Phosphorodiamidate Morpholino Oligonucleotide (PMO) Characterization

For over a decade, Novatia has offered Routine MS Services for Oligonucleotide characterization. These services have included LC-MS profiling and nucleotide sequencing. With our experience providing complex API sameness characterizations, it is conceivable that NMR fingerprinting and evaluation of aggregation by DLS for PMO products may be required by regulatory agencies in the not-so-distant future.

An example characterization study using MS, DLS and NMR of a 24-mer PMO is detailed below.

LCMS Profiling: 

We offer state of the art UPLC-UV-HRMS methods for detailed impurity profiling of PMO samples. This provides data that is crucial in confirming lot-to-lot consistency as well as providing putative identification of PMO based impurities with high mass accuracy.

MS Characterization: 

Mass Spectrometry (MS) can provide crucial information to help confirm the identity of a PMO. High resolution accurate mass spectrometry (HRAM) will provide confirmatory data to support the PMO base composition (Figure 1).

Figure 1: De-isotoped spectrum of the 24mer PMO by ProMass

Sequence confirmation for standard oligonucleotide species relies on efficient MS/MS fragmentation along the phosphate backbone. While PMO’s have a dramatically different backbone structure from that of a standard oligonucleotide, under the appropriate MS conditions, will still undergo fragmentation along the methylenemorpholino backbone enabling sequence confirmation of the PMO (Figure 2).   

Figure 2: Sequence coverage report for the MS/MS spectrum of a 24mer PMO

Aggregation Characterization: 

Dynamic Light Scattering (DLS) and Diffusion Ordered Spectroscopy (DOSY) NMR analyses can provide a glimpse into the aggregation state of a PMO dependent upon the size. Figures 3-4 show the DLS Autocorrelation function and DOSY NMR Spectrum, respectively.

Figure 3: DLS Autocorrelation Function of 24-mer PMO. *Inset – Intensity histogram.

Figure 4: 2D DOSY NMR Spectrum of 24-mer PMO.

Following the diffusion coefficient correction based on the known coefficient of water in DMSO-d6, the corrected diffusion coefficient of the DOSY spectrum was similar to that measured by the DLS.

NMR Characterization: 

For PMO products, NMR can provide a fingerprint of the solution structure. Figure 5-6 below shows annotated 1D ¹H and ³¹P NMR spectra of the 24-mer PMO.

Figure 5: ¹H NMR spectra color annotated for characterization purposes.

Figure 6: 1D ³¹P NMR spectrum of 24-mer PMO annotated for characterization.

Due to the number of potential stereoisomers available in a PMO, the lack of signal dispersion of the 31P NMR data does not allow for sequence characterization from one phosphorus to the next. This lack of signal dispersion is also visible in 2D ¹H-³¹P HMBC analyses as shown in Figure 7 below.

Figure 7: 2D ¹H-³¹P HMBC of 24-mer PMO with annotation for characterization.

Although different phosphorus groups within the backbone could not be easily identified, the use of ¹H-¹³C HSQC and HMBC experiments, shown in Figures 8-9, could identify the presence or absence of specific bases within the PMO but could not provide any additional sequential information.

Figure 8: 2D ¹H-¹³C HSQC – DEPT edited spectra of 24-mer PMO annotated for characterization.

Figure 9: 2D ¹H-¹³C HMBC spectra of 24-mer PMO annotated for characterization.

Although NMR may not provide direct sequencing of the PMO, the use of NMR can provide identity of the bases present in the sequence. Additionally, future applications of statistical comparisons of 1D and 2D NMR data could provide a means to evaluate PMO product sameness.