Methods for seamless nucleic acid assembly
Inventors
NUGENT, Rebecca • Chen, Siyuan • LEE, Elian • RAYNARD, Nathan
Assignees
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Publication Number
US-12270028-B2
Publication Date
2025-04-08
Expiration Date
Abstract
Provided herein are methods, systems, and compositions for seamless nucleic acid assembly. Methods, systems, and compositions as provided herein provide for efficient assembly of nucleic acids without primer removal. Methods, systems, and compositions for seamless nucleic acid assembly comprise use of an endonuclease or exonuclease, optionally in conjunction with additional enzymes to assemble nucleic acids or polynucleotides.
Core Innovation
The invention relates to seamless nucleic acid assembly in which a plurality of polynucleotides are annealed in a processive predetermined order based on a complementary sequence between adjacent polynucleotides. The plurality of polynucleotides do not comprise a terminal region of sequence homology to another polynucleotide of the plurality of polynucleotides, addressing primer removal avoidance in the assembly workflow.
In one aspect, the assembly is performed by mixing the plurality of polynucleotides with a 3′ to 5′ exonuclease, a thermostable endonuclease, a high fidelity polymerase, and a thermostable ligase. The use of a high fidelity polymerase together with ligase supports formation of assembled nucleic acid products after annealing in the predetermined order defined by complementarity between adjacent polynucleotides.
A core focus of the described system is the use of endonucleases, including flap endonuclease, notably flap endonuclease 1, also referred to as exonuclease 1, XPG, Dna2, or GEN1, in combination with the 3′ to 5′ exonuclease, polymerase, and ligase to carry out seamless nucleic acid assembly. Mechanistic schematics are described for flap endonuclease-mediated assembly, including optional nucleic-acid bridge/adapter-driven universal primer binding, to enable efficient assembly while maintaining primer-avoidant operation.
Claims Coverage
The coverage is centered on an independent method claim with dependent claims that narrow or specify the inventive features by selecting specific exonuclease and endonuclease identities and adding defined operating constraints. Overall, the claims cover a seamless nucleic acid assembly approach based on providing polynucleotides lacking terminal sequence homology and assembling them in a processive predetermined order using a multi-enzyme mix.
Polynucleotides lacking terminal homology and processive predetermined order annealing
A method for nucleic acid assembly comprising providing a plurality of polynucleotides, wherein each of the polynucleotides do not comprise a terminal region of sequence homology to another polynucleotide of the plurality of polynucleotides; and annealing the plurality of polynucleotides in a processive predetermined order based on a complementary sequence between adjacent polynucleotides.
Multi-enzyme seamless assembly mix using 3′ to 5′ exonuclease, thermostable endonuclease, high fidelity polymerase, and thermostable ligase
Mixing the plurality of polynucleotides with a 3′ to 5′ exonuclease, a thermostable endonuclease, a high fidelity polymerase, and a thermostable ligase.
Exonuclease III as the 3′ to 5′ exonuclease
The 3′ to 5′ exonuclease is exonuclease III.
Flap endonuclease 1 / exonuclease 1 / XPG / Dna2 / GEN1 as the thermostable endonuclease
The thermostable endonuclease is flap endonuclease 1, exonuclease 1, XPG, Dna2, or GEN1.
Temperature range of about 30°C to about 60°C for step (b)
Step (b) occurs at a temperature of about 30°C to about 60°C.
Enzyme concentration ranges for the 3′ to 5′ exonuclease, thermostable endonuclease, high fidelity polymerase, and thermostable ligase
A characterized method in which concentrations of the 3′ to 5′ exonuclease, the thermostable endonuclease, the high fidelity polymerase, and the thermostable ligase fall within specified ranges.
Stoichiometric ratio between the thermostable endonuclease and the 3′ to 5′ exonuclease
The method is performed with a specified ratio of a thermostable endonuclease to a 3′ to 5′ exonuclease.
Thermostable endonuclease produces a 5′ overhang
Mixing a plurality of polynucleotides with a thermostable endonuclease to produce a 5′ overhang.
Across the independent and dependent claims provided, the inventive coverage combines polynucleotides without terminal sequence homology, annealing in a processive predetermined order based on complementary sequence between adjacent polynucleotides, and seamless assembly using a 3′ to 5′ exonuclease, a thermostable endonuclease, a high fidelity polymerase, and a thermostable ligase. Dependent claims further specify particular enzyme identities, along with additional constraints such as step temperature, enzyme concentration ranges, stoichiometric ratios, and production of a 5′ overhang.
Stated Advantages
Primer removal avoidance in the assembly workflow.
Efficient assembly while maintaining primer-avoidant operation.
Documented Applications
Seamless nucleic acid assembly.
Flap endonuclease-mediated assembly.
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