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Biosynthesis of Nucleic Acids

Biosynthesis of Nucleic Acids

Nucleic acid biosynthesis is a fundamental biochemical process that produces the purine and pyrimidine nucleotides essential for DNA and RNA synthesis. This pathway maintains a balanced nucleotide pool, preventing imbalances that could jeopardize genetic integrity and cellular function. Given the crucial role of nucleotides, their synthesis is tightly regulated to ensure proper cellular homeostasis.Purine BiosynthesisThe biosynthesis of purine nucleotides begins with ribose-5-phosphate, a...

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Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS

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Base composition analysis of nucleosides using HPLC.

A Andrus¹, R G Kuimelis

¹PE Applied Biosystems, Foster City, California, USA.

Current Protocols in Nucleic Acid Chemistry

|April 23, 2008

Summary

This protocol uses nuclease digestion, dephosphorylation, and HPLC to analyze nucleobase modifications in oligonucleotides. It precisely quantitates nucleoside composition and modifications, including conjugated and carbohydrate moieties.

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Area of Science:

Biochemistry
Analytical Chemistry
Molecular Biology

Background:

Oligonucleotides are crucial in molecular biology and therapeutics.
Accurate analysis of oligonucleotide modifications is essential for quality control and research.
Existing methods may lack the sensitivity or scope for diverse modifications.

Purpose of the Study:

To present a robust protocol for the comprehensive analysis of oligonucleotide modifications.
To enable precise quantitation of nucleoside composition and modified bases.
To validate the method for various oligonucleotide modifications, including conjugated and carbohydrate moieties.

Main Methods:

Oligonucleotide digestion using nucleases.
Enzymatic dephosphorylation to yield monomers.
High-Performance Liquid Chromatography (HPLC) analysis on a reversed-phase C18 column.
Quantitation using integrated peak areas and relative extinction coefficient cofactors.

Main Results:

Successful detection and quantitation of a wide range of nucleobase modifications.
Precise determination of nucleoside composition based on chromatogram analysis.
Demonstrated utility for analyzing oligonucleotides with conjugated and carbohydrate modifications.

Conclusions:

The described protocol provides a reliable method for oligonucleotide analysis.
It is versatile for assessing various types of nucleobase, conjugated, and carbohydrate modifications.
This technique enhances the quality control and characterization of modified oligonucleotides.