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While the world of biomedicines is largely made up of proteinaceous platforms, therapeutic oligonucleotides are becoming more prominent and more diverse each year. These classes of biomedicines are synthetic molecules with a chemical backbone structure composed of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) bases. These biomedicines can be designed to target specific mRNAs, miRNAs and proteins related to various diseases. While having the targeted specificity of antibody therapeutics, they can also be synthesized chemically like “low-molecular weight” drugs which reduces manufacturing costs compared to the biological process of protein-based biomedicines. Therapeutic oligonucleotides combine some of the best properties of “low-molecular weight” therapies and biopharmaceuticals.
The functions of nucleic acid medicines are various: small interfering RNA (siRNA) to control protein synthesis by coupling with mRNA, miRNA to strengthen miRNA functions, aptamers that bind to a protein to inhibit its functions, and ribozymes to directly cleave the target RNA are but a few. Their basic structure is a chain comprising a few dozen (deoxy)nucleotides including adenine, thymine, guanine, cytosine, and uracil, which are components of DNA and RNA.
Though these nucleic acid-based medicines can be chemically synthesized without the need to culture cells as in antibody medicines, they still have a range of complex molecular properties to be monitored. These molecules are medium-sized having a molecular weight ranging from several thousands to tens of thousands. The confirmation whether or not synthesized nucleic acid medicines are arranged in the intended base sequence is a critical quality characteristic to ensure the action of pharmaceuticals. Because natural oligonucleotides are very susceptible enzymatic degradation in a biological host, these biomedicines are modified to improve in vivo stability, target specificity, and overall potency. Some of these modifications on Therapeutic oligonucleotides include chemically modification, and phosphorothioation (oxygen atom replaced with sulfur atom on the phosphate moiety) , and even conjugation with N-acetylgalactosamine (GalNac).
With the complexity brought on by their chemical modifications and synthesis, many analytical techniques are employed to monitor both the progress and final product. Shimadzu offers a wide array of analytical technologies and workflows to enable the synthetic chemist and analytical scientist to meet their challenges.
Oligonucleotide Therapeutics Solution Guide
Oligonucleotide therapeutics are nucleic acid polymers generally comprised of a few to several dozen bases (including modified bases) linked together. They are produced by chemical synthesis and act directly on organisms without being translated into proteins. Oligonucleotide therapeutics are characterized by the ability to target specific diseases. Another advantage is that it takes less time than conventional methods to find new therapeutic candidates because oligonucleotides are easy to design and synthesize. However, it is a practical problem that oligonucleotide therapeutics are degraded and excreted rapidly after administration by exonucleases and endonucleases that are abundant in blood and cells. This problem is being addressed by the introduction of modified oligonucleotides to improve chemical stability in vivo and by the development of lesion-targeted DDS (drug delivery systems) technologies.
Oligonucleotides Analysis by Ion Exchange Chromatography and Effects of pH Changes in the Mobile Phase on Separation
In this article, we introduce an analytical method for the separation of oligonucleotides of different length by ion-exchange chromatography, assuming that shorter length components are impurities derived from the synthesis process. In order to achieve optimal analytical performances an inert UHPLC system was used. The Nexera XS inert, which is designed to suppress the adsorption of metal-coordinating compounds containing phosphate groups. We also report the effect of changes in mobile phase pH on analytical results.
Achieving Improved Sensitivity and Reliable Analytical Performances in Nucleotides Analysis
Stainless steel is commonly used in HPLC due to its pressure proof and corrosion resistant. However, it can interact with compounds containing phosphate group(s) by metallic affinity. This is a factor that negatively affecting the shape and intensity of the peak. In order to suppress metal adsorption, cleaning flow path with phosphoric acid, addition of chelating agents to mobile phase, or repeated injections of the target compounds are often performed. Nevertheless, it is not easy to obtain highly reproducible results.
This article introduces the use of the Nexera XS inert ultra-high performance liquid chromatograph, which utilizes a metal-free flow path, and a metal-free column for the accurate and reproducible analysis of nucleotides.
