Related Experiment Video
Updated: Jan 26, 2026
Nucleoside Triphosphates - From Synthesis to Biochemical Characterization
Published on: April 3, 2014
Additive-Free Enzymatic Phosphorylation and Ligation of Artificial Oligonucleotides with C-Nucleosides at the
Yutaro Oda1, Junya Chiba1, Fumihiro Kurosaki1
1Graduate School of Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan.
Abstract:
We report enzymatic phosphorylation and additive-free ligation of DNAs containing unnatural C-nucleotide residues through the action of T4 polynucleotide kinase and T4 DNA ligase. The artificial units are each made up of an alkynyl deoxyribose component and one of the unnatural nucleobases D*, T*, G*, and C*, corresponding-from a viewpoint of hydrogen-bonding patterns-to natural A, T, G, and C, respectively. Phosphorylation progressed quantitatively at the 5'-end in the cases of all of the artificial units in the chimeric DNAs. Ligation also smoothly progressed at the 5'-end in the cases of the D* and G* nucleotide residues, but only negligibly in those of their T* and C* counterparts. Chemical redesign of the last two units successfully improved the ligation efficiency, so that enzymatic ligation worked well for all of the artificial units in every 3'-natural⋅5'-artificial, 3'-artificial⋅5'-natural, and 3'-artificial⋅5'-artificial terminal combination at the nicks.
Related Concept Videos
01:02Phosphorylation
During phosphorylation, protein kinases transfer the terminal phosphate group of ATP to specific amino acid side chains of substrate proteins. Serine, threonine, and tyrosine are the most commonly...
02:15Preparation of Alcohols via Addition Reactions
The acid-catalyzed addition of water to the double bond of alkenes is a large-scale industrial method used to synthesize low-molecular-weight alcohols. An acidic atmosphere is required to allow the hydrogen in the water molecule to act as an electrophile and attack the double bond in an alkene. The addition of a proton to the double bond creates a carbocation intermediate. The proton preferentially bonds to the less substituted end of the double bond to create a more stable carbocation...
01:08Base-Catalyzed Aldol Addition Reaction
01:15Acid-Catalyzed Aldol Addition Reaction
02:24Introduction to Electrophilic Addition Reactions of Alkenes
Addition and elimination...
01:27Conjugate Addition (1,4-Addition) vs Direct Addition (1,2-Addition)
Conjugate addition results in a thermodynamically stable product. The reaction retains the stronger C=O bond at the expense of the weaker C=C π bond. The process is slow as the β carbon is less electrophilic than the carbonyl carbon.
Direct addition products are...