Modified Nucleotides as Substrates of Terminal Deoxynucleotidyl Transferase

Abstract

The synthesis of novel modified nucleotides and their incorporation into DNA sequences opens many possibilities to change the chemical properties of oligonucleotides (ONs), and, therefore, broaden the field of practical applications of modified DNA. The chemical synthesis of nucleotide derivatives, including ones bearing thio-, hydrazino-, cyano- and carboxy groups as well as 2-pyridone nucleobase-containing nucleotides was carried out. The prepared compounds were tested as substrates of terminal deoxynucleotidyl transferase (TdT). The nucleotides containing N4-aminocytosine, 4-thiouracil as well as 2-pyridone, 4-chloro- and 4-bromo-2-pyridone as a nucleobase were accepted by TdT, thus allowing enzymatic synthesis of 3’-terminally modified ONs. The successful UV-induced cross-linking of 4-thiouracil-containing ONs to TdT was carried out. Enzymatic post-synthetic 3’-modification of ONs with various photo- and chemically-reactive groups opens novel possibilities for future applications, especially in analysis of the mechanisms of polymerases and the development of photo-labels, sensors, and self-assembling structures.

N 4-acyl-2′-deoxycytidine-5′-triphosphates for the enzymatic synthesis of modified DNA

“…As to the template-independent DNA synthesis, TdT is known to use a wide variety of nucleotide analogues, hence it may be applied for a robust tail-labelling without the necessity for a precise quantification of the labels. TdT uses different base-modified nucleotides for the enzymatic synthesis of artificial DNA ( 65–68 ), nucleotides bearing steric aromatic pyrene residues for the generation of fluorescent oligomers ( 69 , 70 ) or pyridone/imidazole-based analogues for the specific metal coordination and immobilization ( 71–73 ). Our experiments confirm that TdT utilizes dC Acyl TPs to a similar or even greater extent than natural nucleotides.…”

Characteristics of the excited triplet states of thiolated guanosine derivatives and singlet oxygen generation

“…For instance, 6-thioguanine (6TG) and 4-thiouracil have been used as carcinostatic agents, leukemia drugs, antithyroids and DNA markers among others. [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] Thio-bases have a strong absorption in UVA (320-400 nm) region, [36][37][38][39][40][41][42][43][44][45][46] in which native bases are transparent. Thio-bases localizing in proliferating cells have been reported to generate reactive oxygen species (ROS) when exposed to UVA light, thus leading to cell Please do not adjust margins Please do not adjust margins deaths.…”

Template-Independent Enzymatic Oligonucleotide Synthesis (TiEOS): Its History, Prospects, and Challenges

“…Unlike Pol β, which preferentially binds Mg 2+ , TdT can accept multiple nucleotide binding and catalytic divalent cations (Figure , steps 4–7); these include Mg 2+ , Co 2+ , and Zn 2+ (allosteric cofactor). The specificity of nucleobase incorporation by TdT appears to be largely ion-dependent as demonstrated by Fowler and Suo: G > A > C > T and T > C > G > A, respectively [Mg 2+ (purines) and Co 2+ (pyrimidines)]. , It has been shown, too, that micromolar quantities of Zn 2+ increase the efficiency of nucleotide incorporation for both Mg 2+ and Co 2+ (generally at millimolar concentrations per reaction). , Chang and Bollum identify Zn 2+ as a nonessential allosteric cofactor for terminal transferase, which loosely interacts with the initiator and TdT binding site to induce conformational changes that increase the rate of catalysis . Whether Mg 2+ or Co 2+ competes for the active site when present in the same reaction, tailoring the TiEOS coupling reaction buffers separately for purines (Mg 2+ and Zn 2+ ) and pyrimidines (Co 2+ and Zn 2+ ) may be advisable for improving dNTP incorporation efficiency.…”