DNA oligonucleotide labeling using 4-thiouridine

The enzyme “terminal deoxynucleotidyl transferase” (TdT) adds several ribonucleotide residues to DNA fragments or primers. TdT can add deoxynucleotidyl triphosphates (dNTPs) randomly to the 3’-hydroxyl group (3’-OH) of single-stranded DNA. TdT catalyzed enzymatic polymerization is an alternative to conventional enzymatic and solid-phase DNA synthesis but allows combination with conventional enzymatic and solid-phase DNA synthesis. This approach is useful for the design and production of novel synthetic polymeric materials used in nanotechnology.

During its natural reaction, TdT adds randomly one to ten nucleotides to DNA but prefers adding dGTP and dCTP. GC regions added to DNA oligonucleotides promote efficient annealing of single-stranded DNA in subsequent ligation reactions. The enzyme also allows the synthesis of sequenced polynucleotides.

Figure 1: Schematic representation of the TdT catalyzed deoxynucleotide addition to ssDNA. Oligonucleotides with free 3’-hydroxyl termini are essential for polymerization initiation by TdT. The enzyme can use either a single-stranded DNA (ssDNA) initiator or double-stranded DNA (dsDNA) with a four-nucleotide 3’-overhang. A TdT-catalyzed enzymatic polymerization reaction needs an initiator oligodeoxynucleotide, natural or unnatural dNTPs, the enzyme TdT, and a buffer supplemented with a metal ion cofactor. The presence of secondary structural motifs at the 3’-ends, such as i-motifs or G-quadruplexes, can hinder access of the catalytic site of TdT, thereby limiting the addition of nucleotides. TdT can incorporate a wide range of natural and unnatural dNTPs, where the preferred order of incorporation is dGTP>dCTP>dTTP>dATP.

Eshaghpour et al., in 1979, reported the specific labeling of DNA fragments with 4-thiouridine at the 3’-ends using a combination of enzymatic and chemical reactions.

Figure 2: Enzymatic addition of 4-thiouridine residues.

Figure 3: α-Haloacetamido derivatives allow modification of the enolic form of 4-thiouridine. A variety of haloacetamides are available for chemical labeling. Chemical moieties such as 5-iodo-acetamidofluroescein (IAF), 5-iodoacetamidoeosin (IOESIN), and azido-bromoacetanilide (ABA) as well as others, can be attached to DNA ends. Many unnatural dNTPs allow adding other functional groups. These groups include biotin, amino-, azide-, alkyne-, dibenzoclyclooctyne-, digoxigenin-, dideoxy dNTPs, fluorescent dyes, as well as photocrosslinking molecules to oligonucleotides.  

Bio-Synthesis provides a full spectrum of high quality custom oligo modification services by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides containing backbone, base, sugar and internucleotide linkages. Bio-Synthesis specialize in complex oligonucleotide modifications using phosphodiester backbone, purine and pyrimidine heterocyclic bases, and sugar modified nucleotides such as our patented 3rd generation Bridged Nucleic Acids.

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