EP3973075A1 - Procédé de contrôle de qualité de synthèse d'oligonucléotides - Google Patents

Procédé de contrôle de qualité de synthèse d'oligonucléotides

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Publication number
EP3973075A1
EP3973075A1 EP20728173.4A EP20728173A EP3973075A1 EP 3973075 A1 EP3973075 A1 EP 3973075A1 EP 20728173 A EP20728173 A EP 20728173A EP 3973075 A1 EP3973075 A1 EP 3973075A1
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EP
European Patent Office
Prior art keywords
phosphate
nucleic acid
pyrophosphate
nucleotide
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP20728173.4A
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German (de)
English (en)
Inventor
Michael Chun Hao CHEN
Gordon Ross MCINROY
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Nuclera Ltd
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Nuclera Nucleics Ltd
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Publication date
Application filed by Nuclera Nucleics Ltd filed Critical Nuclera Nucleics Ltd
Publication of EP3973075A1 publication Critical patent/EP3973075A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase

Definitions

  • the invention relates to methods and compositions for the quality control of the synthesis of oligonucleotides during cycles of template independent enzymatic extension.
  • Nucleic acid synthesis is vital to modern biotechnology. The rapid pace of development in the biotechnology arena has been made possible by the scientific community's ability to artificially synthesise DNA, RNA and proteins.
  • DNA synthesis technology does not meet the demands of the biotechnology industry. Despite being a mature technology, it is highly challenging to synthesise a DNA strand greater than 200 nucleotides in length in viable yield, and most DNA synthesis companies only offer up to 120 nucleotides routinely.
  • an average protein-coding gene is of the order of 2000- 3000 contiguous nucleotides
  • a chromosome is at least a million contiguous nucleotides in length and an average eukaryotic genome numbers in the billions of nucleotides.
  • Known methods of DNA sequencing use template-dependent DNA polymerases to add 3'-reversibly terminated nucleotides to a growing double-stranded substrate.
  • each added nucleotide contains a dye, allowing the user to identify the exact sequence of the template strand.
  • this technology is able to produce strands of between 500-1000 bps long.
  • this technology is not suitable for de novo nucleic acid synthesis because of the requirement for an existing nucleic acid strand to act as a template.
  • TdT terminal deoxynucleotidyl transferase
  • dNTP deoxynucleotide triphosphate
  • TdT has not been shown to efficiently add nucleoside triphosphates containing 3'-0- reversibly terminating moieties for building up a nascent single-stranded DNA chain necessary for a de novo synthesis cycle, and thus the synthesis of long strands is inefficient.
  • a primary source of error in solid-phase nucleic acid synthesis - both enzymatic and phosphoramidite-based - comes from the failure to add a nucleotide to the desired sequence. Such failure results in a deletion, which results in frameshift mutations in biological sequences.
  • the ability to assess the nucleotide coupling efficiency and thus the quality with which a particular nucleic acid was synthesized is critical regardless of the method used to synthesize said nucleic acid.
  • Coupling efficiency in phosphoramidite nucleic acid synthesis is measured in-situ by virtue of the colour of deprotected 5'-dimethoxytrityl (DMT) groups.
  • DMT deprotected 5'-dimethoxytrityl
  • the 5'-DMT groups act as reversible terminators in phosphoramidite synthesis, controlling the addition of one and only one amidite per synthesis cycle.
  • the orange colour of the DMT cation released from the 5'-DMT group is eluted; such colour can be quantitated via a spectrophotometer to determine coupling efficiency per phosphoramidite synthesis cycle.
  • non-templated enzymatic nucleic acid synthesis does not result in the production of any groups readily detectable via spectrophotometric means.
  • enzymatic nucleic acid synthesis results in the production of inorganic pyrophosphate and a proton.
  • inorganic pyrophosphate is detectable through a variety of means, such as enzyme-coupled reactions.
  • the preference to rapidly remove inorganic pyrophosphate due to the capability of TdT to perform strand dismutation reactions complicates the detection of inorganic pyrophosphate as a means to quantitate coupling efficiency.
  • reactions often resynthesise nucleoside triphosphate molecules, which is problematic in reactions where nucleotide triphosphates are the TdT substrates.
  • the monitoring is performed on one or more of the nucleotide monomer reaction cycles.
  • the monitoring can be performed on each cycle.
  • the term each cycle refers to monitoring directly after a nucleotide has been added.
  • the quality measurements are performed during or after extension cycles before the completed strand is assembled.
  • the term refers to monitoring after nucleotide extension, rather than just at the end of the synthesis, and does not require that every cycle is monitored. Thus if a 100 cycles of extension are performed, monitoring every other cycle is within the scope of the invention. Similarly if 50 cycles are performed only 49 can be monitored.
  • the claim is merely requiring that the quality of the synthesis is determined as the synthesis progresses not just at the end, not that every cycle must be monitored. Monitoring each and every cycle is within the scope of the claimed invention, but is not essential.
  • Described is a technique for detecting the incorporation of one or more nucleotides into a growing chain by template-independent synthesis.
  • the described method relies on detection of changes in heat, pH, phosphate concentration and/or pyrophosphate concentration as a result of template- independent DNA synthesis.
  • dNTP nucleotide
  • G Guanine
  • A Adenine
  • T Thymine
  • C Cytosine as incorporated into a growing DNA strand.
  • the nucleotide need not be limited to dNTPs (i.e., 2'-deoxy NTPs). They include any polyphosphate species that can be incorporated by an enzyme, including ribonucleoside 5'-triphosphates.
