Analysis of double-stranded poly(A).poly(U) molecules by reversed-phase high-performance liquid chromatography

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Synthetic Poly(A).Poly(U) consists of large, double-stranded molecules.
• Characterizing batch variations in synthetic nucleic acids is crucial for research and applications.

Purpose of the Study:

• To investigate the behavior of synthetic Poly(A).Poly(U) using reversed-phase high-performance liquid chromatography (HPLC).
• To correlate HPLC elution patterns with molecular properties and assess HPLC's utility for routine analysis.

Main Methods:

• Reversed-phase high-performance liquid chromatography (HPLC) was employed to analyze different batches of synthetic Poly(A).Poly(U).
• Conventional methods including electrophoresis, centrifugation, fusion analysis, and enzymatic digestion (S1 nuclease) were used for comparative analysis.

Main Results:

• HPLC revealed differences in elution patterns between Poly(A).Poly(U) batches, correlating with molecular size.
• Contaminants from synthesis were detectable under specific HPLC conditions.
• Chromatograms indicated the transient presence of single-stranded portions in Poly(A).Poly(U) molecules, confirmed by S1 nuclease digestion.

Conclusions:

• Reversed-phase HPLC is a sensitive method for distinguishing between batches of synthetic Poly(A).Poly(U).
• The technique can detect variations related to molecular size and transient single-stranded structures.
• HPLC offers a rapid, reproducible, and easy method suitable for routine analysis of Poly(A).Poly(U).