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Techniques for Oligonucleotide Analysis

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Oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are increasingly being used in diagnostics and therapeutics. For instance, DNA can be introduced into immune cells to genetically engineer them to express chimeric antigen receptor proteins for cell-based immunotherapy.1 They are also being used as DNA origami to mimic viral particles for designing molecular vaccines.2 A variety of RNA, including messenger RNA (mRNA) and small interfering RNA (siRNA), are also being used for transient expressions of proteins and interfering with protein expressions respectively for therapeutic applications.

The need for high purity oligonucleotides

According to
Balasubrahmanyam Addepalli, research associate professor at the University of Cincinnati, despite the increasing use of oligonucleotides, it is still challenging to purify them due to "failure sequences and final products with intact protective groups."

The synthesis of oligonucleotides using techniques like solid-state synthesis can introduce tiny amounts of impurities at each step of the synthesis cycle. As the length of the oligonucleotide increases, the yield of the pure product decreases. Modifications to oligonucleotides – such as attachment of polyethylene glycol to enhance stability and bioavailability – can also introduce impurities during the synthesis process.

The more common impurities are:

  • n-1 shortmers due to reaction failures

  • n+1 longmers due to single coupling step by successive addition of two molecules of phosphoramidite and

  • Residual amount of phosphodiester from incomplete sulfurization and side reaction.

review by Goodchild eloquently summarizes the established technique for oligonucleotide synthesis which has been automated since the 1970s.3 As the use of oligonucleotides increases, it becomes increasingly important to adopt efficient methods to purify, analyze and characterize them.

Purification of oligonucleotides

Polyacrylamide gel electrophoresis
(PAGE) is a standard method that can be used to separate oligonucleotides based on their size.4 Polyacrylamide gels are first formed by the polymerization of acrylamide in the presence of a cross-linking agent, resulting in a mesh-like gel network. Electrical fields are then applied, causing migration of negatively-charged oligonucleotides to move towards the positive electrode. Depending on the size of the oligonucleotide, the smaller molecules move through the gel network more easily and quickly than their larger counterparts. After a set amount of time, oligonucleotides of different sizes would have migrated different distances in the polyacrylamide gel, allowing them to be extracted and purified based on size.

However, PAGE offers low separation resolution if the oligonucleotides are similar in size, with some modifications even being inseparable. Furthermore, PAGE separation is laborious and time-consuming. These limitations have motivated the switch from PAGE to liquid
chromatographic (LC) approaches for oligonucleotide purification.5

There are a variety of LC approaches for purification of oligonucleotides. For instance, ion exchange LC separates ions and charged molecules such as oligonucleotides based on their affinity to the ion exchanger. However, the structures i.e. secondary folding, tertiary folding, dimers and aggregates of oligonucleotides must first be denatured with organic solvents, high pH buffers or high temperature (55-65 oC).

The most popular technique to purify oligonucleotides is
ion-paired reversed-phase high-performance (IP RP HP) LC.6 In this technique, long-chained alkyl amine is added at low concentration where it binds to negatively-charged oligonucleotides in the mobile phase of LC. The retention and elution of the oligonucleotides in the LC column are affected by factors such as charge of oligonucleotides and length of alkyl chain in the ion pairing reagent such as triethylammonium acetate. For instance, retention time generally increases in proportion to the charges of oligonucleotides and hydrophobicity of the long alkyl chain in the ion-paring reagent. Typically, researchers have to optimize parameters such as column length, mobile phase flow rate, separation temperature and ion pairing reagent buffer composition for oligonucleotide separation and purification.7 A key advantage of IP RP HPLC is that it can also be coupled to a mass spectrometer directly for detailed mass characterization of oligonucleotides.

Mass characterization of oligonucleotides

A way to analyze the purity of oligonucleotides is to analyze their mass using mass spectrometry (MS). One of the common methods is
matrix-assisted laser desorption/ionization-time of flight (MALDI TOF) MS.This technique uses laser light with a chemical matrix to ionize the oligonucleotide sample before accelerating the ions through a flight tube to the detector which measures particle counts as a function of time. The TOF is directly proportional to the mass of the molecule. MALDI TOF MS offers high throughput and is ideally used to analyze oligonucleotides below 50 bases, as the ionization efficiency and separation resolution decreases. There is also a risk that modified oligonucleotides which are photosensitive may be damaged by the strong laser source.