  • the nucleotides can be reversibly blocked such that only one monomer per strand is incorporated. The block may be anywhere on the monomer, including optionally at the 3'- position.
  • the nucleotides can also be reversibly blocked at the nitrogenous base by molecular entities such as a small molecule, peptide, oligosaccharide, polymer, or protein.
  • the nucleotide may also be amine-masked to mask the amino groups on the nitrogenous base and prevent hydrogen bonding. The amino-groups may subsequently be unmasked to reveal a free amino (NH 2 ) group.
  • the nucleotides are generally unlabelled in order to synthesise a non-modified strand, but the nucleotides can optionally be labelled. In order to control the desired sequence, a single nucleotide species is generally added per cycle, but more than one nucleotide can be added if the desired strand sequence is degenerate.
  • the DNA polymerase can be a terminal deoxynucleotidyl transferase.
  • the enzyme can be modified to increase the incorporation of 3'- blocked nucleotides.
  • the incorporation of the nucleotide in the above reaction is monitored, by monitoring changes in heat, pH, levels of pyrophosphate or levels of phosphate, to provide quality control information.
  • the T in the above reaction is about 22 kT or ⁇ 570 meV per nucleotide incorporation, and may be measured in accordance with the present invention, as well as DrH.
  • FIG. 1 One embodiment of phosphate detection.
  • Phosphate generated from non-templated enzymatic nucleic acid synthesis is quantified through methods such as enzyme binding, enzyme- coupled reaction, and/or inorganic molecule binding.
  • the aforementioned methodologies generate a detectable signal to quantitate the level of nucleotide incorporation.
  • Figure 2 Coupling of pyrophosphatase and phosphate-binding protein labelled to a fluorophore to monitor and quantitate reversibly terminated nucleotide incorporation by engineered TdTs.
  • Figure 3 Pyrophosphate detection as a method to monitor and quantitate reversibly terminated nucleotide incorporation by engineered TdTs.
  • nucleotide incorporation results in an increased temperature in solution generated by released pyrophosphate.
  • the splitting of pyrophosphate into two inorganic phosphate molecules, accomplished for example with the addition of pyrophosphatases, will produce further temperature change.
  • changes in temperature are monitored by a suitable sensitive instrument to detect if successful nucleotide incorporation has occurred. pH:
  • a method for monitoring the incorporation of one or more nucleotides into a growing chain by template-independent synthesis by monitoring pH change is disclosed herein.
  • Nucleotide incorporation results in an increased negative charge in solution generated by released pyrophosphate and inorganic phosphate.
  • the splitting of pyrophosphate into two inorganic phosphate molecules, accomplished for example with the addition of pyrophosphatases, will further lower the charge in solution.
  • changes in pH are monitored by a suitable sensitive instrument such as an ion-sensitive field-effect transistor (ISFET) to detect if successful nucleotide incorporation has occurred.
  • ISFET ion-sensitive field-effect transistor
  • pyrophosphate is detected by interaction, chelation, binding, or association with a small molecule.
  • pyrophosphate is detected by interaction, chelation, binding, or association with a biomolecule.
  • the biomolecule may be formed from protein, DNA, RNA, or a combination thereof. These interactions result in a detectable signal.
  • the small molecule is or contains a chelated Zn(ll) complex.
  • the small molecule is a terpyridine-Zn(ll) complex.
  • the terpyridine-Zn(ll) complex is carbazole or amino modified.
  • the molecule containing a terpyridine-Zn(ll) complex interacts with pyrophosphate and generates a detectable signal, such as a change in absorbance, chemiluminescence, and/or fluorescence.
  • real-time bioluminometric detection of released pyrophosphate is detected as a result of successful nucleotide incorporation.
  • the released pyrophosphate is converted to ATP by ATP sulfurylase from adenosine 5'-phosphosulfate and the level of ATP is sensed by a luciferase producing a proportional light signal, which is detected by photo sensing devices.
  • the reacted addition solution is removed from the immobilized oligonucleotide and the released pyrophosphate in the removed addition solution is then detected by the bioluminescence regenerative cycle involving ATP sulfurylase and luciferase.
  • the advantage of detecting pyrophosphate in removed addition solution is that there is no potential for the reagents of the bioluminescence regenerative cycle (e.g., addition of the ATP sulfurylase product, ATP, by TdT to the oligonucleotide initiator) to interfere with the enzymatic nucleic acid synthesis process.
  • the reagents of the bioluminescence regenerative cycle e.g., addition of the ATP sulfurylase product, ATP, by TdT to the oligonucleotide initiator
  • incorporation of one or more nucleotides into a growing chain by template-independent synthesis is monitored using a bioluminescence regenerative cycle (BRC).
  • BRC bioluminescence regenerative cycle
  • steady state levels of bioluminescence result from processes that produce pyrophosphate.
  • Pyrophosphate reacts in the presence of ATP sulfurylase and adenosine 5'-phosphosulfate to produce ATP.
  • the ATP reacts with luciferin in a luciferase-catalysed reaction, producing light and regenerating pyrophosphate.
  • the pyrophosphate is recycled to produce ATP and the regenerative cycle continues.
  • kits for use in monitoring changes in pyrophosphate concentration there is provided a kit for use in monitoring changes in pyrophosphate concentration.
  • the nucleotide may carry a reversible block.
  • the nucleotide may carry a 3'-block.
  • the 3'-block is a protecting group on the 3' hydroxyl which can be removed to release the 3'-OH, in which case the block acts as a reversible terminator.
