Methods of nucleic acid synthesis comprising polyphosphate containing species
Inventors
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Assignees
Nucleara Nucleics Ltd • Nuclera Ltd
Member
NucleraNuclera develops automated benchtop platforms and integrated systems for rapid protein expression, optimization, and purification, utilizing cell-free synthesis, digital microfluidics, and software-driven workflows. Their technology enables miniaturized and scalable protein prototyping—including challenging targets such as membrane proteins—directly at the lab bench. Nuclera serves academic and industrial researchers, focusing on reducing turnaround time for functional protein access and streamlining screening and production. The company has secured significant funding to enable broad commercialization, expanded their leadership team to support scale-up, and continues to drive advancements in drug discovery, proteomics, and experimental automation.
Nuclera develops automated benchtop platforms and integrated systems for rapid protein expression, optimization, and purification, utilizing cell-free synthesis, digital microfluidics, and software-driven workflows. Their technology enables miniaturized and scalable protein prototyping—including challenging targets such as membrane proteins—directly at the lab bench. Nuclera serves academic and industrial researchers, focusing on reducing turnaround time for functional protein access and streamlining screening and production. The company has secured significant funding to enable broad commercialization, expanded their leadership team to support scale-up, and continues to drive advancements in drug discovery, proteomics, and experimental automation.
Publication Number
US-11623940-B2
Publication Date
2023-04-11
Expiration Date
Abstract
The invention relates to the use of polyphosphate containing species in a method of nucleic acid synthesis, to methods of nucleic acid synthesis, and to the use of kits comprising said polyphosphate containing species. The invention also relates to the use of polyphosphate containing species for the capping of 3′-terminal hydroxyl moieties using terminal transferases.
Core Innovation
The invention relates to the use of polyphosphate containing species in a method of nucleic acid synthesis, to methods of nucleic acid synthesis, and to the use of kits comprising said polyphosphate containing species. The invention also relates to the use of polyphosphate containing species for the capping of 3′-terminal hydroxyl moieties using terminal transferases.
The background identifies a limitation of current DNA synthesis technology in that it is practically impossible to synthesise DNA strands greater than 120-200 nucleotides in acceptable yields using phosphoramidite chemistry, and that one error type in synthesis is addition failure which leads to deletions and contaminating products that are challenging to separate. The background further states that chemical capping approaches used in phosphoramidite synthesis are less suitable for enzyme mediated DNA synthesis because the capping target is the 3′-hydroxyl and enzymatic methods are performed in aqueous solution, and concludes there is a need to identify novel methods for the capping of 3′-terminal hydroxyl groups to enable error correction strategies in de novo enzymatic DNA synthesis.
The invention provides a novel capping method for aqueous de novo enzymatic nucleic acid synthesis in which polyphosphate containing species are used as permanent capping agents to block the terminal 3′-hydroxyl (for example by 3′-phosphorylation or formation of a phosphodiester bond to a capping species) so that sequences that suffer addition failure are rendered incompatible with further addition. The patent describes that some terminal transferases are able to incorporate polyphosphate containing species, such as tripolyphosphate (for example sodium tripolyphosphate), providing an enzymatic route for 3′-phosphorylation of DNA and RNA oligonucleotides and enabling the capping approach in enzymatic nucleic acid synthesis.
Claims Coverage
Independent claim: one independent method claim reciting a nucleic acid synthesis workflow composed of provision of an initiator sequence, addition of a 3′-reversibly blocked nucleoside triphosphate in the presence of a terminal transferase, addition of a capping polyphosphate containing species, and subsequent reagent removal and cleavage steps.
Addition of reversibly blocked nucleoside triphosphates by terminal transferase
Adding a 3′-reversibly blocked nucleoside triphosphate to an initiator sequence in the presence of a terminal transferase or a functional equivalent or fragment thereof.
Capping with a polyphosphate containing species
Adding a capping polyphosphate containing species to cap any remaining initiator sequence, wherein the polyphosphate containing species functions as a permanent capping agent.
Reagent removal and blocking-group cleavage cycle
Removal of capping reagents from the initiator sequence, cleaving the blocking group from the added 3′-reversibly blocked nucleoside in the presence of a cleaving agent, and removal of the cleaving agent to permit iterative repetition of the cycle.
The independent method claim centers on (1) enzymatic addition of 3′-reversibly blocked nucleoside triphosphates by a terminal transferase, (2) enzymatic capping of non-extended initiator sequences with polyphosphate containing species as a permanent cap, and (3) cycles of reagent removal and cleavage to enable iterative nucleic acid synthesis.
Stated Advantages
Capping addition-failure strands prevents those sequences from participating in further addition cycles, thereby reducing the prevalence of erroneous (near full-length) products.
Reducing the prevalence of errors in a product pool has significant industrial value because errors reduce the efficacy of DNA products in downstream applications and increase costs.
The method can use the same terminal transferase for both addition and capping, providing the advantage of not requiring the use of multiple enzymes.
3′-phosphorylation conveys 3′-exonuclease resistance and typically blocks elongation by a polymerase or ligation by a ligase, providing additional utility beyond use as a cap.
The enzymatic approach described avoids the need to use strong organic solvents used in chemical synthesis methods.
Documented Applications
Use of polyphosphate containing species as a permanent capping agent in methods of nucleic acid synthesis and in kits for nucleic acid synthesis.
Enzymatic capping of 3′-terminal hydroxyl moieties using terminal transferases to prevent further addition during de novo enzymatic DNA or RNA synthesis.
Use of the method in template independent nucleic acid synthesis to add defined sequences and to introduce adapter sequences to a nucleic acid library (library preparation for next-generation sequencing).
Use of tripolyphosphate (for example sodium tripolyphosphate) and a terminal transferase to phosphorylate the 3′-end of a nucleic acid species.
Use of 3′-phosphorylated species to block fluorogenic DNA/RNA primers from being extended and to provide greater control over ligation-mediated nucleic acid assembly (directional ligation).
Provision of kits comprising a terminal transferase and a polyphosphate containing species, optionally together with initiator sequences, 3′-reversibly blocked nucleoside triphosphates, inorganic pyrophosphatase, a cleaving agent, and instructions for use.
Execution of the method in various formats including flow instruments, plate or microarray setups, printing methods (for example inkjet), and digital microfluidic devices.
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