Analytical Technologies Accelerating the Research andDevelopment of Oligonucleotide Therapeutics(Ultra-high Performance LC-MS, NMR, and CD Analyses)
In the R&D of oligonucleotide therapeutics, the analysis of impurities is essential for evaluating medicinal
actions properly. The higher-order structure and complex formation of oligonucleotide therapeutics areimportant evaluation items because they probably have impacts on quality. The following are the examples of
the nucleic acid analyses using ultra-high performance LC-MS, NMR, and CD.

Analysis of single-strand phosphorothioate oligonucleotides
using ultra-high performance LC-MS (high-resolution, accurate-mass Orbitrap mass spectrometry)

UV

15 merMass spectrum

14 mer10 merUnknowne.g. Degradants, Impurities

2428m/zTime [min]Sample: a single-strand phosphorothioate oligonucleotides (S-Oligo,5´CACGTTGAGGGGCAT3´) and the 3 kinds of base deleted S-Oligo
equally mixed solution
Conditions：0.6 nM DNA, 5 mM HFIP in MeOH, 5 mM HFIP in Water

Deconvoluted mass spectra
Relative intensity

yIntensit

Relative Abundance

TICC

2015 mer
14 mer12 mer10 mer

4854.4852

Mass

Mass spectrum

16S-Oligo

Deconvoluted mass spectram/z

12 merMonoisotopic mass

Phosphorothioate oligonucleotides (15 mer)Intensity

Relative Abundance

Orbitrap Fusion Lumos

IntactRelative intensity

Analysis of nucleic
acids using highresolution, accuratemass spectrometry

4838.5051

Mass

The peak of an unknown component with a
MW* different from insufficiently separated
intact were successfully detected with the LC.
*: molecular weightCalculated
exact mass
4854.4896
4534.4664
3900.4081
3210.3487

Measuredaccurate mass
4854.48524534.4632
3900.4054
3210.3491

Deviation
(ppm)
-0.9
-0.7
-0.7
0.1

Deviations from the calculated exact
mass were less than 1 ppm!
Estimation of unknown component
structure is also possible bycalculating accurate-mass using
deconvolution tool.
From the comparison of exact mass
and accurate mass, it was presumed
that a part of 15 mer
phosphorothioate oligonucleotides
were not phosphorothiated andremained as oxygen.
Analysis of impurities, estimationof metabolites, and quantitative
determination using accuratemass.

Analysis of double-strand nucleic acids
using Nuclear Magnetic Resonance (NMR)

CD spectrum sensitively reflects the higher-order structure of
nucleic acids. By measuring the CD spectrum while changing
the temperature, the melting process of double-strand
structure by heat is trucked to calculate the melting
temperature (Tm) at which 50% of double-stranded DNAs
dissociate and the double-strand structures turn to single strand
structures.

When 1H NMR spectrum of nucleic acids is analyzed, the signals of
the imino proton forming hydrogen are observed at low magnetic
field of around 12 to 14 ppm.

CD [mdeg]

Analysis of double strand nucleic acids
using Circular Dichroism (CD)

Hydrogen bonds
between base pairs innucleotides

AromaticringRibose

Spectrum change along withtemperature rise at 251 nm

Tm=339 K（66℃）☜ Tm value: spectrum
inflection point

70℃
55℃
40℃

300

320

340

Temperature [K]Sample ：d(CGCGAATTCGCG)2
Conditions：0.2 mg/mL DNA, 100 mM NaCl, 10 mM NaH2PO4, pH=7360

Using the melting temperature (Tm), the heat stability ofnucleic acid higher order structure is evaluated.
Characterization analysis: The Tm of nucleic acids
changes dependent on the sequence andconcentration of nucleic acids, and solvent, and it is
an important evaluation item to characterizeoligonucleotide therapeutic products.

25℃
10℃

Sample：d(CGCGAATTCGCG)2
Conditions：10%D2O, 8.0 mg/mL DNA, 100 mM NaCl,
10 mM NaH2PO4, pH=7; 500MHz

Characterization/Structur
al Analysis: Observation of
the signals of the imino
proton forming hydrogenbonds enables todetermine the presence ofdouble strands and theequivalence of higherorder structure.

Formation of double strands
can be judged by signal observation.
As temperature rises, more hydrogen bonds cleave, and the double
strand dissociation ratio of nucleic acids rises. The imino proton
signals disappear at a temperature higher than the Tm value ofrespective nucleic acid.
P02006バイオメディカル分析室20190802-2