  • the nucleotide may carry a reversible terminator attached to the nitrogenous base.
  • the base-blocking group is a protecting group which can be removed to enable addition of the nucleotide by a template independent enzyme such as a TdT.
  • the nucleotide may be covalently or non-covalently bound to a template independent enzyme such as a TdT.
  • Each mention of the term block includes optionally a 3'-block.
  • a method of enzymatic nucleic acid synthesis which includes:
  • nucleic acid transferase or template independent polymerase such as an engineered terminal deoxynucleotidyl transferase (TdT);
  • pyrophosphate-sensing reagents such as a terpyridine-Zn(ll) complex or the simultaneous presence of all of the following: adenosine 5'-phosphosulfate, ATP sulfuryase, luciferin, and luciferase;
  • Steps (b) - (f) can be repeated to add greater than one nucleotide to the above DNA initiator sequence.
  • a further method of enzymatic nucleic acid synthesis could include:
  • nucleic acid transferase or template independent polymerase such as an engineered terminal deoxynucleotidyl transferase (TdT);
  • reagents for the quantification of pyrophosphate can include a terpyridine-Zn(ll) complex, adenosine 5'-phosphosulfate, ATP sulfuryase, luciferin, luciferase, or a combination thereof;
  • Steps (b) - (h) can be repeated to add greater than one nucleotide to the above nucleic acid initiator sequence.
  • the further method of enzymatic nucleic acid synthesis directly mentioned above has the advantage of segregating the incorporation of a blocked nucleoside triphosphate from the detection of a produced pyrophosphate molecule.
  • This segregation is advantageous as the pyrophosphate detection reagents may interfere with the incorporation of a blocked nucleoside triphosphate.
  • ATP is generated when using adenosine 5'-phosphosulfate, ATP sulfuryase, luciferin, luciferase, or a combination thereof to detect pyrophosphate through bioluminescence.
  • ATP can be incorporated by a TdT to the 3'-end of a nucleic acid initiator in a method of enzymatic nucleic acid synthesis. This incorporation thus represents a mutation, which is detrimental to the accuracy of nucleic acid synthesis.
  • Disclosed herein is a method for monitoring the incorporation of one or more nucleotides into a growing chain by template-independent synthesis by monitoring the phosphate concentration over time.
  • the method involves the use of an inorganic pyrophophatase enzyme to catalyse the conversion of pyrophosphate, one molecule of which is released during the successful addition of one nucleotide, into two phosphate ions.
  • the presence of the phosphate ions, and hence the presence of the original pyrophosphate molecule can be detected and information on the success or failure of nucleotide addition gained.
  • inorganic pyrophosphatase which converts one molecule of inorganic pyrophosphate to two molecules of inorganic phosphate.
  • a phosphate-sensing assay we couple the reaction of inorganic pyrophosphatase, which converts one molecule of inorganic pyrophosphate to two molecules of inorganic phosphate.
  • the presence of phosphate ions is detected using a phosphate binding protein (PBP).
  • PBP phosphate binding protein
  • the phosphate binding protein is natural, in another embodiment of the invention the phosphate binding proteins sequence is modified. In one embodiment of the invention, the phosphate binding protein is E. coli phosphate binding protein.
  • the phosphate binding protein is conjugated to a fluorescent tag.
  • a kit for use in monitoring changes in phosphate concentration there is provided a kit for use in monitoring changes in phosphate concentration.
  • the method of nucleic acid synthesis involves the use of TdT in non-templated enzymatic DNA synthesis with 3'-0-reversibly terminated 2'-deoxynucleoside 5'-triphosphates (dNTPs).
  • dNTPs 3'-0-reversibly terminated 2'-deoxynucleoside 5'-triphosphates
  • dNTPs 3'-0-reversibly terminated 2'-deoxynucleoside 5'-triphosphates
  • dNTPs 3'-0-reversibly terminated 2'-deoxynucleoside 5'-triphosphates
  • N is known a priori as the quantity of free 3'-OH ends supplied by
  • 5'-immobilized oligonucleotides and N+l is quantified through a phosphate-sensing assay.
  • the phosphate sensing assay above utilizes a phosphate binding protein (PBP), such as E. coli PBP containing the mutation A197C, covalently coupled to fluorescent dye, such as N- [2-(l-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) or tetramethylrhodamine (rho).
  • PBP phosphate binding protein
  • MDCC N- [2-(l-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide
  • rho tetramethylrhodamine
  • E. coli PBP the MDCC or rho is covalently coupled to PBP.
  • MDCC is covalently coupled to PBP through a thioester bond at A197C.
  • the phosphate-sensing assay is based on reaction of inorganic phosphate with maltose, in the presence of an enzyme, to produce glucose. The glucose is then specifically oxidized to generate a product that reacts with a probe to generate fluorescence.
  • the enzyme is maltose phosphorylase which converts maltose, in the presence of inorganic phosphate, to glucose-l-phosphate and glucose. Then, glucose oxidase converts the glucose to gluconolactone and H 2 O 2 . Finally, with horseradish peroxidase (HRP) as a catalyst, the H 2 O 2 reacts with Amplex Red reagent to generate resorufin. The resulting increase in fluorescence or absorption is proportional to the amount of inorganic phosphate in the sample.
  • HRP horseradish peroxidase
  • the phosphate-sensing assay consists of complex formation of inorganic phosphate with ammonium molybdate, which is detectable as a colour change
  • the phosphate-sensing assay is based on quantification of the green complex formed between Malachite Green, ammonium molybdate and free phosphate.
