The use of high-performance liquid chromatography in the isolation and analysis of oligoribonucleotides synthesized by the T4 RNA ligase reaction

✍ Scribed by Larry W. McLaughlin; Elena Romaniuk

• Publisher: Elsevier Science
• Year: 1982
• Tongue: English
• Weight: 664 KB
• Volume: 124
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(82)90216-0

✦ Synopsis

High-performance liquid chromatography (HPLC) with both amino-propyl-silyl ion-exchange and octadecasilyl reverse-phase columns has been used to simultaneously analyze all substrates, intermediates, and products involved in the T4 RNA ligase reaction. Both singlestep ligations (e.g., UpApA + pCp) as well as the coupling of oligonucleotide blocks (e.g., UpApA + pUpUpUpCp) have been examined. A general one-step synthesis of donor oligonucleotide blocks is described. A nucleoside-3',5'-bisphosphate can be ligated to a trinucleoside diphosphate, producing a tetramer with a 3'-terminal phosphate. Subsequent addition of polynucleotide kinase (lacking the 3'-ph~phata~ activity) to this reaction mixture results in a tetra-nucleotide containing 3'-and S-terminal phosphates. RNase T2 digestion and resolution of the resulting nucleosides and nucleotide 3'-monophosphates by HPLC has been used for identification of oligoribonucleotides.