Structure-specific ribonucleases for MS-based elucidation of higher-order RNA structure

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• High-throughput sequencing technologies enable genome-wide nucleic acid structure mapping.
• Structure-specific nucleases are valuable biochemical probes for this purpose.
• Mung bean (Mb) and V1 nucleases selectively target single- and double-stranded nucleic acid regions.

Purpose of the Study:

• To evaluate Mung bean (Mb) and V1 nucleases as structural probes combined with mass spectrometry (MS) detection.
• To explore the benefits and limitations of these nucleases for genome-wide nucleic acid structure analysis.
• To assess the compatibility of these enzymes with ammonium-based solutions for electrospray ionization (ESI).

Main Methods:

• Application of Mung bean (Mb) and V1 nucleases to nucleic acid substrates.
• Analysis of enzymatic cleavage products using direct infusion electrospray ionization (ESI) and tandem mass spectrometry (MS/MS).
• Investigation of factors influencing cleavage accuracy, including ionic strength and Mg(II) presence.

Main Results:

• Both nucleases functioned in ammonium-based solutions compatible with ESI-MS.
• MS/MS confirmed mapping assignments but revealed ambiguities in distinguishing isobaric products.
• Ladder-type product series aided region assignment but showed uncertainty at paired/unpaired boundaries.
• Structural stabilization factors improved accuracy but did not eliminate deviations.

Conclusions:

• Extreme caution is advised when interpreting results from Mb and V1 nucleases for nucleic acid structure probing.
• Potential artifacts necessitate repeating experiments under diverse conditions to increase confidence in structural data.
• These nucleases, while useful, require rigorous validation for reliable genome-wide structure mapping.