  • the phosphate-sensing assay consists of a phosphorylase and a substrate that becomes detectable upon phosphorylation. Examples include utilizing a phosphorylase in the presence of 7-methylguanosine or 2-amino-6-mercapto-7-methylpurine.
  • a method of enzymatic nucleic acid synthesis could include:
  • nucleic acid transferase or template independent polymerase such as an engineered terminal deoxynucleotidyl transferase (TdT);
  • Steps (b) - (f) can be repeated to add greater than one nucleotide to the above nucleic acid initiator sequence.
  • a further method of enzymatic nucleic acid synthesis could include:
  • nucleic acid transferase or template independent polymerase such as an engineered terminal deoxynucleotidyl transferase (TdT);
  • Steps (b) - (h) can be repeated to add greater than one nucleotide to the above nucleic acid initiator sequence.
  • the further method of enzymatic nucleic acid synthesis directly mentioned above has the advantage of segregating the incorporation of a blocked nucleoside triphosphate from the detection of produced phosphate molecules. This segregation may be advantageous if the phosphate detection reagents interfere with the incorporation of a blocked nucleoside triphosphate.
  • references herein to an 'initiator sequence' refer to a short oligonucleotide with a free 3'-end which the nucleotide monomers can attach.
  • the initiator sequence is a DNA initiator sequence.
  • the initiator sequence is an RNA initiator sequence.
  • the strands are synthesised using enzymatic means.
  • the cycles of extension can be performed using a nucleic acid transferase or template independent polymerase, such as an engineered terminal deoxynucleotidyl transferase (TdT), where the nucleotide monomers are nucleoside triphosphates.
  • TdT engineered terminal deoxynucleotidyl transferase
  • the reversibly blocked nucleoside triphosphates can be 3'-reversibly blocked nucleoside triphosphates.
  • the 3'-reversible block can be selected from 3'-0-CH 2 N 3 , 3'-0-CH 2 CHCH 2 , 3'-0- CH 2 CH 2 CN or 3'-0-NH 2 .
  • references herein to a 'DNA initiator sequence' include a small sequence of DNA which a blocked nucleotide triphosphate can be attached to, i.e., DNA will be synthesised from the end of the DNA initiator sequence.
  • the initiator sequence is single-stranded.
  • the initiator sequence is double-stranded. It will be understood by persons skilled in the art that a 3'-overhang (l.e., a free 3'-end) allows for efficient addition.
  • the initiator sequence is immobilised on a solid support.
  • the initiator sequence may be attached to a solid support stable under aqueous conditions so that the method can be easily performed via a flow setup.
  • the initiator sequence is immobilised on a solid support via a reversible interacting moiety, such as a chemically-cleavable linker, an antibody/immunogenic epitope, a biotin/biotin binding protein (such as avidin or streptavidin), or glutathione-GST tag. Therefore, in a further embodiment, the method additionally comprises extracting the resultant nucleic acid by removing the reversible interacting moiety in the initiator sequence, such as by incubating with proteinase K.
  • a reversible interacting moiety such as a chemically-cleavable linker, an antibody/immunogenic epitope, a biotin/biotin binding protein (such as avidin or streptavidin), or glutathione-GST tag. Therefore, in a further embodiment, the method additionally comprises extracting the resultant nucleic acid by removing the reversible interacting moiety in the initiator sequence, such as by incubating with proteinase K
  • the initiator sequence contains a base or base sequence recognisable by an enzyme.
  • a base recognised by an enzyme such as a glycosylase, may be removed to generate an abasic site which may be cleaved by chemical or enzymatic means.
  • a base sequence may be recognised and cleaved by a restriction enzyme.
  • the resultant contiguous oligonucleotide sequence is released from being immobilised. In one embodiment, this release occurs by removing a non-canonical base from one or more of the immobilised oligonucleotides and cleaving the strands at the resultant abasic site.
  • the non-canonical base is uracil, which is removed by uracil DNA glycosylase. In an alternate embodiment, the non-canonical base is 8-oxoquanine, which is removed by formamidopyrimidine DNA glycosylase.
  • the initiator sequence is immobilised on a solid support via a chemically-cleavable linker, such as a disulfide, allyl, or azide-masked hemiaminal ether linker. Therefore, in one embodiment, the method additionally comprises extracting the resultant contiguous oligonucleotide sequence by cleaving the chemical linker through the addition of tris(2- carboxyethyljphosphine (TCEP) or dithiothreitol (DTT) for a disulfide linker; palladium complexes or an allyl linker; or TCEP for an azide-masked hemiaminal ether linker.
  • TCEP tris(2- carboxyethyljphosphine
  • DTT dithiothreitol
  • the cycles of extension are performed using a polymerase enzyme and the nucleotide monomers are nucleoside triphosphates. In one embodiment, the cycles of extension are performed using a template independent polymerase and the nucleotide monomers are nucleoside triphosphates. In one embodiment, the cycles of extension are performed using a template independent polymerase and the nucleotide monomers are reversibly blocked nucleoside triphosphates.
  • the cycles of extension are performed using a template independent polymerase and the nucleotide monomers are 3'-reversibly blocked nucleoside triphosphates.
  • the 3'-blocked nucleoside 5'-triphosphate can be blocked by any chemical group that can be unmasked to reveal a 3'-OH.
  • the 3'-blocked nucleoside triphosphates can be blocked by a 3'-0-azidomethyl, 3'- aminooxy, 3'-0-allyl group, 3'-0-cyanoethyl, 3'-0-acetyl, 3'-0-nitrate, 3'-0-phosphate, 3'-0-acetyl levulinic ester, 3'-0-tert butyl dimethyl silane, 3'-0-trimethyl(silyl)ethoxymethyl, 3'-0-ortho- nitrobenzyl, and 3'-0-para-nitrobenzyl.
