OR2, NH2, wherein R2 is selected from hydrogen, (1-4C)alkyl, (2-4C)alkenyl or (2- 4C)alkynyl; and
B is a nucleobase or nucleobase analogue.
A process according any preceding claim, wherein the second oligonucleotide comprises a terminal nucleotide analogue selected from one of the following:
Figure imgf000063_0001
Rc, Rd and Re are as defined in claim 1 ;
Rc' and Rd' are independently selected from hydrogen or (1-4C)alkyl;
Z is selected from hydrogen, halo, (1-4C)alkyl, (1-2C)haloalkyl, OR2, NH2, wherein R2 is selected from hydrogen, (1-4C)alkyl, (2-4C)alkenyl or (2- 4C)alkynyl; and
B is a nucleobase or nucleobase analogue.
7. A process according to claim 2, wherein n and m are 1 and p is 0.
8. A process according to any preceding claim, wherein Ra, Rb, Rc, Rd and Re are hydrogen.
9. A process according to claim 5 or 6, wherein Ra', Rb', Rc' and Rd' are hydrogen.
10. A process according to any preceding claim, wherein the reaction is carried out in the presence of one or more peptide coupling reagents.
1 1. A process according to claim 10, wherein the one or more peptide coupling reagent is a carbodiimide based coupling agent.
12. A process according to claim 11 , wherein the one or more peptide coupling reagent is selected from Ν,Ν'-dicyclohexylcarbodiimide (DCC), Ν,Ν'-diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), N-cyclohexyl-N'- isopropylcarbodiimide (CIC) or Ν,Ν'-dicyclopentylcarbodiimide (CPC).
13. A process according to any preceding claim, wherein the reaction is carried out in the presence of an activating agent.
14. A process according to claim 13, wherein the activating agent is selected from
hydroxybenzotriazole (HOBt), N-hydroxy 2-phenyl benzimidazole (HOBI), 1-hydroxy-7- azabenzotriazole (HOAt), N-hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (Sulfo-NHS), 4-dimethylaminopyridine (DMAP) or ethyl cyano(hydroxyimino)acetate (Oxyma Pure®).
15. A process according to any preceding claim, wherein the reaction is carried out in the presence of a template oligonucleotide.
16. A process according to claim 15, wherein the template oligonucleotide is a single
stranded oligonucleotide or oligonucleotide analogue.
17. A process according to any preceding claim, wherein the functional group of Formula A is attached to the 3' position of the first oligonucleotide.
18. A process according to any preceding claim, wherein the functional group of Formula B is attached to the 5' position of the second oligonucleotide.
19. A process according to claim 1 , wherein Q1 is O and LG1 and LG2 are selected from halo (e.g. CI), heteroaryl (e.g. imidazolyl) or haloalkoxy (e.g. OCCU).
20. A process according to claim 1 , wherein the coupling agent is carbonyldiimidazole.
21. An oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C shown below:
Figure imgf000065_0001
Formula C
wherein:
Ra and Rb 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
Rc, Rd and Re are independently selected from hydrogen or (1-4C)alkyl;
Y is O or NH;
V is selected from O, S or NRX, wherein Rx is selected from hydrogen or (1- 4C)alkyl;
Q is selected from S or O;
m and n are integers independently selected from 0 to 2; and
p and q are integers independently selected from 0 to 1 ;
with the proviso that:
1) the sum of integers m, n, p and q is equal to or greater than 2; and
2) when p is 1 , q is 0;
3) when q and p are 0 and the oligonucleotide comprises only one phosphodiester backbone mimic of Formula C, the oligonucleotide comprises greater than or equal to 20 nucleotide and/or nucleotide analogue monomers;
4) when q and p are 0 and the oligonucleotide comprises two or more phosphodiester backbone mimics of Formula C, the phosphodiester backbone mimics are separated by at least 10 nucleotide and/or nucleotide analogue monomers.
An oligonucleotide according to claim 21 , wherein the one or more phosphodiester backbone mimics are of Formula C1 shown below:
Figure imgf000066_0001
Formula C1
wherein:
Ra, Rb, Rc, Rd, Re and Rx 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 ;
with the proviso that n + m + q = 2 or 3;
and with the proviso that:
a) when q is 0 and the oligonucleotide comprises only one phosphodiester backbone mimic of Formula C1 , the oligonucleotide comprises greater than or equal to 20 nucleotide and/or nucleotide analogue monomers; or
b) when q is 0 and 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.