Determination of Molecular Mass and Quantification of Oligonucleotide Therapeutics Using Quadrupole Time-of-Flight Mass Spectrometer LCMS-9030
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. This article introduces an example of analysis using the Q-TOF mass spectrometer, LCMS-9030. As an oligonucleotide therapeutic, the 2’-MOE modified oligonucleotide having 20 bases was used. Accurate mass spectrometry determined the molecular mass of the therapeutic with an error of 3 mDa (0.05 ppm). When a calibration curve was prepared using the MRM mode on the LCMS-9030 mass spectrometer, linearity was observed within a range of 1 - 1000 ng/mL.
Oligonucleotide Mass Analysis and In-source Decay Sequencing on a MALDI TOF Mass Spectrometer
Here we will present a simple and speedy method of sequencing oligonucleotides that requires no further sample preparation and little expertise.
Climbing the oligonucleotide ladder toward rapid and wide-ranging oligonucleotide analysis using MALDI-MS
Biopharmaceutical and precision medicine technologies continue to transform drug design, life science research, and clinical diagnostics, demanding mass spectrometry (MS) techniques for fast, high-throughput oligonucleotide analysis requiring mass and sequence confirmation. Matrix-assisted laser desorption/ionization (MALDI-MS), provides a departure from toxic, time-consuming verification using gel electrophoresis and ethidium bromide. MALDI-MS techniques are amenable to rapid sample preparation and high-throughput studies, able to provide results in seconds.
DNA/ RNA Analysis
The analysis of nucleic acids, their characterization gets more and more important nowadays. Beside forensic and ancestry matters, medical diagnostic and specialized treatment in therapy are the driving forces. Verifying the structure of synthesized medicine, its sequence is one task to cover.
Three-Dimensional Spectra Measurement of Fluorescent Probes used for DNA Detection
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 MicroVolume BioSpec-nano Spectrophotometer
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.
Synthesis Confirmation for Nucleic Acid Medicines - Rapid Sequence Confirmation Using a MALDI-TOF Mass Spectrometer
Medicines utilizing nucleic acids such as DNA and RNA that control genetic information are called "nucleic acid medicines". These nucleic acid medicines allow targeting of molecules such as messenger RNA (mRNA) and microRNA (miRNA) which cannot be targeted with traditional low-molecular-weight drugs and antibody medicines, and are expected to be innovative next generation pharmaceuticals for the treatment of genetic disorders which have been difficult to treat so far.
LC/MS Analysis of Nucleic Acid-Related Compounds
The bases and nucleotides are generally separated by ion-exchange or reverse-phase mode HPLC and detected by UV absorbance detection.
In the example introduced here, nucleic acid-related compounds were analyzed by LC/MS, which offers mass information and enables high-sensitivity analysis.
Analysis of Peptide Nucleic Acids (PNAs) using the MALDI-8020 benchtop linear MALDI-TOF mass spectrometer
Matrix assisted laser desorption/ionisation time-of- flight mass spectrometry (MALDI-TOF MS) is gaining popularity due to the ability of MALDI-MS to quickly provide information on accurate molecular mass using a simple sample preparation workflow. The MALDI-TOF MS can be used to confirm and guide synthesis of PNA oligomers, either in QC or R&D laboratories. Here, we demonstrate the analysis of three PNA samples using MALDI-8020 benchtop linear MALDI-TOF mass spectrometer.
Thermal Melt Analysis System for Nucleic Acids
The TM analysis system is an accessory to UV-VIS spectrophotometers (BioSpec-Mini, UV-1800, UV-2450/UV-2550 series, UV-3600), and is comprised of an thermoelectrically temperature controlled micro multi-cell holder employing a Peltier element and PC software specially designed for the purpose. The Tm analysis system measures the temperature at which the double strands which form nucleic acids (DNA, RNA) and nucleic acid analogs (PNA, S-oligo nucleic acid) separate (the melting temperature, Tm). It also makes possible more detailed analysis (analysis of thermodynamic parameters).