  • the 3'-blocked nucleoside 5'-triphosphates can also be blocked by any chemical group that can be directly utilized in chemical ligations, such as copper-catalyzed or copper-free azide-alkyne click reactions and tetrazine-alkene click reactions.
  • the 3'-blocked nucleoside triphosphates can include chemical moieties containing an azide, alkyne, alkene, and tetrazine.
  • the 3'-reversible block is selected from 3'-0-CH 2 N 3 , 3'-0-CH 2 CHCH 2 , 3'- 0-CH 2 CH 2 CN or 3'-0-NH 2 .
  • the template independent polymerase is a terminal deoxynucleotidyl transferase (TdT).
  • the template independent polymerase is a modified TdT.
  • the TdT is added in the presence of an extension solution comprising one or more buffers (e.g., Tris or cacodylate), one or more salts (e.g., Na + , K + , Mg 2+ , Mn z+ , Cu 2+ , Zn 2+ , Co 2+ , etc. all with appropriate counterions, such as Cl) and inorganic pyrophosphatase (e.g., the Saccharomyces cerevisiae homolog).
  • buffers e.g., Tris or cacodylate
  • salts e.g., Na + , K + , Mg 2+ , Mn z+ , Cu 2+ , Zn 2+ , Co 2+ , etc. all with appropriate counterions, such as Cl
  • buffers and salts depends on the optimal enzyme activity and stability.
  • the use of an inorganic pyrophosphatase helps to reduce the build-up of pyrophosphate due to nucleoside triphosphate hydrolysis by TdT. Therefore, the use of an inorganic pyrophosphatase has the advantage of reducing the rate of (1) backwards reaction and (2) TdT strand dismutation.
  • References herein to 'nucleoside triphosphates' refer to a molecule containing a nucleoside (i.e. a base attached to a deoxyribose or ribose sugar molecule) bound to three phosphate groups.
  • nucleoside triphosphates that contain deoxyribose are: deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP) or deoxythymidine triphosphate (dTTP).
  • nucleoside triphosphates that contain ribose are: adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP) or uridine triphosphate (UTP).
  • Other types of nucleosides may be bound to three phosphates to form nucleoside triphosphates, such as naturally occurring modified nucleosides and artificial nucleosides.
  • nucleosides may include any functional equivalents of nucleosides including amine-masked nucleoside 5'-triphosphates, such as 6-azido-adenosine, 4-azido-cytidine, or 2-azido-guanosine.
  • references herein to '3'-blocked nucleoside triphosphates' refer to nucleoside triphosphates (e.g., dATP, dGTP, dCTP or dTTP) which have an additional group on the 3' end which prevents further addition of nucleotides, i.e., by replacing the 3'-OH group with a protecting group.
  • nucleoside triphosphates e.g., dATP, dGTP, dCTP or dTTP
  • references herein to '3'-block', '3'-blocking group' or '3'-protecting group' refer to the group attached to the 3' end of the nucleoside triphosphate which prevents further nucleotide addition.
  • This method uses reversible 3'-blocking groups which can be removed by cleavage to allow the addition of further nucleotides.
  • irreversible 3'-blocking groups refer to dNTPs where the 3'-OH group can neither be exposed nor uncovered by cleavage.
  • the 3'-blocked nucleoside 5'-triphosphate can be blocked by any chemical group that can be unmasked to reveal a 3'-OH.
  • the 3'-blocked nucleoside triphosphate can be blocked by a 3'-0- azidomethyl, 3'-aminooxy, 3'-0-allyl group, 3'-0-cyanoethyl, 3'-0-acetyl, 3'-0-nitrate, 3'-0- phosphate, 3'-0-acetyl levulinic ester, 3'-0-tert butyl dimethyl silane, 3'-0- trimethyl(silyl)ethoxymethyl, 3'-0-ortho-nitrobenzyl, and 3'-0-para-nitrobenzyl.
  • the 3'-blocked nucleoside 5'-triphosphate can also be blocked by any chemical group that can be directly utilized in chemical ligations, such as copper-catalyzed or copper-free azide-alkyne click reactions and tetrazine-alkene click reactions.
  • the 3'-blocked nucleoside triphosphate can include chemical moieties containing an azide, alkyne, alkene, and tetrazine.
  • references herein to 'cleaving agent' refer either to a substance which is able to cleave the 3'- blocking group from the 3'-blocked nucleoside triphosphate or to a substance which is able to cleave immobilised oligonucleotides from the solid support.
  • the cleaving agent is a chemical cleaving agent.
  • the cleaving agent is an enzymatic cleaving agent.
  • cleaving agent is dependent on the type of 3'-nucleoside blocking group used.
  • tris(2- carboxyethyl)phosphine (TCEP) or tris(hydroxypropyl)phosphine (THPP) can be used to cleave a 3'-0- azidomethyl group
  • palladium complexes can be used to cleave a 3'-0-allyl group
  • sodium nitrite can be used to cleave a 3'-aminoxy group. Therefore, in one embodiment, the cleaving agent is selected from: tris(2- carboxyethyl)phosphine (TCEP), a palladium complex or sodium nitrite.
  • the cleaving agent is added in the presence of a cleavage solution comprising a denaturant, such as urea, guanidinium chloride, formamide or betaine.
