Oligonucleotide Analysis
Oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are increasingly being utilized in diagnostics and therapeutics. For instance, DNA can be introduced into immune cells to genetically modify them, enabling the expression of chimeric antigen receptor (CAR) proteins for cell-based immunotherapy. Various types of RNA, including messenger RNA (mRNA) and small interfering RNA (siRNA), are also employed for transient protein expression and protein expression interference, respectively, for therapeutic applications. As the use of oligonucleotides increases, adopting efficient methods for their purification, analysis, and characterization has become increasingly important.
Oligonucleotide Purification Methods
1. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE):
SDS-PAGE is a standard method for purity analysis and can be used to separate oligonucleotides based on their size. Polyacrylamide gel is first polymerized by acrylamide in the presence of a crosslinker to form a mesh-like network. An electric field is then applied, causing the negatively charged oligonucleotides to move towards the positive electrode. Smaller molecules traverse the gel network more easily and quickly than larger ones. After a set period, oligonucleotides of different sizes will migrate different distances within the polyacrylamide gel, allowing them to be extracted and purified based on size.
2. Ion-Pair Reversed-Phase High-Performance Liquid Chromatography (IP-RP-HPLC):
IP-RP-HPLC is one of the most widely used techniques for oligonucleotide purification. In this method, low concentrations of long-chain alkylamines are added to bind negatively charged oligonucleotides in the liquid chromatography (LC) mobile phase. The retention and elution of oligonucleotides on the LC column are influenced by factors such as the oligonucleotide's charge and the alkyl chain length in ion-pairing reagents (e.g., triethylammonium acetate). For example, retention times typically increase proportionally to the oligonucleotide's charge in the ion-pairing reagent and the hydrophobicity of the long alkyl chains. A key advantage of IP-RP-HPLC is its ability to be directly coupled with mass spectrometry (MS) for detailed mass characterization of oligonucleotides.
Oligonucleotide Mass Characterization
One method for analyzing oligonucleotide purity is through mass spectrometry (MS). A common technique is Matrix-Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) MS. This technique uses a laser to ionize the oligonucleotide sample in the presence of a chemical matrix, after which the ions are accelerated through a flight tube to the detector. The detector measures the particle count as a function of time, where the time-of-flight (TOF) is proportional to the molecular mass. MALDI-TOF MS has high throughput and is ideal for analyzing oligonucleotides with fewer than 50 bases, as ionization efficiency and resolution decrease for larger molecules. However, there is a risk of photochemically modifying the oligonucleotides, especially for those with light-sensitive modifications, due to the strong laser source.
Oligonucleotide Structural Characterization
1. X-ray Crystallography
The most powerful method for determining the structure of oligonucleotides is X-ray crystallography, which has been indirectly associated with many Nobel Prizes. The wavelength of X-rays is similar to the size of atomic bonds in molecules, approximately 1.5 Å, and can provide highly detailed structural information about oligonucleotides. By analyzing the scattering of X-rays, the electron density distribution within the sample can be determined, allowing for the reconstruction of the internal molecular arrangement.
2. Nuclear Magnetic Resonance (NMR)
Another popular method for characterizing the structure of oligonucleotides is Nuclear Magnetic Resonance (NMR). The advantage of NMR over X-ray crystallography is that the molecule does not need to be crystallized. When atomic nuclei in a strong, constant magnetic field are subjected to a weak oscillating magnetic field, they respond by emitting electromagnetic signals at frequencies characteristic of their nuclear magnetic field. This occurs when the oscillation frequency of the external field matches the inherent frequency of the atomic nuclei, resulting in resonance. Thus, NMR spectra provide structural information based on specific resonance characteristics from different atomic nuclei within the sample. Depending on the experimental objectives (e.g., resolution), different NMR settings are used.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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