Nucleotide Analysis

The main dogma of molecular biology states that DNA is transcribed into RNA, which than is translated into the amino acid sequence of an expressed protein. Knowledge about the DNA structure and sequence is important to get a better understanding about genes, protein expression and the complex regulation mechanism in cells. Analyzing the nucleic base sequence and structure determining elements of DNA and RNA is often the starting point.
Diagnosis of gene defects as cause for rare diseases and their treatments are the driving force for the development of new generation of medications, such as gene therapy. After synthesis of the special DNA or RNA pieces, the verification of their structure and sequence are the tasks to cover.
Modern vaccines based on mRNA and their productions once more underline the importance of having state of the art techniques and solutions for fast and precise analysis’.

Applications for MALDI & Q-TOF

Analysis of Oligonucleotide Therapeutics using MALDI-8030 and LCMS-9030

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.

Negative Mode Analysis of Synthetic Oligonucleotides using the MALDI-8030 Dual Polarity Benchtop MALDI-TOF Mass Spectrometer

Nucleotide Analysis

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.

Determination of Molecular Mass and Quantification of Oligonucleotide Therapeutics Using Quadrupole Time-of-Flight Mass Spectrometer LCMS™-9030

Nucleotide Analysis

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.

Applications for LC/LCMS

Simple Analysis of Impurities in Oligonucleotide Therapeutics Using a Single Quadrupole Mass Spectrometer

Nucleotide Analysis

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.

Efficient Method Development of Oligonucleotides by Reversed-Phase Ion-Pair Chromatography

Nucleotide Analysis

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.

Quantitative Analysis and Determination of Molecular Weight of siRNA Type Oligonucleotides by LCMS™-8060

Nucleotide Analysis

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.

Oligonucleotides analysis by Ion Exchange Chromatography and Effects of pH Changes in the Mobile Phase on Separation

Nucleotide Analysis

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.

Applications for Spectro

Three-Dimensional Spectra Measurement of Fluorescent Probes used for DNA Detection

Nucleotide Analysis

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.

Quantitation of dsDNA Using the Micro-Volume BioSpec-nano Spectrophotometer

Nucleotide Analysis

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.

Oligonucleotide Therapeutics