  • a denaturant such as urea, guanidinium chloride, formamide or betaine.
  • the cleavage solution comprises one or more buffers. It will be understood by the person skilled in the art that the choice of buffer is dependent on the exact cleavage chemistry and cleaving agent required.
  • the inventors have previously developed a selection of engineered terminal transferase enzymes, any of which may be used in the current process.
  • Terminal transferase enzymes are ubiquitous in nature and are present in many species. Many known TdT sequences have been reported in the NCBI database http://www.ncbi.nlm.nih.gov/. The sequences of the various described terminal transferases show some regions of highly conserved sequence, and some regions which are highly diverse between different species.
  • the inventors have modified the terminal transferase from Lepisosteus oculatus TdT (spotted gar) (shown below). However the corresponding modifications can be introduced into the analagous terminal transferase sequences from any other species, including the sequences listed above in the various NCBI entries.
  • the amino acid sequence of the spotted gar ( Lepisosteus oculatus) is shown below
  • SEQ ID 1 wild type spotted Gar TdT
  • the inventors have identified various regions in the amino acid sequence having improved properties. Certain regions improve the solubility and handling of the enzyme. Certain other regions improve the ability to incorporate nucleotides with modifications at the 3'-position.
  • modified terminal deoxynucleotidyl transferase (TdT) enzymes comprising amino acid modifications when compared to a wild type sequence SEQ ID NO 1 or a truncated version thereof or the homologous amino acid sequence of a terminal deoxynucleotidyl transferase (TdT) enzyme in other species or the homologous amino acid sequence of RoIm, RoIb, RoIl, and RoIq of any species or the homologous amino acid sequence of X family polymerases of any species, wherein the amino acid is modified at one or more of the amino acids:
  • K392, F394, 1397, K398, K400 K401, E402, L403, A404, A405, G406, R407, D411, A421, P422, P423,
  • Modifications which improve the incorporation of modified nucleotides can be at one or more of selected regions shown below. Regions were selected according to mutation data, sequence alignment, and structural data obtained from spotted gar TdT co-crystal I ized with DNA and a 3'- modified dNTP.
  • the second modification can be selected from one or more of the amino acid regions VAIF, MG A, MENHNQI, SEGPCLAFMRA, HAISSS, DQTKA, KGFHS, QADNA, HFTKMQK, SAAVCK, EAQA, TVRLI, GKEC, TPEMGK, YYDIV, DHFQK, LAAG, APPVDNF, FARHERKMLLDNHALYDKTKK, and
  • references to particular sequences include truncations thereof. Included herein are modified terminal deoxynucleotidyl transferase (TdT) enzyme comprising at least one amino acid modification when compared to a wild type sequence SEQ ID NO 1 or a truncated version thereof, or the homologous amino acid sequence of a terminal deoxynucleotidyl transferase (TdT) enzyme in other species, wherein the modification is selected from one or more of the amino acid regions WLLNRLINRLQNQGILLYYDIV, VAIF, MG A, MENHNQI, SEGPCLAFMRA, HAISSS, DQTKA, KGFHS,
  • TdT modified terminal deoxynucleotidyl transferase
  • Truncated proteins may include at least the region shown below (SEQ ID NO 2)
  • TdT modified terminal deoxynucleotidyl transferase
  • SEGPCLAFMRA HAISSS, DQTKA, KGFHS, QADNA, HFTKMQK, SAAVCK, EAQA, TVRLI, GKEC, TPEMGK, DHFQK, LAAG, APPVDNF, FARHERKMLLDNHALYDKTKK, and DYIDP of the sequence:
  • Sequence homology extends to all modified or wild-type members of family X polymerases, such as DNA RoIm (also known as DNA polymerase mu or POLM), DNA RoIb (also known as DNA polymerase beta or POLB), and DNA RoIl (also known as DNA polymerase lambda or POLL).
  • DNA RoIm also known as DNA polymerase mu or POLM
  • DNA RoIb also known as DNA polymerase beta or POLB
  • DNA RoIl also known known as DNA polymerase lambda or POLL.
  • TdT DNA polymerase mu
  • POLB DNA polymerase beta
  • DNA RoIl also known known as DNA polymerase lambda or POLL
  • family X polymerases can be trivially modified to either display template-dependent or template-independent nucleotidyl transferase activities. Therefore, all motifs, regions, and mutations demonstrated in this patent can be trivially extended to modified X family polymerases to enable modified X family polymerases to incorporate 3'-modified nucleotides, reversibly terminated nucleotides, and modified nucleotides in general to effect methods of nucleic acid synthesis.
  • Modifications which improve the solubility include a modification within the amino acid region WLLNRLINRLQNQGILLYYDIV shown highlighted in the sequence below.
  • Modifications which improve the incorporation of modified nucleotides can be at one or more of selected regions shown below.
  • the second modification can be selected from one or more of the amino acid regions VAIF, EDN, MG A, ENHNQ, FMRA, HAI, TKA, FHS, QADNA, MQK, SAAVCK, EAQA, TVR, KEC, TPEMGK, DHFQ, LAAG, APPVDN, FARHERKMLLDNHA, and YIDP shown highlighted in the sequence below.
  • a modified terminal deoxynucleotidyl transferase (TdT) enzyme comprising at least one amino acid modification when compared to a wild type sequence or the homologous amino acid sequence of a terminal deoxynucleotidyl transferase (TdT) enzyme in other species, wherein the modification is selected from one or more of the amino acid regions WLLNRLINRLQNQGILLYYDI, VAIF, EDN, MG A, ENHNQ, FMRA, HAI, TKA, FHS, QADNA, MQK, SAAVCK, EAQA, TVR, KEC, TPEMGK, DHFQ LAAG, APPVDN, FARHERKMLLDNHA, and YIDP of the sequence of a wild type sequence or the homologous regions in other species.
