Modified template-independent enzymes for polydeoxynucleotide synthesis
Inventors
Efcavitch, J. William • Tubbs, Julie L. • Sinha, Prem • Stec, Boguslaw
Assignees
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Publication Number
US-10760063-B2
Publication Date
2020-09-01
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 document describes identification and use of modified, template-independent terminal deoxynucleotidyl transferase (TdT) and related polymerases that can incorporate nucleotide analogs bearing removable 3′-O-blocking moieties. The modified enzymes add a nucleotide analog to the 3′-OH of a nucleic acid initiator without a nucleic acid template.
The described approach uses an enzymatic stepwise synthesis cycle in which 3′-O-blocked dNTPs are incorporated onto the nucleic acid initiator, followed by cleavage/deblocking of the 3′-O group, and repetition of the cycle. TdT is selected because it is available with active-site/structural information, and computational docking is used to explain reduced activity with 3′-O-blocked dNTPs by targeting residues and motifs and their effects on electrostatics.
The document reports docking analysis involving motifs such as GGFRR and TGSR and residues including Arg336/Arg454 and related positions, and it describes screening TdT variants to increase incorporation of 3′-O-blocked analogs. The independent claim specifies modified TdT containing mutations selected from a defined set, and dependent claims further refine mutation combinations as well as performance constraints.
Claims Coverage
The claims are centered on one independent claim (clm-00001) that defines a modified TdT by a specified set of amino-acid mutations and requires template-independent addition of a nucleotide analog with a removable 3′-O-blocking moiety. Dependent claims refine the invention by selecting specific mutation combinations and by adding functional or constraint-based limitations.
Modified TdT for template-independent 3′-O-blocking moiety addition
A modified terminal deoxynucleotidyl transferase (TdT) comprising a mutation selected from the group consisting of E180D and W450H, M192E, E180K, E180K and R454A, R454K, M192K, M192K and E180K, Q455I, M192W, E180R, E180L, M192R, N474R, R461V, E180K and R454I, E180D and M192E, E180D and M192E and R454T, and E180K and W450H, 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 in the absence of a nucleic acid template.
Specific multi-site mutation combinations
The modified TdT includes specified mutation combinations selected among examples such as E180D and W450H and E180D and M192E.
Increased incorporation rate of modified nucleotides
The modified TdT has a higher incorporation rate of modified nucleotides than native TdT.
Temperature range for modified-nucleotide incorporation
The modified TdT incorporates modified nucleotides at reaction temperatures between 30°C and 80°C.
Nucleic-acid-sequence identity constraint
The modified TdT is defined to include a nucleic acid sequence at least 90% identical to one of SEQ ID NOS. 2, 4, 6, 8, or 9.
Overall, the claim coverage is directed to a modified TdT enzyme defined by selected mutation sets, with template-independent incorporation of nucleotide analogs bearing removable 3′-O-blocking moieties onto a nucleic acid initiator. The dependent claims further specify mutation combinations and add constraints relating to incorporation performance, reaction temperature, and sequence identity to specified SEQ ID NOS.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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