Oligonucleotide synthesis is a complex process that requires more than one hundred sequential chemical reactions to make a single, 25-base sequence. Contemporary synthesis chemistry is robust and modern synthesis platforms are reliable and highly automated. Still, each oligonucleotide synthesized at IDT is evaluated for quality before shipping to ensure that the correct sequence was made. The best method available to assess compound identity in a high throughput environment is mass spectrometry (MS).
What is mass spectrometry?
In MS analysis, a small amount of a synthesized oligonucleotide is ionized and the ions are propelled into a mass detector/analyzer where molecular weight is measured. The analysis compares the calculated molecular weight of the given sequence to the measured molecular weight. Two methods of mass spectrometry are routinely used. These are MALDI−TOF (matrix-assisted laser desorption ionization−time of flight) and ESI (electrospray ionization) MS. (Read more about these MS methods in the Technical report, Mass spectrometry analysis of oligonucleotide syntheses).
Interpreting MS results
MALDI-TOF (Figure 1) and ESI (Figure 2) MS results both have a main peak representing the synthesized oligonucleotide. MS analysis at IDT can detect deletions, additions, or substitutions. IDT will remake oligonucleotides with a significant amount of these products, or with significant secondary peaks. However, there can be, and often are, additional peaks present in the final traces. During MALDI-TOF analysis, depurination of the oligonucleotide can occur as a result of heating (laser ionization) in an acidic environment (the matrix). Depurination also can occur during ESI analysis because of heating in the transport region of the ESI instrument.
Figure 1. MALDI-TOF mass spectrogram of a 22 nt oligonucleotide. The synthesized sequence is shown at the bottom and the expected and observed molecular weights (Da) are presented above the sequence.
Figure 2. ESI mass spectrometry trace (raw data) of an HPLC-purified, 60 nt oligonucleotide with a 5’ spacer modification. Multiple peaks are seen at different m/z ratios, representing various amounts of deprotonation.
Depurination can create secondary peaks having approximately 135 (dA) or 151 (dG) mass units less than the major peak. These species are created by the process as the sample is measured, but are not present in the product itself.
In addition, synthetic oligonucleotides made using phosphoramidite chemistries employ protecting groups on the primary amines in dA, dC, and dG phosphoramidites in order to prevent branching and other undesired side reactions during chain elongation. Protecting groups are cleaved off post-synthetically during the final steps. Incomplete removal of these side groups results in additional masses that are easily detected by MS.
Finally, oligonucleotide modifications add mass to the product. Modifications commonly used in oligonucleotides are well characterized and their masses are taken into account in the final mass spectrograms produced. A list of mass contributions for the most often requested modifications of DNA and RNA oligonucleotides are listed on the IDT Modifications page. Further, the anhydrous molecular weight of both unmodified and modified oligonucleotides can be calculated using OligoAnalyzer Tool.