  • Homologous refers to protein sequences between two or more proteins that possess a common evolutionary origin, including proteins from superfamilies in the same species of organism as well as homologous proteins from different species. Such proteins (and their encoding nucleic acids) have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or by the presence of specific residues or motifs and conserved positions.
  • a variety of protein (and their encoding nucleic acid) sequence alignment tools may be used to determine sequence homology. For example, the Clustal Omega multiple sequence alignment program provided by the European Molecular Biology Laboratory (EMBL) can be used to determine sequence homology or homologous regions.
  • EMBL European Molecular Biology Laboratory
  • a first modification is within the amino acid region WLLNRLINRLQNQGILLYYDI of the sequence of a wild type sequence or the homologous region in other species;
  • a second modification is selected from one or more of the amino acid regions VAIF, EDN,
  • MG A ENHNQ FMRA, HAI, TKA, FHS, QADNA, MQK, SAAVCK, EAQA, TVR, KEC, TPEMGK, DHFQ LAAG,
  • APPVDN, FARHERKMLLDNHA, and YIDP of the sequence of a wild type sequence or the homologous regions in other species The modification within the region WLLNRLINRLQNQGILLYYDIV or the corresponding region from other species help improve the solubility of the enzyme.
  • the modification within the amino acid region WLLNRLINRLQNQGILLYYDIV can be at one or more of the underlined amino acids.
  • W-Q N-P R-K, L-V, R-L, L-W, Q-E, N-K, Q-K or l-L.
  • the sequence WLLNRLINRLQNQGILLYYDIV can be altered to QLLPKVINLWEKKGLLLYYDLV.
  • the second modification improves incorporation of nucleotides having a modification at the 3' position in comparison to the wild type sequence.
  • the second modification can be selected from one or more of the amino acid regions VAIF, EDN, MG A, ENHNQ, FMRA, HAI, TKA, FHS, QADNA, MQK, SAAVCK, EAQA, TVR, KEC, TPEMGK, DHFQ, LAAG, APPVDN, FARHERKMLLDNHA, and YIDP of the sequence of a wild type sequence or the homologous regions in other species.
  • the second modification can be selected from two or more of the amino acid regions VAIF, EDN, MGA, ENHNQ, FMRA, HAI, TKA, FHS, QADNA, MQK, SAAVCK, EAQA, TVR, KEC, TPEMGK, DHFQ, LAAG, APPVDN, FARHERKMLLDNHA, and YIDP of the sequence of a wild type sequence or the homologous regions in other species shown highlighted in the sequence below.
  • the identified positions commence at positions V32, E74, M108, F182, T212, D271, M279, E298, A421, L456, Y486.
  • Modifications disclosed herein contain at least one modification at the defined positions.
  • the modified amino acid can be in the region FMRA.
  • the modified amino acid can be in the region QADNA.
  • the modified amino acid can be in the region EAQA.
  • the modified amino acid can be in the region APP.
  • the modified amino acid can be in the region LDNHA.
  • the modified amino acid can be in the region YIDP.
  • the region FARHERKMLLDNHA is advantageous for removing substrate biases in modifications.
  • the FARHERKMLLDNHA region appears highly conserved across species.
  • the modification selected from one or more of the amino acid regions FMRA, QADNA, EAQA, APP, FARHERKMLLDNHA, and YIDP can be at the underlined amino acid(s).
  • the positions for modification can include A53, V68, V71, D75, E97, 1101, G109, Q115, V116, S125, T137, Q143, N154, H155, Q157, 1158, 1165, G177, L180, A181, M183, A195, K200, T212, K213, A214,
  • Amino acid changes include any one of A53G, V68I, V71I, D75N, D75Q, E97A, 1101V, G109E, G109R, Q115E, V116I, V116S, S125R, T137A, Q143P, N154H, H155C, Q157K, Q157R, I158M, 1165V, G177D,
  • N352Q N360K, Q361K, I363L, Y367C, H389A, L403R, G406R, D411N, A421L, A421M, A421V, P422A, P422C, V424Y, N426R, R438K, F447W, R452K, L455I, and/or D488P.
  • Amino acid changes include any two or more of A53G, V68I, V71I, D75N, D75Q, E97A, 1101V, G109E, G109R, Q115E, V116I, V116S, S125R, T137A, Q143P, N154H, H155C, Q157K, Q157R, I158M, 1165V,
  • the modification of QADNA to KADKA, QADKA, KADNA, QADNS, KADNT, or QADNT is advantageous for the incorporation of 3'-0-modified nucleoside triphosphates to the 3'-end of nucleic acids and removing substrate biases during the incorporation of modified nucleoside triphosphates.
  • the modification of APPVDN to MCPVDN, MPPVDN, ACPVDR, VPPVDN, LPPVDR, ACPYDN, LCPVDN, or MAPVDN is advantageous for the incorporation of 3'-0-modified nucleoside triphosphates to the 3'- end of nucleic acids and removing substrate biases during the incorporation of modified nucleoside triphosphates.
  • FARHERKMLLDRHA WARHERKMILDNHA, FARHERKMILDNHA, WARHERKMLLDNHA, FARHERKMLLDRHA, or FARHEKKMLLDNHA is also advantageous for the incorporation of 3'-0-modified nucleoside triphosphates to the 3'-end of nucleic acids and removing substrate biases during the incorporation of modified nucleoside triphosphates.
