A: 5′-adenylation is the process of adding an adenosine monophosphate (AMP) group to the 5′ phosphate of a DNA or RNA molecule.
A: 5′ adenylated linkers are commonly used in small RNA-seq, miRNA-seq, and other next-generation sequencing workflows that rely on adapter ligation to 3′ ends of RNA. By adenylating in-house, users can generate custom pre-adenylated linkers compatible with T4 RNA Ligase 2 (truncated), enabling ligation without ATP. This reduces concatemer formation and other ligation artifacts. The Adenylator™ DNA/RNA 5′-Adenylation Kit allows the user to efficiently prep these linkers from inexpensive 5′ phosphorylated oligos, offering a cost-effective and flexible alternative to ordering pre-adenylated linkers.
A: The Adenylator™ Enzyme is a non-ligase based proprietary enzyme optimized for 5’-adenylation. Therefore, it cannot catalyze undesirable side products that can be produced in some ligase-based 5′-adenylation methods. It converts 5′-monophosphorylated oligos into 5′-adenylated substrates compatible with T4 RNA Ligase 2 truncated or truncated/K227Q-based reactions.
A: Yes, the adenylation reaction can be scaled up without loss of efficiency. This allows for the preparation of larger quantities of adenylated DNA or RNA oligos for extensive applications.
A: For 5’-adenylation of single-stranded RNA oligonucleotides, it is recommended to include ScriptGuard™ RNase Inhibitor in the reaction to protect RNA integrity during the process.
A: The efficiency of the 5′-adenylation reaction can be assayed by running the adenylated and unadenylated oligos side-by-side on an 8M Urea, 20% polyacrylamide gel. The adenylated oligo will show a one nucleotide band shift.
A: Yes, a Control Phosphorylated Oligo that is 23-b DNA oligonucleotide is supplied in the Adenylator™ DNA/RNA 5′-Adenylation Kit.
