A plurality of terminal deoxynucleotidyl transferases (TdTs) and variants thereof are used for de novo synthesis of polynucleotides having controlled sequences and efficiently controllable synthesis of nucleic acid molecules without depending on a template. It is found that some amino acid residues in the TdT catalytic domain can be specifically modified to improve the ability of such modified TdTs to synthesize polynucleotides.

This disclosure provides electrochemically-cleavable linkers with cleavage potentials that are less than the redox potential of the solvent in which the linkers are used. In some applications, the solvent may be water or an aqueous buffer solution. The linkers may be used to link a nucleotide to a bound group. The linkers include a cleavable group which may be one of a methoxybenzyl alcohol, an ester, a propargyl thioether, or a trichloroethyl ether. The linkers may be cleaved in solvent by generating an electrode potential that is less than the redox potential of the solvent. In some implementations, an electrode array may be used to generate localized electrode potentials which selectively cleave linkers bound to the activated electrode. Uses for the linkers include attachment of blocking groups to nucleotides in enzymatic oligonucleotide synthesis.

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.

Provided herein are methods and compositions related to non-human animals that express exogenous Terminal Deoxynucleotidyltransferase (TdT).

Provided herein are methods of nucleic acid synthesis. In some embodiments, a method of nucleic acid synthesis comprises selectively activating and deactivating an engineered terminal deoxynucleotidyl transferase (TdT). In some embodiments, a method of nucleic acid synthesis comprises activating a pH sensitive enzyme to digest and remove a TdT.

The invention relates to engineered terminal deoxynucleotidyl transferase (TdT) enzymes or the homologous amino acid sequence of Pol, Pol, Pol, and Pol of any species or the homologous amino acid sequence of X family polymerases of any species and their use in a method of nucleic acid synthesis, to methods of synthesizing nucleic acids, and to the use of kits comprising said enzymes in a method of nucleic acid synthesis. The invention also relates to the use of new terminal deoxynucleotidyl transferases and 3-blocked nucleoside triphosphates in a method of template independent nucleic acid synthesis.

Provided herein are methods, and compositions for the detection of protein synthesis. The methods are applicable to monitoring and purification of protein synthesis on a microfluidic device.

The invention provides improved methods for synthesizing polynucleotides, such as DNA and RNA, using enzymes and specially designed nucleotide analogs. Using the methods of the invention, specific sequences of polynucleotides can be synthesized de nova, base by base, in an aqueous environment, without the use of a nucleic acid template. Because the nucleotide analogs have an unmodified 3 OH, i.e., as found in natural deoxyribose and ribose molecules, the analogs result in natural polynucleotides suitable for incorporation into biological systems.

The present disclosure provides modified terminal deoxynucleotidyl (TdT) polymerases and compositions (e.g., conjugates) comprising modified TdT polymerases. The present disclosure also provides circularly permuted terminal deoxynucleotidyl (TdT) polymerases as well as modified TdT polymerases and compositions (e.g., conjugates) comprising such cpTdT or modified TdT polymerases. Also provided are nucleic acids, vectors, and host cells encoding modified TdT polymerases, as well as methods including methods of producing modified TdT polymerases, and methods of using modified TdT polymerases (e.g., methods of nucleic acid synthesis).

This disclosure provides electrochemically-cleavable linkers with cleavage potentials that are less than the redox potential of the solvent in which the linkers are used. In some applications, the solvent may be water or an aqueous buffer solution. The linkers may be used to link a nucleotide to a bound group. The linkers include a cleavable group which may be one of a methoxybenzyl alcohol, an ester, a propargyl thioether, or a trichloroethyl ether. The linkers may be cleaved in solvent by generating an electrode potential that is less than the redox potential of the solvent. In some implementations, an electrode array may be used to generate localized electrode potentials which selectively cleave linkers bound to the activated electrode. Uses for the linkers include attachment of blocking groups to nucleotides in enzymatic oligonucleotide synthesis.