MS sequence maps of mRNA using partial RNase T1

A short digest with bead-bound RNase T1 plus tandem MS covers more than 80% of long mRNA sequences in one run, beating complete T1 maps.

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What coverage does partial immobilized T1 + MS achieve?

>80% sequence coverage of large RNAs and mRNA therapeutics from one analysis.

How does this compare to complete RNase T1 digestion?

Significant improvements in sequence coverage versus conventional complete T1.

Which clinically relevant mRNA was mapped?

mRNA for the SARS-CoV-2 spike protein.

What digest amounts/times are used?

20–40 μg RNA with 1.25–5 μL immobilized T1 for 2–15 min at 37 or 60 °C.

What LC buffers are used for intact mRNA?

100 mM TEAA pH 7.0 vs TEAA with 25% acetonitrile.

What flow/temperature/UV for the RNA gradient?

0.4 mL/min, 50 °C, 260 nm.

Why immobilize T1 on magnetic beads?

Timed partial digestion that can be stopped and automated in plates.

Do random control sequences match?

High coverage with no or low matches against random control RNA sequences.

What else can the workflow flag?

Chemically modified mRNA and impurities.

Why is large RNA mapping hard?

Limited methods exist for RNA >1000 nucleotides, including mRNA vaccines.

How is RNA concentration measured?

NanoDrop absorbance at 260 nm.