23. An oligonucleotide according to claim 22, wherein integers m and n are 1.
24. An oligonucleotide according to claim 23, wherein q is 0.
25. An oligonucleotide according to claim 24, wherein, when the oligonucleotide comprises two or more phosphodiester backbone mimics of Formula C1 , the phosphodiester backbone mimics are separated by at least 25 nucleotide and/or nucleotide analogue monomers.
26. An oligonucleotide according to claim 24, wherein, when the oligonucleotide comprises two or more phosphodiester backbone mimics of Formula C1 , the phosphodiester backbone mimics are separated by at least 50 nucleotide and/or nucleotide analogue monomers.
An oligonucleotide according to claim 21 , wherein the terminal nucleotides of the first and second oligonucleotides and the phosphodiester backbone mimic is selected from one of the following formulae:
Figure imgf000067_0001
wherein:
Z and Z' are independently selected from Z is selected from hydrogen, halo, (1- 4C)alkyl, (1-2C)haloalkyl, OR2, NH2, wherein R2 is selected from hydrogen, (1- 4C)alkyl, (2-4C)alkenyl or (2-4C)alkynyl; and
B and B' are each independently a nucleobase or nucleobase analogue.
28. An oligonucleotide according to claim 21 , wherein the terminal nucleotides of the first and second oligonucleotides and the phosphodiester backbone mimic is selected from one of the following formulae:
Figure imgf000068_0001
4C)alkyl, (2-4C)alkenyl or (2-4C)alkynyl; and
B and B' are each independently a nucleobase.
The use of an oligonucleotide comprising one or more phosphodiester backbone of Formula C shown below:
Figure imgf000068_0002
Formula C
wherein each of Q, V, Y, Ra, Rb, Rc, Rd, Re, n, m, p and q are as defined in claim 21 above;
with the proviso that:
1) the sum of integers m, n, p and q is equal to or greater than 2; and
2) when p is 1 , q is 0; 3) when q and p are 0 and the oligonucleotide comprises only one phosphodiester backbone mimic of Formula C, the oligonucleotide comprises greater than or equal to 20 nucleotide and/or nucleotide analogue monomers;
4) when q and p are 0 and the oligonucleotide comprises two or more phosphodiester backbone mimics of Formula C, the phosphodiester backbone mimics are separated by at least 10 nucleotide and/or nucleotide analogue monomers;
or an oligonucleotide according to any one of claims 21 to 28;
as a template for amplification in a polymerase chain reaction (PCR).
The use of an oligonucleotide comprising one or more phosphodiester backbone of Formula C shown below:
Figure imgf000069_0001
Formula C
wherein each of Q, V, Y, Ra, Rb, Rc, Rd, Re, n, m, p and q are as defined in claim 21 above;
with the proviso that:
1) the sum of integers m, n, p and q is equal to or greater than 2; and
2) when p is 1 , q is 0;
3) when q and p are 0 and the oligonucleotide comprises only one phosphodiester backbone mimic of Formula C, the oligonucleotide comprises greater than or equal to 20 nucleotide and/or nucleotide analogue monomers;
4) when q and p are 0 and the oligonucleotide comprises two or more phosphodiester backbone mimics of Formula C, the phosphodiester backbone mimics are separated by at least 10 nucleotide and/or nucleotide analogue monomers;
or an oligonucleotide according to any one of claims 21 to 28; as a template in a DNA replication process.
The use of an oligonucleotide comprising one or more phosphodiester backbone of Formula C shown below:
Figure imgf000070_0001
Formula C
wherein each of Q, V, Y, Ra, Rb, Rc, Rd, Re, n, m, p and q are as defined in claim 21 above;
with the proviso that:
1) the sum of integers m, n, p and q is equal to or greater than 2; and
2) when p is 1 , q is 0;
3) when q and p are 0 and the oligonucleotide comprises only one
phosphodiester backbone mimic of Formula C, the oligonucleotide comprises greater than or equal to 20 nucleotide and/or nucleotide analogue monomers;
4) when q and p are 0 and the oligonucleotide comprises two or more phosphodiester backbone mimics of Formula C, the phosphodiester backbone mimics are separated by at least 10 nucleotide and/or nucleotide analogue monomers;
or an oligonucleotide according to any one of claims 21 to 28;
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.
