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Core Concepts
Scientific Fundamentals Explained


Two single-stranded nucleic acid fragments can be covalently linked (ligated) by T4 RNA ligase. In the presence of ATP, ligase adenylates the 5’ end of single-stranded nucleic acids, which can then be ligated to the 3’ OH of other single-stranded sequences. However, when ATP is absent, synthetically adenylated oligonucleotides act as direct substrates for T4 RNA ligase, with the pyrophosphate linkage providing the energy required for the reaction and eliminating the need for ATP [1]. IDT custom synthesizes adenylated oligonucleotides using the chemical adenylation method of Unrau and Bartel [2]. Adenylated oligonucleotides can be used in numerous applications, including miRNA library construction, next generation sequencing, 5’ end labeling, and ribosome assembly experiments.

To add 5’ adenylation to an oligonucleotide order, select 5’ Adenylation from the 5’ mods tab on the oligo entry page. Note that this modification requires HPLC purification, and a 3’ blocking group to prevent oligonucleotide circularization. Preferred blocking groups are C3 Spacer (/3SpC3/) or Dideoxy-C (/3ddC/).


  1. England TE, Gumport RI, and Uhlenbeck OC (1977) Dinucleoside pyrophosphate are substrates for T4-induced RNA ligase. Proc Natl Acad Sci USA, 74:4839–4842.
  2. Unrau PJ and Bartel DP (1998) RNA-catalysed nucleotide synthesis. Nature, 395:260–263.

Author: Jeremy Pritchard is a Technical Support Representative at IDT.