Modified template-independent enzymes for polydeoxynucleotide synthesis
Inventors
Tubbs, Julie L. • Sinha, Prem • Stec, Boguslaw • Wilson, Christopher • Efcavitch, J. William • Sammond, Deanne W.
Assignees
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Publication Number
US-11390858-B2
Publication Date
2022-07-19
Expiration Date
Abstract
The invention includes methods for identifying polymerases, such as modified terminal nucleotidyl transferases (TdT), that are capable of binding nucleotides comprising removable 3′-O-blocking moieties to a nucleic acid initiator, without the use of a template. The invention further includes the identified polymerases, and methods of using the polymerases for de novo synthesis of predetermined oligonucleotide sequences.
Core Innovation
The invention relates to a template-independent, modified terminal deoxynucleotidyl transferase (TdT) that adds nucleotide analogs to a nucleic acid initiator in the absence of a nucleic acid template. The nucleotide analogs comprise removable blocking moieties at the 3′-Oxygen of the analog and are added to a 3′-OH of the nucleic acid initiator.
The background addresses that native TdT cannot incorporate 3′-O-blocked dNTPs, which prevents incorporation of these analogs during template-free synthesis. Gel and electrophoresis evidence states that commercial TdT does not extend with 3′-O-blocked analogs, while engineered TdT variants can incorporate 3′-O-blocked analogs.
To create the modified TdT variants, the disclosed strategy combines computational modeling with mutagenesis and screening. Active-site docking and computational modeling guide selection of mutations, followed by saturation mutagenesis and screening to generate TdT variants with improved incorporation of 3′-O-blocked analogs.
The invention further includes a removable 3′-O blocking strategy to enable iterative cycles for longer polynucleotides. 3′-O blocking can be removed enzymatically, notably using Shrimp Alkaline Phosphatase (SAP), to enable cycles with additional nucleotide addition.
Claims Coverage
The provided independent claim covers one modified TdT with one or more specific mutations, and it requires template-independent addition of a removable 3′-O-blocking-bearing nucleotide analog to a 3′-OH of a nucleic acid initiator.
Mutation-defined modified TdT for template-free 3′-O-blocked analog addition
A modified terminal deoxynucleotidyl transferase (TdT) comprising one or more mutations selected from E33K, E180L, E180K, M192E, M192K, M192W, W303H, L381K, L381Q, L381R, L381V, W450H, R454I, R454T, R454K, E457K, R461V, R461Q, N474R, and N474K, capable of adding a nucleotide analog comprising a removable blocking moiety at a 3′-Oxygen of the analog to a 3′-OH of a nucleic acid initiator in the absence of a nucleic acid template.
The claim coverage centers on mutation-defined modified TdT that performs template-independent transfer of nucleotide analogs bearing removable 3′-O blocking moieties to a nucleic acid initiator 3′-OH.
Stated Advantages
Enables template-independent addition of nucleotide analogs comprising a removable blocking moiety at the 3′-Oxygen to a nucleic acid initiator 3′-OH.
Engineered TdT variants can incorporate 3′-O-blocked nucleotide analogs where native/commercial TdT cannot extend with such analogs.
Supports stepwise de novo oligonucleotide synthesis and iterative cycles for longer polynucleotides by using removable 3′-O blocking that can be removed enzymatically.
Allows aqueous, reduced-waste synthesis of longer polynucleotides.
Provides increased rate compared to native TdT for addition or processing of the 3′-O-blocked analogs.
Documented Applications
Stepwise template-independent de novo oligonucleotide synthesis using nucleotide analogs with removable 3′-O blocking moieties, including iterative cycles to produce longer polynucleotides.
Enzymatic removal of 3′-O blocking moieties, notably using Shrimp Alkaline Phosphatase (SAP), to support iterative addition cycles for longer polynucleotides.
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