  • the modification can be selected from one or more of the following sequences FRRA, QADKA, EADA, MPP, FARHERKMLLDRHA, and YIPP. Included is a modified terminal deoxynucleotidyl transferase (TdT) enzyme wherein the second modification is selected from two or more of the following sequences FRRA, QADKA, EADA, MPP, FARHERKMLLDRHA, and YIPP. Included is a modified terminal deoxynucleotidyl transferase (TdT) enzyme wherein the second modification contains each of the following sequences FRRA, QADKA, EADA, MPP, FARHERKMLLDRHA, and YIPP.
  • composition for monitoring the quality of template-independent nucleic acid synthesis comprising measuring the amount of incorporated nucleotide monomer on each reaction cycle comprising inorganic pyrophosphatase, a terminal deoxynucleotidyl transferase (TdT), a 3'-0- reversibly blocked dNTP and a fluorescently labelled phosphate binding protein.
  • composition for monitoring the quality of template-independent nucleic acid synthesis comprising measuring the amount of incorporated nucleotide monomer on each reaction cycle comprising; a terminal deoxynucleotidyl transferase (TdT), a 3'-0-reversibly blocked dNTP and a terpyridine-Zn(ll) complex.
  • TdT terminal deoxynucleotidyl transferase
  • a 3'-0-reversibly blocked dNTP and a terpyridine-Zn(ll) complex.
  • Example 1 Coupling of pyrophosphatase and phosphate-binding protein labelled to a fluorophore to monitor and quantitate reversibly terminated nucleotide incorporation by engineered TdTs.
  • PBP-MDCC (Thermo Fisher Scientific) was tested as a means to monitor non-templated enzymatic DNA synthesis.
  • PBP-MDCC (0.5 mM), engineered TdT (0.04 mg/ml), pyrophosphatase (0.01 mg/m!), dATP-ONH 2 (0.25 mM), fluorescently labelled oligonucleotide initiator (purchased from IDT; SEQ IDS 662 and 663 in Table 1; 5 mM) (Seq IDs 3 and 4), and required buffer components were mixed together and incubated at 37 °C for 10 minutes. Reactions were monitored at an excitation and emission wavelength of 430 and 460 nm, respectively, and measured 60 s after reaction initiation.
  • Phosphate mopping refers to incubation of solutions with purine nucleoside phosphorylase (Sigma-Aldrich; from microbial sources). PNPase was used at 1 unit/ml and 7-methylguanosine (MEG) was used at 200 mM final concentration (37 °C for 10 minutes).
  • “Spike Mop” in the figure above only contains buffer components and 20 mM phosphate, which were subsequently phosphate mopped.
  • "GA(ddC)” 1 and 2 contain oligonucleotide SEQ NO 662 from Table 1 whereas “GAC” 1 and 2 contains oligonucleotide SEQ NO 663 from Table 1.
  • GA(ddC) and GAC are fully constituted mixtures with all components necessary for reversibly terminated nucleotide addition.
  • No mop contains GA(ddC) reaction components, but no phosphate mop was performed previous to reaction initiation.
  • "Spike” contains only buffer components and 20 mM phosphate.
  • Example 2 Pyrophosphate detection as a method to monitor and quantitate reversibly terminated nucleotide incorporation by engineered TdTs.
  • Engineered TdT (only present if indicated; 0.04 mg/ml), pyrophosphatase (only present if indicated; 0.01 mg/ml), dTTP-ONH 2 (only present if indicated; 0.25 mM), immobilized oligonucleotide initiator (only present if indicated; 1 pmol), and required buffer components were mixed together and incubated at 37 °C for the indicated amount of time.
  • Reaction conditions were (1) buffer + pyrophosphate + immobilized oligonucleotide (0.1 mM); (2) buffer + pyrophosphate + immobilized oligonucleotide (0.5 mM); (3) buffer + pyrophosphate + immobilized oligonucleotide (1.0 mM); (4) buffer + engineered TdT + dTTP-ONH2 + immobilized oligonucleotide (0 min); (5) buffer + engineered TdT + dTTP-ONH 2 + immobilized oligonucleotide (10 min); (6) buffer + dTTP-ONH 2 + immobilized oligonucleotide (10 min); and (7) buffer + dTTP-ONH 2 + immobilized oligonucleotide + pyrophosphatase (10 min).
  • nucleotide incorporation over time can be monitored by the ATP sulfurylase - !uciferase bioluminescence regenerative cycle. If pyrophosphatase is included in the reaction mixture, then no luminescence is detected, confirming that the assay is specific for pyrophosphate.

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Abstract

L'invention concerne des procédés et des compositions pour assurer la qualité de la synthèse d'oligonucléotides pendant des cycles d'extension de transférase terminale indépendante de la molécule matrice. La quantité de nucléotides incorporés peut être déterminée pour chaque cycle d'extension, confirmant ainsi que le nucléotide fourni a été efficacement incorporé pour étendre le brin.
EP20728173.4A 2019-05-22 2020-05-22 Procédé de contrôle de qualité de synthèse d'oligonucléotides Withdrawn EP3973075A1 (fr)

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GBGB1907209.9A GB201907209D0 (en) 2019-05-22 2019-05-22 Method of quality control of oligonucleotide synthesis
PCT/GB2020/051250 WO2020234605A1 (fr) 2019-05-22 2020-05-22 Procédé de contrôle de qualité de synthèse d'oligonucléotides

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