Methods for sequencing oligoribonucleotides and RNA by high performance liquid chromatography

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Accurate separation and quantification of ribonucleosides and oligonucleotides are crucial for molecular biology research.
• Existing methods may be time-consuming or require larger sample quantities.
• Ribonucleic acid (RNA) digestion analysis requires precise identification of nucleotide components.

Purpose of the Study:

• To establish conditions for the separation and quantitative determination of ribonucleosides and oligonucleotides using high-performance liquid chromatography (HPLC).
• To identify products of RNase digestions of oligonucleotides and 5S ribosomal RNA (rRNA) efficiently.
• To develop a rapid analytical method for nucleotide analysis.

Main Methods:

• Utilized high-performance liquid chromatography (HPLC) on AS-Pellionex SAX and AL-Pellionex WAX columns.
• Integrated a high-speed UV spectrum monitor with the HPLC apparatus for continuous analysis.
• Identified nucleotide compounds based on UV spectra and retention times.

Main Results:

• Successfully established conditions for separating and quantifying ribonucleosides, mono-, and oligo-ribonucleotides.
• Identified products from RNase digestion of oligonucleotides and 5S ribosomal RNA (rRNA) using combined spectral and chromatographic data.
• Achieved rapid analysis, with each chromatography run taking only 10 to 30 minutes.
• Required minimal sample amounts, less than 0.01 A260 unit per nucleotide peak.

Conclusions:

• The developed HPLC method provides an efficient and rapid approach for the analysis of ribonucleosides and oligonucleotides.
• This method is suitable for identifying products of RNA digestion, including those from 5S ribosomal RNA (rRNA).
• The technique's speed and low sample requirement make it valuable for nucleotide analysis in molecular biology.