Variants of terminal deoxynucleotidyl transferase and uses thereof
Inventors
Champion, Elise • SOSKINE, Mikhael • Ybert, Thomas • Delarue, Marc
Assignees
Centre National de la Recherche Scientifique CNRS • DNA Script SAS • Institut Pasteur
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Publication Number
US-12071638-B2
Publication Date
2024-08-27
Expiration Date
Abstract
The present invention relates to variants of Terminal deoxynucleotidyl Transferase (TdT), each of which (i) has an amino acid sequence similarity to SEQ ID NO: 2. 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35 with corresponding amino acid substitutions, (ii) is capable of synthesizing a nucleic acid fragment without a template and (iii) is capable of incorporating a modified nucleotide into the nucleic acid fragment.
Core Innovation
The invention relates to terminal deoxynucleotidyl transferase (TdT) variants defined by amino acid sequences that are at least 90% identical to specified SEQ ID NO sequences, together with an amino acid substitution of cysteine at position 173, including functionally equivalent residues. A BRTC-like domain is removed as part of defining the variant set centered on SEQ ID NO:2 and identity-based equivalents. The variants are capable of synthesizing a nucleic acid fragment without a template and of incorporating a 3′-O-modified nucleotide into the nucleic acid fragment.
The disclosure emphasizes improved incorporation of 3′-O-modified nucleotides relative to wild type TdT, including incorporation at a rate greater than wild type TdT. The document further expands to nucleic acids encoding the TdT variants, expression cassettes, vectors, and host cells for producing the variants, and to modified nucleotide chemistries usable in nucleic acid synthesis cycles. Example modified nucleotide chemistries include 3′-O-amino, azidomethyl, allyl, propargyl, and 3′-(2-nitrobenzyl) nucleoside triphosphates.
The description also includes 3′-O-blocked nucleoside triphosphates and controlled cycles of addition and potentially deprotection/repeat for sequencing-style synthesis. Comparative results are reported showing greater than 10-fold activity increases and improved elongation versus wild-type or prior variants, with efficiency increases relative to wild type TdT in the range of 110% to 200%.
Claims Coverage
The independent claim defines a TdT variant with four inventive features: at least 90% sequence identity to specified SEQ ID NO sequences, a cysteine substitution at position 173, template-independent nucleic acid fragment synthesis, and incorporation of a 3′-O-modified nucleotide at a rate greater than wild type TdT. Dependent claims further add specific identity tightening, specify eligible 3′-O-modified nucleotide chemistries, define the incorporation target as a free 3′-hydroxyl, and specify additional amino-acid substitutions at defined positions.
Template-independent synthesis with improved 3′-O-modified nucleotide incorporation
A terminal deoxynucleotidyl transferase (TdT) variant comprising an amino acid sequence at least 90% identical to SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:29, or SEQ ID NO:31 with an amino acid substitution of cysteine at position 173, wherein the TdT variant is capable of synthesizing a nucleic acid fragment without a template and is capable of incorporating a 3′-O-modified nucleotide into the nucleic acid fragment, and wherein the TdT variant incorporates the 3′-O-modified nucleotide at a rate greater than that of a wild type TdT.
Specified 3′-O-modified nucleotide chemistries
The TdT variant incorporates a 3′-O-modified nucleotide that is one of the named 3′-O-modified nucleoside triphosphates.
Incorporation onto a free 3′-hydroxyl of a nucleic acid fragment
The TdT variant incorporates a modified nucleotide onto a free 3′-hydroxyl of a nucleic acid fragment.
Tightened sequence identity for the TdT variant
The TdT variant has an amino acid sequence that is at least 95% identical to one of the specified SEQ ID sequences.
Additional amino-acid substitution at a defined residue position
The TdT variant includes a substitution of arginine at position 328 relative to SEQ ID NO:29.
Overall, the claim set is centered on a TdT variant defined by sequence identity to specific SEQ ID NO sequences plus a cysteine substitution at position 173 that enables template-free nucleic acid fragment synthesis and faster incorporation of 3′-O-modified nucleoside triphosphates than wild type TdT. The dependent claims narrow eligible 3′-O-modified nucleotide chemistries, define the incorporation target, tighten sequence identity thresholds, and specify additional residue substitutions.
Stated Advantages
Improved incorporation of 3′-O-modified nucleotide compared to wild type TdT (incorporation at a rate greater than wild type TdT).
Template-independent synthesis of nucleic acid fragments.
Increased affinity/activity for modified nucleotides.
Quantitative incorporation of 3′-O-modified nucleoside triphosphates.
Greater than 10-fold activity increases versus wild-type or prior variants.
Improved elongation versus wild-type or prior variants.
Efficiency increases relative to wild type TdT are described as 110% to 200%.
Documented Applications
Encoding sequences, expression vectors, and host cells are described in association with the TdT variants and modified nucleotide incorporation.
Processes for template-free synthesis via successive addition of 3′-O-modified nucleotides to nucleic acid fragments are described.
Kits for nucleotide incorporation reactions are described, associated with incorporation of modified nucleotides by the TdT variants.
Nucleic acid synthesis cycles for sequencing-style synthesis, using modified nucleotides with controlled addition and potentially deprotection/repeat cycles.
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