Modified template-independent DNA polymerase
Inventors
Magyar, Andrew P. • Cavanagh, Peter • Rogers, Miles • McFarland, Kirsty A. • Sprachman, Melissa M. • Billings-Siuti, Amanda Nicole • Raustad, Nicole E.
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Assignees
Charles Stark Draper Laboratory Inc
Member
DraperDraper is an independent nonprofit engineering innovation company with a legacy spanning over 90 years, dedicated to delivering transformative solutions for national security, prosperity, and global challenges. Renowned for its pioneering work in guidance, navigation, and control (GN&C) systems, Draper partners with government, industry, and academia to engineer advanced technologies in space, defense, biotechnology, and electronic systems. The company leverages multidisciplinary expertise, digital engineering, and a collaborative approach to provide field-ready prototypes, mission-critical systems, and innovative research. Draper’s mission is to ensure the nation's security and prosperity by delivering sustainable, cutting-edge solutions that address the toughest problems of today and tomorrow, while fostering an inclusive and diverse workforce. Draper also invests in the next generation of innovators through robust educational programs, including internships, co-ops, and the Draper Scholars Program, integrating academic research with real-world problem-solving.
Draper is an independent nonprofit engineering innovation company with a legacy spanning over 90 years, dedicated to delivering transformative solutions for national security, prosperity, and global challenges. Renowned for its pioneering work in guidance, navigation, and control (GN&C) systems, Draper partners with government, industry, and academia to engineer advanced technologies in space, defense, biotechnology, and electronic systems. The company leverages multidisciplinary expertise, digital engineering, and a collaborative approach to provide field-ready prototypes, mission-critical systems, and innovative research. Draper’s mission is to ensure the nation's security and prosperity by delivering sustainable, cutting-edge solutions that address the toughest problems of today and tomorrow, while fostering an inclusive and diverse workforce. Draper also invests in the next generation of innovators through robust educational programs, including internships, co-ops, and the Draper Scholars Program, integrating academic research with real-world problem-solving.
Publication Number
US-12227775-B2
Publication Date
2025-02-18
Expiration Date
Abstract
Described herein are genetically engineered template-independent DNA polymerases, specifically terminal deoxynucleotidyl transferases, and methods of using these polymerases to control DNA synthesis by adding a single nucleotide (mononucleotide) at a time to the 3′ end of a growing single-stranded DNA polynucleotide.
Core Innovation
The invention encompasses genetically engineered template-independent DNA polymerases, specifically terminal deoxynucleotidyl transferases (TdT), and methods of using these polymerases to control DNA synthesis by adding a single nucleotide (mononucleotide) at a time to the 3′ end of a growing single-stranded DNA polynucleotide. The present invention describes methodologies for engineering the TdT protein/enzyme in order to control the addition of nucleotides to a growing nucleotide strand, including modifications to the enzyme's amino acid sequence and the introduction of photoswitchable moieties such as azobenzene derivatives to enable photo control of the enzymatic polymerase activity.
The background identifies that wild-type TdT displays template-independent activity but adds nucleotides in an uncontrolled manner and therefore has not yet been successfully used for the synthesis of sequence-specific DNA. The invention addresses this problem by providing engineered TdT variants and compositions that regulate or gate nucleotide entry, translocation/ratcheting, or catalytic progression—for example via bifunctional azobenzene derivatives attached through non-naturally occurring amino acids—to enable controllable, single-nucleotide addition and thereby enable synthesis of sequence-specified DNA, RNA, or other nucleic acid molecules.
Claims Coverage
Three independent claims are identified (claims 1, 21, and 27). The main inventive features below are extracted from those independent claims.
Genetically-engineered TdT incorporating a non-naturally occurring amino acid
A genetically-engineered terminal deoxynucleotidyl transferase (TdT), wherein a wild-type TdT has been mutated at a single amino acid residue site to incorporate an azide or cyclooctene non-naturally occurring amino acid selected from the group consisting of: 4-Azido-L-phenylalanine (AzF), N-Propargyl-Lysine (PrK), Cyclooctene-L-Lysine (TCO-K) or Cyclooctyne-Lysine (SCO-K).
Bifunctional azobenzene derivative with two orthogonal functional domains
The non-naturally occurring amino acid is modified with a bifunctional azobenzene derivative comprising two orthogonal functional domains, wherein the first functional domain comprises a click reactive group for attachment to an affinity tag peptide for purification, and the second functional domain comprises a click reactive group whereby the bifunctional azobenzene derivative is attached to the non-naturally occurring amino acid.
TdT modified for reversible conformational control of mononucleotide addition
Resulting in a TdT modified with the bifunctional azobenzene derivative capable of a reversible conformational change for controlled addition of a mononucleotide to the 3′ end of a single-stranded polynucleotide.
Immobilized TdT method for template-independent polynucleotide synthesis
A method of template-independent polynucleotide synthesis comprising contacting mononucleotide with the genetically-engineered TdT of claim 1, wherein the genetically-engineered TdT is immobilized on a solid support and is capable of attaching the mononucleotide to the 3′ end of a single-stranded polynucleotide under conditions suitable for the incorporation of a mononucleotide to the 3′ end of the single-stranded polynucleotide.
Kit comprising genetically-engineered TdT and synthesis reagents
A kit comprising reagents for template-independent polynucleotide synthesis and the genetically-engineered TdT of claim 1, wherein the genetically-engineered TdT is capable of attaching a mononucleotide to the 3′ end of a single-stranded polynucleotide under conditions suitable for the incorporation of a mononucleotide to the 3′ end of the single-stranded polynucleotide.
The independent claims define (1) a genetically-engineered TdT bearing a site-specific non-naturally occurring amino acid modified with a bifunctional azobenzene derivative that provides reversible conformational control for single-nucleotide addition, (2) a method using such an engineered TdT immobilized for template-independent polynucleotide synthesis, and (3) a kit comprising the engineered TdT and reagents for template-independent polynucleotide synthesis.
Stated Advantages
Enables controllable synthesis of DNA of a desired sequence by controlling the addition of nucleotides to a growing nucleotide strand.
Provides control of the enzyme for the synthesis of sequence-specified DNA, RNA, or other nucleic acid molecules.
Enables photo control of enzymatic polymerase activity (e.g., use of light to control DNA synthesis) via photoswitchable moieties such as azobenzene derivatives.
Provides an improved, accurate and cost-effective method of in vitro polynucleotide synthesis and enables new DNA synthesis strategies not previously possible.
Documented Applications
Poly A tailing of DNA (explicitly stated as an existing in vitro application of TdT).
In vitro DNA synthesis using TdT, including arrayed synthesis of DNA strands by light-control.
Synthesis of sequence-specified DNA, RNA, or other nucleic acid molecules by controllably adding mononucleotides one at a time.
Photo-controlled polymerase activity for gating nucleotide entry, translocation/ratcheting, or catalytic steps using photoswitchable azobenzene derivatives.
Purification of enzymes containing azobenzene molecules via attachment of an affinity tag peptide (enabling purification of enzymes only containing azobenzene molecules).
Kits for template-independent polynucleotide synthesis comprising the genetically-engineered TdT and reagents (e.g., dNTPs or analogs thereof).
Fluorescently-verified and photo-controlled synthesis (verification/detection of nucleotide incorporation using detectable labels as described).
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