To overcome the limitation of MS, scientists are also combining techniques. Kimura and colleagues recently combined deep sequencing and MS to characterize transfer RNA (t)RNA.9Deep sequencing is a high-throughput method that reads an incredible number of RNA sequences in a single run. However, deep sequencing cannot identify the chemical properties of predicted modifications. In contrast, MS analyzes the composition and position of modifications of oligonucleotides. tRNA is the most heavily modified RNA molecules and historically, MS is used to identify their modification profiles. Thus, in our paper, we took the benefit of both analyses: taking deep sequencing for predicting modification sites and conducting MS analysis for detailed characterization of the predicted modified sites,” says Kimura.

The other popular method is
electro-spray ionization (ESI) MS. This technique applies high voltage to generate aerosol from liquid samples, ionizing target molecules into multiple charge states represented by different mass spectra that can be deconvoluted into parent peaks to identify oligonucleotides and potential impurity. ESI MS is an excellent tool to analyze oligonucleotides greater than 50 bases and as it uses milder ionization condition, it is suitable for oligonucleotides which are photosensitive. Nevertheless, it has lower throughput than MALDI TOF MS. Therefore, depending on how urgent the characterization is and the tolerance for error, either of these methods can be used.

Structural characterization of oligonucleotides

The most powerful way to resolve the structure of oligonucleotide is using X-ray crystallography which has been indirectly linked to
many Nobel prizes.10 X-rays have a wavelength in the same dimensions as interatomic bonds in molecules of about 1.5 angstrom and can provide highly-detailed structures of oligonucleotides. Through analyzing the scattered X-rays, the electron density distribution in the sample can be determined to reconstruct the internal molecular arrangement. Nevertheless, reconstruction is not always easy due to loss of phase information which has to be resolved using computational methods like Fourier maps. To overcome the problem of loss of phase information, methods to synthesize nucleoside and oligonucleotide analogs with selenium have been developed.11 These methods make use of the heavy atom, selenium, as an anomalous scattering center or reference to enable phase determination.

Another major problem in sample preparation during X-ray crystallography is to prepare the crystalized structures which require great care and is time-consuming. To enhance the probability of crystallization,
racemates have been added to increase the number of molecular contacts.12 Racemic crystal structures of various DNA sequences and folded conformations, including duplexes and quadruplexes have even been demonstrated to be suitable for structure elucidation. RNA crystallography is especially challenging due to paucity of surface chemical diversity and poor flexibility- not ideal for crystal packing. To address this issue, Sherman and colleagues created the Fab (fragments of antibody) chaperon assisted RNA crystallography to facilitate crystal packing and expediate phase determination.13

The other popular method to characterize the structure of oligonucleotides is
nuclear magnetic resonance (NMR).14 The advantage of NMR over X-ray crystallography is that molecules do not have to be crystallized. When a nucleus which is in a strong constant magnetic field is being perturbed by a weak oscillating magnetic field, it responds by emitting an electromagnetic signal with a frequency characteristic of the magnetic field at the nucleus. This happens at the resonance frequency when the oscillation frequency of the external field matches the intrinsic frequency of the nuclei. NMR spectra therefore provide information on structures derived from specific resonance properties of different atomic nuclei in the samples. Depending on the exact goals of the experiments such as resolution, different NMR set-ups are used. For instance, two-dimensional nuclear overhauser effect spectroscopy (NOESY) NMR spectrum can resolve sample structure to a resolution of five angstrom.

Future outlooks

Oligonucleotides are an important resource for diagnostics and therapeutics, but in order for them to be clinically useful without adverse side effects, their synthesis and characterization have to be rigorous. The use of PAGE or LC followed by MS is a crucial step to separate and purify oligonucleotides. However, the structures of oligonucleotides also play an important role in their uses such as exploiting their conformations to mimic viral particles. Therefore, techniques like X-ray crystallography and NMR are essential to elucidate the molecular structure of oligonucleotides which will help inform their structure-function relationship, and to propel the use of molecular design of oligonucleotides to enhance  greater use in  biomedical settings. The characterization of oligonucleotides has also focused largely on genomic DNA, but
RNA modifications play important role in physiology as well. With greater investigations, more RNA modifications and their roles will also be elucidated.


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2.       Veneziano R, Moyer TJ, Stone MB, et al. Role of nanoscale antigen organization on B-cell activation probed using DNA origami. Nature Nanotechnology. 2020;15(8):716-723. doi:10.1038/s41565-020-0719-0.

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9.      Kimura S, Dedon PC, Waldor MK. Comparative tRNA sequencing and RNA mass spectrometry for surveying tRNA modifications. Nature Chemical Biology. 2020;16(9):964-972. doi:10.1038/s41589-020-0558-1.

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