The use of a RNA oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C shown below:
Figure imgf000071_0001
Formula C
wherein each of Q, V, Y, Ra, Rb, Rc, Rd, Re, n, m, p and q are as defined in claim 21 above;
with the proviso that:
1) the sum of integers m, n, p and q is equal to or greater than 2; and
2) when p is 1 , q is 0;
3) when q and p are 0 and the oligonucleotide comprises only one
phosphodiester backbone mimic of Formula C, the oligonucleotide comprises greater than or equal to 20 nucleotide and/or nucleotide analogue monomers;
4) when q and p are 0 and the oligonucleotide comprises two or more
phosphodiester backbone mimics of Formula C, the phosphodiester backbone mimics are separated by at least 10 nucleotide and/or nucleotide analogue monomers;
or an oligonucleotide according to any one of claims 21 to 28;
as template in a translation process to produce a corresponding protein or peptide.
The use of an oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C shown below:
Figure imgf000071_0002
Formula C
wherein each of Q, V, Y, Ra, Rb, Rc, Rd, Re, n, m, p and q are as defined in claim 21 above;
with the proviso that: 1) the sum of integers m, n, p and q is equal to or greater than 2; and
2) when p is 1 , q is 0;
3) when q and p are 0 and the oligonucleotide comprises only one
phosphodiester backbone mimic of Formula C, the oligonucleotide comprises greater than or equal to 20 nucleotide and/or nucleotide analogue monomers;
4) when q and p are 0 and the oligonucleotide comprises two or more
phosphodiester backbone mimics of Formula C, the phosphodiester backbone mimics are separated by at least 10 nucleotide and/or nucleotide analogue monomers;
or an oligonucleotide according to any one of claims 21 to 28;
as a guide RNA in a CRISPR-Cas process (e.g. in a CRISPR-Cas9 gene editing process).
The use of an oligonucleotide comprising one or more phosphodiester backbone mimics of Formula C shown below:
Figure imgf000072_0001
Formula C
wherein each of Q, V, Y, Ra, Rb, Rc, Rd, Re, n, m, p and q are as defined in claim 21 above;
with the proviso that:
1) the sum of integers m, n, p and q is equal to or greater than 2; and
2) when p is 1 , q is 0;
3) when q and p are 0 and the oligonucleotide comprises only one
phosphodiester backbone mimic of Formula C, the oligonucleotide comprises greater than or equal to 20 nucleotide and/or nucleotide analogue monomers;
4) when q and p are 0 and the oligonucleotide comprises two or more
phosphodiester backbone mimics of Formula C, the phosphodiester backbone mimics are separated by at least 10 nucleotide and/or
nucleotide analogue monomers;
or an oligonucleotide according to any one of claims 21 to 28
as a donor DNA template in a CRISPR-Cas mediated homology directed repair (HDR) process (e.g. a CRISPR-Cas9 mediated homology directed repair (HDR) process).
35. A method for amplifying an oligonucleotide sequence, the method comprising the steps of:
1) providing an oligonucleotide prepared according to the process defined in any one of claims 1 to 20, or an oligonucleotide as defined in any one of claims 21 to 28; and
2) carrying out a polymerase chain reaction (PCR) using the oligonucleotide of step 1 as a template.
36. A method for replicating an oligonucleotide sequence, the method comprising the steps of:
1) providing an oligonucleotide prepared according to the process defined in any one of claims 1 to 20, or an oligonucleotide as defined in any one of claims 21 to 28; and
2) carrying out a replication reaction using the oligonucleotide of step 1 as a template.
37. A method for producing a ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)
sequence comprising the steps of:
1) providing an oligonucleotide prepared according to the process defined in any one of claims 1 to 20, or an oligonucleotide as defined in any one of claims 21 to 28; and
2) transcribing the oligonucleotide of step 1 to form a ribonucleic acid (RNA) transcript or deoxyribonucleic acid (DNA) transcript.
38. A method for preparing a protein or peptide comprising the steps of:
1) providing a RNA oligonucleotide prepared according to the process defined in any one of claims 1 to 20, or a RNA oligonucleotide template as defined in any one of claims 21 to 28; or a RNA oligonucleotide prepared according to claim 37; and
2) translating the oligonucleotide of step 1 to form the protein or peptide.
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