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Scientific Fundamentals Explained

Adenylated oligonucleotides provide direct substrates for T4 RNA ligase

Creating direct substrates for T4 RNA ligase

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 oiigo applications 

Adenylated oligonucleotides can be used in numerous applications. These include miRNA library construction, next generation sequencing, 5’ end labeling, and ribosome assembly experiments.

Quick facts:

Availability: DNA

Location: 5'

Scales: 100 nmol–10 µmol

Purification: HPLC required

IDT Ordering Symbol: /5rApp/

Ordering adenylated oligos

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.

Additional reading

DNA Oligonucleotide Resuspension and Storage
Upon receiving newly synthesized oligonucleotides, researchers must decide how to resuspend and store the product. Here are some guidelines and recommendations.

Oligonucleotide Modifications: Choosing the Right Mod for Your Needs
Guidelines on selecting oligonucleotide modifications and a discussion of how they can help you in your research.

Oligo Modification—Post-Synthesis Conjugation Explained
A description of addition of NHS esters and of amino and alkene modifications through use of click chemistry. We also provide answers to common questions regarding post-synthesis conjugation.

Which Biotin Modification to Use?
A discussion of the applications for each of the different biotins available through IDT.

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

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