Polymer synthesis using electrowetting devices
Inventors
Chen, Michael • Huang, Jiahao • Lazar, Radu • McInroy, Gordon
Assignees
Publication Number
US-12129501-B2
Publication Date
2024-10-29
Expiration Date
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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 provides alternating-phase polymer synthesis methods comprising providing a monomer immobilised to a support moiety via a cleavable linker, providing a polymer of length (N), providing components to couple the polymer to the immobilised monomer to create an immobilised, coupled polymer of length (N+1), removing any uncoupled polymers, and cleaving the immobilised, coupled polymer of length (N+1) from the support moiety. The invention further provides nucleic acid synthesis methods comprising providing a deoxynucleotide triphosphate (dNTP) or nucleotide triphosphate (NTP) immobilised to a support moiety via a cleavable linker, providing an initiator nucleic acid sequence of length (N), adding a nucleic acid synthesis catalyst to couple the initiator sequence to the immobilised dNTP/NTP to create an immobilised, coupled sequence of length (N+1), removing any uncoupled initiator nucleic acid sequences, and cleaving the immobilised, coupled sequence of length (N+1) from the support moiety.
Aspects include process variants that incorporate a nucleic acid trap strand immobilised to the same support moiety as the dNTP/NTP, variants in which the dNTP/NTP is immobilised to a mobile phase support moiety while the initiator is immobilised to a solid phase support moiety, an immobilised dNTP/NTP of formula (I) in which a cleavable linker connects the nucleotide to a support moiety, the use of immobilised dNTP/NTPs in nucleic acid synthesis methods, and nucleic acid synthesis kits comprising immobilised dNTP/NTPs, initiator nucleic acid sequences, nucleic acid synthesis catalysts and instructions. The methods may be implemented using devices including microfluidic devices, electrowetting-on-dielectric devices, inkjet devices and column-based devices.
The problem addressed is that current solid-phase DNA synthesis retains nascent molecules that have not successfully reacted, producing heterogeneous product pools containing X−1, X−2, . . . species which are undesirable and challenging to remove during purification; existing mitigations such as exposure to a large excess of reagent or the use of a capping step do not fully remove this heterogeneity. The invention aims to provide improved methods of nucleic acid synthesis that overcome the problems associated with currently available methods.
Claims Coverage
The independent claim defines one inventive feature directed to droplet-based electrowetting polymer synthesis with immobilised monomers and cleavable linkers.
Droplet-based electrowetting polymer synthesis
A method for polymer synthesis in a droplet on an electrowetting-on-dielectric device comprising coupling an immobilised monomer to a polymer of length (N) to provide a polymer of length (N+1), wherein the method comprises actuating a droplet to move the droplet by electrowetting, and wherein the polymer is immobilised to a support moiety via a cleavable linker.
The claims are directed to a droplet-actuated electrowetting method for N to N+1 polymer coupling where monomers or polymers are immobilised to a support moiety via a cleavable linker and droplet movement is effected by electrowetting.
Stated Advantages
Error correction by eliminating X−1, X−2, etc., species because strands that fail N to N+1 conversion are not immobilised and are washed away, avoiding contaminating shorter species.
Controlled exposure to reagent by immobilising the nucleotide triphosphate so that unintended additional reactions and insertions are precluded, enabling controlled addition of one nucleotide per cycle.
Avoiding polymerase steric gate clashes by immobilising nucleotide triphosphates via the base, which allows polymerases such as TdT to accept base modifications more readily than modifications at the sugar 2′ or 3′ positions.
Documented Applications
Synthesis of polymers, biopolymers and nucleic acids using alternating-phase methods with immobilised monomers or immobilised dNTPs/NTPs.
Enzymatic de novo nucleic acid synthesis using immobilised dNTP/NTPs and nucleic acid synthesis catalysts (e.g., TdT, poly(U) polymerase, poly(A) polymerase, PolQ).
Process variants for nucleic acid synthesis including trap-strand-mediated alternating-phase synthesis, mobile-phase dNTP/NTP with solid-phase initiator variants, and subsequent nucleic acid assembly of synthesized oligonucleotides into longer fragments.
Use in devices and platforms including microfluidic devices, electrowetting-on-dielectric (EWOD) devices, inkjet devices and column-based devices, including droplet movement schemes that carry initiator strands over defined tracks of immobilised dNTPs/NTPs.
Nucleic acid synthesis kits comprising immobilised dNTP/NTPs, initiator nucleic acid sequences, nucleic acid synthesis catalysts and instructions to use said kit in accordance with the disclosed methods.
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