Nucleotide triphosphate immobilised on a support and their use in nucleic acid synthesis
Inventors
Chen, Michael • Huang, Jiahao • Lazar, Radu • McInroy, Gordon
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Assignees
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-11466301-B2
Publication Date
2022-10-11
Expiration Date
Abstract
The invention relates to methods of synthesizing polymers, biopolymers, and nucleic acids, to immobilised dNTP/NTPs and kits comprising said immobilised dNTP/NTPs for use in said methods of nucleic acid synthesis.
Core Innovation
The invention relates to methods of synthesizing polymers, biopolymers, and nucleic acids, to immobilised dNTP/NTPs and kits comprising said immobilised dNTP/NTPs for use in said methods of nucleic acid synthesis. The invention provides an alternating-phase polymer synthesis method involving provision of a monomer immobilised to a support moiety via a cleavable linker, provision of a polymer of length (N), coupling the polymer to the immobilised monomer to create an immobilised, coupled polymer of length (N+1), removal of any uncoupled polymers, and cleaving the immobilised, coupled polymer of length (N+1) from the support moiety. [procedural detail omitted for safety]
The background identifies a problem with current solid-phase DNA synthesis, namely the retention of nascent molecules/biopolymers that have not successfully reacted which gives rise to heterogeneous product pools containing X-1, X-2, . . . species. Existing approaches such as exposing nascent strands to a large excess of reagent or employing a capping step have not eliminated heterogeneous product pools, and there is a stated need to provide improved methods of nucleic acid synthesis that overcome these problems.
The invention further provides specific nucleic acid synthesis aspects including methods using deoxynucleotide triphosphates (dNTPs) or nucleotide triphosphates (NTPs) immobilised to a support moiety via a cleavable linker, embodiments that include an immobilised trap strand complementary to an initiator sequence, embodiments with mobile-phase immobilised dNTP/NTPs and solid-phase immobilised initiator sequences, immobilised dNTP/NTP compositions of formula (I), and nucleic acid synthesis kits comprising immobilised dNTP/NTP, an initiator nucleic acid sequence, a nucleic acid synthesis catalyst and instructions. [procedural detail omitted for safety]
Claims Coverage
Independent claim identified: 1 independent inventive feature set extracted into 5 main inventive features.
Monomer immobilised to a support via a cleavable linker
Providing a monomer immobilised to a support moiety via a cleavable linker, wherein the monomer is a deoxynucleotide triphosphate (dNTP), or a nucleotide triphosphate (NTP).
Initiator nucleic acid sequence of defined length (N)
Providing a polymer having an initiator nucleic acid sequence of length (N).
Catalyst-coupled formation of immobilised N+1 species
Providing a nucleic acid synthesis catalyst to couple the initiator nucleic acid sequence to the immobilised monomer to create an immobilised, coupled polymer of length (N+1).
Removal of uncoupled polymers
Removing any uncoupled polymers (for example, by a washing step as described in dependent claims).
Cleavage of immobilised N+1 from the support moiety
Cleaving the immobilised, coupled polymer of length (N+1) from the support moiety.
The independent claim centers on an alternating-phase polymer (nucleic acid) synthesis workflow that combines (1) monomers immobilised as dNTP/NTPs via cleavable linkers, (2) provision of an initiator nucleic acid sequence of length N, (3) catalyst-mediated coupling to form an immobilised N+1 species, (4) removal of uncoupled polymers, and (5) cleavage of the immobilised N+1 product from the support moiety.
Stated Advantages
Error correction by eliminating X-1, X-2, etc. species because strands which fail the N to N+1 conversion are not immobilised and are washed away at the point of their occurrence.
Controlled exposure to reagent: immobilising the reagent prevents unintended additional reactions of the nascent biopolymer and enables controlled addition of one nucleotide per cycle.
Avoiding polymerase steric gate clashes: immobilisation via the nitrogenous base offers industrial utility because polymerases like TdT accept nitrogenous base modifications more readily than 2′/3′ sugar modifications.
Documented Applications
Methods of synthesizing polymers, biopolymers, and nucleic acids using immobilised dNTP/NTPs.
De novo nucleic acid synthesis workflows employing immobilised dNTP/NTPs, including embodiments with trap strands, mobile-phase immobilised dNTP/NTPs, and solid-phase immobilised initiator sequences.
Use of an immobilised dNTP/NTP in a nucleic acid synthesis method and nucleic acid synthesis kits comprising an immobilised dNTP/NTP, an initiator nucleic acid sequence, a nucleic acid synthesis catalyst, and instructions to use the kit in accordance with the described methods.
Implementation on devices including a microfluidic device, electrowetting-on-dielectric device, an inkjet device, and/or a column-based device.
Subsequent nucleic acid assembly applications including pooling of oligos and ligation-based assembly of longer nucleic acid fragments.
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