Compositions and methods related to non-templated enzymatic nucleic acid synthesis
Inventors
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Assignees
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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-11505815-B2
Publication Date
2022-11-22
Expiration Date
Abstract
The invention relates to the use of an amine masked moiety in a method of enzymatic nucleic acid synthesis. The invention also relates to said amine masked moieties per se and a process for preparing nucleotide triphosphates comprising said amine masked moieties.
Core Innovation
The invention relates to the use of an amine masked moiety in a method of enzymatic nucleic acid synthesis and also relates to said amine masked moieties per se and a process for preparing nucleotide triphosphates comprising said amine masked moieties. According to the invention, there is provided the use of an amine masked derivative of a nitrogenous heterocycle, such as adenine, guanine, cytosine, isoguanine, isocytosine and 2,6-diaminopurine, in a method of enzymatic nucleic acid synthesis, and there is provided the use of a compound of formula (I) in a method of enzymatic nucleic acid synthesis.
The background identifies a problem that current DNA synthesis technology does not meet the demands of the biotechnology industry because it is practically impossible to synthesise a DNA strand greater than 200 nucleotides in length by phosphoramidite chemistry, and that existing reversible terminator chemistry using a —ONH2 moiety requires deprotection conditions (for example sodium nitrite under acidic conditions) that lead to oxidative deamination of adenine, guanine and cytosine, producing mutations and limiting the practical utility of the —ONH2 reversible terminator, particularly in non-templated enzymatic DNA synthesis.
The detailed description and summary state that the invention provides compounds and methods in which an amine masking group (R3) on the nitrogenous heterocycle and a moiety at the 3′-position (R1) and an X group endowing competence for enzymatic addition together produce nucleotide analogs that protect the amino group from mutation during enzymatic nucleic acid synthesis, and that certain amine masking groups (for example azide, acetyl, benzoyl, benzyl, methyl, anisoyl, allyl and others) are exemplified and can be unmasked by appropriate means to reveal the amino group.
Claims Coverage
The independent claims disclose two inventive features corresponding to (A) a method of non-templated enzymatic nucleic acid synthesis involving a compound of formula (I), and (B) a compound of formula (I)a.
Method of non-templated enzymatic nucleic acid synthesis using a compound of formula (I)
A method of non-templated enzymatic nucleic acid synthesis comprising providing a compound of formula (I).
Compound of formula (I)a
A compound of formula (I)a as recited in the claims, wherein R1, R2, X, R3 and B are defined as in the specification.
The independent claims cover (1) a method of non-templated enzymatic nucleic acid synthesis comprising providing a compound of formula (I) and (2) a compound of formula (I)a with defined R1, R2, X, R3 and B substituents.
Stated Advantages
Provides a solution to the problem of oxidative deamination of adenine, guanine and cytosine in the presence of reagents such as sodium nitrite under acidic conditions.
Offers utility in a method of enzymatic DNA synthesis by protecting amino groups on nitrogenous heterocycles during enzymatic nucleic acid synthesis.
Documented Applications
Reversibly terminated enzymatic nucleic acid synthesis.
Non-templated de novo enzymatic nucleic acid synthesis, for example utilizing a PoIX family polymerase such as terminal deoxynucleotidyl transferase.
Templated nucleic acid synthesis, including sequencing-by-synthesis.
Preparation and use of nucleotide triphosphates comprising amine masked moieties in enzymatic nucleic acid synthesis.
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