Separation of oligo-RNA by reverse-phase HPLC

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Complex mixtures of oligoribonucleotides require efficient analytical and purification methods.
• Existing chromatographic techniques may lack speed, reproducibility, or versatility for diverse nucleic acid fragments.

Purpose of the Study:

• To develop a rapid, highly reproducible chromatographic technique for analyzing and purifying oligoribonucleotides.
• To establish a versatile method capable of separating a wide range of nucleic acid sizes, from monomers to larger oligomers.

Main Methods:

• Utilized a reversed-phase high-performance liquid chromatography (RP-HPLC) system.
• Employed a column packed with microparticulate porous silica beads derivatized with octadecylsilyl (ODS) groups.
• Eluted with gradients of acetonitrile/water/ammonium acetate at pressures between 1500-300 psi.

Main Results:

• Achieved rapid separations of oligoribonucleotides, typically completed within 5-15 minutes.
• Demonstrated high reproducibility, with better than 1% absolute and 0.1% relative retention time reproducibility.
• Successfully separated mononucleosides, mononucleotides, and oligoribonucleotides up to 20-mers on a single column.
• Established a detection limit of approximately 1 picomole (pmole) of base for analytical separations.
• Showcased the capability for purifying approximately 1 milligram (mg) of oligonucleotide in 10-30 minutes.

Conclusions:

• The developed ODS-silica based chromatographic method provides a rapid and highly reproducible solution for oligoribonucleotide analysis and purification.
• This technique is versatile, accommodating a broad spectrum of nucleic acid sizes and offering sensitive detection limits.
• The method's efficiency and reproducibility make it suitable for both analytical and preparative applications in molecular biology and biochemistry.