What are oligonucleotides?
Oligonucleotides, which cover both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are increasingly demonstrating their central value in clinical practice. A notable example is the introduction of customized DNA sequences into immune cells that are genetically engineered to drive these cells to express chimeric antigen receptors (CARs), triggering a new era of cell-based mediated immunotherapy. At the same time, a variety of RNA molecules, such as messenger RNA (mRNA) and small interfering RNA (siRNA), are also cleverly used to achieve instant protein synthesis and specific protein expression inhibition, respectively, opening up new ways of therapeutic intervention. In view of the increasing role of oligonucleotides in medical intervention, it is becoming increasingly important to adopt effective methods for purification, analysis and characterization of oligonucleotides.
BOC Sciences has been committed to providing high-quality oligo-nucleic acid synthesis services for global partners over the years. We provide standard purification, PAGE purification, and HPLC purification from milligram level to kilogram level accordingly.
| Custom DNA Oligo | Provide standard unmodified DNA synthesis & multitype modified DNA synthesis. It includes standard oligos, micro-scale oligos, large-scale oligos, degenerate primers, and long oligos. |
| Custom RNA Oligo | Provide unmodified RNA, modified RNA, chimeric oligonucleotides with mixed DNA and RNA bases, 2′-OMe-RNA, 2′-F-RNA, and others with various labeling, modifications, and scales. |
| Custom Oligo Synthesis | Provide a variety of chemistry, modification, specifications, and purified oligos for biology, diagnostics, and drug discovery. |
| Antisense Oligos Synthesis | Strictly comply with QC testing standards and HPLC purity detection is used to ensure the high-quality output of all our ASO products. |
| Oligo Modifications | Comprehensive oligo modification and labeling services, such as backbone modification, modified bases, inverted base, oligo modified linker attachment chemistry, spacers, fluorescent labeling, phosphorylation, etc. |
| GMP Oligonucleotide Production | Our team consists of a group of experienced professionals with advanced technology and equipment to produce oligonucleotides of various sizes and types, including short, medium and long chain and modified oligonucleotides. |
| Long Oligo Synthesis | Using state-of-the-art biotechnology and equipment, we are able to synthesize and deliver oligonucleotides in the length range of 40-300 bases with consistent quality. |
| GMP Oligonucleotide Production | Our team consists of a group of experienced professionals with advanced technology and equipment to produce oligonucleotides of various sizes and types, including short, medium and long chain and modified oligonucleotides. |
Oligonucleotide purification method
Polyacrylamide gel electrophoresis (PAGE)
Polyacrylamide gel electrophoresis is a standard purity analysis method, which is particularly suitable for the separation of oligonucleotides based on fragment length. The process begins with the polymerization of polyacrylamide with the aid of a crosslinker to produce a three-dimensional gel matrix. Subsequently, under the action of an electric field, the oligonucleotide fragment carried by the negative charge migrates towards the anode. Due to spatial structural limitations, small molecules exhibit faster penetration rates due to lower resistance compared to larger molecules. Over time, oligonucleotides of various sizes exhibit differentiated migration distances in the gel matrix, which makes it possible to classify and purify them by length.
Ion pair reversed phase high performance chromatography (IP-RP-HPLC)
IP-RP-HPLC is the most popular oligonucleotide purification technique, in which low concentrations of long-chain alkylamines are added to bind negatively charged oligonucleotides in the LC mobile phase. The retention and elution of oligonucleotides in LC columns are influenced by the charge and ion of the oligonucleotides on the alkyl chain length in reagents such as triethylammonium acetate. For example, the retention time usually increases in proportion to the charge of the oligonucleotide and the hydrophobicity of the long alkyl chain in the ion-pairing reagent. A key advantage of IP-RP-HPLC is that it can also be coupled directly to a mass spectrometer for detailed mass characterization of oligonucleotides.
Qualitative characterization of oligonucleotides
One way to analyze the purity of oligonucleotides is to analyze their quality using mass spectrometry (MS). One common method is matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) MS. The technique uses a laser with a chemical matrix to ionize a sample of oligonucleotides and then accelerates the ions through a flight tube to a detector, which measures the particle count as a function of time. TOF is proportional to the mass of the molecule. MALDI TOF MS has a high throughput and is well suited for analyzing oligonucleotides below 50 bases, as ionization efficiency and separation resolution are reduced. There is also a risk that photosensitive modified oligonucleotides may be damaged by strong laser sources.
Structural analysis of oligonucleotides
X-ray crystallography
X-ray crystallography is undoubtedly the most authoritative and detailed means to reveal the structure of oligonucleotides, and its brilliant history is inseparable from the birth of several Nobel prizes. Given that the wavelength of X-rays is similar to the length of the internal bonds of molecules – about 1.5 angstroms – this technique provides unmatched structural detail with high precision. Through the precise interpretation of X-ray diffraction patterns, scientists were able to reconstruct the three-dimensional layout of oligonucleotide molecules and gain insight into their electron density distribution.
Nuclear magnetic resonance spectroscopy (NMR)
Another popular approach to structural analysis is nuclear magnetic resonance (NMR) spectroscopy, which is unique in that it can be analyzed without the need for the sample to form crystals. In a stable strong magnetic field, the nucleus is excited by a weak fluctuating magnetic field and releases electromagnetic waves reflecting the characteristic frequency of its magnetic field. When the oscillation frequency of the external magnetic field coincides with the natural frequency of the nucleus, that is, the resonance phenomenon is produced. The NMR spectra are rich in specific resonance information from the different nuclei in the sample, and the NMR Settings can be flexibly adjusted according to the resolution required for the experiment.
Circular dichroism (CD) spectrum
In the exploration of secondary structure of oligonucleotides, circular dichroism (CD) spectrum plays a key role. The technique is particularly suitable for identifying structures such as double helices, hairpins, and G-quadruplets, each of which presents a unique peak shape and signal on the CD spectrum. CD spectroscopy is not only good at identifying different secondary structures, but also tracking the conformational evolution of oligonucleotides in response to environmental changes (such as temperature, pH, salt concentration). Especially under temperature-controlled conditions, the melting behavior of oligonucleotides can be accurately described and their thermodynamic stability can be quantified. These insights are invaluable in elucidating the biological activity mechanism of oligonucleotides.
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Reference
Gilar, M., et al. Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides: Retention prediction. Journal of Chromatography A. 2002, 958(1-2): 167-182.