Oligonucleotide Characterization

SMART Digest Ribonuclease T1 Magnetic Resin           60120-101
SMART Digest Ribonuclease A   Magnetic Resin            60120-102
SMART Digest Ribonuclease T1 Column                             60140-101
SMART Digest Ribonuclease A   Column                             60140-102

•  Large RNA molecules have emerged as an important new class of therapeutics

• Furthermore, a growing number of cell and gene therapies utilize large RNA molecules to deliver gene editing tools

• It is important to assess quality and repeatability of RNA manufacturing as well as impurities that may be formed during manufacturing and storage

• Enzymatic digestion of large oligonucleotides is required prior to mass spectrometric analysis

• Soluble enzymes over digest large oligonucleotides resulting sequences that are too short to provide a useful characterization

• Solution - SMART Digest Ribonuclease T1 in column and mag bead formats

“The automation of the digestion and analysis drastically reduces the labor, potential human errors, and RNase contamination.” Anal. Chem. 2024, 96, 21, 8674–8681
“This novel approach demonstrated significant improvements in sequence coverage compared to conventional complete RNase T1 digestion.” Anal. Chem. 2022, 94, 7339-7349

 Example protocols

SMART RNase Kits

• Partial RNase digestions were performed using 20-40 μg of RNA incubated with 1.25-5 μL of immobilized RNase T1 (Part no. 60120-101) at either 60 or 37 °C for 2-15 min in a volume of 50 μL of the SMART digest RNase buffer.

• Reactions were stopped by the magnetic removal of the immobilized RNase T1.

• Automated RNase digestions were performed using an automated robotic liquid handling system (KingFisher Duo Prime System, Thermo Scientific)

 SMART RNase Columns (2D-LC workflow)

• The two cartridges were attached to two 6 positions/7 port column selectors (Part nos. 60140-101 and 60140-102). The immobilized RNase cartridge was set in-line with the HILIC column from 0 to 10 min when digesting the mRNA.

• The resulting acetonitrile content after combining the 1D aqueous flow at 0.035 mL/min and 2D mobile phase A containing 97% acetonitrile (v/v) via the mixing tee is greater than 86% (v/v) (a high acetonitrile content is required to retain the digestion products on the 2D HILIC column).

• Mobile phase A contained the RNase digestion buffer composed of 200 mM ammonium acetate in water (unadjusted pH). The cartridge temperature was set at 37 °C, and the flow rate was set at 0.035 mL/min.

• The injection volume was 10 μL (10 μg of mRNA injected into the column). 

 Select Publications

• Vanhinsbergh, C. J., et al. (2022). Characterization and sequence mapping of large RNA and mRNA therapeutics using mass spectrometry. Analytical Chemistry, 94(20), 7339–7349

• Hermon, S. J., et al. (2024). Quantitative detection of pseudouridine in RNA by mass spectrometry. Scientific Reports, 14(1).

• Goyon, A., et al. (2024). Online nucleotide mapping of mRNAs. Analytical Chemistry, 96(21), 8674–8681.

• Goyon, A., et al. (2022). Characterization of impurities in therapeutic RNAs at the single nucleotide level. Analytical Chemistry, 94(48), 16960–16966.