Determination of 7-methylguanosine-containing 5′-termini of messenger ribonucleic acid by NaB[3H]4 labeling and high-performance liquid anion-exchange chromatography

✍ Scribed by Hans-Joachim Breter; Lawrence T. Malek; Gary M. Hellmann; Robert E. Rhoads

• Book ID: 102985426
• Publisher: Elsevier Science
• Year: 1979
• Tongue: English
• Weight: 757 KB
• Volume: 98
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(79)90712-7

✦ Synopsis

A method is described for the determination of 5'-terminal methylated (cap) structures in unlabeled mRNA based on oxidation with NaIO.,, reduction with NaB["H],. cleavage with P, nuclease. and separation on a strong anion-exchange resin by high-performance liquid chromatography (hplc). Model compounds (cap I dinucleotides) were used to show that no structural alteration other than cleavage of the ribose ring of 7-methylguanosine occurred under the conditions used for oxidation and reduction. It was shown that the enzyme tobacco acid pyrophosphatase could be used to cleave cap dinucleotides containing unmodified or ring-opened ribose moieties and could also be used to release [:'H]pm'G' from NaB["H].,-labeled rabbit globin mRNA. All five known cap I dinucleotides were resolved by hplc. The cap of rabbit globin mRNA was identified as m'Gpppm"Am, in agreement with other methods of determination.