Enzymatic synthesis and modification of high molecular weight DNA using terminal deoxynucleotidyl transferase

Abstract

The recognition that nucleic acids can be used as polymeric materials led to the blossoming of the field of DNA nanotechnology, with a broad range of applications in biotechnology, biosensors, diagnostics, and drug delivery. These applications require efficient methods to synthesize and chemically modify high molecular weight DNA. Here, we discuss terminal deoxynucleotidyl transferase (TdT)-catalyzed enzymatic polymerization (TcEP) as an alternative to conventional enzymatic and solid-phase DNA synthesis. We describe biochemical requirements for TcEP and provide step-by-step protocols to carry out TcEP in solution and from surfaces.

The Possible Involvement of Apoptotic Decay of Terminal Deoxynucleotidyl Transferase-Positive Lymphocytes in the Reutilization of the Extracellular DNA Fragments by Surrounding Living Cells

“…Among four deoxynucleosides triphosphate (dNTP), the concentrations of dGTP in dividing cells are 5 -8 fold lower than in tree others dATP, dCTP, and dTTP [18] [25] [30]. Surprisingly, the preferred order of incorporation by TdT is dGTP > dCTP > dTTP > dATP [31], seeming as a process for compensation of intracellular deficit of dGTP. Tight control of the dNTP pool is essential for cellular homeostasis, especially for deoxyguanosine nucleotides during radiation [32] or chemical damages, for example, the therapeutic toxicant gemcitabine [33].…”

The Protein‐Templated Synthesis of Enzyme‐Generated Aptamers

“…The template protein may also act to inhibit further elongation of the polynucleotides through competition for binding with the TdT enzyme. We would also expect a kinetic bias for the incorporation of each nucleotide by TdT enzyme [12] . The kinetics of incorporation and the bias of TdT can effectively be controlled through the incorporation of different divalent metal ions, the time of incubation and the ratios of initiator sequence to each type of nucleotide [12] …”

“…We would also expect a kinetic bias for the incorporation of each nucleotide by TdT enzyme. [12] The kinetics of incorporation and the bias of TdT can effectively be controlled through the incorporation of different divalent metal ions, the time of incubation and the ratios of initiator sequence to each type of nucleotide. [12] In the case of the divalent metal ions used in the reaction buffer, the kinetics of TdT incorporation of nucleotides can change remarkably.…”

“…[12] The kinetics of incorporation and the bias of TdT can effectively be controlled through the incorporation of different divalent metal ions, the time of incubation and the ratios of initiator sequence to each type of nucleotide. [12] In the case of the divalent metal ions used in the reaction buffer, the kinetics of TdT incorporation of nucleotides can change remarkably. For instance, in most commercial buffers, the use of Co 2 + results in the formation of longer polynucleotides with a broader range of sizes compared to Mg 2 + ions.…”

“…[10] This serves to reduce the rate of incorporation and slow down the kinetics of incorporation to a point that each strand of polynucleotide elongates at a different kinetic rate and deviates from the living polymerisation mechanism observed when incorporating a single type of nucleotide. [11,12] The template protein may also act to inhibit further elongation of the polynucleo-tides through competition for binding with the TdT enzyme. We would also expect a kinetic bias for the incorporation of each nucleotide by TdT enzyme.…”