Quantitative measurement of mRNA cap 0 and cap 1 structures by high-performance liquid chromatography

Viral and eukaryotic mRNA molecules have a unique 5'-end. The 5'-terminus consists of m7G(5')ppp(5')N'(m)pN''(m), which is termed a "cap" structure. The study of these cap structures has led to the development of many methods of identification and analysis. Many of the methods have been time-consuming or have not been able to distinguish between the different caps, and they are quantifiable only by employing radiolabels. This paper presents the use of reversed-phase high-performance liquid chromatography as a rapid and efficient tool for the separation, identification and quantitation of caps. An ion-exchange enrichment procedure was also developed for the isolation of cap 0 and cap 1 structures from unfractionated RNAs. The recoveries of different caps ranged from 83 to 99%, with a relative standard deviation range of 1.3-4.4%. In this method, caps were released from commercially obtained rabbit globin mRNA by nuclease P1 digestion. The products of digestion were treated with alkaline phosphatase and separated on an octadecylsilyl column using stepwise or gradient elution. Cap structures and any internal modified nucleosides were identified by their retention times and UV spectra relative to reference compounds. The amount of each cap 0 or cap 1 structure was determined by its UV absorbance relative to a known quantity of reference compound. This method allows the quantitation of 0.2 nmol or more of cap 0 and cap 1 structures. Total UV spectra can be obtained for 0.5 nmol or more of cap. This methodology permits investigations on viral and eukaryotic mRNA cap biosynthesis and turnover during viral transformation, differentiation, cap synthesis in the cell cycle, etc.