Modified template-independent enzymes for polydeoxynucleotide synthesis
Inventors
Efcavitch, J. William • Tubbs, Julie L. • Sinha, Prem • Stec, Boguslaw
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-10774316-B2
Publication Date
2020-09-15
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 template-independent de novo oligonucleotide synthesis using a modified terminal deoxynucleotidyl transferase (TdT). The modified TdT contains mutations in a GGFRR amino acid motif and a TGSR amino acid motif, and is capable of adding a nucleotide analog bearing a removable 3′-O-blocking moiety to the 3′-OH of a nucleic acid initiator. This enables formation of predetermined oligonucleotide sequences without relying on a template.
A core problem addressed is that native TdT lacks activity for 3′-O-blocked substrates. The disclosure presents the rationale for modifying the TdT active-site motifs GGFRR and TGSR to overcome the lack of activity with 3′-O-blocked dNTPs. Computational docking and screening of TdT variants are described as supporting selection of modifications expected to improve incorporation of specific removable 3′-O-blocked dNTPs.
The disclosure further describes nucleotide analogs with removable 3′-O-blocking moieties and discusses blocking-group options and engineered residue substitutions/combinations for improved activity. The overall aim is aqueous-phase, lower-waste, cost-reduced synthesis, including controlled stepwise extension with a deblocking/recycling cycle on solid support to produce longer polynucleotides.
Claims Coverage
The independent claim is directed to a modified TdT with mutations in the GGFRR and TGSR amino acid motifs that can incorporate nucleotide analogs carrying a removable 3′-O-blocking moiety onto the 3′-OH of a nucleic acid initiator.
Motif-mutated TdT for 3′-O-blocked nucleotide incorporation
A modified terminal deoxynucleotidyl transferase (TdT) comprising a mutation in a GGFRR amino acid motif and a TGSR amino acid motif, said modified TdT capable of adding a nucleotide analog comprising a removable 3′-O-blocking moiety to the 3′-OH of a nucleic acid initiator.
Across the provided claim set, the inventive concept centers on altering the GGFRR and TGSR motifs in TdT to enable incorporation of nucleotide analogs that include a removable 3′-O-blocking moiety onto a nucleic acid initiator.
Stated Advantages
Enables template-independent de novo synthesis of predetermined oligonucleotide sequences.
Overcomes lack of activity of TdT with 3′-O-blocked substrates.
Supports aqueous-phase, lower-waste, cost-reduced synthesis.
Enables production of longer polynucleotides via controlled stepwise extension with a deblocking/recycling cycle on solid support.
Documented Applications
Template-independent de novo oligonucleotide synthesis of predetermined oligonucleotide sequences using a nucleic acid initiator and a modified TdT capable of incorporating removable 3′-O-blocked nucleotide analogs.
Solid support, stepwise extension with deblocking/recycling to produce longer polynucleotides.
Interested in licensing this patent?