Enhanced Diagnostic Tools
Rapid progress has been made in recent years in high-throughput next-generation sequencing of small RNAs, which has increased the demand for 5’-adenylated DNA linkers or adapters. 5’ adenylation is an important biochemical step needed for the following applications:
Structure of 5’-adenylated RNA (5’ AppRNA).
The enzyme DNA ligase forms two covalent phosphodiester bonds between the 3' hydroxyl end of one nucleotide and the 5' phosphate end of another. ATP is required for the three step ligation reaction:
Structure model of a DNA ligase.
The helix-hairpin-helix (HhH) domain of Thermus filiformis(Tfi) DNA ligase is shown. The HhH domain of the NAD+-dependent DNA ligase from Thermus filiformis has four helix-hairpin-helix motifs arranged on one surface with a similar twofold symmetry. The nicked DNA from the DNA ligase I structure is positioned above these DNA-binding elements, indicating that they are properly spaced to bind to the minor groove.
DNA, with a 5’-adenylpyrophosphoryl cap (5’-adenylated DNA; AppDNA), is an activated form of DNA that is the biochemical intermediate of the reactions catalyzed by DNA ligase, RNA ligase, polynucleotide kinase, and other nucleic acid modifying enzymes. 5’-Adenylated DNA is also useful for in vitro selection experiments, therefore, an efficient preparation of 5’-adenylated DNA is desirable for several biochemical applications.
RNA ligases are involved in repair, splicing, and editing pathways that either reseal broken RNAs or alter their primary structure. RNA ligases join 3’-OH and 5’-PO4 RNA termini through a series of three nucleotidyl transfer steps similar to DNA ligases.
RNA Ligase-Nucleic Acid Complex Structures.
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