Nucleotide Analysis

Molecular characterization of nucleic acids using mass spectrometry is of current growing interest, because oligonucleotide therapeutics are a promising medicine to cure some diseases by working at upper stream of action mechanism with fewer side effects. Whereas a high-resolution accurate ESI mass spectrometer enables an exact intact mass measurement, routine oligo sequence analysis is a still hurdle, using ESI instruments. The complete internal oligo sequence is rarely obtained using typical ESI-MS/MS technique. On the other hand, in-source decay (ISD) using MALDI-TOFMS was reported as a useful method to conduct sequence analysis, although the instruments often employed do not have sufficient specification for exact mass measurement. Here, we report the characterization of oligonucleotide therapeutics using ESI-QTOF, LCMS-9030, and a dual polarity benchtop linear MALDI-TOFMS, MALDI-8030.

Synthetic oligonucleotides are short DNA or RNA sequences which find different applications in molecular biology, such as primers used in DNA sequencing and amplification by the polymerase chain reaction (PCR). Recently, synthetic oligonucleotides have also been explored for therapeutic and diagnostic purposes, and DNA-based diagnostic test kits, in several conditions. Cystic fibrosis is an example of a condition which develops at the DNA level. It is the commonest autosomal recessive disorder among Caucasians and caused by mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is located on chromosomal region 7q31.2 and contains 27 exons.

Oligonucleotide therapeutics are synthetic oligonucleotides that demonstrate their medical efficacy through binding to target genes or target proteins that may be responsible for a range of diseases. To date, eight types of oligonucleotide therapeutics have been approved, many of which have a length of approximately 20 bases. As an oligonucleotide therapeutic, the 2’-MOE modified oligonucleotide having 20 bases was used.

Oligonucleotide therapeutics have attracted attention in recent years as a new modality for drug discovery, because they can be used to create disease-specific therapeutic agents and can be designed easily by chemical synthesis. Typically, they are composed of oligonucleotides with about a dozen to several dozen bases (including modified bases). Quality control requires analyzing impurities, such as by-products, unreacted residues, and degradation products, in addition to the principal components. HPLC-UV is commonly used for purity confirmation, but if impurities are detected, they must be checked to confirm whether they are known impurities or not.

Nucleic acid drugs, such as antisense oligonucleotides, exert their effect by interacting with targets (genes and proteins) inside and outside of cells. Nucleic acid drugs are produced through chemical synthesis, but the synthesis process can introduce impurities such as shorter and longer length of products and protection groups. Proper separation of the target oligonucleotide is required. For LC separation, one commonly used mode is reversed-phase ion-pair chromatography (RP-IP). 

This article describes how to achieve the optimal separation of oligonucleotides and related impurities efficiently by utilizing LabSolutions MD, a dedicated software for supporting method development, through initial screening and optimization phase respectively.

Nucleic acid drugs are synthetic oligonucleotides which are designed to bond specifically with target RNA or proteins. Although most nucleic acid drugs approved to date are the antisense type, the aptamer type and siRNA type have also been approved. Many nucleic acid drugs consist of around 20 bases and have molecular weights on the order of 6,000. Precision mass analysis techniques such as MALDI-TOF type and Q-TOF type LC/MS are used in measurements of the molecular weight of drug substances. On the other hand, the triple quadrupole mass spectrometer is generally used in quantitative analysis such as analysis of the fate of drugs in the blood, as this instrument offers high sensitivity in combination with a wide dynamic range.

Nucleic acid drugs, such as antisense oligonucleotides, exert their efficacy by interacting with target genes inside and outside cells. Unlike conventional small molecule drugs, they are capable of targeting disease causes at the genetic level and are attracting attention as a next-generation drug. Nucleic acid drugs are mainly produced through chemical synthesis, but the synthesis process also produces many impurities such as shorter length components and protecting groups, so proper separation and purification of the target oligonucleotide is required.

DNA probes labeled with fluorescent dye (below referred to as fluorescent probes) are used extensively to detect and identify specific DNA when conducting life science studies. The mechanism involves the selective binding of the probe to specific DNA, thereby permitting the detection of that DNA. However, due to the wide variety of fluorescent dyes, it is important to know the exact wavelength at which the probe fluoresces to ensure DNA detection. Here, using the three-dimensional spectral measurement feature of the RF-6000 Spectrofluorophotometer, we introduce examples of fluorescence measurement of two types of fluorescent probes.

The Shimadzu BioSpec-nano is a low-maintenance micro-volume spectrophotometer designed for the modern life science laboratory. It offers superior detection limits, up to 10 times better compared to the competition, making it the perfect instrument for quantitation of DNA, RNA, Protein analysis, and photometric measurements. The “Drop and Click” design combined with easy sample mounting and automated cleaning offers a rapid 3 second analysis time and a 10 second cycle time between samples.